w ‘ ----m-----__—= - P-GLYCOLATE PHOSPHATASE AND ACONTTASE FROM TOBACCO LEAVES Thesis for the Degree of Ph. D. MTCHIGAN STATE UNIVERSITY DONALD E. ANDERSON 1969 jnfiSTb 0-169 h'uia»..&;.L- _ .. 1 ‘ t it LIB R x.’ K 1" Michigan Stat-c Ulll'v'Clb ty r . ~ "FM-":- '1»- Inn-- This is to certilg that the thesis entitled P-GLYCOLATE PHOSPHATASE AND ACONITASE FROM TOBACCO LEAVES presented by DONALD E. ANDERSON has been accepted towards fulfillment of the requirements for £129;— degree in Ms C r Y 1 mm Major professor Date February 19, 1969 Imam? ammunm'fic. 66in...“ fun-i}. ABSTRACT P-GLYCOLATE PHOSPHATASE AND ACONITASE FROM TOBACCO LEAVES by Donald E. Anderson In eXploratory studies from this laboratory (unpub- lishedL it was found that P-glycolate phOSphatase from wheat leaves was associated with cis-aconitate, which stabilized the enzyme. Citrate or isocitrate also stabil— ized the phOSphatase from wheat. The investigations described here extend these observations to the phoSpha- tase from tobacco leaves, and in addition are concerned with relationships between the phOSphatase, aconitase, and the tricarboxylic acids. P-glycolate phOSphatase from tobacco leaves was purified 1000 fold to a Specific activity of 333 umoles of substrate hydrolyzed per minute per mg of protein. During the last purification step, which was gel filtra- tion chromatography, the phOSphatase became unstable, as determined by heating the enzyme to 45° for 1 hour. It could be restabilized with fractions which emerged after the phoSphatase from the column. The endogenous stabil- izing factors in these fractions were identified as citrate, isocitrate, and cis-aconitate. Commercial Donald E. Anderson - 2 citrate, isocitrate, and cis-aconitate, as well as trans- aconitate, also stabilized the enzyme, but mono and di- carboxylic acids did not. Cis-aconitate was found to be an inhibitor, com— petitive'with reSpect to P-glycolate, of the phOSphatase. inn apparent KI for cis-aconitate was 5.0 x 10'3M when +2 +2 the Hg was 2 x 10’31'1. Increasing the Mg '2 to 2 x 10 M. with cis-aconitate at 10'2M, did not significantly affect the apparent K The data suggest that cis-aconitate did I' not cause inhibition by complexing the Mg+2 required as an activator of the phoSphatase. In fractions from the next-to-last purification step, which was DEAE-cellulose chromatography, one of the two isocitrate peaks approximately coincided with the phosphatase peak. .At this stage of purification, approxi- mately 1 mole of isocitrate per 10 to 15 moles of amino acid of the phOSphatase fractionated with the enzyme. Furthermore, comparison of the sizes of the Spots on paper chromatograms suggests that more citrate than isocitrate fractionated with the phosphatase. In most exPeriments, aconitase and P-glycolate phosphatase fractionated in parallel. However, when field grown tobacco leaves were kept in darkness for 3 to 4 rmmrs before harvest and homogenization. the phOSphatase and.aconitase were separated during their purification. In the latter case, isocitrate no longer fractionated with Donald E. Anderson - 3 tmm phOSphatase. The evidence suggests that the fraction- ation of the endogenous tricarboxylic acids with the phos- phatase was closely related to the fractionation of aconi- tase with the phOSphatase. Although it is not known whether the parallel frac- tionation of the two enzymes was artifactual or of physio- logical significance, it is of interest that the tricar- boxylic acids are stabilizers of aconitase, and that any one tricarboxylic acid would be eXpected to competitively inhibit the interconversion of the other two. Thus, aconitase and P-glycolate phOSphatase seem to possess at least two similarities, i.e. stabilization by the tricar- tmxylic acids and competitive inhibition by cis-aconitate. Partial purification and characterization of aconi- tase from tobacco leaves are described. In particular, partially purified aconitase was inactivated by Sephadex G-25 chromatography and was reactivated by fractions which emerged after aconitase from the column, or by sulfates or chlorides. The activation of aconitase by sulfates or chlorides is consistent with the concept that the enzyme may possess groups, other than at the active center, capable of binding citrate, isocitrate, or cis-aconitate. In the course of the above investigations, it was discovered that P-glycolate phosphatase in fresh extracts was unstable toward dilution.of 20 to 30 fold at 30° for 1 hour, and that under aerobic conditions, the enzyme in .p. r,. R.- on Donald E. Anderson- 1+ the extracts slowly became stable toward this dilution at 30°. A variety of treatments of the extracts including mixing under air or 02 (but not N2 or 002), or a 10 minute preincubation with 10'2M c-hydroxy-2-pyridinemethanesul- fonate, a specific inhibitor of glycolate oxidase, quickly and completely stabilized the phOSphatase toward dilution at 30°. The stabilized enzyme could be partially and rapidly reconverted to the unstable but Just as active enzyme by preincubation of the oxygenated extracts with glycolate. The sulfonate completely inhibited, while anaerobic conditions enhanced, this glycolate dependent conversion. Glycolate also inhibited the stabilization toward dilution at 30° of the phosphatase by 02. The data suggest that the phoSphatase can exist in an oxidized or in a reduced state,and that in the extracts, glycolate oxidase could be involved in the interconversion between the two state 8 . P-GLYCOLATE PHOSPHATASE AND ACONITASE FROM TOBACCO LEAVES By .. Ii .0 ‘ ‘ Donald EEOAnderson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1969 Dedicated to my wife, Adrienne 11 ACKNOWLEDGMENTS I thank Dr. N. E. Tolbert for the thesis problem, and for his guidance, support, and patience during the course of the work. I acknowledge the technical help received from D. Glen Aitken, Ruth Allen, and P. L. Young- blood. The support for a period of three years of the National Science Foundation in the form of a Graduate Fellowship is acknowledged. I thank my mother, father, brothers, and sister for their financial help, interest, and patience during this research program. 111 TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . IJTEBATURE REVIEW . . . . . . . . . . . . . . . . . Glycolate Biosynthesis . . . . . . . . . . . Photosynthetic C02 Fixation . . . . . . . . Glycolate from C02 . . . . . . . Effect of Light on the Biosynthesis of Glycolate . . . . . . . . Effect of Oxygen on the Biosynthesis of Glycolate . . . . . . . . . . . 8 Effect of CO on the Biosynthesis of Glycolate . . . . . . . . . . . . . . 10 -o \acnp er he Warburg Oxygen Effect . . . . . . . 10 Glycolate From the Photosynthetic Carbon Cycle . . C O O O O O O O O O O O O 0 1“ P-Glycolate as the Precursor for Glycolate . 25 G13colate from Isocitrate . . . . . . . . . 26 Glycolate from Acetate . . . . . . . . . 29 The de Novo Synthesis of Glycolate from 30 Co . g Q 0 O O 0 O Similarities Between 02 Evolution and Glycolate Formation . . . . . . . . . . . 33 The Glycolate Pathway . . . . . . . . . . . . . . 34 P-G1y001ate Phosphatase o o o o o o o o o o o o 37 Inhibition and Activation of Various Enzymes by the Tricarboxylic Acids . . . . . . . . . . #2 Aconitase O O o o o o o o o o o o o o o o o o o [4'8 Introduction and History . . . . . . . . . . #9 The Function of Aconitase . . . . . . . . . 49 The Intracellular Location of Aconitase . . 51 The Possible Role of Aconitase in Metabolic Control . . . . . . . . . . . . 55 The Purification of Aconitase . . . . . . . 60 Evidence that Aconitase is One Enzyme . . . 62 The Effect Of Ions . o o o o o o o o o o O 0 64 PH Optimum , , . . . o . 65 The Effect of Metals, Chelating, and 6 Bedu01ng Agents 0 o o o o o o o O 0 0 ° ° 5 Inhibitors . . . . . . . . . . . . . . 7i Substrate Specificity . . . . . . . . . 7 Binding of Tricarboxylic Acids to 4 Aconitase . . . . . . . . . 7 Th6 K Values for the Substrates and 5 th KI for Trans-aconitate . . . . . . . 7 iv Substrate as a Competitive Inhibitor of Aconitase . . . . . . . . . . . . . . The Mechanism of Act on of Aconitase . . HATER IAI-s AND METHODS O O O O O O O O O O O O 0 Plants 0 O O O O O O O O O O O O I Preparation of Ex racts fro Leaves Measurement of Light Intensities . Water and Temperature . . . . . . . The Gassing of Enzyme Preparations Mixing of Enzyme Preparations by "Bu NaCl Determinations . . . . . . . 3 Protein Determinations . . . . . . 260/280 Method . . . . . . . A280 MethOdo o o o o o o o o o o o O Lowry's Modified Folin-Ciocalteu Method Inorganic PhOSphate by a Modified Method of Fiske and Subbarow . . . . . . . . . . . . Inorganic Phosphate by an Isobutanol Benzene Extraction Method . . . . . . . zin oooNooooo é" Determination of Total Phosphorous . . . . . Determination of Glycolic Acid . . . . . . . Enzyme Assays . . . . . . . . . . . . . . . . Standard P-glycolate PhoSphatase Assay . Aconitase Assays . . . . . . . . . . . . Recovery and Yield . . . . . . . . . . . The Stability of P-glycolate PhOSphatase at H The Stability of P-glycolate PhOSphatase towa Dilution at 30° 0 o o o o o o o o o 0 I O The Enzymatic Determination of Isocitrate . . Paper Chromatography . . . . . . . . . . . . General 0 O O O O O O O O p .. O O O O O Solvent Systems . . . . . . . . . . . . Detection of Compounds on Paper . . . . Purity and Characterization of P-glycolic A01 RESULTS AND DISCUSSION 0 o o o o o o o o 0‘ O 0 0 The.Adequacy of P-glycolate PhOSphatase . . . Influence of Leaf Size and Position on P-8137colate Phosphatase Activity . . . . . Effect of the Homogenization Method on P‘slycolate PhOSphatase . . . . . . . . . Stability of P-glycolate PhOSphatase as a Function of pH . . . The Purification of P-glycolate PhOSpha ass and the Stability of the PhOSphatase at the Different Stages of Purification . . . . . Extractooooooooooooooo'O HUI 9. 00000000 Page 118 119 120 122 127 127 128 129 133 137 138 Extract . . . . . . . . . . . First Acetone Fractionation . Second Acetone Fractionation Third Acetone Fractionation . DEAE-Cellulose Chromatography Bio-Gel P-60 Chromatography . . Rechromatography on DEAE—Cellulose Discussion of the Purification Procedures . . First Acetone Fractionation . . . . . . . . 139 Second Acetone Fractionation . . . . . . . 1&0 Third Acetone Fractionation . . . . . . . . 140 DEAE-Cellulose Chromatography . . . . . . . 1A2 Concentration of the Pooled Fracti ns . . . . 142 Bio-Gel P-60 Chromatography . . . . . . . . 143 c P-glycolate PhOSphatase . . . . . 148 o The Best Single Purification Sequen e for O O O O O O O O C O O 0 O O O O O H 4: \o 154 00000000.... Further Data and Observations on the Purificati n of P-glycolate Phosphatase . . . . . . . . . 158 Preparation of Acetone Powders . . . . . 158 Observations on the Acetone Fractionations . 160 Optimum pH for DEAE-Cellulose Chromatog- raphy O O O O O O O O 169 Suspected Inactivation of P-glycolate PhOSphatase by Metal . . . . . . . . 170 The Effect of Preincubation with Mn++, Fe++, or Fe+++. . . . . . . . . 171 Gel Filtration Chromatography . . . . . . . . 173 Protein Determinations . . . . . . 174 Stability of P-glycolate PhOSphatase. toward Freezing and Thawing . . . . . . . 175 Stability of P-glycolate PhOSphatase toward Dilution at 30° and toward Dialysis . . . . . . 176 Storage Characteristics of P-glycolate. PhOSphatase . . . . . . . . . . 177 Purification by Other Methods . . . . 178 Identification of the PhoSphatase Stabilizing FaCtors O O I O O O O O O O O O O O O O O O O I 181 Purification . . . 181 Identification of Tricarboxylic Acids . . . . 183 Stabilization of the PhOSphatase by the Tri- carboxylic Acids . . . . . . . . 192 Coincidence of the Tricarboxylic Acids with the PhOSphatase in Fractions from DEAE- Cellulose . . . - . . . 195 Competitive Inhibition of the PhOSphatase by Cis-aconitate . . . . . - . . . 202 Discussion of the P-glycolate PhOSphatase, Tricarboxylic Acid Relationship . . . . . . . . 206 vi Investigations on the Stability Toward Dilution at 30° of P-glycolate PhoSphatase in Fresh Extracts from Tobacco Leaves . . . . . . . . . Stabilization by Time After Homogenization . Stabilization as a Diurnal Function . . . . Stabilization by Mixing in a Waring Blend-or O O O O O O O O O O O O O O O 0 Stabilization by Oxygen . . . . A Diurnal in the Stabilization by Buzzing Effect of Sephadex G- 25 Chromatography . Stabilization by Acetone Precipitation . Effect of Dilution . . . . . . . . . . . Effect of Metals . . . . . . . . Effect of OHPMS and of Glycolate . . . . Protection of the Phosphatase by Cis- aconitate . . . . . . . . . Effect of Arsenite and Cd++ . . . . . . . Reversal of the Stabilized PhOSphatase to an Unstable Enzyme . . . . Discussion of the Stability Toward Dilution at 300 of P-glycolate PhOSphatase in Fresh Extracts from Tobacco Leaves . . . Aconitase, and Relationships Between Aconitase and P-glycolate PhOSphatase . . . . . . . . . pH Optimum . . . . . . . . . . . . . . . . . Activators . . . . . . . . . Relative Reaction Rates with Citrate or Isocitrate . . . . . . . . . . . . . . The Adequacy of Aconitase . . . . . . . . . The Fractionation of Aconitase and P-glycolate PhOSphatase . . . . . Evidence for Aconitase Activity . . The Stability of Aconitase . . . . . Partial Reactivation of Aconitase . The Effect of Divalent Metal Ions on the Activity of Aconitase . . . . . . . . . . Speculation about an Endogenous Aconitase Inhibitor(s). . . . . . Similarities Between the Substrates and Reaction Mechanisms of P—glycolate PhOSphatase and Aconitase . . . . . . . . Discussion of Similarities, Differences, and Relationships Between Aconitase and P-glycolate PhOSphatase . . . . . . . Further Observations on the Stability of P-glycolate PhOSphatase . . . . . . . . . . . SUMMARY 0 o o o o o o o o o o o o o o o o o 0 o O o BIBLIOGRAPHY o o o o o o o o o o o o 0 0 0 ' 0 ' ' ° Page 208 208 211 215 215 218 222 225 225 229 232 237 240 242 251 278 278 281 289 289 290 308 310 321 333 334 337 3112 358 361 365 Table No. 1 10 11 12 13 14 LIST OF TABLES Influence of Leaf Size and Position on P-glycolate PhOSphatase Activity . The Effect of Dilution at 0° and 30° . Purification and Stability of P-glyco- late Phosphatase . . . . . . . . . Purification of P-glycolate PhOSphatase The Acetone Concentration Required to Precipitate P-glycolate PhOSpha- tase as a Function of Post Homog- enization Time . O O O O O O O O O The Acetone Concentration Required to Precipitate P-glycolate PhOSpha- tase as a Function of pH . . . . . Improvement in Substrate Specificity by the First Acetone Fractionation . The Effect of Preincubation with Mn++, Fe++, or Fe+++ on the Activity and Stability of P-glycolate Phos- phatase . . . . . . . . . . . . . PhOSphatase Stabilizing Factor(s) in the Ether Extracted Fraction . . . Identification of Tricarboxylic Acids . Effect of MgSO on the Stability of the PhOSphatase . . . . . . . . . . . Effect of High Concentrations of Mgsou on the PhOSphatase . . . . . . . . A.Diurnal in the Stabilization by Buzz ins o o o o o o o o o o o o 0 Effect of Dilution on the Stability of the Phosphatase . o p o o o o o 0 viii Page 130 134 144 157 164 166 168 172 184 186 195 206 221 228 Table No. 15 Effect of EDTA or Orthophenanthroline 16 Activation of the PhOSphatase by Arsenite and Cd++. . . . . . . . 1? Correlation Between the Extent of the Reversal of the Stabilized Phos- phatase to the Unstable Enzyme, and the Extent of the Stabiliza- tion 0 O O O O O O O O 0 O O O 0 1x 246 LIST OF FIGURES Figure No. Page 1 The Stereochemistry of the Aconitase Reactions . . . . . . . . . . . . . . 78 2 Inorganic PhOSphate by the Isobutanol Benzene Extraction Method . . . . . . 99 3 P1 Released in the Standard P—glycolate PhOSphatase Assay as a Function of the Concentration of Enzyme . . . . . 104 4 The Effect of Mg“ on P-glycolate Phos- phataseooooooooooooooolorz 5 Initial Reaction Rates for Aconitase as a Function of the Concentration of Enzyme O O O O O O O O O O O O O O O 111 6 Inactivation of P-glycolate PhOSphatase . 115 7 Stability of the PhOSphate-Glycolate Bond of P-glycolic Acid . . . . . . . 125 8 Effect of the Homogenization Method on the Stability Toward Dilution at 30° of P-glycolate PhoSphatase . . , 131 9 Stability of P-glycolate PhOSphatase in Extracts as a Function of pH . . . . 135 10 Resolution of P-glycolate PhOSphatase into an Unstable PhOSphatase Frac- tion and a Stabilizing Fraction during Bio-Gel P-60 Chromatography 146 11 Final DEAR-Cellulose Chromatography of P-glycolate PhOSphatase . . . . . . . 155 12 The Arrangement Used for Making Large Scale Acetone Fractionations . . . . 162 13 Identification of the Tricarboxylic Acids in the n-Butanol-Ethyl Acetate-Formic Acid System . . . . . 188 Figure No. Page 14 Identification of Citric and Isocitric A01d8 O O O O O O O O O O O O O O O O 190 15 Stabilization of PhOSphatase by Tricar- boxylic ACIdS O O O O O O O O O O O O 193 16 Isocitrate and the PhOSphatase in Frac- tions from DEAE-Cellulose . . . . . . 197 17 A.Magnified Plot of Figure 16 . . . . . . 199 18 Competitive Inhibition of the PhOSpha- tase by Cis-aconitate . . . . . . . . 204 19 Stabilization by Time After Homogeniza- tion 0 O C O 0 O O O O O O O O O O O 209 20 Stabilization as a Diurnal Function . . . 212 21 Stabilization by Mixing in a Waring Blendor O O O O I O O O O O O O O O O 216 22 Stabilization by Oxygen . . . . . . . . . 219 23 Effect of Sephadex c-25 Chromatography . . 223 24 Stabilization of the PhOSphatase by Acetone Precipitation . . . . . . . . 226 25 Effect of OHPMS and of Glycolate . . . . . 233 26 Effect of Glycolate and Time After .Homogenization . . . . . . . . . . . 235 27 Protection of the PhOSphatase by Cis- aconltate o o o o o o o o o o o o o o 238 28 Reversal of the Stabilized PhOSphatase to an unStable EnZyme o o o o o o o o 243 29 Reversal of the Stabilized Phosphatase to the Unstable Enzyme as a Func- tion of the Preincubation Time with Glycolate, and the Time Dependent Restabilization of the PhOSPhatase.............252 30 A Model for the Stabilization by Oxida- tion of P-glycolate PhOSphatase. and for the Conversion by Glyco- late of the Stable Phosphatase to 265 the Unstable Enzyme . . . . . . . . . xi Figure No. Page 31 Activation of Aconitase by MgSO or by Factor(s) Removed by Sepha ex G-1O Column Chromatography . . . . . . . . 282 32 Activation of Aconitase by Sulfates or Chlorides . . . . . . . . . . . . . . 284 33 Activation with Citrate, Isocitrate, or Cis-aconitate as Substrate . . . . . 286 34 Acetone Fractionation of P-glycolate Phosphatase and Aconitase (Leaves Harvested from the Field During Daylight) O O O O O O O O O O O O O O 293 35 Acetone Fractionation of P-glycolate PhOSphatase and Aconitase (Leaves Harvested from the Field During the Night) 0 o o o o o o o o o o o o 295 36 Acetone Fractionation of P-glycolate PhoSphatase and Aconitase from Swiss Chard Leaves . . . . . . . . . 297 37 DEAE-Cellulose Chromatography of P-glycolate PhOSphatase and Aconi- tase (Leaves Harvested from the Field During Daylight) . . . . . . . 299 38 DEAR-Cellulose Chromatography of P-glycolate PhoSphatase and Aconi- tase (Leaves Harvested from the Growth Chamber During the Dark) . . . 302 39 DEAE-Cellulose Chromatography of P-glycolate PhOSphatase, Aconitase, and Isocitrate (Leaves Harvested From the Field During the Night) . . 305 40 Inactivation Rate of Aconitase . . . . . . 312 “1 Effect of Gasses of the AtmoSphere on Aconitase o o o o o o O o o o o o o o 315 42 The Effect of the Precipitation by Acetone on Aconitase and P-glyco- 8 late Phosphatase o o o o o o o o o o 31 “3 Reactivation of Aconitase as a Function of the pH During Preincubation . . . 324 xii Figure No. 44 Reactivation of Aconitase as a Function of Cysteine During Preincubation . 45 Reactivation of Aconitase as a Function of Preincubation Time . . . . . . 46 Reactivation of Aconitase as a Function of the Iron Concentration During Preincubation . . . . . . . . . . 47 Similarities Between the Substrates and Reaction Mechanisms of P-glycolate PhOSphatase and Aconitase . . . . xiii Page 326 328 330 338 BAL CM-cellulose DCMU DEAE FDP OHPMS PGA RuDP SDP TEAE TES TPP LIST OF ABBREVIATIONS British anti-lewisite; 2,3-dimercapto-1- propanol carboxy methyl cellulose 3-(3,4-dichlor0phenyl)-1,1-dimethylurea diethyl amino ethyl fructose-1,6-diph03phate c-hydroxy-Z-pyridinemethanesulfonate; 2-pyridylhydroxymethanesulfonate 3-ph08phoglycerate ribulose-1,5-diph08phate sedoheptulose-i.7-diph03phate triethyl amino ethyl N-tris (hydroxymethyl) methyl-Z—amino- ethanesulfonic acid thiamine pyrophOSphate xiv INTRODUCTION As elaborated in the literature review, under cer- tain environmental conditions, a substantial percent of the newly fixed 14 C02 appears as glycolate and products of the glycolate pathway during photosynthetic CO2 fixa- tion. As further elaborated in the literature review, although the source of glycolate remains unknown, circum- stantial evidence suggests that glycolate comes from the photosynthetic carbon cycle, that P-glycolate should be. the precursor of glycolate, and that P-glycolate phospha- tase should be the enzyme which catalyzes the conversion of P-glycolate to glycolate 13.1112. Purification of P-glycolate phOSphatase from tobacco leaves by (NH4)2804, calcium phosphate gel, and DEAE- cellulose chromatography yielded an enzyme Specific for P-glycolate (202). Because some of the purification steps proved troublesome, a different procedure was developed based on acetone precipitation followed by DEAR-cellulose chromatography (265). When purified in this way, the phosphatase from wheat was associated with cis-aconitate, which stabilized the enzyme. Commercial citrate or iso- citrate also stabilized the wheat enzyme (Tolbert and Yu, unpublished data). The investigation described in this thesis was 2 inaugurated for the further purification of the phOSpha— tase from tobacco leaves, and for a study of the relation- ship between the enzyme from tobacco leaves and endogenous tricarboxylic acids. The study of the latter relationship led to a study of a possible relationship between P-glyco- late phosphatase and aconitase from tobacco leaves. As discussed in the literature review, at least five other relationships may exist between glycolate metabolism and the metabolism of the tricarboxylic acids. These relationships, which are listed below, make the study of the relationships between the phOSphatase and the tricarboxylic acids and between the phOSphatase and aconitase of greater interest. 1. Citrate may be important for the inhibition of phoSphofructokinase, which should be inhibited during the gluceogenic flow of carbon through glycolate to sucrose. 2. Evidence is reviewed which suggests that glyco- late or glyoxylate may participate in inhibition of the tricarboxylic acid cycle. 3. The concentrations of d-ketoglutarate and glutamate build up significantly in tobacco leaves in the light, and it may be that these acids function catalytically in the conversion of glyoxylate to hydroxypyruvate. 4. .Although isocitrate is not thought to be a pre- cursor of glyoxylate in the leaves of higher plants, it is in algae under certain conditions. The reverse, that 3 glyoxylate may be a precursor of isocitrate and glutamate in tobacco leaves, is discussed. 5. Malate dehydrogenase has been found in leaf peroxisomes, which are concerned with the metabolism of glycolate and related compounds. During the course of the investigations, it was discovered that oxygen had a pronounced effect on the stability of P-glycolate phOSphatase. The investigation of the effect of OXygen on the phOSphatase was of particu« lar relevance because, as outlined in the literature review, of the known requirement for 02 for the synthesis of P-glycolate and glycolate, and possibly for the excre- tion of glycolate from the chloroplast. LITERATURE REVIEW Glycolate Biosynthesis Some of the research described in this thesis con- cerns relationships between the tricarboxylic acids and P-glycolate phosphatase, and between oxygen and the enzyme. This first section of the literature review represents an attempt to at least partially discuss some questions which were suggested by some of the results described in this thesis. Some of these questions are as follows. In tobacco, could glycolate come from the tricarboxylic acids and in particular from isocitrate? What is the most prob- able source of glycolate? What effect does oxygen have on the synthesis of glycolate and on C0 fixation? Might 2 such effects be related to the observed effect that oxygen had on P-glycolate phOSphatase? Is the phOSphatase neces- sary for the synthesis of glycolate? Photosynthetic CO9 Fixation The status of the classical photosynthetic carbon cycle has been reviewed by Bassham (15). There has been further clarification of the details, but no alteration of the framework of the classical cycle as originally con- ceived (260). 5 Recently. certain higher plants have been character— ized as having a Cu-dicarboxylic acid pathway in which the first products of 114C02 fixation are dicarboxylic acids. P-pyruvate is carboxylated to give C4 labeled oxalacetate which is in rapid equilibrium with aSpartate and malate. It is thought that the newly incorporated 1I‘I'COZ is trans- ferred from one of these dicarboxylic acids to an unknown acceptor molecule to give labeled PGA. A cyclic mechanism involving oxalacetate, pyruvate, and P-pyruvate is thought to substitute for ribulose diphosphate carboxylase, which is present in inadequate amounts in these plants. The subsequent Spread of label from PGA follows a pattern con- sistent with the Operation of the classical photosynthetic carbon cycle. In these plants, enzymes of the latter cycle, other than ribulose diphOSphate carboxylase, are present in adequate amounts. The high activity of P- ribulokinase in these plants is indicative of a role for RuDP, but whether it functions as an acceptor for the transcarboxylation reaction, which has been prOposed, remains to be determined (109, 110, 111. 112, 221, 222). Besides low ribulose diphOSphate carboxylase activ- ity. plants in which the Cu-dicarboxylic acid pathway represents the main carboxylation mechanism during photo- synthesis are characterized by high activities of P-pyru_ vate carboxylase and.P-pyruvate synthetase, a unique type of leaf anatomy, and high rates of photosynthetic CO2 .6 fixation (109). Most of the plants characterized by the Cu-dicarboxylic acid pathway belong to one of two main subgroups of Gramineae. Although these plants are mono- cotyledons, not all monocotyledons contain the Cu-dicar— tmxylic acid pathway. Furthermore, several dicotyledons lmwe recently been found to contain this new method of C02 fixation (109, 111). The dicotyledonous plant Nicotiana (tobacco), which was used for most of the investigations of this thesis, fixes CO2 by the classical photosynthetic carbon cycle (111, 117). If the dicarboxylic acid pathway is considered a modification of the photosynthetic carbon cycle, then with but few exceptions, this cycle is the only demon- strable mechanism for the total de novo synthesis of com- pounds from 002. The fluctuations in the level of ribulose diphosphate carboxylase activity with autotrophic and heterotrophic conditions, as well as the loss of C02 fix- ing ability in a mutant of Chlamydomonas reinhardii devoid of the carboxylase, are strong evidence for its obligatory role in organisms which contain the classical photosynthe- tic carbon cycle (94, 260). PGA and the sugar phOSphates clearly account for most of the 1LLC found in individual compounds following a few seconds of photosynthesis with 14002. Nonetheless, as pointed out by Bassham, one might ask whether or not other important pathways of C02 reduc- tion not involving these compounds have been overlooked. 7 Such a pathway would have to include substances which are so small in concentration as not to be seen, or which are so unstable as:not to be isolated by the methods of paper chromatography. These possibilities were tested by Bassham and Kirk, who compared the rate of uptake of external 11+COZ with the rate of appearance of lLAC in indi- vidual compounds. With Chlorella pyrenoidosa, it was found that labeling of PGA and the sugar phosphates l4C accounted for at least 70% of the externally measured uptake between 10 and 40 seconds after the introduction of 14002. The small pool of 11+C which was not accounted for was not more than the equivalent of 5 seconds of photosynthesis. Bassham suggests that this small pool was intracellular C02 and enzyme-bound C02. If an unknown path to carbohydrates through a pool of such small Size existed, the carbohydrates would have become labeled much more rapidly than the eXperiments Show (16). Glycolate from 009 The source of glycolate formation during CO2 fixa- tion in photosynthesis remains unknown, although under certain conditions over 50% of the total fixed carbon may in passing through this metabolite (117, 233). Effect of Light on the Biosynthesis of Glycolate High light intensities are required for maximum 8 glycolate synthesis (233). It is not synthesized in the dark by tobacco (268) or excreted in the dark by Chlorella pyrenoidosa (120). Effect of Oxygen on the Biosynthesis of Glycolate Evidence has been presented that the biosynthesis of glycolate by Chlorella pyrenoidosa is a function of the concentration of externally applied oxygen (21, 26, 255). For this alga, the rate of glycolate synthesis, which was nearly zero under anaerobic conditions,1 was found to be an approximately linear function of the concentration of externally applied oxygen from 0% to 99.97% (21, 255). The synthesis of P-glycolate was also stimulated by exter- nally applied oxygen (21). Whittingham et al (256) reported that the synthesis of glycolate in the light by Chlorella from radioactive glucose was stimulated 10 fold in 99.97% 02 compared to the control in air. Tolbert and 2111 (238) found that Chlorella pyrenoidosa would synthe- size but not excrete glycolate under nitrogen1 while it would synthesize and excrete glycolate under a 99:1 mix- ture of N2 and 02. Their data suggest that the excretion of glycolate may itself require aerobic conditions. The requirement of oxygen for the excretion of glycolate by 1Since these experiments were conducted in the light, some oxygen must have been present from photosynthesis. Therefore,complete inhibition of glycolate synthesis could not be eXpected. ' 9 Chlorella.pyrenoidosa was confirmed by Miller et al (161) and Hess et al (120), but these latter eXperiments did not indicate whether OXygen is required at the site of excre- tion as well as at the site of synthesis. Some direct evidence has also been presented which suggests that externally applied 02 is necessary for the synthesis of glycolate in the leaves of higher plants. Using OHPMS, an inhibitor of glycolate oxidase, Zelitch and Walker (274) demonstrated that the accumulation of glycolate in Nicotiana tabacum was greatly enhanced in air as opposed to N2 containing 0.03% 002. The authors concluded that an anaerobic atmosphere inhibits the synthesis of glycolate in tobacco leaves (125). Data on photoreSpiration indirectly suggest that oxygen is required for the biosynthesis of glycolate in the leaves of higher plants. PhotoreSpiration (defined as the light dependent evolution of C02) exhibits a depen- dence on the concentration of externally applied oxygen, falling off to nearly zero at an oxygen concentration of zero (88). Contrary to dark respiration which is essen- tially saturated at very low oxygen partial pressures, photoreSpiration is not saturated even in an atmosphere of 100% oxygen. In tobacco, photoreSpiration is a func- tion of glycolate metabolism (272). Although the C02 fixation by leaves of Solidago multiraciata remained high, lowering the concentration of externally applied 10 oxygen did not result in an accumulation of glycolate (30), which it Should have if the only site for the enhancement of photoreSpiration by oxygen were glycolate oxidase. Effect of 002 on the Biosynthesis of Glycolate Formation of JJAG-labeled glycolic acid in photo- synthesis is favored by low C02 pressure (15). For Chlorella, the maximum glycolic acid production occurs when the gas phase (at one atmosphere) contains 0.1% 002 (250, 256). Whittingham et al estimated that the effec- tive C02 concentration at the alga surface is about one- tenth of the C02 concentration in the gas phase. Thus the 002 concentration at the cell surface, for maximum glycolate production, would be about 0.01%. The synthe- sis of glycolate or glycolate products in the leaves of rugher plants is also favored by Similarly low C02 par- tial pressures (232, 233). Warburg Oxygen Effect Photosynthetic 002 fixation and 02 evolution in some intact higher plants are inhibited about 30% in an atmoSphere of 21% oxygen and 0.03% C02 as compared to nitrogen and C02. This inhibition by oxygen, referred to as the Warburg oxygen effect, is rapidly produced and is usually rapidly and fully reversible (29. 9“. 95). There is disagreement among investigators concern- 11 ing the effect of light and CO2 concentration on this inhibition by oxygen. Although it has been reported that high light intensities and low 002 partial pressures (con- ditions which increase glycolate synthesis) increase the inhibition by oxygen (95), Bjcrkman (29) has presented data showing that light intensity is without effect on the inhibition by oxygen and that CO2 partial pressure is probably without pronounced effect. Bjcrkman (29) reported that algae do not show the inhibition by oxygen, but other investigators have reported that algae do Show the inhibition (95). Appar- ently the intensity of the oxygen inhibition is influenced by the nutrition of the algal cultures (94). Egle and Pack (74) found that the reduction by oxygen of net CO2 Uptake, though present, was much less pronounced in algae than in higher plants. A possibly related observation was made by Hess and Tolbert (118), who reported that 5 strains of algae did not metabolize glycolate under their culture conditions. Corn, sugar cane, and Amaranthus edulis are capable of reducing the CO2 content in a closed system to zero, which suggests that these plants lack photoreSpiration, a phenomenon thought to be a function of glycolate metabo- lism. These plants do not exhibit the Warburg oxygen effect at 21% c2 (30). The Warburg oxygen effect may be the result of an 5‘ 12 increased loss of carbon as glycolate from the sugar phos- phate pools of the photosynthetic carbon cycle (21, 53, 94, 255) and/or the reduction of net 002 uptake and 02 evolution because of CO2 production and 02 uptake from glycolate metabolism. However, the Warburg effect is not a function of temperature in the leaves of Solidago and Mimulus while photoreSpiration was found to be strongly affected by temperature in tobacco (30). This and other arguments led Bjtrkman to prOpose that the Warburg effect is not an eXpression of photoreSpiration. Inhibition by oxygen in some ways mimics inhibition by DCMU in that both inhibi- tors increase fluorescence and decrease the reduction of plastocyanin. This and other data led Bjcrkman (29) to postulate that the site of 02 inhibition is in the elec- tron carrier chain between the two photosystems. oh the other hand, Fewson et a1 (81) reported that the production of ATP coupled to the reduction of NADP+ is not inhibited by oxygen. - Gibbs et al (95) have reported an essentially com- plete (and'reversible) inhibition of 1L’cciz fixation by whole chlorOplasts eXposed to 21% oxygen. Thus,the metabolism of glycolate by the glycolate pathway should not be the only cause of the Warburg effect,since at the _most.only i of the newly fixedco2 should be evolved by munia.mechanism. The finding of Tolbert et al (237) 1.; V 13 that glycolate oxidase from Spinach leaves is associated with excess catalase in the peroxisomes makes it doubtful that glycolate products are completely converted to C02 by reacting with H202. However, complete inhibition of CO2 fixation by oxygen could be the result of an increased loss of carbon as glycolate from the photosynthetic carbon cycle. High levels of oxygen concurrently produce increased rates of formation of glycolate and diminished levels of the intermediates of the photosynthetic carbon cycle in Chlorella (20, 21. 53. 255)- The finding that ascorbate and cysteine, but not fructose diphOSphate, reversed the inhibition of luCOZ fixation by 1.5% oxygen in whole Spinach chloroplasts suggests that there may be an interference by molecular oxygen with some component of the photochemical act (95). Since present knowledge of the chain transferring elec- trons from water to NADP points to Spinach ferredoxin as the carrier most readily influenced by oxygen, Gibbs et al (95) favor the hypothesis that oxygen interferes with the formation of NADPH by reoxidizing reduced ferredoxin. In the presence of saturating levels of ferredoxin and large concentrations of NADP+, air does not inhibit the formation of ATP or of NADPH by Spinach chloroplasts. It has been established that reduced ferredoxin is auto- oxidizable but that in the presence of NADP+ and molecular 02. electrons are transferred to the former rather than to 14 the latter. Therefore, in the presence of sufficient accepter, the formation of NADPH is not affected by oxygen (95). After presenting and reviewing evidence on the mechanism of inhibition of photosynthesis by high partial pressures of oxygen in Chlorella, Coombs and Whittingham (53) proposed that the oxidation leading to the formation of glycolate from the sugar phosphates of the photosynthe- tic carbon cycle is accomplished by H202 formed by a Mehler reaction between oxygen and ferredoxin. In the presence of normal photosynthesis with C02, the level of reduced ferredoxin is presumed to be too low to react with O2 to produce H O 2 2' absence of CO2 or at very low levels of COZ, photosynthe- It was postulated that in the sis would not utilize the reduced ferredoxin rapidly and its level would increase to a point where it could react with oxygen. This proposal is consistent with the finding that high light intensity and the presence of oxygen increased the rate of glycolate production (20). A pos- sibly related observation that FMN or FAD stimulated the synthesis of glycolate from fructose diphosphate by chloroplast preparations has been made by Bradbeer and Anderson (35). Glycolate From the Photosynthetic Carbon Cycle 14 The kinetics of C accumulation in labeled glyco_ .1. ‘\ 15 late by algae (183) and by tobacco leaves (117) suggests that glycolate is formed as a product from one or more components of this cycle. The plot of the percentage of 1.”C in phOSphate esters (183) or PGA (117) extrapolates to nearly 100% at zero time, while the percentage in glycolate extrapolates to zero before zero time. Feeding studies with 14c labeled intermediates of the photosynthetic carbon cycle have indicated that gly- colate may be formed from this cycle (35, 233). However, the recoveries of glycolate from intermediates of the cycle were low. Tolbert (233) has pointed out that small yields with crude enzymes are suSpect, particularly since RuDP nonenzymatically decomposes into a multitude of products including C2 pieces. In one such feeding study, Bradbeer and.Anderson (35) concluded that "since there appears to be no strong evidence in favor of a sugar phos- phate being the starting point of the pathway of glycolate synthesis in green plants, serious consideration must be given to the possibility of a de novo C02 fixation being the major pathway." However, studies have also been made with radioactive glucose fed to Chlorella pyrenoidosa in the light. For that fraction which was converted to gly— colate, the C1 or C6 of glucose gave rise almost exclu- sively to the C2 of glycolate while the CZ of glucose gave rise almost exclusively to the C1 of glycolate (256), The results are consistent with cleavage between C2 and 16 C3 of an intermediate of the photosynthetic carbon cycle so that carbon 2 of the intermediate became the carboxyl of glycolate. With oxygen as the gas phase and in the presence of isonicotinyl hydrazide, as much as 50% of the glucose incorporated may be converted to glycolate (255). That glucose can be fed into the photosynthetic carbon cycle and converted to glycolate under conditions of low CO2 concentration and high oxygen partial pressure could account for the high glycolate production reported by Warburg and Krippahl (250, 53) and for the high produc- tion of glycolate by Chlorella pyrenoidosa in the light under 02 in the absence of H00 ' (120). 3 Calvin and coworkers found that when glycolate—1- 140 was administered to Scenedesmus during 10 minutes of photosynthesis with %% C02 in air or N2, a pattern of photosynthetic intermediates was found similar to that h 14 obtained during photosynthesis wit C02. Upon degrada- tion of the PGA, it was found that less than 10% of the radioactivity was in the carboxyl carbon. Thus,with this alga under the conditions used, some of the glycolate was incorporated into normal intermediates of the photosyn- thetic carbon cycle without preliminary conversion to C02, since so little 140 was found in the carboxyl of PGA1 (45), M . 1The alpha and beta carbons of PGA were found to be eQually labeled.as though the pathway from glycolic acid to these carbon atoms involves a randomization of the label. This could mean that along this pathway there is a symetrical intermediate,or that an intermediate is in rapid reversible equilibrium with a symetrical compound (45), 17 Calvin et al and Schou et a1 degraded glycolate and PGA obtained from barley leaves and from Scenedesmus that had photosynthesized for a few seconds in the presence of 14C02 or HluC03-. The alpha and beta carbon atoms of PGA were found to be always about equal to each other in radioactivity and always less than the carboxyl carbon of PGA, until such time (1 to 5 minutes) as all three carbon atoms were completely labeled. The two carbon atoms of glycolate were always about equal to each other in radio- activity (45). Glycolate from tobacco leaves consistently showed about equal distribution of label in both carbon atoms after various Short periods of photosynthesis with 13002 (117). ' Hess and Tolbert found that in glycolate formed by 3 different algae in the first 5.and 10 seconds of I"LACOZ fixation, the 02 was at least twice as radioactive as Ci’ A similar skewed labeling was evident between C and C2 3 of PGA in the same eXperiments1 (118). Such a relation- 1The Gibbs effect,in which carbon atom 4 of hexose is more highly labeled than carbon atom 3 while carbons 1 and 2 are more highly labeled than carbons 5 and 6,13 not inconsistent with the photosynthetic carbon cycle. Con- sideration of the two eXplanations for the Gibbs effect will show that the size of the effect will depend on pool sizes and the rate of the net forward reaction as compared with the rate of the reverse reactions. It is not sur- prising therefore that sometimes the Gibbs effect is observed and sometimes it is not (15). Sedoheptulose phosphate from soy bean leaves which had been exposed to CO for very short times contained about 3 times more labe in carbon 3 than carbon 4, which is consistent with the Gibbs effect (15). Since carbon 3 of sedoheptulose becomes carbon 1 of pentose while carbon 4 becomes carbon 2. the labeling pattern noted by Hess and Tolbert is not inconsistent with the photosynthetic carbon cycle. 0‘ an; 18 ship between the labeling of glycolate and.PGA is consis- tent with the concept that glycolate is formed from one or more intermediates of the photosynthetic carbon cycle,and suggests as possible precursors the following moieties of intermediates of the cycle: carbons 1 and 2 of the pentose, hexose. and heptose mono and diphosphates, the terminal two carbons of these compounds, and carbons 2 and 3 of the C3 intermediates. Certain tropical grasses, which lack a detectable photorespiration (which has been shown to be a function of glycolate metabolism (272)), are characterized by the C4‘ dicarboxylic acid pathway for C62 fixation (111). These plants contain a nonclassical photosynthetic carbon cycle inthat ribulose diphosphate carboxylase activity is inade- quate. Whether or not the reduced photoreSpiration in the tropical grasses is due to a changed cycle in general or deficient ribulose diphoSphate carboxylase activity in particular, remains to be demonstrated. A Cz-TPP intermediate formed in the photosynthetic carbon cycle from fructose-é-P, sedoheptulose-7-P, and xylulose-5-P by transketolase may exist in a common pool (16). Bassham et a1 (17) demonstrated that carbons i and 2 of ribulose, fructose, and sedoheptulose were uniformly labeled; these carbons are the precursors of the C2 moiety transferred by transketolase. Wilson and Calvin (258), following their observation of glycolate accumula- ’O ‘1 19 tion at low CO2 pressure, suggested that this glycolalde- rmde moiety transferred by transketolase is the source of glycolic acid. The enzymatic oxidation of glycolaldehyde- TPP (called more precisely 2-(1,2-dihydroxy ethyl)-TPP) to free glycolate has been demonstrated using artificial oxidants (86, 116). Such a reaction has not been demon- strated with natural oxidants or with an enzyme from photosynthetic tissue. However. further work with the pdg heart enzyme. hydroxypyruvate decarboxylase, has revealed that it will oxidize glycolaldehyde-TPP to gly- colyl CoA plus NADH + Hf (87). The conversion of acetyl CoA to acetyl phOSphate by phospho-trans acetylase from g. 321i (254) suggests that an analogous formation of glycolyl phosphate from glycolyl CoA is possible. Calvin and Bassham have Speculated that glycolaldehyde-TPP may eliminate water to give acetyl-TPP which could undergo phOSphoroclastic cleavage to give acetyl phOSphate and TPP (45). To my knowledge, elimination of two hydrogens rather than the elements of water, and subsequent phos- phoroclastic cleavage to give glycolyl phOSphate has not been reported. Sugar diphOSphates are not known to be cleaved by transketolase. Furthermore,RuDP does not have the neces- sary trans configuration.of the hydroxyl groups between carbons 3 and 1+ as is required by aldolases. transaldolases and transketolases; however,FDP and SDP do have the neces- e.— 20 sary trans configuration. P-glycolaldehyde might be a pre- cursor for P-glycolate. Aldolase has been shown to cata- lyze the formation of xylulose diphosphate from glycol- aldehyde phoSphate plus dihydroxyacetone phosphate. The reverse reaction would produce the CZ-phoSphate from car- bons # and 5 of the pentose. During photosynthesis these 14C. However, two carbons are also equally labeled with no data exist which implicates xylulose diphosphate in photosynthesis (233). .A transaldolase type of reaction could similarly produce the Cz-phosphate from xylulose-S— phosphate. Although the substrates for transaldolase are monophosphates, to my knowledge xylulose-S-phOSphate has not been shown to be cleaved by this enzyme. Bassham and Kirk (21) have proposed that RuDP may be the precursor of P-glycolate. Ribulose diphosphate carboxylase cleaves the pentose between carbons 2 and 3. According to these authors it may be that at some early stage in this reaction mechanism, the moiety composed of carbons 1 and 2 can be oxidized, giving rise to P—glyco- late. That glycolate synthesis is favored by low COZ par- tial pressures is consistent with RuDP being the possible Precursor of glycolate, since RuDP increases in concentra- tion under this condition (15). Furthermore, high 02 partial pressures, which favor the synthesis of glycolate, greatly decrease the pool of RuDP (20. 21). The administration of 8-methyl-lipoic acid to I: A II. V . Fifi ..a I - 21 Chlorella pyrenoidosa which was fixing 14002 was found to cause a rapid and great increase in the pool sizes of FDP and SDR,as though fructose diphOSphatase was inhibited.ig zizg by this compound. The evidence suggests that ribulose diphOSphate carboxylase and ATP formation were also strongly inhibited. Inhibition of ATP synthesis was accompanied by an essentially unchanging pool of RuDP even though the carboxylase was inhibited. An increase in glycolate label- ing matched these changes in the concentrations of inter- mediates of the photosynthetic carbon cycle (19). FDP or SDP are thus suggested as possible precursors for glycolate. Although the RuDP pool did not change appreciably, the inhibition of the carboxylase is consistent with the pos- sibility that RuDP might also be the precursor for glyco- late. Lipoic, octanoic, and methyl octanoic acids pro- duced effects similar to those of 8-methyl-lipoic acid (19, 188). Gibbs has hypothesized that the Warburg oxygen effect is a function of glycolate synthesis (9b) and the reoxidation of reduced ferredoxin (95). Evidence has been presented that the fructose diphoSphatase of the photosyn- thetic carbon cycle is activated by reduced ferredoxin but not by oxidized ferredoxin (40, #1). Loss of activation kw oxidation of ferredoxin might thus give the same pat- tern of phOSphatase inhibition and enhanced glycolate syn- thesis as that resulting from the application of 8-methy1— lipoic acid. 22 Results from experiments with light dark transients also seem to favor one of the diphoSphates as a glycolate precursor. The kinetics of incorporation of luCO into 2 glycolate by Chlorella pyrenoidosa during a light to dark transition were very much like the kinetics of incorpora- tion into RuDP and somewhat like the kinetics of incorpor- ation into FDP and SDP, while the curves for the other intermediates of the photosynthetic carbon cycle do not look like the glycolate curve (189). Davies et al (56) have postulated a biosynthetic route to uniformly labeled glycolate from PGA through 3 P-hydroxypyruvate, P—glycolaldehyde, and P-glycolate but very little evidence in support of the pathway is pre- sented. Bradbeer and Anderson (35) reported from a survey of possible sources of glycolate, that hydroxypyruvate and.P-hydroxypyruvate gave good yields of glycolate with chloroplast preparations. Further investigations estab- lished that these high yields resulted from a non-enzyma— tic reaction which required hydroxypyruvate, Mn++ , FMN or FAD, oxygen, and light. The large amount of glycerate dehydrogenase found in chloroplasts has prompted Gibbs et al to propose a pathway for the synthesis of glycolate from PGA to glycerate to hydroxypyruvate to glycolate (95). A pathway for photosynthetic serine biosynthesis by chloroplasts from P-glycerate and glycerate through Imfiroxypyruvate has been postulated by Chang and Tolbert. ‘- so; cl '3' A»; 23 The a and B carbons of hydroxypyruvate would be uniformly labeled but less highly labeled than the carboxyl carbon (48). Hydroxypyruvate labeled in this way would be decarboxylated by hydroxypyruvate decarboxylase of pig heart to give uniformly labeled glycolyl-00A (87). Although this enzyme has not been isolated from plant tissue, it has been reported that Scenedesmus converted rmdroxypyruvate-Z-lnc to glycolate-1-1uc in the light. In the same eXperiments, no pyruvate-Z-luc was converted to glycolate (160). The 01-02 fragment of any intermediate of # car- bons or longer or the terminal two carbons of any photo- synthetic carbon cycle intermediate could serve as the precursor of essentially uniformly labeled glycolate. These moieties from the erythrose, pentose, hexose, and heptulose intermediates would all be at the glycolalde- hyde level and would require the removal of two electrons per molecule of glycolate synthesized. PGA, after the removal of C1 by decarboxylation, would require the removal of n electrons per molecule of glycolate synthesized, while the trioses,after decarboxylation,would require the removal of 6 electrons. Thus,every possible glycolate precursor of the photosynthetic carbon cycle would require at least one oxidation step in the course of the synthesis of glycolate. 0n the basis of the amount of oxidation required, the C intermediates do not seem to be likely 3 . A 2h glycolate precursors. Furthermore, conversion of the 03 intermediates to glycolate would require evolution of 1/3 of the newly fixed 002 by decarboxylation. That the higher algae synthesize and excrete large quantities of glycolate, yet do not consistently exhibit CO2 exchange with the surrounding gas phase in the light (30, 74), might rule out the C intermediates of the photosynthetic carbon cycle as glycilate precursors.1 Removal of the top two carbons of erythrose-h-phOSphate as glycolate would leave a remainder which is not an intermediate of the cycle. However, removal of the bottom two carbons as gly- colate would leave a remainder which is an intermediate of the cycle, would not unbalance the cycle, and would obviate the need for sedoheptulose mono and diphOSphate and ribose-5-phosphate. But to my knowledge there is no precedent for such an enzymatic cleavage of erythrose-u— phosphate. Removal of the terminal two carbon atoms of the pentose, hexose, and heptulose intermediates would also leave compounds which are not intermediates of the cycle. Removal of a Ci'CZ fragment of the pentoses, hexoses, or heptuloses would leave compounds which are intermediates of the cycle, and removal of any of these 1When Coombs and Whittingham fed labeled glucose to Chlorella under CD free 0 in the light, C02 evolu- tion paralleled glycolate syn hesis. To my knowledge, glycolate production by algae without a concomitant pro- duotion of 002 has not been demonstrated in the same experiment. a». .— q». a: 25 moieties as glycolate would not unbalance the operation of the cycle. Thus, on the basis of labeling, oxidation level, conservation of carbon, the nature of the remain- ing compound, and the balanced operation of the photosyn- thetic carbon cycle, a Cl-C2 fragment of the 5, 6, or 7 carbon intermediates seems the most likely glycolate pre- cursor. If P-glycolate is the precursor of glycolate, a 01-0 fragment of FDP, SDP, or RuDP could be considered 2 as the most likely precursor of glycolate. P-Glycolate as the Precursor for Glycolate The labeling of P-glycolate at early times1 during photosynthesis in 14002 (18, 21, 271L together with the discovery of a Specific and highly active phOSphatase for this compound (202),suggest that the precursor of glyco- late could be P-glycolate which in turn could come from carbons 1 and 2 of an intermediate of the photosynthetic carbon cycle (233). That five strains of algae were found to contain P-glycolate phOSphatase but not glycolate oxi- dase (118), together with evidence that algae are capable of excreting large quantities of glycolate (120, 250), 1The inconsistent observation of P-glycolate in photosynthesis experiments is possibly a reflection of the resistance of P-glycolate phOSphatase to inactivation by methanol. Ullrich (243) has reported that terminating eXperiments in methanol does not prevent some hydrolys1s of phOSphate esters, and in particular the hydrolysis of P-glycolate. a.) 7, pr 26 suggest that P-glycolate may well be the precursor for glycolate. A similar argument is suggested from evidence that spinach chloroplasts excrete glycolate (131) and contain P-glycolate phOSphatase (230, 236, 265),but not glycolate oxidase (236, 237)- The above evidence is also consistent with the rmpothesis that P-glycclate phOSphatase may be part of a permease system for the excretion of glycolate from the chloroplasts (202, 260, 265). Furthermore, evidence that inorganic phoSphate is transferred only slowly across the outer chloroplast membrane.and that a major portion of the phOSphorylated compounds still are present in the chloro- plasts after 10 minutes of photosynthesis even though much carbon has been moved into the cytoplasm during this 10 minutes.has led to the proposal that phOSphatases are 1 and oriented in present in the outer chlorOplast membrane such a way that carbon is released into the cytoplasm while phOSphate is released back into the chloroplast (182)° Glycolate from Isocitrate Asada and Kasai (7) reported that in tobacco leaves, there is a link between isocitrate and glyoxylate,but lP-glycolate phoSphatase has not been directly studied as part of a membrane system. Adenosine triphos- phatase is one example of a phoSphatase which has been directly studied in membrane fragments of various tis- sues (32). 27 their evidence leads to the conclusion that glyoxylate is a precursor of isocitrate in this tissue in the light rather than the reverse. Harrop and Kornberg (107) found that isocitrate lyase was a constitutive enzyme in the Brannon no. 1 strain of Chlorella vulgaris and that the alga excreted labeled glycolate during growth in the dark on cold glucose in the presence of H14 003-. The evidence supported the view that the glycolate excreted under these heterotrophic conditions in the dark derived ultimately from the cleavage of isocitrate and not, as postulated for algae growing autotrophically in the light, from the cleavage of pentose phosphate or possibly from direct condensation of C1 units derived from C02. Results obtained‘with[:1-luC] acetate, added to similar cultures growing on glucose in the dark (or on C02 in the light), indicated that the glyoxylate cycle did not function under these conditions. 'In contrast,the organism would not excrete labeled glycolate during growth in the dark on cold acetate in the presence of H1”)+ 603‘, and the incor- poration of isotope from[:1-1”C] ethanol by this alga growing on ethanol aerobically in the dark was consistent with the Operation of the tricarboxylic acid cycle and glyoxylate cycle. No differences were observed between the properties of the isocitrate lyase purified from cells grown on acetate and glucose. But whereas isocitrate lyase was wholly found in a soluble fraction of the organ- .‘a' A. ’0 .3 28 ism after growth on glucose or on carbon dioxide, acetate grown cells contained a major portion of their isocitrate lyase in a dense particulate fraction. Since the excre- tion of labeled glycolate ceased after transfer of the cells to acetate growth medium, it can be presumed that the glyoxylate formed from isocitrate now reacted with malate synthetase and acetyl CoA as part of the operation of the classical glyoxylate cycle. The results suggest that in the Brannon no. 1 strain, isocitrate lyase par- ticipates in the glyoxylate cycle when it is incorporated into a particulate fraction but it participates in glyco- late synthesis and excretion when it is in the soluble form, and that culture of the cells on 02 metabolites is necessary for the incorporation of isocitrate lyase into the particulate fraction. In marked contrast to the Brannon no. 1 strain of §.'vulgaris, isocitrate lyase was not a constitutive enzyme in the Pearsall's strain of Chlorella or in Chlamydomonas reinhardii, and experiments with Pearsall's strain of Chlorella indicated that this alga would not excrete labeled glycolate during growth in the dark on cold glucose “003' (107). Thus, although iso- in the presence of H1 citrate apparently can be an important precursor for gly- colate in at least one strain of Chlorella under certain conditions of growth, isocitrate as an important precur- sor of glycolate would seem to be the exception rather than the rule. 29 Glycolate from Acetate Two pathways have been proposed for the formation of uniformly labeled acetyl 00A from the photosynthetic carbon cycle (16). EXperiments by Goulding and Merrett (100) with 3H-luC-acetate revealed glycolate as an early product of its photoassimilation in Chlorella pyrenoidosa,since the 3H and 14C in glycolate reached a steady total dpm for state in early samples. However, the conversion of acetate to glycolate resulted in a 2% fold increase in the ratio of 3H to 14C,presumably because of simultaneous incorporation of.12CO2 into glycolate. This significant. change in ratio was cited as evidence against a direct conversion of acetate to glycolate. Data was also pre- sented which tended to exclude the conversion of acetate to glycolate by the glyoxylate cycle or the decomposition of acetate to 002 and refixation through the photosynthe— tic carbon cycle to glycolate. The possibility of a photo- chemically produced reducing compound formed by removal of hydrogen from acetate reacting with carbon dioxide to pro- duce glycolate was not ruled out by the experiments. Jagow et al (126) have reported that in rabbits, acetic acid may be hydroxylated to glycolic acid after Ewing bound to p-aminopropiophene by microsomal hydroxy- lation of (h-propionyl)-acetanilide and hydrolysis of the hydroxylation product. To my knowledge, no direct conver- 30 sion of acetate to glycolate by oxidation has been reported in plants. The de Nova Synthesis of Glycolate from 002 Several investigators have concluded that glycolate biosynthesis may not originate with the photosynthetic car- bon cycle. From electron spin resonance signals on mangan- ese-deficient Chlorella, Tanner et;al (229) suggested that glycolate is a product formed directly from the condensa- tion of 2 002 molecules. Warburg and Kripphal (250) observed that Chlorella, during 1 hour of photosynthesis, converted 92% of the fixed C02 into glycolate. These results, however, may be in error owing to nonphotosyn-‘ thetic metabolism of the algal carbohydrates to glycolate (53, 120%. Zelitch (271) reported that after C02 fixation by tobacco leaves for 2 to 5 minutes, the glycolate was uniformly labeled and the Specific activity of these car- bon atoms was greater than the Specific activity of the carboxyl carbon of PGA. Therefore,he suggested that gly- colate must originate from a previously undetected C02- fixation pathway. In contrast, the possibilities that glycolate might be labeled before PGA or by a separate cog-fixation process in tobacco leaves were not indicated by the data of Hess and Tolbert (117). The reverse, that PGA could be the precursor of glycolate, is consistent with their data. The latter authors point out that 31 Zelitch required 20 to 30 seconds to move leaf tissue from the 1&002 fixation vessel through air containing 12CO2 to the killing solution and that during this time, cold 12co2 would have replaced a Significant amount of 1”C from the carboxyl group of PGA. Stiller (226) has presented argu- ments favoring a proposal for the de novo synthesis of a CZ fragment from C02, which would condense with a triose to form a pentose directly, and she has prOposed that this 02 fragment is the precursor of glycolate. However, Bassham (15) has presented convincing arguments negating Stiller‘s proposals. Thus,in the euchariotic cells of higher plants and algae, there is no well documented defi- nitive evidence for the total de novo synthesis of glyco- late from 002. Nor in these cells is there evidence for the total de novo synthesis of any compound from 002 by a mechanism other than the photosynthetic carbon cycle pro- vided that the Cu-dicarboxylic acid pathway is considered a modification of this cycle. Nevertheless, until the enzymes for glycolate biosynthesis are known in these cells, the possibility will remain that CO2 fixation for glycolate formation differs from that for PGA formation. As Stiller (226) correctly pointed out, the slower label- ing of glycolate than PGA does not rule out the independent synthesis of glycolate. The observation that glycolate can be forced to accumulate in the absence of 002 does not argue against the de novo synthesis of glycolate,since 32 such reasoning makes the invalid assumption that glycolate can be formed either de novo or not at all. In procaryotic cells, two mechanisms other than the photosynthetic carbon cycle have been discovered for the total de novo synthesis of compounds from C02. A ferre- doxin dependent carbon reduction cycle has been reported in.the photosynthetic bacterium, Chlorobium thiosulfato- philum, by Evans et a1 (78) that is essentially a reversal of the citric acid cycle. Two, 3, 4, 5, or 6 carbon com- pounds are synthesized de novo from C02 by the cycle. The presence of isocitrate lyase in this organism would make possible the de novo synthesis of glyoxylate from two molecules of C02, but isocitrate lyase was not reported. Secondly, the total synthesis of acetate from 002 has been reported in the anaerobe,Clostridium thermoaceticum,even though the reductive pentose cycle does not Operate in this organism. {Available evidence suggests that a Co- methylcorrinoid and.a Co-carboxymethylcorrinoid are inter- mediates in the pathway from C02 to acetate. It is most probable that the conversion occurs via intermediate com- pounds linked directly to the cobalt of the corrinoids which are enzyme bound. Therefore,no intermediate com- pound occurs free in solution which may be detected by the usual tracer techniques such as employed by Calvin and others in the study of photosynthesis. The intermediate cobalt complexes are degraded in the light by photolysis 33 (1&6). It is of interest that the corrinoids, which are coenzyme B12 derivatives, are structural analogs of chlorOphyll. The de novo synthesis of acetate via cor- rinoid intermediates thus brings to mind the well docu— mented 002 requirement for the Hill reaction, the proposal by Warburg that in_zizg_the proposed cyclic participation of COZ in the photochemical reactions is unbalanced so that there is a net reduction of carbon (226), and the binding of 602 by chlorophyll (251). Anderson and Fuller (2) have reported that glyco- late appears to be the first stable product of C02 fixa- tion in the procaryotic cell R. rubrum cultured photo- heterotrophically with L-malate,and that unlike auto- trophically grown H, rubrum, the contribution of the photosynthetic carbon cycle to the fixation of C02 is nil. However, the 002 fixation rates through glycolate were low,so that L-malate was a far more important source of cell carbon than was carbon dioxide. Similarities Between 02 Evolution and Glycolate Formation Oxygen evolution and glycolate biosynthesis share several similarities. Manganese is required for glycolate synthesis (118) and oxygen evolution (132). The culture of Chlamydomonas or Chlorella for several days in blue light resulted in the increased synthesis of glycolate and an increased chlorophyll b to a ratio (119). Chloro- . .r" in n . ..',i. u.- 93.. C... ,.' i r ‘ 4F. nIv ll. 34 phyll b is associated with photosystem II in higher plants, which is the photosystem closest to the site of O2 evolu- tion (132). Talbert (233) has reviewed other similarities which exist between glycolate synthesis and 02 evolution.1 However, since glycolate synthesis is greatly decreased under anaerobic conditions while CO2 fixation and 02 evolu- tion are increased (21, 95, 255, 27%), it would seem that glycolate synthesis is not an obligate step in oxygen .evolution. Oxygen synthesis could be an obligate step in glycolate synthesis, but even here the apparent requirement for external oxygen for the synthesis of glycolate (21, 255, 27h).as opposed to oxygen or oxidants generated during photosynthesis, is not consistent with the concept of a close relationship between oxygen evolution and glycolate synthesis. The Glycolate Pathway This pathway2 describes the path from.P-glycolate through glycolate, glyoxylate, glycine, serine, hydroxy- pyruvate, glycerate, and phosphoglycerate in that order. The enzymes of the pathway, except for P-glycolate phos- phatase, have been thought to occur in the cytoplasm, 1The effect of phosphate on glycolate synthesis was later shown by Orth et al (183) to be an effect of pH and not a Specific effect of phosphate. 2For a figure of the glycolate pathway, see the Ph.D. thesis of Hess (116). 35 which is consistent with the excretion of glycolate by chloroplasts. Presumably, conversion of PGA from the glycolate pathway to sucrose could occur in the cyto- plasm by the gluceogenic functioning of the appropriate enzymes,including some of the enzymes of glycolysis (233). The rather extensive evidence in favor of the present formulation of the glycolate pathway has recently been reviewed (116). The eXperiments of Hess and Tolbert (117) with tobacco leaves are fully consistent with the operation of the pathway in that plant. Recent discover- ies that the plants which possess the dicarboxylic acid pathway for 002 fixation do not exhibit photorespiration (111) or inhibition of photosynthesis at high oxygen concentrations (72) suggest that the glycolate pathway may not be as important in these plants as it is in plants such as wheat, tobacco, and soybean which possess the classical photosynthetic carbon cycle and exhibit photorespiration. Similarly, the glycolate pathway as it exists in these latter higher plants may not be as important in the higher algae (116, 118). Although some of the enzymatic details for the conversion of glyoxylate to hydroxypyruvate through glycine and serine have been investigated, many remain to be elucidated. EXperiments performed by Cossins and Sinha (5“) with leaves of 15 day old wheat seedlings, which had been grown in a regime of light and dark periods 36 of 12 hours duration, provided evidence which strongly suggests that in this tissue, glycine is cleaved directly to yield N5N10-methylene-tetrahydrorolate, and that the C-2 of glycine can give rise to the C-3 of serine. It was proposed that glycine + tetrahydrofolate react to produce N5N1o-methylenetetrahydroyfolate + NH3 + CO2 and that the activated C1 unit formed could then be utilized in the serine-hydroxymethyl transferase reaction, result- ing in the formation of serine. These same authors (55), using nonphotosynthetic tissue, i.e. endOSperm of 5-day- old castor bean seedlings which had been germinated in darkness for 5 days, conducted eXperiments which provided a background for a model of serine synthesis from glyoxy— late in which half the glyoxylate is decarboxylated yield- ing the a carbon of glyoxylate as formate, which is then activated to a tetrahydrofolate adduct. It was proposed that the activated C1 then condenses with glycine through a hydroxymethyl transferase reaction to form serine. Both of the above schemes for serine synthesis are in accord with the findings of Tolbert and Cohan (23“) that the C-2 of glyoxylate is incorporated into the C-3 of serine in wheat and barley leaves, and with the finding of Hess and Tolbert (117) that in tobacco leaves at early times, the Cl and C2 of serine were uniformly'labeled from 1""C 02, but C3 was less highly labeled, presumably because in the synthesis of serine the C3 carbon had to pass through at “:.mv_—————-r_v 4. a “‘I u. .a n,, (In a‘ ‘\ 37 least one more pool than did the C1 and 02 carbons. Kinetic studies using 1“'0 and 15N suggest that the primary site of ammonia incorporation in Chlorella pyrenoidosa is principally into glutamic acid, presumably via the reductive amination of alpha-ketoglutaric acid (20, 22). By the recycling of ammonia released in the conversion of glycine to serine, alpha-ketoglutarate and glutamate may thus play a catalytic role in the turnover of the glycolate pathway in the leaves of higher plants. Because alpha ketoglutarate is only one reaction removed from isocitrate, the latter hypothesis is relevant to the present thesis. P-Glycolate Phosghatase cHZ-Coo' cHZ-coo' 0 + H20 —————9’ 0H + P1 ‘o-P-OH -5 P-glycolate phOSphatase was discovered in tobacco leaves by Richardson and Tolbert (202) in 1960. The enzyme purified from.these leaves by ammonium sulfate and calcium phOSphate gel fractionation was found to contain an.endogenous divalent metal ion necessary for activity, and to have a pH optimum of 6.3 in the presence of this endogenous metal. After an additional DEAE purification step, the enzyme was active only toward P-glycolate and 38 completely inactive toward 21 other phoSphate esters and anhydrides.1 The purified enzyme, which released stoichio- metric amounts of glycolate and phOSphate from its sub- strate, exhibited a rather unique spectrum of reactivity toward Sulfhydryl reagents and.sulfhydryls. It was found to be strongly inhibited by p-chloromercuribenzoate but (was unaffected by iodoacetate. 0n the other hand, cysteine and glutathione gave strong inhibition. An observation which may be related to this behavior toward sulfhydryl reagents and sulfhydryls is that at low concentrations, zinc has a considerably greater activating effect than does magnesium.2 1The pronounced specificity of the hydrolases of phOSphoryl compounds toward either polyphosphates, or phosphomonoesters, or phosphodiesters contrasts sharply with the lack of Specificity shown by some phos homono- esterases toward various phOSphomonoesters (213 . Thus, there is generally great Specificity for one particular classification of phosphoryl compound.but often a lack of Specificity within this classification. Some phospho- monoesterases however, eSpecially those acting on sugar phoSphates, are quite highly Specific (69 ). Fructose diphOSphatase from spinach chloroplasts is another example of a phosphatase active in photosynthesis which is highly Specific toward its substrate (194). 2Zinc shows a relatively greater affinity for sul- fur ligands than does magnesium (57). From the laws of chelation and atomic structure, there are reasons to believe that Cd++ and Hg++ will chelate with protein or coenzyme ligands that ordinarily chelate with Zn++ (216), Zn'"+ is likely to be coordinated to sulfur and nitrogen at physiological pH, but not to oxygen. Mg++ on the other hand is unlikely to be coordinated to anything other than oxyanions (75). It is known that zinc can bind to thiol groups in proteins ( 70 ),and sulfhydryl groups are thought to bind zinc to liver alcohol dehydro- genase (246). On the other hand, in the case of serum mercaptalbumin, zinc tends to be bound by imidazole grows (129). C" v.-' . a key 39 Yu et al (265) discovered that P-glycolate phoSpha- tase is present in large amounts in the green leaves of Spinach, wheat, and alfalfa,as well as tobacco. The enzyme is also highly active in five of the higher algae (118). Little activity was detected in etiolated leaves or roots of wheat, but during greening of etiolated tis- sue in the light, the phOSphatase activity developed, which would implicate the enzyme in photosynthesis. It was found that the phosphatase from all higher plants tested except tobacco was unstable toward ammonium sul- fate fractionation.l In contrast to the tobacco enzyme purified by the method of Richardson and Tolbert, the wheat enzyme purified by acetone precipitation and DEAE- cellulose chromatography was completely inactive without added metal (265). Richardson and Tolbert (202) found that the pH optimum of the tobacco enzyme purified by their procedure was significantly shifted downwards from 6.3 in the presence of Mn++, Zn++, or Cult,which suggests that the endogenous phOSphatase metal is not one of these. Ca++ is excluded by the kinetic evidence (202). in et al (265) found that the pH optimum of the enzyme purified by acetone precipitation and DEAE-cellulose chromatography was 6.3 with Mg++ as the added metal. This pH optimum is 1There seems to be no basis for the report that ammonium sulfate precipitation resulted in a shift in the pH optimum from 6.3 to 5.0 (265). Different metal cofac- tors shifted the pH Optimum,however (202). Ecil in] P!- 40 the same as that for the tobacco phosphatase purified by the method of Richardson and Tolbert which still contained endogenous metal. Taken together,these data suggest that Mg?"+ is the endogenous metal cofactor for the phOSphatase.l’2'3 0n the basis of kinetic evidence, Mg++ or Mn++ were pro- posed as the possible endogenous metal by Richardson and Tolbert (202). The effect of Fe++ on the pH optimum has not been determined and kinetic data (202) does not exclude it as the endogenous phosphatase metal cofactor. All phOSphate monoesterases which have so far been studied cleave the P-O bond of the substrate (70,.213, 260). In this respect, the mechanism of phOSphate monoester cleavage resembles the nonenzymatic hydrolysis of mono- ionized phOSphoric esters by water. The mono-ionized phos- phoric anion predominates at slightly acidic pH (127, 213). 1It is of interest that Mg++ serves as an allo- Steric effector of the two photosynthetic carbon cycle enzymes, ribulose 1,5-diphOSphate carboxylase (227) and fructose diphOSphatase (194). Mg++ shifts the pH opti- mum of both enzymes from the alkaline to the neutral range and greatly increases the Vmax values and decreases the Km values of both enzymes at neutral pH. 2Chloroplasts isolated by the organic solvent tech- nique were found to contain'50 to 72% of the total Mg++ of bean and tobacco leaves, respectively. The magnesium content of bean and tobacco leaves was determined to be 1 and 2% of the dry weight, reSpectively (194). 3The idea has been proposed that the known rapid and large scale light dependent fluxes of Mg++ could os- sibly have a bearing on the activities of various Mg+ dependent enzymes of spinach leaf cells (66). ‘ ‘0‘ ..¢ .. g in. 11) . I rl'lkili... .iI-i. , 41 The P-glycolate phosphatase isolated from wheat was found to contain endogenous cis-aconitate after it was purified by acetone precipitation followed by DEAE-cellu- lose column chromatography (Tolbert and Yu, unpublished data). The cis-aconitate could be removed by passage of the partially purified wheat enzyme through a Sephadex G-50 column. The enzyme without sis-aconitate was unstable but could be restabilized by adding back commer- cial citrate,isocitrate, or cis-aconitate. Cysteine par- tially stabilized the defactored wheat enzyme but was not as effective as the three tricarboxylic acids. Other acids tested were without significant stabilizing effect so that the effect of the three tricarboxylic acids appears to be rather Specific. The evidence suggests that cis-aconitate may be associated with the wheat enzyme 3g,zizg. .Although an equilibrium mixture would contain about 91% citrate, 6% isocitrate, and 3% cis-aconitate (137), wheat contains almost twice as much aconitate as citrate. Only 2% of the aconitate is estimated to be in turnover pools in wheat while 15% of the citrate is esti- mated to be in such pcols(199). The concentration of most of the intermediates of the citric acid cycle in various rat tissues was found to be of the order of 10'4M. Much higher concentrations (up to 0.1M) can occur in plant tissues. Cells of baker's yeast were found to contain intermediate levels (about 10'3M) of these acids (139). 42 Inhibition and.Activation of Various Enzymes by the Tricarboxylic Acids Citrate has been shown to inhibit a number of enzyme systems, including rat liver pyruvate carboxylase, DPNH- cytochrome c reductase, aldehyde oxidase, aSpartase, and fumarase, but the effects were not Specific for the tri- carboxylic acid. Binding of protein bound metal at the active enzyme site appears to be the mechanism of inhibi- tion of DPNH-cytochrome c reductase, and possibly aSpar- tase (176). In the case of DPNH-cytochrome 0 reductase, the inhibition by citrate (or by perphosphate) was com- petitive with reSpect to cytochrome c (150). The inhibi- tion of fumarase by citrate was competitive as was inhibi- tion by trans-aconitate (159). Citrate was an inhibitor of inorganic perphosphate-glucose phoSphotransferase and glucose-6-phosphatase activities which are thought to be catalyzed by the same enzyme. The inhibition was compe- titive with reSpect to the two phosphate substrates, glucose-6-phosphate and pyrophosphate, and non competitive with reSpect to glucose. Isocitrate was less effective and cis-aconitate was completely ineffective as an inhib- itor of the latter enzyme (176). It was later found that "immediate" inhibition (i.e. no preincubation of enzyme with chelating agents) of microsomal glucose-6-phoSphatase, competitive with reSpect to glycose-6-phosphate and pyro- phOSphate and non competitive with reSpect to glucose, QI' v.4. 0', '1'- 7A. or. i l 43 could be obtained with sodium azide, sodium cyanide, sodium oxalate, and 1,10-phenanthroline. "Immediate" inhibitions were prevented by titration of the metal binding agents with divalent cations prior to exposure of the former to the enzyme. An irreversible time dependent inhibition was noted in preincubation eXperiments with 1,10-phenanthroline, sodium azide, diethyldithiocarbamate , and. 3-hydroxy- quinoline. These observations suggest that glucose-6- phOSphatase is a metaloenzyme, and that protein bound metal may participate in the binding of phosphate substrates, but not glucose, to the enzyme (175). These observations also suggest that as has been proposed for DPNH-cytochrome 0 reductase and aSpartase, binding of protein bound metal at the active enzyme site was the mechanism of inhibition of glucose-6-ph08phatase by citrate. Cennamo et al (47) found that citrate and fumarate Specifically inhibited pig heart mitochondrial malate dehydrogenase but did not inhibit pig heart supernatant malate dehydrogenase, as measured by oxalacetate reduction. Neither of the dehydrogenases were inhibited by 3.3 mM DL-isocitrate or cis-aconitate, or by the same concentra- tion of a variety of other acids. Although citrate competitively inhibited phospho- glucomutase (275) and intestinal alkaline phosphatase (79), the inhibition of these two enzymes could be reversed by the addition of relatively high concentrations of Mg++. 44 It was therefore hypothesized that citrate exerted its effect by complexing the Mg++ required by each of the two enzymes as an activator (79, 275). Citrate was found to be necessary for the activa- tion of acetyl CoA carboxylase from animal tissue. Iso- citrate was also very effective and malonate was effective. Fumarate was effective to an intermediate extent while cis- aconitate was completely ineffective as an activator. Activation was accompanied by an ordered aggregation of the enzyme. Phosphate was also effective in causing the ordered aggregation of the animal enzyme (103, 245). In contraSt, wheat germ acetyl CoA carboxylase, unlike its counterpart in animal tissues, was not stimulated by di- carboxylic and tricarboxylic acids (43). Deoxyribose phosphate aldolase from rat liver has also been found to be activated by di and tricarboxylic acids. Citrate, isocitrate, sis-aconitate, and trans- aconitate were about equally effective. Malate was about 2/3 as effective as the tricarboxylic acids while succinate, fumarate, and d-ketoglutarate were found to be somewhat effective in the activation of this aldolase (128). Although the purified aldolase showed a marked tendency to aggregate to form a dimer and trimer, the presence of an activating carboxylate ion did not influ- ence the observed aggregation (104). Very high concentrations of citrate (1-1.5M) 45 activated y—glutamyl transpeptidase of kidney bean fruit. The possibility that association of subunits or dissocia- tion to subunits occured in the presence of citrate was eliminated by the finding that the presence of citrate did not affect the sedimentation behavior of the enzyme. Other data indicate that citrate promoted a conformational change favorable for catalytic action. The activating effect was not confined to citrate alone, but was also shown by salts of some dicarboxylic acids and by EDTA (99). Lanchantin et al (140) reported that very high concentrations of citrate (25% weight per volume) acti- vated human prothrombin. Citrate was found to be an important inhibitor of the phosPhofructokinase from mammalian tissue and from yeast (209, 261). ATP and citrate cooperated in the inhi- bition of the yeast phosphofructokinase so that physio- logical concentrations of ATP increased the sensitivity of phoSphofructokinase to inhibition by citrate (209). Similar data have been obtained for the liver enzyme (260). Isocitrate was similarly inhibitory of the yeast enzyme while c-ketoglutarate was not (209). Nevertheless, there is conflicting evidence in assessing the regulating agents in the inhibition of glycolysis under aerobic conditions (the “Pasteur effect) in yeast. In contrast to the find- ings of Pye and.Eddy, evidence has been presented that a transition from anaerobic to aerobic conditions resulted 46 in a great increase in the concentration of citrate plus isocitrate while the ATP concentration remained unchanged. Citrate was therefore proposed as the Pasteur effect agent in yeast (260). End product inhibition Of mammalian and yeast phOSphOfructOkinase activity by citrate (and ATP) is believed to constitute an important feedback mechanism for the flux from glucose-6-phosphate via pyruvate into the citrate cycle (176). Switching mechanisms in the light and in the dark would seem to be required for fructose diphosphatase and phosphofructokinase from plants. The net effect of the uninhibited Operation of both enzymes in the light would be activity equivalent to an ATPase resulting in the hydrolysis Of photosynthetically produced ATP (20). Chloroplasts are supposedly not capable of glycolysis, (94) and to my knowledge there is no evidence for a phos- phofructokinase in the chloroplasts. Fructose diphOSpha— tase activity in the light is required in the chloroplast for the operation of the photosynthetic carbon cycle and presumably in the cytoplasm for the gluceogenic synthesis of sucrose from glycolate. In gluceogenesis, the balance between phOSphOfructOkinase and fructose diphosphatase plays an important role in control (260). Two fructose diphOSphatases have been reported, one in the chloroplast, and the other apparently in the cytoplasm (226). The phosphofructokinase of the cytoplasm would have access to e'flmrA—r .lvw a f...- ‘- ‘1 O. '1 ‘r "be ’r in. ‘ I 47 fructose-6-phOSphate from the cytoplasmic phosphatase. Furthermore, since many of the intermediates of the photo- synthetic carbon cycle seem to be exchangeable with the cytoplasm (20), even the chloroplast phosphatase could act with the cytoplasmic kinase to give ATPase activity if it were not for inhibition of the kinase in the light. Light dark kinetic studies with algae cells indicated that there was an inhibition of phosphofructokinase in the light and a release from inhibition in the dark. Although ATP has been shown to be an inhibitor of phos- phOfructokinase in mammalian and yeast cells, the level of ATP remained essentially constant in these light and dark experiments with algae (20, 189). PhOSphOfructokinase from carrots was completely inhibited by 10 mM ATP and almost completely by 10 mM citrate. ATP and citrate showed a distinctly synergistic inhibition. Inorganic phosphate partially reversed the inhibitions by ATP and citrate, but unlike the enzyme from adipose tissue, citrate inhibition was not allevi- ated by AMP, ADP or 3',5'-AMP (61). Experiments with phosphofructokinase from the leaves of Brussels Sprout yielded similar results. The authors concluded that P1 is probably the positive and citrate the negative effec- tor for the plant enzyme (62). 48 Aconitase Data is presented in the results section concern- ing similarities between aconitase and.P-glycolate phos- phatase, and a possible association between the two enzymes. This section of the literature review, on aconitase, represents an attempt to discuss some questions which were suggested by some of the results. Some of these questions are as follows. Is aconitase found out- side Of the mitochondria 12,1132? Might the enzyme have functions other than participation in the tricarboxylic acid and glyoxylate cycles? Is the enzyme under metabolic control? IS aconitase stabilized by the tricarboxylic acids? How stable is aconitase to freezing and thawing, precipitation by (NH4)ZSOu or acetone, and acid or alka- line conditions Of pH? Night there be an evolutionary relationship between aconitase and.P-glycolate phospha- tase?What anions activate aconitase? What are the Km values for the tricarboxylic acids? What is the effect of trans-aconitate on the enzyme? Does the enzyme have binding sites, other than the active site(s), for its substrates? What is its pH Optimum? What are the effects Of cations, sulfhydryl reagents, and inhibitors on the enzyme? What is the substrate Specificity of aconitase? What is its mechanism of action? Introduction and History Aconitase (or cis-aconitate hydratase, or citrate (isocitrate) hydrolyase, E.C. 4.2.1.3), is an enzyme catalyzing the interconversion of citrate, cis-aconitate, and isocitrate, and was discovered by Martius and Knoop in 1937 (156). It was first found in higher plants (kidney beans, cucumber seeds) by Martins in 1939 and was later shown to occur in leaves of cabbage by Jacobsohn and Soares in 1940,and of rhubarb by Morrison and Still in 1947 (11). The Function of Aconitase The two most Obvious requirements for cell mainte- nance and growth are a source of energy and a source of carbon skeletons. In aerobic organisms, including micro- organisms and higher plants, both energy and carbon Skele- tons are supplied by the reactions of, and ancillary to, the citric acid cycle (33). In this cycle, the pathways Of protein, fat, and carbohydrate catabolism are united. Oil bearing seeds make use of the glyoxylate cycle for the conversion of fat to carbohydrate during germina- tion. Other than enzymes of the citric acid cycle, iso- citritase and.malate synthetase are required to establish the cycle (23). In higher plants,isocitritase is present in those tissues which are actively converting fate to carbohydrates, and the malate synthetase activity of Oil 50 seeds rises sharply during germination (33). Some malate synthetase activity has been detected.in mature leaves of tomato, tobacco, and barley by Yamamoto and Beevers,but Carpenter and Beevers were unable to detect Significant isocitritase in extracts of the leaves of tobacco, barley seedlings, or bean seedlings (270). Although the glyoxy- late cycle is not thought to be in leaves, some Specula- tion continues on this possibility. Thus Asada and Kasai (7) suggest that isocitritase may function 13 3113 in tobacco leaves.and evidence has been presented which sug- gests the possible participation of the glyoxylate cycle in S3932 leaves (200). The glyoxylate cycle is also neces- sary for the replenishment of the intermediates of the citric acid cycle when a 2 carbon compound such as acetate serves as the sole carbon source for growth. Such an application of the glyoxylate cycle is well established in E, ggli_(135), and under the appropriate conditions acetate apparently induces the Operation of the glyoxylate cycle in several algae (167). Aconitase seems to be an indiSpensible enzyme in the citric acid cycle, the glyoxylate cycle, and the ferredoxin dependent carbon reduction cycle (p. 32). There is evidence that the path through aconitase may be the only path available between citrate and isocitrate. Aconitaseless mutants have been isolated from the yeast, saccharomyces, which are capable of converting various 51 substrates to the level of acetate,but which are incapable of degrading two carbon substrates to 002 via the citric acid cycle. The block at aconitase leads to citrate accum- ulation when glucose is metabolized. The inability to reach a-ketoglutarate via the citric acid cycle is expressed as a total growth requirement for glutamate in the presence Of 18 other common amino acids (180). Mutants of Bacillus subtilus, devoid of aconitase, were asporogenic, and trans- fer Of the defective aconitase gene to Sporogenic strains by transformation caused the recipient strains to become asporogenic (228). Aconitase is Often required even under anaerobic conditions. An.anaerobic citric acid cycle has been observed in the photosynthetic bacterium, Rhodospirillum rubrum (92, 93). All the reactions of the citric acid) cycle, with the exception of 2~oxoglutarate dehydrogenase, have been demonstrated in the green Obligately photosyn- thetic anaerobic bacterium,Chloroseudomonas ethylicum, where they act in a biosynthetic capacity (44). The anaerobic bacterium,ClOstridium klgyzeri,possesses aconi- tase and other enzymes Of the upper half of the citric acid cycle,which are necessary for the synthesis Of glutamate from.citrate and precursors of citrate (224). The Intracellular Location Of Aconitase In rabbit brain, aconitase is localized in the ."B -0!- A A ‘4 u. 1“ 1|- q" 1‘ ) 52 mitochondria (214). In rat liver, however, over 80% of the aconitase activity is found in the soluble fraction after differential centrifugation in cold 0.25M sucrose (65, 197). The relatively small percentage of aconitase which is associated with rat liver mitochondria cannot be leached out by repeated washings with cold 0.25M sucrose (65). Schneider (214) pointed out that since small mole- cules such as citrate are kept within the mitochondria during isolation, it seems unlikely that large molecules such as aconitase should leak out, and the fact that citrate rather than any other citric acid cycle inter- mediate is found in the mitochondria might be interpreted in favor Of a low level Of aconitase in mitochondria (19?). A case can be made for a functional role of a naturally occuring extramitochondrial aconitase. It may act in conjunction with extramitochondrial isocitrate dehydrogenase, in a shuttle of tricarboxylic and dicar- boxylic acids to and from the mitochondria, to regulate the rate of oxidation. At the same time, aconitase and isocitrate dehydrogenase may function as a reductive sys- tem for extramitochondrial NADP (197). It is well docu- mented that citrate is excreted from the mitochondria of mammalian tissues in significant quantities (9, 167). Thus, although there is suggestive evidence in favor of a functional extramitochondrial aconitase in mammalian tissue, we cannot decide at present whether it Fi d C- t... -o' A. I A; ‘- 53 is an artifact of preparation or not (197). Histochemical methods conducted with the human parasite,Trichomonas vaginalis,were able to demonstrate aconitase only in some Of the organisms, where aconitase appeared in some of the cytoplasmic granules (presumably mitochondria) and along the nuclear membrane and para- basal body (218). Eighty-five to ninety-five percent of the aconitase activity was found in the supernatant fraction after crude extracts from the leaves of broad bean, cabbage, and mus- tard were centrifuged at 40,000 g for periods up to one hour (11). Similarly, Pierpoint (193) found that most of the aconitase activity of tobacco leaf extracts was found in the supernatant fraction after differential centrifu- gation. On the other hand, Brummond and Burris (38) found that aconitase from the leaves Of three weeks Old Lupinus alggg (Lupine), which had been ground in 0.2M sucrose, was found entirely in the fraction centrifuged down by 25,000 g for 20 minutes. In a preliminary communication,Breiden- bach and Beevers (36) reported that glyoxysomes, isolated from castor bean endosperm by sucrose gradient centrifuga- tion after isolation in a sucrose containing solution, contained all the enzymes needed for the net synthesis of succinate from acetyl COA, with the exception of aconitase. The glyoxysomes were separated from the mitochondria,but the aconitase was present in neither the mitochondria nor 54 the glyoxysomes; rather it was found in the soluble frac- tion. In a note added in proof in a later, more complete report on glyoxysomes from the same tissue source, the authors (37) repOrted that "By providing SH protectants in the sucrose gradient we have now shown that aconitase is indeed associated both with mitochondria and glyoxy— somes." Chloroplasts do not contain a cytochrome c oxidase system,and the prevailing view seems to be that chloro- plasts do not contain a citric acid cycle (94). Leech (143) and Zelitch (270) have reported that some of the cellular malate dehydrogenase from photosynthetic tissue was associated with chloroplasts. More recently, Mukerji and Ting (168) found that malate dehydrogenase was associated with chloroplasts from cactus. Both aqueously and nonaqueously isolated chloroplasts, after washing and purifying by aqueous or nonaqueous gradient centrifugation, were found to contain the enzyme. How- ever, Yamazaki and Tolbert (262), using the technique Of isopycnic centrifugation in a sucrose gradient, found that part of the malate dehydrogenase from the leaves of 6 different plants fractionated with the peroxisome and mitochondrial fractions,but not with the chloroplast fraction. Ogren and Krogmann (177) and Leech (143) have presented evidence that isocitrate dehydrogenase may he present in chlorOplasts. Although the absence of :l AW' . "6.. .i It 55 cytochrome c oxidase in chloroplasts should rule out the classical functioning of tricarboxylic acid cycle enzymes which might be in the chloroplast, such enzymes could be used for the purpose of biosynthesis. The Possible Role Of Aconitase in Metabolic Control A common type of metabolic control involves regula- tion Of an enzyme catalyZLng the first reaction after a metabolic branch point (8). In animal tissues, citrate is an important precursor of fatty acid synthesis (167), SO that in these tissues, citrate is at a metabolic branch point. In plants, the importance of citrate as a precur- sor for fatty acid synthesis is unknown (167). Not all controlled enzymes have their substrates at a metabolic branch point. For example, the affinity of fumarase (an enzyme similar to aconitase) for fumarate is very strongly decreased by ATP even though fumarate is not known to occupy a metabolic branch point of any significance (8). Evidence that the conversion of citrate to gluta- mate is blocked in the light in mung bean leaves has been Obtained by Graham and Cooper (101). Cook and Carver (52) found that light decreased the activity of aconitase, but not isocitrate dehydrogeanse, by 50% in green Euglena, but not in the permanently bleached mutant. On the other 14 hand, when pyruvate 3- C was supplied to the cut ends of Wheat seedlings in the light, the label in glutamate con- 1!. I... v. A a .4 56 firmed the Operation of the Krebs cycle in this tissue in the light (172). A possible physiological role of glyoxylate in the control of the citric acid cycle is suggested by the work of Ruffo et al (205, 206), Payes and Laties (187), and Laties (141). These workers have demonstrated that oxalo- malate, the product of the condensation of glyoxylate and oxalacetate under physiological conditions, and y-hydroxy- a-ketoglutarate, the decarboxylation product of oxalomalate, are remarkably effective inhibitors of citrate oxidation. Both compounds are competitive inhibitors of aconitase,so that control by these compounds would not be an example Of the type of control in which the concentration Of a modi- fier affects the substrate affinity of an enzyme. Never- theless, oxalomalate at 1 mM inhibits aconitase and iso— citrate dehydrogenase almost completely (206). Zelitch and Barber (273) found that glycolate or glyoxylate inhibited the oxidation of Krebs cycle acids by particles from Spinach leaves. Marsh et al (154) found that light had no detectable effect on the turnover of the citric acid cycle in the alga, Scenedesmus obliquus. However, Hess and.TOlbert (118) presented evidence suggesting the absence of glycolate oxidase, and an incomplete glycolate pathway in algae. The enhanced production of glycolate in the light may partially explain the inhibition of the Krebs cycle that is sometimes observed in some plants in 57 the light (73. 273). The work Of Hoch et al (123) and Forrester et al (88, 89) provides evidence that dark reSpiration may indeed be inhibited in the light. But the work of Santarius and Heber (210) suggests that the inhibition Of glycolysis and of dark reSpiration in leaf cells in the light may be because of the changed ratio of ATP to ADP, both Of which are permeable to the chlorOplast membrane. Furthermore, the work of Graham and Walker (102) demonstrates that the ratio of malate to oxaloacetate is significantly increased in mung bean leaves in the light as a consequence Of the increased ratio of NADH to NAD, and.Mudd (167) has sug- gested that the apparent inhibition Of the tricarboxylic acid cycle in the light may be due to the diversion Of acetyl COA to fatty acid synthesis in the presence of ample reductant. Citrate synthetase, which seems to be the locus of the slow rate determining step for the turnover of the citric acid cycle (139), is inhibited by high ATP concen- trations in tobacco and in mammalian tissue (8), and the concentration Of pyruvate, which is freely permeable to the chlorOplast membrane, is known to decrease in the light and increase in the dark (210). Graham and Walker (102), using mung bean leaves, found that the concentration of citrate, which had been labeled by the dark fixation of 14'C02, decreased Significantly after 30 minutes in the light and again increased after 30 minutes in a following dark period, "W1 . l! Hp .lb. ‘7 58 while the concentration Of the labeled glutamate changed - relatively little during the same transitions. Thus, control of the catabolic functioning of the citric acid cycle by the inhibition Of aconitase and/or isocitrate dehydrogenase would seem inconsistent with the latter findings, and at least partially superfluous in 3 view of the possible control of glycolysis and dark reSpiration by other mechanisms. Dark reSpiration may be completely inhibited in the light in corn (89), yet the negligable photoreSpiration in this plant (89), together with evidence that photoreSpiration is a function of gly- colate metabolism (272), suggest that glycolate metabo- lism may not be important in the control of dark reSpira- tion in corn. The possible inhibition of aconitase in the light, which is suggested by the work of Graham and COOper (101), Cook and Carver (52), Ruffo et al (205, 206).Payes and Laties (187), Laties (141), and Zelitch and Barber (273), either by the condensation products of glyoxylate and oxalacetate or otherwise, might be for a less Obvious reason than the control of the catabolic functioning of the citric acid cycle. The concentrations of glutamate and alpha keto- glutarate in tobacco leaves were found to follow diurnal variations. The maximum glutamate concentration per gram fresh weight, which occured between 10 A.M. and 2 P.M., was NIC’ —‘l R“' ib- f‘“ p no—ll .,, U. in u. to 59 about 4 times greater than the minimum which occured between midnight and 4 A.M. (173). The maximum alpha ketoglutarate concentration per gram dry weight also occured during daylight hours (174). Burns et a1 (42) have recently Obtained labeling patterns for glutamate in the leaves of Nicotiana rustica after eXposure of the lucoz'in the light. The investigators thought plants to that the labeling data for glutamate were not easily eXplained by a combination of the photosynthetic carbon cycle and tricarboxylic acid cycle.1 The data were also not compatible with 4 other pathways to glutamate which had previously been proposed. The authors pointed out that their data could be eXplained by assuming a rapid formation of symmetrically labeled glycolate and its subsequent conversion to glutamate, either via glyoxylate, oxalomalate, Y-hydroxy-c-ketoglutarate, and a-ketogluta- rate, or by condensation of glyoxylate with pyruvate to give y-hydroxy-d-ketoglutarate directly. Since it is assumed that isocitrate lyase is absent in the leaves of higher plants, the proposal of Burns et a1 (based on experiments with tobacco leaves in the light) might eXplain the observations of Asada and Kasai (7) that 1The data and prOposals of Burns et al, which were in a preliminary communication, have not been con- firmed by the authors Or by groups from other laborator- ies. The labeling times were 3 minutes and 18 minutes, which seem long for kinetic labeling experiments. r»' We an. .I: 60 glyoxylate was a precursor of isocitrate in tobacco leaves in the light,and that the labeled isocitrate was apparently not converted to labeled citrate. The Purification Of Aconitase To date, most work has been done with aconitase from pig heart. It remains as one Of the major metabolic enzymes which has not been obtained as a stable homogene- ous preparation. The purification Of aconitase has been greatly hampered by its apparent instability (162). Krebs and Eggleston found that glycerol stabilized crude enzyme extracts, but Buchanan and Anfinsen reported that glycerol was without effect in stabilizing purified preparations of aconitase (39). Substrate was found to be the most effective stabilizer of aconitase (5, 39, 114, 162). Henson and Cleland (114) found that for 20-fOld purified beef liver aconitase, both citrate and ammonium sulfate were required for good recovery of activity after storage at -20°. Aconitase from pig heart has been purified 24- fold by Morrison (162) by low temperature ethanol and ammonium sulfate fractionation combined with heat frac- tionation. Citrate was included in all purification steps except the first two ethanol fractionations. Electrophoresis Of the final preparation permitted its purity to be estimated at 75-80%. When the enzyme was 2‘... .1... -.. n u Avid 9: .3 o . Ad ‘5 n- c: '- 61 dissolved in 4 x 10'3M citrate, it was stable for 15 minutes at any temperature up to 51°. From 51° to 55.50. 15 minutes Of heating destroyed the activity so that no activity survived at 55. 5°. Aconitase from mustard leaves has been purified about 200 fold by ammonium sulfate fractionation, DEAE- cellulose chromatography, and gel filtration chromatog- raphy (185). Citrate was included in all purification steps. The best preparation was homogeneous in the ultracentrifuge with 520 = 4.78. Sedimentation of the enzyme after partial decay of activity provided no evi- dence that its instability is due to a major change in the protein structure. The final Specific activities of the mustard aconitase were roughly 10% of those obtained with the pig heart aconitase purified by Morrison. Marked loss of pig heart aconitase activity occured when the pH of the clarified crude extract was adjusted bSIOW‘pH 5.0 (162). The pig heart enzyme was unstable toward freezing at the earliest stage of purification,but the purified enzyme was unaffected by repeated freezing and thawing or by freeze drying (162). In contrast, the ZOO-fold purified aconitase from mustard was totally inactivated by freezing or freeze drying (185). Morrison reported that the fractionation Of purl- fied aconitase from pig heart with acetone resulted in a marked loss of activity even though carried out between ) I n .v» A: ‘- t. p) 62 -5° and -10° (162). The previous purification included two ammonium sulfate fractionations; however the final preparation had been dialysed against 0.004M citrate to remove salt. This finding is in contrast to that Of Buchanan and Anfinsen (39) who reported that aconitase may be precipitated by alcohol or acetone without loss of activity, provided the temperature is kept suffi- ciently low. These authors used ammonium sulfate frac- tionation as a last step after fractionation by alcohol. They also reported that such high concentrations of alcohol are required to precipitate the enzyme in the presence of salts that denaturation results. Thus,it may be that aconitase from pig heart is stable to organic solvents before ammonium sulfate treatment, but not after, even if the salts are removed. Eyidence that Aconitase is One Enzyme Morrison found that during the purification Of aconitase from pig heart, the ratio Of the activities, measured by the conversion of isocitrate to citrate and cis-aconitate to citrate, stayed constant. Furthermore, the partially purified aconitase,when further isolated as an electrophoretically homogeneous protein,was cap- able of converting both isocitrate and sis-aconitate to citrate (162). Morrison's results were a confirmation of the same findings by Buchanan and Anfinsen (39). In IV. 1"- U.- f.” .01 An- '- (t) \“ 63 addition, the latter authors reported that the activities of the enzyme,with reSpect to both citrate and isocitrate formation from cis-aconitate,decayed at the same rate. Similarly, during the ZOO-fold purification Of aconitase from mustard, the ratio Of the activities,measured by the conversion of citrate and of isocitrate to ole-aconitate, stayed constant,and these two activities decayed at the same rate (185). Ogur et al (180, 181) have isolated two mutants (glt1_1 and glt2_1) from the yeast,Saccharomygg§,which exhibited a blocked citric acid cycle due to the lack Of aconitase and which were dependent on added glutamate. When the two mutant strains were mated, glutamate inde- pendent hybrids were always Obtained. Tetrad analysis of the four-Spored asci Obtained from these diploid hybrids showed a high frequency of prototrophic recombinants and the independent assortment of the glt and gltz markers 1 predicted for unlinked genes. Attempts to obtain comple- mentation.in,zlggg| by mixing cell free preparations from the two mutants were negative. Although the work of Ogur et a1 suggests that aconitase in yeast may include two proteins, the work of Tomizawa (240) with isolated acon- itase frOm yeast suggests that it is one protein. Neilson (169, 170, 171) has reported an enzyme from.AsEergillus niggr which catalyzes the interconversion of cis-aconitate and citrate,but which is inactive in the interconversion A\~ Dr '0‘ HA 64 of cis-aconitate and isocitrate. The enzyme (E.C. 4.2.1.4 aconitic hydrase, citrate hydrolyase, also called citrate dehydratase) is also present in Penicillium chrysogenum and to a lesser extent in cucumber leaves (171). Aconitase is present in Aspergillus giggg_but can be separated from aconitic hydrase (169, 170, 171). To my knowledge, no enzyme has been reported which catalyzes only the inter- conversion of ole-aconitate and isocitrate. The Effect Of Ions The pH Optimum of aconitase isolated from mammalian tissue is influenced by the ions present in solution. For the isocitrate to citrate, isocitrate to cis-aconitate, and cis-aconitate to citrate reactions, the pH optima were the same in the presence of a particular buffer and were altered to the same extent in different buffers. The range in the pH optima for the 4 buffers tested was 7.5 to 8.6, while the pH Optimum with no buffer present was estimated at 8.0. For each of the three reactions, the absolute rates were approximately the same at the pH Optimum for each buffer (165). Thomson et al (231) found that aconitase isolated from beef liver had a pH Optimum 0f 7 in 0.02M glycine buffer,but that the pH Optimum was shifted to 8 when 0.2M NaCl was included. Furthermore, the maximum activity with NaCl present was 1.4 times greater than the maximum activity when it was absent. .i‘— u .1 (lililllillrlll L. r 65 Peters (191) found that increasing amounts of NaCl dra- matically increased the activity of aconitase from pig heart,while KCl at low concentrations increased the activity and at high concentrations decreased the activ- ity. Aconitase isolated from mustard is reported to have a pH Optimum Of 8.0 at low isocitrate concentrations, and 8.5 at high isocitrate concentrations. Increasing the isocitrate concentration also had an activating effect (185). pH Optimum For the interconversion Of the tricarboxylic acids, rat liver mitochondria exhibited a biphasic pH Optima curve, with Optima at pH 5.8 and about 7.3 (65). Aconi- tase which could be released from the mitochondria exhibited the same single alkaline pH optimum as the soluble aconitase. The authors favored the idea that the lower pH optimum was a consequence of increased permeabil- ity Of substrate through the mitochondrial membrane, although the alternative that the structural binding of aconitase may alter its pH Optimum was not excluded. The Effect of Metals, Chelating, and Reducinnggents Iron binding agents such as O-phenanthroline and “cm-bipyridyl inhibit the animal aconitase, which is con... sistent with the concept that the native enzyme contains :‘1'4.’ '0 r;- AIV -'i 66 iron which is necessary for activity (64). Chelating agents affect the animal aconitase only after preincuba- tion. The inhibition can be reversed by excess Fe++ but not by excess substrate (115). Thus, it appears that sub- strate prevents inhibition by chelating agents but will not reverse it. Dickman and Cloutier (64) found that aconitase from pig heart, which had lost activity during purifica- tion, could be reactivated by preincubation with Fe++ plus cysteine or ascorbate. NO other metal would sub- stitute for iron. Glutathione was about half as effec- tive as the other two reducing agents. Morrison (162, 163) confirmed and extended these findings with the enzyme from pig heart. With reSpect to its ability to convert both isocitrate and cis-aconitate into citrate, the addi- tion of Fe++ and cysteine increased the two activities equally. The activating effect Of Fe++ and cysteine during the early stages of purification was small and remained reasonably constant. After dialysis, the activation was greatly enhanced.1 Fe*+ and cysteine can, therefore, completely replace the dialyzable prosthetic group. It would appear that not only is iron capable of activating 1The dialysis was performed against 0.004M citrate. Apparently, citrate was required to maintain the enzyme in a stable configuration even while it became inactive from loss of Fe++. in '0 o c 0’ \AI v.. ’If 67 pig heart aconitase, but it is also the metal with which the enzyme is associated ig_zizg (162). Buchanan and Anfinsen (39) reported that cysteine alone, in the initial stages of the purification, showed a stabilizing effect On pig heart aconitase,but as the purification of the enzyme continued, cysteine alone strongly inhibited the enzyme.1 A similar Observation was made by Herr et al (115) who found that when cysteine alone was preincubated with a freshly prepared enzyme solution, a marked loss of activ- ity occured. The activation of pig heart aconitase in the presence of reducing agent (10'2M) by Fe++ followed a Michaelis Kenton saturation curve, suggesting that one iron is bound per active Site to form the active enzyme (163). The KA for Fe++ in the presence of cysteine is 3.9 x 10'6M and it is 1.7 x 10'5M in the presence of ascorbic acid. The activation of the enzyme in the presence of Fe++ (5 x 10'uM) by cysteine, thioglycolate, and ascorbate also followed Michaelis Menton saturation curves, suggesting that one molecule Of reductant is required per active site to form the active enzyme. The 1Dickman and Cloutier (64) found that cysteine alone activated pig heart aconitase, but it was later found by Morrison (162) that the preparation of cysteine which gave similar results to those of Dickman and Cloutier contained appreciable amounts of iron. Morri- son (163) found that cysteine alone had no activating effect on the inactive purified dialyzed aconitase. He also found that it was not necessary to add Fe++ and cysteine as stabilizing agents (162). 3" v. ID mu. ’f.’ l..< 0.. 68 KA values are 2.3-3.6 x 10'3M for cysteine, 4.6 x 10'3M for thioglycolate, and 1.2 x 10’3M for ascorbate. The amounts Of the reducing agents present,and the fact that the redox potentials are such that essentially all the iron present, for all concentrations of reductant, was in the Fe++ state, suggest that the reductants must have a function in addition to keeping iron in the Fe++ state. The Vmax was dependent on the nature of the reductant, suggesting that not only Fe++ and enzyme,but also reduc- tant, participate in the active complex, a concept which is strengthened by the finding that the dissociation con- stant of the Fe++ enzyme complex varies according to the reducing agent present. The kinetic evidence that only one reductant was required per active site and the find- ing that arsenite does not inhibit aconitase suggest that the function of the reductant is not the cleavage of S-S on the enzyme (163). Speyer and Dickman (223) postulated that Fe++ and reducing agent take part in an enzyme-metal-substrate- reducing agent complex and also that Fe++ acts as an electron acceptor or Lewis acid which aids in lowering the activation energy in the removal of OH' from citrate or isocitrate. But whether Fe++ participates in the dehydration mechanism directly or indirectly is still unknown (203). The picture is further complicated by evidence that the pig heart enzyme reconstituted by the ‘ I, i iilime‘Fau . 69 addition Of iron and reducing agent may be different from the native enzyme (191). Bacon et al (10) grew mustard plants in nutrient solutions containing different concentrations of iron. The leaves of those plants receiving the least iron, which always showed signs Of iron deficiency, exhibited about one-half the aconitase activity per gram of leaf as those from non-deficient plants. Simultaneous measure— ments of malate dehydrogenase and fumarase showed that these activities were not significantly affected by iron deficiency. The aconitase activity of extracts from the deficient plants could not be increased by treatment with Fe++ and cysteine, and mixtures of extracts from normal and deficient plants did not Show an activity greater than the sum Of the activities measured separately. The authors postulated that the lower aconitase activity in iron deficient plants is due to a lack of the enzyme Sys- tem as a whole and not merely to a lack of iron (10). Palmer (185) found that the ZOO-fold purified aconitase from mustard was not activated by Fe++ and cysteine. Under certain conditions,partially purified preparations could be activated to some extent. Dialysis of the enzyme against citrate buffer did not lead to any measurable loss in activity. Cysteine alone had no effect on the stabil- ity of the plant aconitase. Preincubation with o-phen- anthroline caused a slow loss Of activity compared to the 70 control, which the author postulated may have been due to a nonchelation effect.1 The purified enzyme did not con- tain a significant amount of iron. Palmer (185) concluded that iron does not play a direct role in the activity of plant aconitase and supported the earlier conclusion that the lower activity of aconitase in iron deficient leaves is because of an indirect effect on the enzyme (10). ’ Rahatekar and Rao (198) reported on the Fe++ plus cysteine dependent activation Of aconitase from bacteria, molds and plants. The activation was measured using crude preparations which had been concentrated by ammonium sul- fate precipitation. Aconitase from E. ggli was activated 2.3 to 8-fold by Fe++ plus cysteine, the enzyme from Aspergillus giggg 1.8-fOld, and the aconitase from pea (Piggm sativum) and chOlai (Klfiflé catjang) three day Old etiolated seedlings 1.2 to 2.3-fold. The aconitase from cholai cotyledonous tissue, grown in tissue culture, was (stimulated 3 to 9 fold. The aconitase from all the sources tested showed significant inhibition by phenanthroline. These activations, though Significant, are relatively small compared to those which have been reported for the aconitase from animal tissue (163). Growth of Aspergillus giggg on a chemically defined 1Glutamate dehydrogenase is inhibited by o-phen- anthroline and also by analogs of this compound Which do not possess chelation properties (264). I, h 71 medium containing no manganese yielded a mycelium that apparently was free Of both aconitase and aconitic hydrase, and which gave an accumulation of citric acid. When the medium was made 3 ug percent in manganese, the mycelium contained aconitic hydrase activity but lacked aconitase activity. Doubling the manganese concentration in the medium to 6 us percent yielded a mycleium with some aconitase activity along with aconitic hydrase activity. When manganese was added to extracts from the mycelium grown on the manganese deficient medium, the two enzyme activities were still absent. Thus manganese is appar- ently required for formation Of the two enzymes rather than for direct activation (170). The aconitase of Chlorella vulgaris has been reported to Show a dependence on manganese similar to the dependence found with Agpgg- gillus alga; (28). These same investigators reported that an iron deficient culture medium for Chlorella increased the aconitase activity, which is in contrast to the effect of iron deficient growth conditions on the aconitase of mustard. Morrison (162) reported that incubation of the Fe+f. cysteine activated aconitase from pig heart with phOSphate buffer at pH 7.4 at 30° essentially completely inactivated the enzyme in 30 minutes, presumably due to the formation of the insoluble iron phOSphate complex. The water con- trol lost little activity. Similar results were Obtained 72 with veronal-acetate, borate, and glycero-phOSphate buf- fers at pH 7.4. The inactivation by incubation with buf- fers could be completely prevented with substrate (162, 165). Therefore, when it was desired to maintain activ- ity, reactions were initiated by adding enzyme to the buffer substrate mixture (165). In contrast to Morrison's findings, Dickman and Cloutier (64) found that phosphate did not inactivate the iron-cysteine activated enzyme from pig heart. However, they did find that phOSphate prevented the activation Of aconitase by iron plus cysteine,and that bicarbonate had this same effect. Phos- phate buffer had no inactivating effect on the aconitase of higher plants compared to Tris buffer (11). Inhibitors p-Mercuribenzoate at 0.00053 mM or HgCl2 at 0.0011 mM inhibited aconitase 50%. Iodoacetate at 10.0 mM or arsenite, a reagent for vicinal sulfhydryls (84, 85, 217), at 10.0 mM gave no inhibition of aconitase. The enzyme had been activated by Fe++ and ascorbate and the inhibitors were at the concentrations indicated during the 5 minutes Of preincubation at the reaction temperature of 30°. Inactivation of aconitase from pigeon breast muscle with p-mercuribenzoate could be reversed with glutathione (63). Aconitase from pig heart is strongly inhibited by cyanide and sulfide, possibly because of the presence of Fe++ as s” fl 73 a cofactor, and is also inhibited by low concentrations of Cu?"+ ions (5, 63). Inclusion of Cu'"+ with manganese in the growth medium prevented the formation of both aconi- tase and aconitic hydrase in ASpergil;E§_gigg£ (170). Ten minutes of preincubation of aconitase from mustard with 1 mM iodoacetate had no effect on the activity (185). Aconitase in centrifuged extracts of broad bean, cabbage, tomato, mustard, Heracleum, Hosta, and Sambucus lost 50-75% of its activity when preincubated with 1-10 mM cyanide for 30 minutes at 0° (11). In a study of aconitase from rat kidney, Fanshier et al (80) found that of the four possible fluorocitrates, the isomer from the enzymatic synthesis starting with fluoroacetyl COA and oxalacetate is the inhibitory Species. Two kinetic mechanisms are reSponsible for the inhibition Of this aconitase by fluorocitrate: (a) direct competi- tive inhibition, detectable by initial rate measurements, and (b) time dependent progressive inhibition. With sonic extracts as the enzyme system, fluorocitrate inhibited the 12.2itgg,conversion Of citrate to cis-aconitate, iso- citrate to Sis-aconitate, and citrate to isocitrate, but for some reason did not produce direct competitive inhi- bition of cis-aconitate to a-ketoglutarate when the aconitase reaction was coupled to isocitrate dehydrogen- ase. Peters (191) reported that the time dependent pro- gressive inhibition Of the pig heart aconitase by fluoro- U. V. 'r '1 (‘1‘ f 74 citrate could be partially reversed by high concentrations of substrate. Sycamore aconitase is inhibited by fluoro- citrate at a much higher concentration (about 2000-fold) than that needed to inhibit pig heart aconitase. The inhibition is qualitatively similar to the inhibition of pig heart aconitase in that the inhibition is Slow to develOp, requiring 10 to 20 minutes at 25° to reach a maximum, but once developed, the inhibition of the plant aconitase can be largely reversed by high concentrations Of substrate (242). Substrate Specificity 0f the substances present in biological material, only isocitrate, citrate, and ciS-aconitate are sub- strates for aconitase (63, 139, 219). There appear to be no significant competing reactions in crude pig heart tissue extracts which might lead to error in the assay of aconitase (5). Binding of Tricarboxylic Acids to Aconitase Henson and Cleland (114) reported that citrate, which was added during purification for stability pur- poses, remained bound to beef liver aconitase even after passage through Sephadex G-25. The amount of citrate that was bound to the enzyme was sufficient to require removal before kinetic experiments could be performed. .u nil 75 The activities of aconitase (165, 191, 231) and fumarase1 (158) are both known to be affected by the presence of various anions in the assay solution. Massey (158) found that the effect Of anions on fumarase was not a direct effect at the active center. He suggests that both activating anions and substrate can combine with groups on the protein other than at the active center. Palmer, using the argument of Massey, postulated that the apparent activating effect on purified mustard acon- itase by isocitrate was by combination with groups on the protein other than at the active center (185). The KIn Values for the Substrates and the KT for Trans-aconitate The Km of beef liver aconitase for citrate was found to be 0.95 x 10'3M, for threO-Ds-isocitrate 1.39 x 10'4m, and for cis-aconitate 0.99 x 10’4M (231). The KIn values Of pig heart aconitase (164) and rabbit liver aconitase (239) were found to be Of the same order, and the Km values determined by Racker (196) are likewise of the same order. However, the Km values of beef liver aconitase which were determined by Henson and Cleland (114) are an order of magnitude lower than those deter- mined by the other investigators. Every investigator 1Fumarase is thought to be an enzyme similar to aconitase (164, 179). 76 found that the Km for cis-aconitate is the smallest, the Km for isocitrate is intermediate, and the Km for citrate is the largest. Bacon et al found the Lineweaver and Burk plots Obtained with crude aconitase preparations from higher plants to be not linear (185). At low substrate concen- trations, Palmer (185) found the Km of purified mustard aconitase for citrate to be 4.4 x io-3M, for threO-Ds— isocitrate 1.5 x io'uM, and for cis-aconitate 1 x 10'4M. These values are of the same order as those quoted by most investigators for the enzyme from animal tissue. But the Km values of the purified mustard aconitase were found to be a function Of substrate concentration. The Km for isocitrate was found to increase 100-fold to 15 x 10'3M at high concentrations. Trans-aconitate is a competitive inhibitor Of aconitase from animals and plants (5, 63, 231). The KI Of beef liver aconitase for trans-aconitate was found to be 7.0:x 10'§M which is about the same value as the Km for citrate in the same experiments (231). The isomer threO-LS-isocitrate was found to have a KI of about 4 x io-3M (231). §pbstrate as a Competitive Inhibitor of Aconitase Product inhibition is usually competitive inhibi- tion (149). For the conversion of any one tricarboxylic '. .Xwnfi— 77 acid, the other two tricarboxylic acids should be competi- tive inhibitors of aconitase. Tomizawa (241) demonstrated that DL-isocitrate-Z-lu'c competitively inhibited the forma- tion of unlabeled citrate from unlabeled cis-aconitate. The Mechanism Of Action of Aconitase StereOSpecificity Water can be added in four ways to cis-aconitate to give the 4 stereoisomers of isocitric acid. The two pos- sible trans additions yield the two threo enantiomorphs, while the two possible cis additions yield the two erythro enantiomorphs. The "natural" isomer Of isocitric acid is threo-Ds-isocitrate1 (248).2 From the known geometry of cis-aconitate (151, 152) and the known config- uration of the threo-Dseisomer, the trans nature of the dehydration Of isocitrate to cis-aconitate and of the reverse reaction is established (90) (Figure 1C). 1Until 1959,0nly threo-Ds-isocitrate had been found in living organisms. In 1959, erythro-Ls-isocitrate was discovered as the fermentation product Of Penicillium. The evidence suggests that the path to this fermentation product does not include aconitase (208). TO my knowledge, there is no evidence for any aconitase in nature having as substrate any isomer other than the threo-Ds form. 2Much confusion exists in the early literature with regard to the nomenclature and absolute configuration of the isocitrate isomers. The review by Vickery (248) sum- marizes the history up until 1962. Since 1962, there has been little confusion. A. B. C. 78 Figure 1 The Stereochemistry1 Of the Aconggase Reactions (106) The absolute configuration of enzymatically formed citric acid. Hanson and Rose, using citric acid with one Of the methylenes containing one tritium, and with a known abso- lute configuration with respect to the center carboxyl group, the -OH group, and the methylene group which con- tained tritium, found that all the tritium was released to water by aconitase from pig heart. Thus,the stereo- chemical relationship between the center -COOH group, the -OH group, and the methylene containing the hydrogen exchangeable by aconitase is established in absolute terms. Since the portion of the molecule derived from oxalacetate is known to bear the hydrogen exchangeable by aconitase, the stereochemical course Of citric acid biosynthesis is also established in absolute terms. The symbollcvindicates that the configuration of the carbon atom bearing tritium was unknown. The configuration at the oxalacetate derived methylene group Of citric acid (the determination of which hydrogen exchanges with the medium in the presence Of aconitase). The configuration at the oxalacetate-derived methylene group of citric acid had been correlated with the fumarase reaction by Englard (76),and the trans nature Of the fum- arase reaction had been established by Gawron et al (90) and by Anet (4). ‘fhe stereochemical knowledge of which methylene group of citric acid is derived from oxalace- tate lead Hanson and Rose to summarize Englard's experi- ments as shown. When Englard treated the two citric acids with pig heart aconitase and isocitrate dehydrogenase, the deuterium from fumaric acid -2,3—D appeared in NADPH while the deuterium from D20 did not. The hydrogen which exchanges with the hydrogen of the medium in the presence of aconitase is therefore known and it is established that the hydration of cis-aconitate to citrate in the presence of pig heart aconitase is trans. The stereochemistry Of the aconitase reactions. The hydra- tion of cis-aconitate to form citrate and the hydration to form isocitrate are both trans. Furthermore, a given double bond carbon atom is always attacked from the same side, i.e. the alternative approaches of the proton must be from Opposite sides Of the cis-aconitate plane (106). 1The stereochemical formulas of this figure are Fisher pro— jection formulas. 79 A. COOH I H~C~T I HOOC—c—OH I H-C—H l COOH B. D II COOH COOH COOH COOH H I COOH \ / I I I \ / C DO H-C-D H-C-D H-C-D C n .39 l _9 I __9 I u c H-c-OH c=O HOOC-c-OH C\ I \ I / l HOOC II COOH COOH H-C-H HOOC-CHZI COOH I w COOH OH H 0 COOH COOH COOH cOOH o T COOH \ / \ / c ygo D-C-H D-C-H c-c-H c u .—s I —e I -—e I —e II c. D-C-OH C=O HOOC-C-OH c / \ I I I / I\ HOOC :3 COOH COOH ri-C-+I HOOC-CHZI COOH I \I COOH OH .C. H* OH COOH H I COOH H i COOH COOH I \ / \ / l H-C—H* c c H-c-OH HOOC-C-OH ‘73 c ’ c HOOC-c-H* I / \ / \ I H-c-II HOOC—CHZt apom ommpmsamosa opmHoomHMim you oommmmm pmhaa who; msoapomhm soapomnm msaudaanmpm a one asamamomeOHno omim Hooiofim Medusa scapomam ammpmsamonm manmpmsp as oped ommpmsamosm opmaoomawlm mo scapsaomom 0H mhswam (°/.) paueiii'to M!!!QDlS ° _'_0' AAA O OO O N (010 V 889 l I T l l l O O O .575 .23}: o __. {if-3 3,32 . O O O 0 :6: -( O .C O. (D O .C 0. C + l l 1 L l O O O O O s 0.2 x 2 w luJ/sigun ‘Mmgioo espioquoqd 20 26 32 38 44 50 56 62 Fraction number l4 148 The Best Single Purification Sequence for P-glycolate PhOSphatase A summary of the procedure is given in Table 4. The acetone which was used was reagent grade. Extract About 85 medium sized tobacco leaves were harvested at 4:00 P.M. on 8-4-1966. The 721 grams fresh weight of leaf blades were passed through a Hobart meat grinder fol- lowed by 1442 ml of cold water. The slurry was allowed to stand about 5 minutes before it was strained through a double layer of cheesecloth. After centrifugation, the extract was adjusted from its pH of 6.0 to 5.7 with 0.1N HCl. The final volume of the extract was 1800 ml. First Acetone Fractionation 720 ml of 00 acetone was added to the 1800 ml of extract (which contained 11.7 mg of protein per ml) through one 0.70 mm I.D. teflon tube over a period of 3% hours while the mixture (held in an ice bath) was contin- uously stirred by a magnetic stirrer. The mixture was centrifuged at 15,000 x g for 15 minutes and the precipi- tate was discarded. 360 ml of cold acetone was added as before to precipitate the enzyme. This precipitate was removed by centrifugation at 15,000 x g for 8 minutes and the supernatant solution was drained and rinsed as com- Pletely as possible from the centrifuge cups. The pre- 149 cipitate was taken up in 225 ml of 0.02M cacodylate, pH 6.3, and recentrifuged at 15,000 x g for 8 minutes. The 235 ml of supernatant solution from this last centrifuga- tion, which contained 20-fold purified phOSphatase, was saved and the precipitate was discarded. Second Acetone Fractionation Fifty-one ml of 0° acetone was added as before to 203 ml of the aqueous solution from the first acetone fractionation (which contained 4.7 mg of protein/ml). The mixture was centrifuged at about 6000 x g for 8 minutes and the precipitate was discarded. Then 41 ml of cold acetone was added as before and the mixture was cen- trifuged at about 6,000 x g for 8 minutes to remove the enzyme. After careful removal of as much of the acetone solution as possible, the precipitate was dissolved in 81 ml of cold 0.02M cacodylate, pH 6.3. The mixture was recentrifuged at about 6,000 x g for 8 minutes to remove insoluble protein. The volume of the active supernatant solution, which contained 59-fold purified phosphatase, was 85 ml. Third Acetone Fractionation A pilot fractionation at pH 5.8 was first made. Then 74 ml Of the aqueous solution from the second acetone fractionation was adjusted from pH 6.4 to 5.7 with 0.1N 150 HCl. The volume after the pH adjustment was 80 ml. Twenty-seven ml of 0° acetone (34% of the adjusted volume of the aqueous solution) was added as before to the 80 ml of aqueous solution (which contained 2.5 mg protein/ ml). The precipitate was discarded by centrifugation at 5,900 x g for 8 minutes. Then 16 ml of 0° acetone (20% of the adjusted volume of the aqueous solution) was added as before to precipitate the enzyme. After centrifugation as before, the supernatant was drained and rinsed from the centrifuge cups. The precipitate was dissolved in 18 ml (25% of the 74 ml) of 0.02M cacodylate, pH 6.3. Recentri- fugation was not necessary. The final solution contained 99-fold purified phOSphatase and 5.5 mg of protein per ml in a volume Of 20 ml. DEAE-Cellulose Chromatography One hundred ten days elapsed between the third ace- tone fractionation and the DEAE chromatography during which the enzyme was held at 0-4°. Before the enzyme was added to the DEAE column, an inactive precipitate was removed by centrifugation at 15,000 x g for 10 minutes. 49.5% of the activity was lost in the 110 days of storage, while the Specific activity remained almost constant. The loss in activity during storage is not charged against the purification. DEAE-cellulose (Sigma, 0.80 milliequivalents per 151 gram, medium mesh) was pretreated as recommended by Peterson and Sober (192). 0.1M EDTA was included in the early NaOH washings. The DEAE slurry was adjusted to pH 6.3 and equilibrated with 0.02M cacodylate,pH 6.3.in a column 1.5 cm in diameter and 6.0 cm high. 6.5 ml of the enzyme containing 3 mg of protein/ml was added to the column and eluted with 200 ml of the same buffer contain- ing NaCl in a linear gradient from 0 to 0.25M. The active fractions, 78 through 84, eluted at a NaCl.concentration of 0.13M, contained 450-fold purified phOSphatase and 0.09 mg protein/ml in a volume of 17.3 ml. Bio-Gel P-60 Chromatography A Bio-Gel P-60 (50-150 mesh Calbiochem) column 1.5 cm in diameter x 17.5 cm high was prepared using gel which had been hydrated in 0.02M buffer, pH 6.3, for 2 days. A 1.2 cm thick layer of DEAE-cellulose,prepared as described in the previous step,was layered over the gel and the entire column was washed at pH 6.3. 12.5 ml of the enzyme from the previous step,which was 0.13M in NaCl,was diluted 4-fold with 0.02M cacodylate, pH 6.3, which also contained 10'3M citrate and 10-3M MgSOu. The diluted enzyme prepara- tion was slowly fed onto the top of the column over a period of about 10 hours. The column and enzyme were first washed with about 30 m1 of the buffer containing 0.02M cacodylate, pH 6.3, 10'3M citrate, and 10'3M MgSOg, 152 which effectively removed the NaCl from the column. The enzyme was then eluted with 10 ml of the same buffer which contained, in addition, 0.25M NaCl, and was then further eluted with buffer without NaCl. The most active fractions, 9 through 16, which were shown to be free of NaCl, con- tained 1060-fold purified phOSphatase and 0.023 mg of protein per ml in a volume of 7.45 ml. The method which was used for the concentration of the enzyme seems like it should be generally applicable to situations in which gel filtration chromatography is to follow ion exchange chromatography, providing the enzyme is stable to the dilution which is necessary to decrease the salt concentration of the preparation from the ion exchange column. The valume of the ion exchange pad which is required for the concentration of the enzyme is small in comparison to the volume of the column of gel, and the amount of enzyme which the pad will retain should be independent of the volume in which this enzyme is con- tained. .In the procedure described here,the enzyme,which had origrnally been contained in 12.5 ml containing 0.13M NaCl, had been applied to the column in a volume of 50 ml containing 0.033M NaCl. Even though dilution of the enzyme would be eXpected to occur during the gel filtra- tion chromatography, 85% of the phosphatase which was recovered from the column was in the 7.45 ml from the pooled fractions. Thus,the method which was used for 153 concentrating the enzyme was quite effective. If care is taken in the preparation of the column, the overlying relatively thin ion exchange pad, and in particular the interface between the pad and the gel, it should be pos- sible to elute the enzyme in a relatively sharp horizontal band ideal for further development by gel filtration. As was demonstrated, the procedure is also capable of com- pletely removing all the salt which is present in the enzyme preparation from the ion exchange chromatography step, as well as all the salt used to elute the enzyme from the ion exchange pad. Finally,although the require— ment for sharp elution is somewhat restrictive, further purification of the enzyme on the pad itself is possible. Rechromatography_on DEAE-Cellulose The previous preparation was held 3 days at 0-4°, during which time 20% of the activity was lost and the Specific activity drOpped from 333 to 266. DEAE-cellu- lose, prepared as described above, was equilibrated with a buffer of 0.02M cacodylate, pH 6.3, 10'3M citrate,and 10'3M MgSOu in a column 0.7 cm in diameter and 1.0 cm high. 6.3 ml of the NaCl free enzyme preparation from the previous step was slowly added over a period of % hour to the column,and then eluted with 20 ml of the same buffer-citrate-MgSOu solution containing NaCl in a linear gradient from o to 0.5M. Only one major protein peak, 154 which contained the phOSphatase, was eluted (Figure 11). In the most active fractions 10 and 11, eluted at a NaCl concentration of 0.12M, containing 0.043 mg protein per ml in a volume of 2.2 ml, the Specific activity of the phOSphatase was 214. Discussion of the Purification Procedures The purification of the phOSphatase which was described on pages 137 to 148 was part of experiments which were conducted to test the hypothesis that P-glyco- late phOSphatase from tobacco leaves contains an endo- genous stabilizing factor. The three acetone fractiona- tions may have been especially beneficial in removing other small molecules from the phOSphatase-endogenous stabilizing factor complex. In particular, care had to be taken not to add compounds either for stabilization or as specific'chromatographic eluents. Attempts to further improve the purification procedure for the phOSphatase per se should not be subject to these restrictions. The Specific activity of 333 for the purest prepar- ation (Table 4) is considerably greater than the previ- ously reported high of 1.61 (202). Part of the increase is because the starting specific activity in my procedure (0.314 (Table 4)) was about 10 times greater than the 1480/309.75 = 1.6 (see p. 109 for a discussion of this calculation). 155 .HB .39 maopopa m1 OIIIO .HB pom ommpmsamona opmaooadwtm .Ho mpHQD Dill. ommpmsamosm mumaoohawim no hmammmOpmaoasu omOHSHHoU|m¢mm Hmsflm .3 mhdwflm 156 [LU/5” ‘UlOiOJd l L 1 L L 9 co co asow cacaooam i 158 starting Specific activity in the procedure used by Richardson and Talbert,l perhaps partly because advantage was taken Of the study conducted on the influence of leaf Size and position on the Specific activity of P-glcyolate phOSphatase from tobacco (Table 1). Another part of the increase was because of the use of cacodylate instead of Tris-acetate, with consequent improved buffering and rate increase. Finally, some of the increase was because of the higher fold purification. The final DEAE-cellulose chromatography of the purest preparation (Figure 11) failed to Show significant protein contamination of the phosphatase. However, the enzyme cannot be considered homogeneous on the basis of one criterion of purity. Further Data and Observations on the Purification of P-glycolate Phosphatase Preparation of Acetone Powders When it was desired to store the phOSphatase for long periods of time, the acetone precipitates from the first or second fractionations were converted to a dry powder. The acetone precipitates, without first rinsing the excess acetone from the centrifuge tubes with water, were dried in a dessicator for 15 hours at an ambient temperature of 20-25°. The first 12 hours of the drying lThe Specific activity of the crude tobacco sap after the first centrifugation was 9.5/309.75 = 0.031 (202). 159 period were under a vacuum, from a water pump, of about 15 mm Hg, and the final 3 hours were under a vacuum, from a Cenco vacuum pump, of 1 to 2 mm Hg.1 It was found that rapid drying of the precipitates under a vacuum of less than 1 mm Hg caused the destruction of much of the phos— phatase activity. As discussed elsewhere, the phOSphatase at this stage of purification was found to be unstable to freezing. Inactivation by freezing may eXplain the low recoveries which were Obtained when the acetone precipi- tates were dried rapidly. When making powders of first acetone precipitates, it was profitable with reSpect to time and yield to centri- 1The procedure as described was used for acetone precipitates which had come from extracts which had been prepared by Waring blendor. Although only precipitates from the first acetone fractionation of these extracts were converted to a powder, there is no evidence that precipitates from the second or third acetone fractiona- tions from such extracts could not successfully be con- verted to dry acetone powders. . When the extracts were made by the meat grinder, a modified procedure was used for the preparation of the acetone powders. The phosphatase in such extracts was not as stable as the enzyme in extracts prepared by Waring blendor (p. 129). As discussed in a later section, the unstable enzyme was stabilized by acetone precipitation, but the stabilization was not as great as the stabiliza— tion by Waring blendor (compare Figures 24 and 42, with F1Sures 8 and 21). The modifications included the follow- ing.“ The acetone was first rinsed from the centrifuge tubes with cold water. The initial drying at 15 mm H was for 3 to 4 hours, with the ambient temperature at 0—4 , until the smell of acetone had vanished from the precipi- tates. The next 12 hours of the drying period at 15 mm Hg and a final 12 hours at 1 to 2 mm Hg were also at an ambient temperature of 20-25°. Only precipitates from the second acetone fractionation of extracts which had been made by the meat grinder were converted to a powder. 160 fuge the active precipitates in centrifuge tubes contain- ing precipitates from previous centrifugations until each tube contained precipitates from three centrifugations. After the precipitates were dry, static electric- ity made it difficult to remove the powder from the poly- ethylene centrifuge tubes. This difficulty was overcome with the aid of a polyethylene rod charged by rubbing with a clean cloth. Little or no activity was sacrificed in the drying of theprecipitates.1 When the phOSphatase in the dry acetone powder was stored'at 0-4° in a dessicator over anhydrous CaSOu, it retained all its activity for at least 5% months. When the powder was stored in a poly- ethylene bottle at room temperature, the enzyme retained all its activity for at least one week. When it was desired to redissolve the dry acetone powder, the same volume of cold 0.02M cacodylate, pH 6.3, was used as would have been used had the precipitate not been dried, and insoluble material was removed by centri— fugation at 6,000 x g for 8 minutes. Observations on the Acetone FractiOnations The arrangement used for making large scale acetone 1NO activity was lost when precipitates from extracts which had been made by Waring blendor were con- verted to a powder. A relatively small percent of the activity was lost when precipitates from extracts which had been made by a meat grinder were converted to a powder by the modified method described in the previous footnote, page 159. However, even more activity was lost in the latter case when the unmodified procedure was used. 161 fractionations is Shown in Figure 12. The height of the ice bucket containing the acetone was easily adjusted so as to control the rate of the addition of acetone. Some of the following observations are from a large number of eXperiments designed for other purposes, and therefore were not controlled with reSpect to each observation. Effect of the Homogenization Method and the Amount of H20 Used The phOSphatase from extracts prepared by meat grinder or by mortar and pestle was about 2 times more highly purified by the first acetone fractionation than from extracts prepared by Waring blendor. Part of this advantage was lost during the two subsequent acetone fractionations. The increment of acetone needed to precipitate the phOSphatase during the first fractionation depended on the amount of water used in preparing the leaf extract. When an extract was prepared by the meat grinder without the addition of water, the activity was precipitated over a broad range of acetone concentration. The addition of one weight of water per fresh weight of leaf tissue in the preparation of an extract resulted in virtually all of the activity being precipitated by an increment of acetone that was equal to only 15% of the volume of the extract. Addition of twice as much water did not seem 162 mso mso Hp anomam mQOpoo< madam amped madam: how pom: psoaomsmhhs one NH enemas xotn coon. sotzm Ozocoos. n w 7 .02 E .963 «.36 con 9:th a o c. 0.335 ooaosoo ocoafozoa “a T 2535 ocoaood .. u. a C a do Cut 4 «<4 cucn _ .853 2: co £58 20365 822 3.532... anG 3 6 1 .28.... 8:8 85 act uoaoatmcoo «1 266on j quu or a .393 ocoafiozoa .0 acts. , 2963 320 o c_ .o._ ESQ. o. EENO m 2 _ ocoaooo cacao Eooooa so 2595 623022 164 to contribute further to this pronounced decrease in the range of acetone concentration within which virtually all of the enzyme was precipitated. Post Homogenization Time Since the phOSphatase in extracts prepared by a meat grinder or mortar and pestle slowly changed from an enzyme unstable to dilution at 30° to an enzyme stable to these conditions (Figure 19), the effect of post homogen- ization time on the concentration of acetone required to precipitate thephosphatase1 from an extract, which had been prepared by meat grinder, was tested (Table 5). Post homogenization time had no significant effect on the concentration of acetone required to precipitate the enzyme.1 Table 5 The Acetone Concentration Required to Precipitate P-glycolate PhOSphatase as a Function of Post Homogenization Time W Post homogenization time Maximum activity precipitated hours ml acetone/10 ml of extract 1% 4.8 6 4.8 34 4.7‘ 1The concentration of acetone required to precipi- tate the phOSphatase is defined to include half of the range of acetone concentration within which virtually all of the phOSphatase was precipitated. 165 Effect of pH and Unknown Factors Over a 2 year period, a considerable variation was found in the acetone concentration needed to precipitate the phosphatase. One of the smallest acetone concentra- tions needed to reach the maximum activity which was pre— cipitated was 4.5 ml of acetone per 10 ml of extract,while the greatest was 6.5. Both of these extracts were pre— pared by mortar and pestle using two weights of water per fresh weight of leaf. In the former case, the enzyme was from a pH 5.3 extract.and in the latter the enzyme came from a pH 6.4 extract. Both pH values were those natur- ally occuring in the extracts. An extract which had been prepared by mortar and pestle and in which the pH was 5.9, was divided into 3 aliquots, which were then adjusted to pH 5.3, 5.8 and 6.3. These three aliquots were then individually fractionated with acetone (Table 6). The relationship between the pH of the extract and the concentration Of acetone required to precipitate the phOSphatase (Table 6) was opposite to the relationship which had been found in extracts (pH 5.3 and 6.4) which had originated from two different batches of leaves harvested months apart. Factors other than pH must be more important than pH in determining the concen- tration of acetone required to precipitate the phOSpha- tase. 166 Table 6 The Acetone Concentration Required to Precipitate P-glycolate PhOSphatase as a Function of pH pH Maximum activity precipitated ml acetone/10 ml of extract 5.3 4.6 5.8 4.3 6.3 4.1 Purification did not seem to be better at pH 5.7 than at unadjusted pH values in the range of 5.5 to 6.4. Because of this and because pH control alone did not insure reproducibility in the concentration of acetone required to precipitate the phOSphatase, adjustment of the extract pH before fractionation, as was done in the purification sequence shown in Table 4, is generally not warranted. The main reason for the success of the first ace- tone fractionation seems to be that most of the protein is irreversibly denatured by the treatment. Thus,a 7- fold purification of the wheat phosphatase was Obtained simply by precipitating a Waring blendor extract with 3 volumes of acetone (265). The additional purification is obtained from the fractionation of the remaining proteins at the different acetone concentrations. When the phos- phatase in a fresh tobacco leaf extract made by mortar 167 and pestle was precipitated between 3 ml and 8 ml of ace- tone per 10 ml of extract, the purification was 14-fold. This 5 ml increment of acetone was wide enough to include the considerable variation which was found, over the 2 year period, for the acetone concentration needed to pre- cipitate the enzyme. The additional purification possible by the first acetone fractionation requires at least an occasional pilot fractionation. Effect of Time and Temperature Although the slow addition of acetone through a single 0.7 mm I.D. teflon tube over a period of 3 to 4 hours resulted in excellent enzyme yields, it was found that adding the acetone simultaneously through five 0.7 mm I.D. teflon tubes or four 1.14 mm I.D. polyethylene tubes over only 15-20 minutes also resulted in excellent yields, even though the temperature during the mixing rose to as high as 8°. Even for these short times, the flow of ace- tone through any one tube was not allowed to be quite rapid enough to give a continuous stream. When the final centrifugation of an acetone mixture at 0-4° was delayed for 20 hours, the yield was down to 40% and the fold purification was also low. Acetone precipitation of the phOSphatase at room temperature was not as effective as acetone precipitation of the enzyme with the temperature held between 0 and 8°. 168 Activation of P-glycolate PhOSphatase by Acetone Precipitation It was not unusual to Obtain recoveries in excess of 100% from the first acetone fractionation. Further- more, it was sometimes noted that the activity in the aqueous solutions from the acetone precipitates increased for a time. Substrate Specificity Enzyme specificity for P-glycolate was greatly increased by the first acetone fractionation which was designed to save the P-glycolate phOSphatase activity (Table 7). Table 2 Improvement in Substrate Specificity by the First Acetone Fractionation Relative rate of P1 release Substrate Extract Acetone preparation P-glycolate . 1.000 1.000 3-P-glycerate 0.248 0.046 Phenolphthalein-di-phOSphate 0.481 0.083 Purification by Aging of Extracts When extracts were held overnight, a large precipi- tate develOped. Removal of this precipitate by centrifuga- 169 tion gave about a 1.7-fold purification. Little activity was lost by this procedure. When the recentrifuged extract was further aged, little if any precipitate develOped. That the purification from aging was at least partly addi- tive with the purification from the first acetone frac- tionation is suggested by the turbidity which often gradually develOped in once acetone purified enzyme pre- pared from fresh extracts. Three times acetone purified enzyme stayed clear for long periods of time. Thus it may be that part of the success of the acetone fractionations was due to the elapsed time required for all three frac- tionations. Optimum pH for DEAE-Cellulose Chromatoggaphy Using pH as the variable,an attempt was made to increase the Specific activity of the enzyme by causing a Shift in the eluted peak Of phOSphatase activity rela- tive to the protein elution pattern. The following pH values and buffers were used: 5.0 (acetate); 5.5, 6.3, and 7.0 (cacodylate): 8.0 and 9.0 (glycyl-glycine). A separate aliquot of DEAE-cellulose was used at each pH. For each chromatographic run, the cellulose, pretreated as recommended by Peterson and Sober (192), was equilibrated with 0.02M buffer in a column 0.7 cm in diameter and 2-3 cm high. 0.10 ml of 3 times acetone purified enzyme was added to the column and then eluted 170 with 20 ml of the same buffer, which was used in the prep- aration of the column, containing NaCl in a linear gradient from 0 to 0.5M. All eluents were monitored at 254 mu. Except for the chromatographic run at pH 5.0, the positions of the phOSphatase peaks relative to the A254 patterns were relatively constant, and the NaCl concentra- tions at the maximums of the phOSphatase peaks varied only between 0.12M and 0.16M, so that the shifts were not large enough to take advantage of. At pH 5.0, the phos- phatase was eluted at afiNaCl concentration of 0.07M. However, the recovery of activity was too low to take advantage of this Shift. It was demonstrated that pH and not acetate was reSponsible for this shift. At pH 6.3, 0.02M acetate did not decrease the NaCl concentration (0.15M) necessary to elute the phOSphatase. The highest recoveries (as high as 90%) were obtained with the pH 6.3 cacodylate system. It was concluded that no apparent advantage was to be gained by changing from pH 6.3 for the DEAE-cellulose chromatography. SuSpected Inactivation of P-glycolate PhOSphatase by Metal The following circumstantial observations suggest that contact between the partially purified phOSphatase and metal containers, plumbing, and utensils should be completely and carefully avoided. 1. Higher yields were obtained with reagent grade 171 acetone from glass bottles than with reagent grade acetone from cans. 2. During DEAE-cellulose chromatography, higher yields were obtained when the use of all metal was avoided in the plumbing than when the plumbing contained some metal. This includes the plumbing ahead of the column, including the gradient device, as well as after the column. 3. When a syringe (metal needle) was repeatedly used to remove enzyme from a 3 times acetone purified preparation, there was a concomitant increase in the rate of phOSphatase inactivation. The Effect of Preincubation with Mn++, Fe++, or Fe+++ 'Preincubation with Mn++ had little effect on the activity or the stability toward dilution at 30° of the once acetone fractionated phOSphatase. Preincubation with Fe++ had a significant effect on the activity and some. effect on the stability toward dilution at 30° Of the phOSphatase in the same preparation. Preincubation with Fe‘t“+ had little effect on the activity but had about the same effect as Fe++ on the stability toward dilution at 30° of the once acetone fractionated phOSphatase. Pre- incubation with Fe++ had little effect on the activity or stability toward dilution at 30° of the phosphatase in a buzzed extract (Table 8). .oom as soassaao panacea ll, Rmn Rum .sda om .zaa on zmloa N m.a Emloa +++om R05 &mm .GHE 0N m SmtoH N m.H Smtoa ++mm mmmpmsmmozn Rum Rmoa .Saa om .SHE 3w Emloa N m.a Enloa ++sz OopaQOHpoanm it it it it oeoz oaopood new moon 1. Rob Rum .sHa ma .Saa mu amtoa N m.m Entoa ++mm pomnpwo mum Rona I: It in in 0:02 possum ”w Ill Q a t o a asasdsm assesses soaaodoa season asses soap secure 1. tapaaapapm pd an a a p amspasamosm iaosaoam cahwsm oaap acapmn502HOHm Hopes mo acapmhpaooaoo 1.11.14!!! .00 on who: meadpansOGHOHQ Had a anion w e so H1 mm ao ++oa compasamosa condeoapomnm accused 0:» Spas monspx m.. 9mm 90 H1 00: mo Oopmdmaoo pomhpxo commas one Spas Ohdpxaa soapmnoosd MOOSOQ oeopcoa so on oouaobaoo none was opmpanaOOHa chance on» "00:0 oops as case pomhpwo am we copanamaho omapdnmmosa 6096:0Hpomhm H1 mm + ++2 ++ osopoom one Oh ENIOH mo H1 0 none ++om 029 . iaodposhp has one hoonoap madam: muoa N a mo H1 mm as soameSOeaoHa one .+++oa a toe w N co.daom no .m.o ma .oscaaooowo smo.o co .oahuno mo H: on mo uopmamsOo .omm so an on one .hfim ad common use one cannon use aspnoa an vasomohn was pomapxo one use assesses can so +++ca no .++om .++ss spas cosponsosaoam co access use ooosmmmmosm cedaoowwmia ao Nassaswsm m panda 173 Richardson and Talbert (202) found that an endo- genous metal accompanied P-glycolate phOSphatase during the purification of the enzyme. Arguments have been pre- sented which suggest that the endogenous metal was Mg++, but the evidence did not exclude Fe++ as the metal (literature review, p. 40). The relatively significant inactivation rate of P-glycolate phOSphatase at pH 6.3 and at 0°, in the presence of 10'3w Fe++ (Table 8), sug- gests that the endogenous metal which accompanied the phOSphatase (202) was not Fe++. Furthermore, kinetic evidence excludes Fe+++ as the endogenous metal for the enzyme (202). Gel Filtration Chromatography The phOSphatase Rf (effluent volume/void volume) on Sephadex G-25 was 1.0, on Sephadex G-100 it was 1.6, and on Sephadex G-200 the Rf varied from 2.3 to 3.0. The Rf values on G-100 and G-ZOO suggest that the enzyme is relatively small. The phOSphatase preparations were highly colored (orange-brown) through the 3 acetone steps. This color could not be removed by dialysis. DEAE removed much of the color. Gel filtration chromatography gave a color- less phOSphatase, as the color emerged in a peak far behind the phOSphatase but ahead.of the salts. Whenever the phOSphatase activity was assayed in 174 all the fractions which were eluted, including those later fractions which would normally be expected to contain only very small molecules, no large second phOSphatase peak was ever found. However, a very minor phOSphatase peak, containing 1 to 2% of the activity, usually was found, and it coincided with or slightly followed the color peak. Protein Determinations Determinations on colored preparations by the 260/ 280 method gave very inconsistent results. The 260/280 method was therefore not used. During acetone purifica- tion, the A280 method gave results 3 to 4 times higher than Lowry's method, although both showed about the same fold purification over the 3 acetone purification steps. When the color was removed during gel filtration chroma- tography, the fold purification based on the A280 method was as much as 5 to 6 times greater than when based On Lawry's method. The colored peak absorbed very strongly at 280, 260,and 254 mp. This ‘UV absorption peak was not matched by a major protein peak,as measured by Lowry's method, although a small amount of protein did coincide with the color. Apparentlm.the colored material was reSponsible for undependable protein determinations by either the 260/280 method or the A280 method and the related A25“ method. Under the conditions that prevailed, 1 the Lowry's modified Folin-Ciocalteau method for the 1For a good analysis of Lawry's method and other methods for measuring protein, see the article by Ennis Layne (142). 175 determination of protein seemed the most conservative and dependable. Stability of P-glycolate Phosphatase Toward Freezing and Thaying Freezing and thawing of enzyme which had undergone one or three acetone fractionations resulted in the destruction of 1/4 to 1/3 of the phOSphatase activity.1 When these same preparations were refrozen and rethawed, 1/4 to 1/3 Of the remaining activity was destroyed. By contrast, slow or quick freezing of the Bio-Gel P-60 preparation, which is described in Table 3, caused the destruction of only about 4% of the phOSphatase activity. When neutralized citrate was added to the latter prepara- tion so that it contained 10‘3M citrate, the phOSphatase in the preparation was still stable to freezing and thaw- ing.2 1The enzyme had come from extracts prepared by Waring blendor and had not been powdered. 2The enzyme in the Bio-Gel P-60 preparation was different in at least three ways from the enzyme in those preparations in which it was unstable to freezing and thawing. The enzyme in the Bio-Gel P-6O preparation had come from an extract which had been prepared by meat grinder, it had been made into an acetone powder, and it had been purified beyond the acetone fractionations by DEAE-cellulose and Bio-Gel P-60 chromatography. Without further experiments, it is not possible to know the reason for the stability toward freezing Of this enzyme. However, the data suggest that at early stages of purification, the phOSphatase may be associated with something other than citrate which makes it unstable to freezing. 176 Stability Of P-glycolate PhOSphatase Toward Dilution at 30° and Toward Dialysis The test for the stability of the phOSphatase toward dilution at 30° could cause greater inactivation of the enzyme than the test for the stability of the enzyme at 45°. For example, the enzyme in the third acetone, the DEAE-cellulose, and the DEAE concentrate preparations which are described in Table 3, was com- pletely stable at 45° but was only 45-55% stable toward dilution at 30°. The enzyme in these preparations had been extracted by meat grinder and had been converted to an acetone powder. Enzyme which had come from an extract prepared by Waring blendor, and which had been purified by two ace- tone fractionations, but had not been converted to an acetone powder, was 100% stable toward dilution at 30°. This preparation was then dialyzed overnight against 175 _volumes of 0.0aicacodylate, pH 6.3, in a 4° room while being stirred by magnetic stirrer. The dialyzed enzyme retained 90% of its activity compared to the nondialyzed control. The remaining activity in the dialyzed prepara- tion was 90% stable toward dilution at 30°. Results Obtained with preparations which had been converted to acetone powders suggest that conversion to a powder rendered the enzyme in the preparations less stable toward dilution at 30°. 177 Storage Characteristics of P-glycolate ghggphatase During the time the phOSphatase was stored as an acetone powder, no loss in activity was ever detected (see p. 160). When acetone precipitates or acetone powders at any of the 3 stages of purification were dis- solved in 0.02M cacodylate, pH 6.3, the phosphatase retained all or moSt of its activity at 0-4° for at least 2 weeks. The phOSphatase in those preparations from DEAE- cellulose chromatography in which the enzyme was most stable, last only a little activity in 2 weeks at 0-4°. Following one or more purification steps that included at least one acetone fractionation, enzyme which 'was further purified by Sephadex G-100, Sephadex G-200,or Bio-Gel'P-60 gel filtration chromatography last activity relatively rapidly at 0-4°. This loss occured whether or not the enzyme, before addition to the gel filtration column, had been converted to an acetone powder. Further- more, the loss in activity at 0-4° occured with enzyme which had come from extracts made by Waring blendor, meat grinder, or mortar and pestle. Most of the loss in activ- ity could be prevented by including 10'3M citrate, iso- citrate, Or cis-aconitate in the preparations from gel filtration chromatography (Figure 15). The enzyme in the preparation described as "Bio- Gel 9-60" in Table 3 was stable at -20° for at least 2 weeks. 178 Purification bngther Methods Ammonium Sulfate The phosphatase in extracts from tobacco leaves, which had been prepared by Waring blendor, was purified by the first acetone fractionation. The acetone precipi- tated enzyme, after it had been taken up in 0.02M cacodyl- ate, pH 6.3, was further purified by an (NH4)2804 frac- tionation. The phOSphatase was precipitated between 3.5 grams and 5.0 grams of solid (NHu)2304 per 10 ml of ace- tone purified enzyme, and was redissolved in 0.02M cacodylate, pH 6.3. The yield in the active fraction was 64-74% of the activity from the acetone step. The (NHu)2804 step gave a moderate additional purification and removed much of the color. The phosphatase in the (NH4)ZSOu preparations was more stable toward freezing and thawing than the acetone precipitated enzyme. The phOSphatase in an (N34)2°°4 preparation was 90% stable toward dilution at 30°, and the enzyme retained most of its activity at 0-4° for at least two weeks. Dialyzing this preparation overnight in a 4° room resulted in 50% inactivation of the phospha- tase. The (NHu)2804 fractionated phOSphatase was com- pletely unstablefito further fractionation with acetone. The phOSphatase from plants other than tobacco 179 was unstable to (NH4)2°°4 fractionation (265), and even in tobacco, the recoveries were only moderate (202, 265). The instability of the (NHu)2304 fractionated enzyme from tobacco leaves toward acetone fractionation or dialysis is consistent with these earlier findings. Ultracentrifugation When an extract from tobacco leaves which had been prepared by Waring blendor1 was recentrifuged at 151,000 g for 90 minutes, only 5% Of the phosphatase was found in the small pellet while 95% was in the supernatant solu- tion. Part of the activity in the pellet would be due to trapped solution. The Specific activity in the solu- tion increased 1.3—fold. When 3 times acetone purified phOSphatase (the enzyme had come from an extract prepared by Waring blendor and had been powdered after the first fractionation, or had come from an extract prepared by the meat grinder and had not been powdered) was centrifuged at 122,000 g for 10 to 12 hours, 90% of the activity which was recovered (81 to 91%) was found in the bottom 6 to 10% of the volume. However, not more than 4% was found in the tiny pellets. The Specific activity increased from the tap to the bottom of the centrifuge tubes,but not enough to be profitable 1The extract had been centrifuged at 37,000 g for 5 minutes. 180 for purification. When precipitated enzyme from a 3rd acetone frac- tionation (the enzyme had come from an extract prepared by Waring blendor, and had been powdered after the first fractionation) was taken up in a salt solution with a density of 1.21, and the solution was centrifuged at 122,000 g for 24 hours, the same results were obtained. Most of the phOSphatase was concentrated near the bottom of the centrifuge tube. The method has been used to 1 float lipoprotein with a density less than 1.21 (113). Ion Exchange Chromatography Other Than DEAE All the following results were obtained with 3 times acetone purified enzyme which had come from an extract prepared by the meat grinder and which had not been converted to an acetone powder. 1Freezing and thawing of enzyme which had been purified by 3 acetone fractionations resulted in partial inactivation of the phosphatase and the formation of a thin, Oily, upper phase. Freezing and thawing has been used as a method for the dissociation of lipOproteins (166). It has been determined that the relative effec- tiveness of organic solvents for dissociating lipoprotein complexes in aqueous media and for releasing undenatured protein in general may be eXpressed as follows: (1) most effective: n and iso-butanols; (2) partially effective: sec-butanol, cyclohexanol, and tert-amyl alcohol; (3) ineffective: all other solvents tested, including chloro- form, carbon tetrachloride, toluene, ether, acetone, etc. (166). 181 Chromatography on TEAE-cellulose with the same NaCl gradient at pH 6.3 as used for DEAE-cellulose chromatography gave results very similar to those obtained with DEAE-cellulose. When the enzyme was chromatographed on cellulose- phOSphate at pH 6.3 or CM-cellulose at pH 5.7 or 6.3, most of the protein emerged as the first peak. This peak contained all of the phOSphatase,which was purified no more than 1.5-fold, in 0.02M cacodylate, pH 6.3, which was free of NaCl. The results were not significantly altered on CM-cellulose at pH 6.3 when the eluting cacodylate was reduced from 0.02M to 0.001M. Identification of the PhOSphatase Stabilizing Factors | Purification Fractions 44 through 58 (Figure 10) were pooled to give a 43.0 ml stabilizing fraction. 11.0 ml of this stabilizing fraction was acidified with 10N H2804 to a pH of about 1 (by calculation and pH paper). The acidi- fied fraction was then continuously extracted for 3 days in a soxhlet apparatus with anhydrous diethyl ether (Fisher reagent grade). After the ether and water phases in the soxhlet apparatus were separated by separatory funnel, the combined ether portions were evaporated in a flash evaporator at room temperature to a volume of about 182 0.5 ml. The clear colorless concentrated ether extracted fraction was transferred to a pointed test tube,and the flash evaporator flask was carefully rinsed with two 0.2 ml aliquots of water which were then individually trans- ferred to the pointed test tube. The ether extracted fraction and the remaining aqueous fraction (ether washed fraction) were then exposed to a vacuum of 15 mm Hg until the bubbling ceased and the smell of ether had vanished. The volumes were then 0.8 ml and 8.0 ml for the ether extracted and ether washed fractions respectively. Initial paper chromatography of the ether extracted frac- tion was with the preparation described, but the final paper chromatographic determinations were run after the 0.8 ml ether extracted fraction was diluted 4-fold with water. The most active factor fractions which had been separated from the phOSphatase by Sephadex G-100 chroma- tography, after the phOSphatase had been purified by three acetone fractionations, were also pooled and then continuously extracted with anhydrous diethyl ether in a soxhlet apparatus using a procedure essentially the same as that described above. 14.8 ml of the pooled stabiliz- ing fraction gave a clear colorless 0.6 ml ether extracted fraction. The ether extracted and ether washed fractions Of this extraction were assayed for the presence of the phOSphatase stabilizing factor(s). Twenty-fold diluted 183 ether extracted fraction was more effective in conferring stability than was the ether washed fraction (Table 9). The two fractions together seemed to confer stability additively. The pretreatment with either fraction also gave some activation, and the pretreatment with the two fractions together seemed to confer activation additively. Although the stabilizing factor peak in both gel filtration chromatography runs was behind the color peak, it overlapped with it, so that both pooled factor frac- tions were colored. In both ether extractions, all of the color remained behind in the ether washed fraction. Identification of Tricarboxylic Acids Citric, isocitric, and cis-aconitic acids were identified by paper chromatography of the purified (ether extracted) phosphatase stabilizing fraction (Table 10). The only other acidic Spot which was evident on the ohromatograms was sulfuric acid. Only one unknown organic acid Spot Showed up in the ether-acetic acid- water system. It could have been citric, isocitric, or cis-aconitic acid or a mixture of all three (Table 10). The presence of cis-aconitic acid was evident with the other 3 solvent systems. The n-butanOl-ethyl acetate- formic acid and phenol-water systems did not distinguish between citric and isocitric acids. The major unknown spot in both these systems could have been citric or iso- citric acid Or both. 184 ‘1 llltill‘l . was u woos w mm.mm i one as aoaaaswom possessoo asaHaswam .mooa . m.asa u moss w HaNmoass sa Hoaosoo oo.co assesses soassaaaos .ps0a00nm0 mmOHo CH 0u03 aHma 2000 SH m0sH0> 02p “msoHpmsHaamp0o 03p no 00m0n0>0 090 moHprHpom one .om: um thHHnmpm you o0p00p sons 0h03 msoHpma0a0pa noom HH< .soHpomam o0uoahpx0 H0sp0 p0pSHHp ps0 U0NHH0HpS0: 0:» mo H1 m + :oHpomam 603003 H0su0 pmnHH0Hp50a 0:» mo H1 m A: one .coHpomam O0pomppx0 90:90 popSHHo one oomHHmhp50: 05p mo H1 CH Am .SOHpomam @03003 H0Spo pomHHmapS0: 02» mo H1 0H AN .m.© ma .mpmHmooomo zNo.o mo H1 CH AH ”wsHSOHHom 0s» o0cH0psoo m0sp .soHpHuom SH .005 0hom0n pmsfi poems» 00: ps0 0x003 03p psonm you oomi p0 6H0: S00n p0: .oH 0asmHm sH p0nHH000o .0mmpmsamosa 0:9 .0mmpmsamoca 0Hnmpmss 90 H1 0H ©0SH0psoo £000 m0a3pMHa ps0apmonp0aa HSOQ 0:9 .AM0Q0Q mav 0.5 on m.w psonm :03» 0h03 mposvHHm 03p 0:» MO mosHmb ma 0:8 .093» p00» HHmam pmspocm CH mom 7Ho.o mo H1 0 msHa .m.© ma .0p0Hhooomo zmo.o mo H1 m.NN 59H; o0xHB was soHpompm o0poahpN0 a0£p0 0:» mo H1 m.H .095» 900» HH080 a sa mos 2a co H1 m seas eowas was soaoodaa ocsmws assoc can we H1 on .asawamoa imaoaso oole N0p0£a0m Scam msoHpomaw panama o0Hooa 0:» mo :oHpomhpM0 h0um< :OHuomam ompomapxm ponum 02» :H Amqnopomm mmHNHHHnmpm onenesnmosm m 0HDmB 185 .oH oastm :H pothomov ammumzamona 0:» no Ha\muHsd :H* m.om o.om o.ama woos a .oms so posses spasm 05H mm c.msa co pd oHom msoosow as a.cm o.aa a.maa woos a .oms so possum ma m.mma co or pass scram Am o.mH m.m: 0.00 Moos H .om: pm 00p00m a m.ama co or pass msocsow Am o.o m.mm m.os sacs a .oms as pastor o m.HsH oo as oHom osoz AH a a a oonhomaoo on: p0 SOHp0>Hp0< *mpH>Hpo< :OHpomhm anS mahNa0 cocoa aaaHasmsm aaaHasdsm co ssoaswcasoaa soasodaa Ilul 186 .aoemam was» as om.o wo am so on: on 0 0.0Hw «.BOSOOHnHmov oHoa OHpHsoomtmaaha .md.o mo mm :0 SHHS po 0 HOHHOam 0 new ow.% no mm :0 SPHS poem HowhmH 0 .muoam 03p 0>0m aHom OHpanoomlmHo Hmaohoaaoost .oHom oHpHnoomlmHo H0H0H0aaoo mo pas» 39H: HH03 oonaaaoo zoomnm waHeaoH 0:» mo sOHpHmoa 038 .300030 wsHomoH 0% onsopsH s0 0>0w sSoaMs: 0sat omaopsH mmoH M0HH080 0 one A3H.o u mmv poem BOHH am.o me.o am.o Mammuw . us.o as.o ommuoaco casoaaoaataoswssmrs ea.o ma.o ma.o ma.o ommtmooommouaosom a aa.o mm.o ma.o ma.o_ omniaososa os.o om.o as.o em.o mm.o mooomsoaosmiaoswbsmas esoaxss azosxad aeoaxes OHpHaocdimHo OHHpHOOmH OHHpHo Sopmhm peobHom msHo00A 0Hoon wanwcq modem H0H0H0aaoo hsmmnw09030H£O H0909 .mm m . .eooSHosH no: H mm mpH pS0OH>0 was omHa eHoa OHHaMHSn empospHd .mH oaSme SH o0pHnomoe 000:» 00 0500 0:» ohms mSOHpsHom pHoa OHpHsoosumHo 0S0 .oHom oHapHOOmH .oHos OHapHo .sSosmss 0:9 peace oHHNwosawoaae co soasdoaaaasoeH OH OHDNB 187 When the unknowns were mixed with commercial citric, isocitric, cis—aconitic,or sulfuric acids, the identifica- tion of the acids in the purified phOSphatase stabilizing fraction was further extended (Figure 13). In every case, the intensity, but not the shape, of a Spot was increased by the corresponding acid. The only system which distinguished between citric and isocitric acid was the n-butanol-propionic acid-water system. With this system, the unknown mixture was shown to contain both of these acids (Figure 14). The lagging spot was citric acid. Commercial cis-aconitic acid gave two Spots in this system, with the leading Spot being the larger (Table 10). The Rf of the middle Spot (Figure 14) was such that it could have been the lagging cis-aconitic acid Spot or isocitric acid. But the ratio of Spot sizes for the middle and leading Spots (Figure 14) was opposite to that for cis-aconitic acid. The pattern suggests that the middle spot was predominantly isocitric acid. The Spot Sizes suggest that the concentrations of citric, isocitric, and cis-aconitic acids in the phOSpha- tase stabilizing fraction may have been citric > isocitric)» cis-aconitic acid (Figures 13 and 14). Spot areas are a function of the logarithm of the concentration of the material in each Spot (31). .esoexes 090 0H00 0H959H50 zH.o 90 A>\>v mam 0959NHE 0 0 9o H1 m n o .oaod cahscHsm 29.0 so 91 m spas ecaooam o 090 09009090 0H00 OHpHSOomimHO 90 A>\>v swam 0939NHE 0 M1 as n p + sonar .ssossaa m m ”>9 809wopdao9:o .szosxqs 90 H1 om u D + 090 .SSoame: 90 H1 :0 u D .0900S0pm 0Hom oHpH9000imHo 90 H1 0 n 0H0 ”HHH 809wop0a09:o .esoaxes 0:0 09009000 0H00 0H9pHoowH 90 A>\>v :«m 09SpNHa 0 90 H1 5 u D+OmH .ezoaxes 90 H1 m n D .09009090 0H00 099pHoomH 90 H1 0 n 009 “HH 809wopmao9:o .szonmnd 0:0 09009090 0H00 OH9HHO 90 A>\> a N. .CSOGHSd no H1 w u D .09009090 0900 099pHO 90 H1 : .A0009w s 80:00HnHmov H1 909 0Hos1 9H\H 950:0 0H00 OHpH:oomimHo 0 909 0H 0H0 .A+m N030D :sz 0H00 tH9p .OHHm + H0 .0009w o a0:OOH:H00V H1 909 0Hoa1 om\H 188 H 09:»NHB 0 90 H1 0 u D + o n o “H 809w090809:o 9o w1 oH wsHsHmpnoo 09002090 0:» on 00p90>eoo pHmm SSH0O0 990:0 9O w1 0H 950:0 wanHmp 1:00 0900:0p0 0900 0H9pHoomH :0 909 0H OmH .A.a9oo 0HmoHa0:oon HmaoHpH9pszv H1 909 0Hoa1 om\H props 9O w1 oH msHaHmpaoo 0900:090 0H00 0H9pHO 0 909 0H 0 .h:na9wOpoaoH:o 09090: 0ND :sz 0HO9I: 0005HH0 003 90099N0 Ha m.o 0:9 .0H 09stm :9 009990000 mm :m:o9:u a: 0:0H00099 00Hooa 0:» 90 00099N0 90:90 0:» :9 .mwsHom9p 0:9 90 :a09wopo:a 0 0H 09st9 i950 9H090900ESH 0903 Awsso9mxomn 05H: :900 0 90v 09090 .59000000: 090:3 moo louse .00posHe mH 909 00990 990 0903 0809mopmao9:o 0:9 0:9 .mam9mopmao9:o 0:9 990 :09 amen ps0>Hom 0:9 .0930: 10200000 9: hHmSO0smpHoaHm 00QOH0>00 0903 0809mousao9:o 90 0:309:95 909 009090 D 0:9 .000099 9090H 0:0 00GHH soHHoa made consume H90: 0:9 02 SMIOH N m :0:p 0:0 S009m H0009ooao9: :pHs 0090990 .0090HO .00:0909 0903 :OH:3 90 0090 \m w 909 9:909mopaao9:o weH 09 09090900 9s09 0:9 soomNm pace oaaaoauosmpoow stomuHoswpsmnc one :9 noses oaHaNooadoaaa 0:» 9o coaowoacapsoea HH,0usmam 189 1 vi- 1 come... eOm~r o O o Q O 05.0 ....... Q O o o o s o . 190 .0900:0p0 0Ho0 0H9pHoomH 90 H1 :0 + :30:::: 90 H1 m: .m .0900:090 0900 09999000H 90 H1 mm + :3o:x:s .0900:0p0 0H00 0H9pHO 90 H1 :0 + :3O:H:S 90 H1 w: 0900 0H9pHo 90 H1 mm + :30:::: 90 H1 m: 90 91 m: .s .m .oamoswsm .N .:3o:::: 90 H1 w: .H .MH 09ame 909 00:H90000 00 00H0 0903 0:0993H00 0900:090 0:0 :30:::: 0:9 .MH 09smHm 909 009990000 00 0903 09smH9 0:9 0:9:H0ppo 0:0 0609w090ao9:o 00QOH0>00 0:9 w:HH0:0: 909 009500009: 0:9 .0950: om 930:0 003 0899 w:H:::m .809090 90903I0Ho0 09:0H90991Ho:0p:::: 0:» :H m:a09mOu0ao9:o w:H0:00000 a: 00aOH0>00 003 a09wop0ao9:o 9090: 0:9 mpaow oaaoaoomH use oaaaao 9o soaswoacapsooH :H 09st9 191 192 Stabilization of the PhOSphatase by the Tricarngylic Acids At 10’3M, citrate, isocitrate, cis-aconitate, and trans-aconitate stabilized the phOSphatase (Figure 15). Although all four acids were about equally effective, the data suggest that cis-aconitate was somewhat more effec- tive than isocitrate which was somewhat more effective than citrate. Trans-aconitate was about as effective as citrate. For the other 9 acids which were tested, the phOSphatase activity decreased about the same extent as for a water con- trol. When the test solutions were made 10'3M in MgSOu, the overall stabilizing pattern was essentially the same as shown in Figure 15. The tricarboxylic acids plus M5304 stabilized the phOSphatase while the other 9 acid plus M8304 combinations seemed essentially ineffective (data not shown). The MgSOu + citrate and Mgsou + isocitrate combin- ations gave somewhat more stabilization than these acids without MgSOh, while the reverse was true for cis-aconitate. The MgSOn + trans-aconitate combination was also somewhat more effective than trans-aconitate alone. Mg804 alone at 10-3M had no effect on the stability of the phosphatase (Table 11). 193 Figpre i5 Stabilization Of PhOSphatase by Tricarngylic Acids Stock solutions contained the designated commercial acids at pH 6.3 and 10'2M. To each of 14 individual 62x5” mm test tubes were added 25 111 of". H20 and then 25 ul of a stock acid solution. Another 25 ul of H20 was added tolflm H20 control. Three times acetone purified phOSphatase, which had, originated as an extract prepared by Waring blendor and whnwi had been converted to an acetone powder, was further purified by Sephadex G-100 chromatography using 0.02M cacodylate at pH 6.3 as the eluting buffer. The fractions were assayed for phOSphatase activity as they accumulated and the most actrwe fractions were pooled. At zero time, 200 pl of the pooled phOSphatase was added to each of the test tubes, they were corked and the contents mixed. The concentration of the acid in each tube was then 10-3M. Next, the phosphatase in the pooled frac- tions was immediately assayed for activity to give the 100% value (Li-32 units per m1 - - -).1 After 4 days at 0-40. the contents of the 14 test tubes were assayed for the phOSpha- tase activity which remained. The H20 control retained 3&5 of the original activity (1.47 units per ml ).1 Stability conferred = Activity remaining, units per m11 4 1.12 x 100% 11.32 — 1.47 ° 1“_ _-‘ .1 ___ _ _._fi. -1. 1-! - Ao\ov 00:09:00 3:505 00000 98765mwwmnl00 194 _._____.____—————-——-—- IOO nil 03:00.0 05.0w 05030 0909000000. F 0920030 092004 H 0900.22 H 036:5... F 03502.6 7 03330005.... _ 0.0250005 H 090.5000. 22:0 _,i 9 _ _ _ _ o 5. 0. 5. 0. 3 2 2 l i .00 0:0: 53:00 00090510050 4.0 — 0.5 H _ 5. 3 E werc‘ 6 ft oi a l to i'.‘ i and I: pulf‘r Late 8‘» ,33191 .1 act‘ 2 was 1 the :h “if 030' 30, fr? )spid' 195 Table 11 Effect of Mgsohgon the Stability of the PhOSphatase The conditions were as described in 2Figure 15 except that, where indicated, 25 pl of 10"2 M MgSO% was added to the preincubation mixtures, rather than 5 ul of H20. Thus the concentration of MgSQn during the # days that the enzyme was held at o- no was 10-3M. Stability conferred Acid - MgSOu + MgSOu Citrate 57% 71% Isocitrate 68% 85% Cis-aconitate 76% 65% Trans-aconitate 55% 79% None 0 O Coincidence of the Tricarboxylic Acids with the PhoSphatase in Fractions from DEAE-Cellulose A DEAE-cellulose column was prepared as described on p. 142. The remaining enzyme from the 3rd acetone fractionation (described on pp. 140-141), which had been held 34 days at 0-40, was centrifuged at 5900 g for 8 minutes to remove an inactive precipitate. Eighty percent .of the phOSphatase activity survived the 34 days of stor- age. Thirty-threenfl.of the enzyme was added to the column and eluted with #00 m1 of 0.02M cacodylate, pH 6.3, con- taining NaCl in a linear gradient from 0 to 0.5M. The eluent was continuously monitored at 253.? mu. The phos- phatase recovery was 90%. 196 A paper chromatographic assay for citric and/or isocitric acid and the enzymatic assay for isocitrate revealed a correspondence between isocitrate (and/or citrate) and the phoSphatase in the fractions from the DEAE-cellulose chromatographic fractionation (Figures 16 and 17).1 The results of the paper chromatographic assay were completely consistent with the results of the enzy- matic assay for isocitrate (Figure 17).2 Assuming that the phosphatase with a specific activity of 333 (Table #, Figure 11) was homogeneous, 1 mole of isocitrate per 10 to 15 moles of amino acid resi- due in the.ph03phatase fractionated with the phoSphatase in fraction no. 22. There were 55.3 units of phOSphatase/ ml in fraction no. 22 which calculates to be 55.3/333 units x mg'1 or 0.166 mg of phoSphatase/ml in fraction 22. Using 100 mg/millimole for an average amino acid residue, there were 0.166 x 10"2 millimole or 1.66 umoles of phoSphatase amino acid reside/ml in fraction 22. In 1The heating and recooling, and the filtering methods which were used in the preparation of the frac- tions for the enzymatic determination of isocitrate (Figure 16) did not affect the recovery of isocitrate (Figure 17). 2The n-butanol-ethyl acetate-formic acid system did not distinguish between citric and isocitric acids (Table 10 and Figure 13). Since both citric and iso- citric acids were identified in the purified ether extracted stabilizing fraction (Figure 1%) which had originated from the same batch of 3 times acetone puri- fied enzyme from which the enzyme for the eXperiment of Figure 17 had originated, the citric and/or isocitric acid Spots (Figure 17) probably contained both acids. 197 .000000000 0800 000:0 :0 00000000 0000000000200 0000000000 0308000020 0: 003 00030 0030 000000000 £0023 .00000000 008 000 0000000000 00 00o00000800000 000080000 030 w00000 00000000 0003 £0053 000H0> 0000000000 #:0HD 0:9 .00o0008 :0 009000000 00 0000 003 £0033 00000 000000000 028 .000000000 00000H00 000.00Hooo .000005 030 80 000000 003 20033 .00000000 000000000 000 0000 00 0005.003 000008000|000 .000000000 00030000000000 003 mm .0: 00000000 00 00000 003 00023 0000000 1000 000 .0.0 .Aom00.o u NV 000H0> ¢VAVoz0 00000 000 no 850 020 0020 0000000 Rm 003 0000008 020 000 0000000000.600£B .50000.o n ¢AVV 0000008 000 00 H1 000 0:0 .00mmo.0 n 0000 mm .00 00000000 00 0a 00 .00mmo.o n 0000 0000000000 00000 000 00 H1 00 :0 0008 003 0000000000 00 0000000000000 000080000 00 0009 .030: 0 00:0 0008 000 00 00 0000 003 0000008 009 .00000H00 0000000000 00000 000 00 H1 000 00000 003 .00000H0m 0:0 .00Hooo .0000 00 00000: 003 00 000000 .mm .0: 00000000 00 an 000 09 “03oHHom 00 000000 003 00000000 0 8000 00030000 000000000H .0000008 :0 000000000 00 0003 0000000000 mo 00000000800000 00008000m .000000 000000 0 .oz 0000003 0000000000 0000000 00000000 0000 .0000 000 00 00 00H00000 0H00000 .0000008 on 000 .00050 0000 000 0000 00Hn008 000Hw £003 .n00n 00003 000H0on 0 s0 0000Ha 0000 000 003020 0003 000000000 029 .000: 00 0H0: 0003 000000000 000003000 030 .0008 0003 000000 00000300029 030 00004 .0000 020 00 009000000 00 003 00000300029 0:0 00 0:000000080000 000H0HH001m0mm 0:9 .18 m.mmm 00 0000000000 !Eu .0000000000AYIAQ .00000smmosm 000Hoohamlmfivllmv 000H5HH0olmmma 8000 000000000 :0 00000snmo£m 0:0 000 000000000H 00 003000 198 lw/saloum ‘aumgoos' 80 9.0 2.0 00005: 00:02... no mo 00 00 00 00 00. $0W000~0~v-.~o.~0. 0. 0.. o o u '4 '<"fil Iali¢é q 0 0 ... O o o '00. 30,000. O O 00‘000'0‘. / .. <1. \ I... ..\/... /. :6 know 00 000000034 .. /.. 822000.... 20.00».oi.\\0 . .oflfl. . .0 .0 4 . ....0...\ .... 0 0 m 0 . (04/1: 22:82 1 0. ON 00‘ 1m 19d suun ‘mnuoo esoqusoqd 199 Figure 1 7 A Magnified.Plot of Figure 16 0—0 P-glycolate phoSphatase. A—-—A Isocitrate. The fractions had been heated and recooled, but not filtered. [j_D Isocitrate. The fractions had been heated and recooled, then filtered. ‘QL__§7 Isocitrate. The fractions had not been heated. The plots of the phoSphatase and of the isocitrate con- centrations after the fractions had been heated, recooled, and filtered are taken from Figure 16. The isocitrate concentra- tions were determined before heating, and after heating and recooling but before filtering, in the fractions indicated,tm using the same procedure described for the fractions after they had been heated, recooled, and filtered. The determination of citrate and/or isocitrate by paper chromatography was performed before the eXperiment of Figure 16, i.e. before any of the fractions were heated, cooled, and filtered. 0.50 ml aliquots from the 33 fractions which were assayed for phOSphatase activity were pipetted.into individual pointed test tubes. The aliquots were acidified with 10N H 804 (10 to 30 ul) to a pH of 1-2 (pH paper). The acidified aliquots were placed under 15 mm Hg at room temperature over- night. 50 pl of H O was then added to the dry contents of each tube, the con%ents were mixed, and the tubes were centri- fuged. 12 ul from each tube was spotted on Whatman no. 1 paper as described in the methods section,except 50° air was used to dry the spots. Commercial citric acid was similarly spotted. The ohromatograms were run in the n-butanol-ethyl acetate-formic acid system, air dried, autoclaved, Sprayed with bromocresol green and then 3 x 10'3M Na CO where neces— sary (see methods). Only the fractions indicated showed SPOtS which correSponded in position to the commercial citric acid. -..:‘0 /ml Phosphatase activity, units/ml 200 50— 4a» iv Only fractions I9 throughi 27 showed citric and/or isocitric acid spots. l ‘ l l l L L O.|5 Isocitrate, umoles/ml l8 20 22 24 26 28 Fraction number 201 addition, there was 0.131 umole/ml of isocitrate in frac- tion no. 22 (Figure 16). In fraction no. 22, the iso- citrate molecules/amino acid residue of the phosphatase calculates to be O.131/1.61 or 1 isocitrate/12.5 amino acid residues. If the phoSphatase with a Specific activ- ity of 333 were not pure, the actual ratio of isocitrate molecules/phoSphatase amino acid residue was even greater.1 Furthermore, the chromatographic evidence suggests that more citrate than isocitrate was separated from the phosphatase (Figure 1a), and that,in addition,a relatively small amount of cis-aconitate was also separated from the enzyme (Figure 13). Thus, even after 3 acetone fractiona- tions, at least one tricarboxylic acid per 5 amino acid residues of the phoSphatase could have been present in fraction no. 22 from the DEAE-cellulose chromatography. 1Other than the assumption concerning the purity of the most pure phOSphatase preparation, three other assumptions are implied in the above calculations. a. The average molecular weight of the amino acid residues in the phoSphatase is approximately 100. b. Equal weights of the phoSphatase and of bovine serum albumin would give reasonably comparable results with the Lawry's modified Folin-Ciocalteu method for measuring protein, which was the method used when the Specific activity of 333 was measured. c. The number of substrate molecules transformed per minute per molecule of enzyme was about the same when the Specific activity of 333 was measured as when the activity of the phoSphatase in fraction no. 22 was measured. In both cases, the phoSphatase activity was measured using the standard P-glycolate phoSphatase assay. 202 Competitive Inhibition of the PhoSphatase .py_Cis-aconitate P-glycolate phOSphatase which had been precipitated by acetone was essentially inactive without added metal (footnote, p. 106). When an activator, such as Mg+2 , is required for a reaction, inhibition data must be interpreted with care, since the site of inhibition could be at the activator rather than on the enzyme (252). With the concentration of Mg+2 equal to 2 x 10'3M, kinetic data suggest that cis- aconitate at 10’2M is a competitive inhibitor of the P-glycolate phOSphatase reaction (data not shown).1 1The enzyme had been fractionated once by acetone and then once by ammonium sulfate. Residual ammonium sul- fate was not removed from the enzyme. Kinetic eXperimentS conducted with this enzyme preparation, when it was fresh, suggest that cis-aconitate could be both an activator and a competitive inhibitor of the phOSphatase. After this enzyme preparation had aged, the enzyme was no longer activated by cis-aconitate, but it continued to be inhibited competitively by the acid. Because cis-aconitate is an inhibitor of the phos- phatase competitive with reSpect to the substrate, activa— tion by cis-aconitate in the reaction mixtures was noted only at high substrate concentrations. The value of the abscissa (Figure 18) corresponding to the concentration of substrate which is used in the standard P-glycolate phos- phatase assay is 0.03. It is apparent that at this con- centration of substrate, the inhibition by cis-aconitate is almost completely reversed. It should be noted that when pretreatment with the ether washed and ether extracted factor fractions activated the phoSphatase (Table 9), the enzyme wgs relatively fresh and the factor fractions contained 804 from the H 804 which was added before the extraction “with ether. n this activation, tricarboxylic acids were not added to the phos- phatase reaction mixtures. Therefore, any activation by 203 Under these conditions, it is possible that the competi- tive inhibition might be by the chelation of Mg+2 by cis- aconitate. This objection was overcome by including an excess of Mg+2 in the reaction mixtures. With the concentrations .of cis-aconitate and Mg+2 which were used in the eXperi- ment of Figure 18, excess Mg+2 available for the phOSpha- Vtase reaction, which was not chelated by cis-aconitate, Should have been in excess of 10'2M. Variation of the con- centration of Mg+2 over a range from 10'3M to 10‘1M did not significantly affect the reaction rate (Table 12). The evidence suggests that cis-aconitate is a com- petitive inhibitor of P-glycolate phosphatase, that the inhibition is with reSpect to the substrate, and that the site of inhibition is not at the Mg+2 (Figure 18). When the concentration of Mg+2 during the reactions was 2 x 10*2M, the apparent Km for P-glycolate was 7.5 x 10‘5M and the tricarboxylic acids probably resulted from the pre- treatment with the acids, which were also in the factor fractions. The possible activation of the phOSphatase by cis- aconitate under certain conditions suggests that the enzyme may possess a binding site(s) for the acid which is different from the Site of competitive inhibition. It is of interest that fumarase, an enzyme thought to be similar to aconitase (16k, 179), is activated (158) and inhibited competitively (159) by citrate, and that the site of the activation of fumarase by anions is thought to be different from the active site (158). Further work is required to determine under what conditions the phoSphatase may be activated by the tri- carboxylic acids. 20b Figure 18 Competitive Inhibition of the PhOSphatase by Cis-aconitate O—O No cis-aconitate. D—D Plus cis-aconitate. v = velocity in mumoles of P-glycolate hydrolyzed per 30 seconds per 3 ml reaction mixture. , All reaction mixtures contained 0.50 ml of 0.20M Na cacodylate, pH 6.3, and 0.30 ml of 0.20M Mg304 (reaction concentration of Mg++ was 2.0 x 10‘2M). The tubes with cis- aconitate contained 0.10 ml of 0.30M Na cis-aconitate, pH 6.3, (reaction concentration was 1.0 x 10‘ M). .The amounts of substrate used were 0.30 ml, 0.10 ml. 0.050 ml, 0.025 ml. and 0.0125 ml of Na P-glycolate, pH 6.3, 10.0 umoleS/mlo Water was added to make the final reaction volume 3.00 ml. The reaction mixtures were brought to 30.00 in a water bath and the reactions were initiated with 2.50 ul of the Bio- Gel P-60 preparation described in Table 3. (The enzyme had been held at -20° for 6% months and had been thawed and refrozen several times.) After 30.0 seconds, the reactions were terminated with 0.75 ml of the acid molybdate solution which was used for the isobutanol benzene extraction method. P} was determined on a 2.50 ml aliquot (containing 2.0 m1 0 the reaction mixture + 0.50 ml of the acid molybdate solution) by the iso-butanol benzene extraction method. For every reaction, a zero time control was run by adding the enzyme after the acid molybdate solution had been added. After one of the values, which was obviously in error. was discarded the strai ht least squares, 8 lines were fit by the method of 205 0.|000 )— + Cis-aconitate 0.0750 -- 00500 -- No cis-aconitate 0.0250 l L I 2 (l/(P-glycolate)) x l0"4 206 the apparent KI for cis-aconitate was 6.5 x 10'3M. When the concentration of Mg+2 during the reactions was 2 x 10'3M, the apparent Km for P-glycolate was 6.7 x 10-5M and the apparent KI for cis-aconitate was 5.0 x 10'3M. Table 12 Effect of High Concentrations of MgSOu on the Phosphatase Standard P-glycolate phoSphatase reaction mixtures were used except that the M3304 concentrations were varied. The enzyme (same preparation as the one described in Figure 18) was saturated with substrate during the 10 minute reactions at 30°. Ehch.mixture held 1.0 ul of enzyme. (M3304) ' Activity* M 10-3 0.555 10"2 0.591 10-1 0.520 *umoles P-glycolate hydrolyzed per 10 minutes per 0.75 ml reaction mixture. Discussion of the P-glycolate Phosphatase, Tricarboxylic Acid48elationship The evidence concerning the separation of endogen- ous stabilizing factors from the phOSphatase, after pre- liminary purification by three acetone fractionations and DEAE-cellulose chromatography (Figure 10), and the identi- fication of the stabilizing factors as citrate, isocitrate, and cis-aconitate (Table 10, Figures 13 and 14), suggest 207 that these acids may somehow be associated with the phos- phatase $2.2222- The correSpondence between large quan- tities of the tricarboxylic acids and the phosphatase in fractions from a DEAE-cellulose chromatographic fractiona- tion, after 3 preliminary acetone fractionations (Figures 16 and 17), could have been a coincidence, but considera— tion of the stabilization of the phoSphatase by the tri- carboxylic acids (Figure 15) and the competitive inhibi- tion of the phoSphatase reaction by cis-aconitate (Figure 18) makes it seem that the correspondence between the phosphatase and the tricarboxylic acids was more than a coincidence. The above observations, together with knowledge that aconitase is stabilized by its substrates, that any one substrate is a competitive inhibitor of the intercon- version of the other two tricarboxylic acids by aconitase, and suggestive evidence that relatively large quantities of the tricarboxylic acids may bind to aconitase from plant and animal tissues (literature review),led to the hypothesis that in tobacco leaves, aconitase activity may be associated with P-glycolate phosphatase. Experiments which tested this hypothesis are described in a later sec- tion. 208 Investigations on the Stability Toward Dilution at 30° of P-glycolate PhOSphatase in Fresh Extracts from Tobacco Leaves Unless noted otherwise, each eXperiment was initi- ated with the harvest of 2 to h medium sized tobacco leaves (see Table 1), and the leaf blades were homogenized in a mortar and pestle with sand with two weights of water. Stabilization by Time After Homogenization When extracted from tobacco leaves by mortar and pestle, the phOSphatase in the extract remained active if stored at 0°. However, the enzyme was initially unstable toward dilution at 30°, but with time it became stable to this dilution (Figure 19). The enzyme in the extract from the greenhouse grown tobacco leaf harvested in the winter was initially more stable and became stabilized many times faster than the phOSphatase in the extract from the field grown tobacco leaves harvested in the summer.1 During the summer, the greatest initial stability toward dilution at 30° was about 50%, while during the winter, the smallest 1When water rather than 0.1M cacodylate, pH 7.5, was used as the extraction fluid, the initial stability toward dilution at 30° of the phoSphatase in an extract during the winter was 18% and the stability after only four hours post homogenization time was 93%. Thus, the increased initial stability and the increased rate of stabilization of the phoSphatase in the extract during the winter compared to the enzyme in an extract during the summer (Figure 19) were not because of the use of pH 7.5 cacodylate as the extraction fluid. 209 Figure 19 Stabilization by Time After Homogenization U—D Controls. <3--£> Activity after 33.3-fold (Figure A) or 10—fold.(Figure B) dilution at 30°. F1Sure A: The leaves were harvested from the field at 3:30 P.M. on 8-3-1967 (light intensity = #000 ft—c). The extract was held near 00 in a 100 ml glass beaker open toiflw air during the time of this eXperiment. Its depth in the beaker was 1.8 cm, and it was stirred gently about once an hour. F1Sure B: One leaf was harvested at 5:00 P.M. on 12-30-1964 from the second growth of a plant which.had been grown in the field and tranSplanted back to the greenhouse about Oct. 1. The leaf blades were homogenized in a morta? and pestle with sand and two weights of 0.1M cacodylate. PH 7.5. The extract, which had been centrifuged at 35,000 8 for 5 minutes, was held under air near 00 in a glass beaker during the time of this eXperiment. 8.0 4.0 per ml of extract 2.0 |.5 0.5 Phosphatase activity, umoles Pi per min. 6.0 i— ‘ I0 210 l l 1 1 #1 i 4 8 l2 IS 20 30 32 Post homogenization time, hours __ 41 II firs—=8 After- dilution at F. 1 J L gal | 2 3 T5 Post homogenization time, hours 211 initial stability toward dilution at 30° was about 13%. However, the initial stability toward dilution at 30° of the phoSphatase during the summer was usually less than 10% and was sometimes 0%. In extracts from tobacco leaves harvested in the winter from senescent plants grown in the greenhouse, the initial stability toward dilution at 30° of the phoSphatase was as great as 95%. The data suggest that the more vigorous the leaves, the less the initial stability toward dilution at 300 of the phoSphatase and the more the post homogenization time which is required to stabilize it. Stabilization as a Diurnal Function The phOSphatase in extracts from tobacco leaves harvested at night did not become more stable with time. In contrast, the enzyme did become more stable with time in extracts from leaves harvested during daylight hours - (Figure 20). Even though in the experiment of Figure 20 there was considerably more cloud cover in the afternoon hours than in the morning hours, the optimum hour of harvest for the maximum stabilization rate of the phos- phatase in the extracts was about 3 or h P.M. Thus it seems that the stabilization rate of the phOSphatase in the extracts was a function not only of the light inten- sity before harvest but also of the time of eXposure to light or darkness before harvest. The 6 to 7 minutes of 212 Figure 20 Stabilization as a Diurnal Function Leaves which were selected at random from similar field grown plants were harvested in pairs at the times indicated. Each extract, which had been centrifuged at 15.000 g for 10 minutes, was held near 00 in a glass beaker open to the air long enough for the completion of the assays of that extract. EJ'°'C] pH of the extracts. 0""0 The stability toward dilution at 30° of P-glycolate phOSphatase in each extract 45 minutes after the homogenization. (3--C) The Stability toward dilution at 300 of the phOSpha- tase in each extract 12 hours and #5 minutes after the homogenization. 213 a? , O o O 40 Day Night Day r0 _ 4— o K C. O '2 .2 6 30b- '0 8.. O 3 O .0— 3” o 3 20 D ._ .1: Q. a) .2 Q I ' Q. a) ‘— 2 o 0 IO pH —6.3 e. I N.» _ o -—6.2 0’ d. D o /O\ I/D/ —-6‘ \\ Cr” \\‘0 . u— / ~~"°"”q —-6.0 o \’ / \\ [s > / \3 ’ V“ 5.9 5?; I [\‘EQ—-—¢/ l ‘ \ {’3 6PM. IOPM. 2AM. 6AM. IOAM. 2PM. 6PM. a 7-Ie-66 7"9‘55 Time of harvest 21# exposure to artificial light (10-100 ft-c) immediately before and during the homogenization had no apparent effect on the results, which is consistent with the approximately 3 hour lag between the light intensity maximum and the harvest time for the maximum stabiliza- tion rate of the phOSphatase in the extracts. Even when extracts from field grown tobacco leaves which were harvested at night were held for longer periods of time, the phoSphatase did not become stable toward dilution at 30° (Figure 26). When leaves were harvested during daylight hours but the light intensity was very low (rain and fog,for examplex the phOSphatase in the extracts likewise did not become stable toward dilution at 300 with time (Figure 24). However, in these cases, the phOSphatase retained most of its activity in the extracts stored at 00 (Figures 24 and 26), and it last no more activity in an extract stored at 00 than did the phoSphatase in an extract from leaves harvested during a sunny day (Figure 26). In the experiment of Figure 20, the Specific activity of the phoSphatase in the 7 extracts varied in a random way. There was no apparent relationship between the time of day of harvest and the Specific activity of the enzyme in these extracts (data not Shown). 215 Stabilization by Mixing in a Waring Blendor The phoSphatase extracted from tobacco leaves by mortar and pestle was unstable toward dilution at 300 while the phosphatase extracted by Waring blendor was stable (Figure 8). When an extract which had been pre- pared by mortar and pestle was mixed in a Waring blendor, the unstable phoSphatase was stabilized (Figure 21). Thus, the stabilization (Figure 8) was not dependent on material removed by the cheesecloth filtration or by centrifugation. The results also suggest that the lower activity per gram of leaf for the phOSphatase in the extract prepared by Waring blendor (Figure 8) was pri- marily due to inactivation of extracted enzyme rather than less efficient extraction. The amount of inactiva- tion by Waring blendor (Figure 21) was about equal to the amount of inactivation which accompanied the time dependent stabilization of the phOSphatase (Figure 19). The enzyme in extracts prepared by Waring blendor last no further activity in 24 hours at 00 (data not shown). The data suggest that the time dependent changes in the activity and stability toward dilution at 300 of the phoSphatase (Figure 19) also occured during the Waring blendor treatments, which lasted 2 minutes (Figure 21). Stabilization by Oxygen When fresh extracts were "buzzed" under N2 or C02, 216 Figure 21 Stabilization by Mixing in a Waring Blendor Six leaves were harvested from the field.at 12:30IhM. on 9-7-1965. After removal of an aliquot of the extract, which had been centrifuged at 20,000 g for 10 minutes,tflw remainder of the extract was vigorously mixed in a Waring blendor for 2 minutes. The extract in the Waring blendor was under air during the mixing. The aliquot which was mixed in the Waring blendor was then refiltered through cheesecloth and recentrifuged as before. Open bars: controls. Closed bars: activity after 20-fold dilution at 30°- 217 5.0 0 0. 0. 0. 4. 3 2 l 80.58 “.0 2: nod .26 dog 5 «208: .2328 omoaocamoza Waring Blendor Mortar and Pestle 218 the phOSphatase remained unstable toward dilution at 30°, but if the extracts were "buzzed" under air or 02, the phOSphatase was stabilized (Figure 22). The data suggest that 02 is the component in air which stabilizes the phos- phatase and that the vigorous mixing is necessary only to increase the oxygen concentration near the phoSphatase molecules. In another eXperiment, done once, a fresh extract from tobacco leaves was divided into two aliquots. One was gassed with N2 and the other was left open to the atmOSphere. While the phoSphatase in the extract exposed to air became more stable with time, the phOSphatase in the extract under N2 did not become more stable with time. A Diurnal in the Stabilization by Buzzing1 In general, the stabilization by buzzing of the phoSphatase in fresh extracts followed a diurnal (Table 13) that was qualitatively similar to the diurnal in the stabilization by post homogenization time (Figure 20). ’ The relatively low stability value for the phoSphatase in the extract from the leaves harvested at 11:45 A.M. could be explained by the 100% cloud cover and low light intensity at.the time of harvest. However, the 67% 1The data were collected from 7 different experi- ments conducted for other purposes in Aug. and Sept. of 1967. 219 Figure 22 Stabilization by Oxygen The leaves were harvested from the field at 5:40 PAL on 7-26-67 (light intensity = 1100 ft-c). Five 3.0 ml aliquots of the extract, which had been centrifuged at LSJWO 8 for 15 min., were placed in separate side arm test tubes, filling them to a depth of 1.8 cm. The five were treated as described in the following schedule. 1. Left open to the air; not buzzed. 2. Left open to the air: buzzed. 3. Gassed with N2; buzzed. 4. Gassed with C02: buzzed. 5. Gassed with 02; buzzed. Open bars: controls. Closed bars: activity after 33.3-fold dilution at,%) 220 per ml of extract N 'o :1 per mm. 5.0L 0:9:0:0:0:0.o . ... .i. O O '— 0 0 0 0.0 4.0 0 O0 00. 0 00 . zo:ozozozozozozo:ozozozozozozozozo 0 0.0.0.0 0 0 0 0 0 0 0 0 0 0 0 0 0 2.0 fl ' v 0.0 0 0.0- v V V v 0.0.0.0 9.0.0. 0 0 '0000 0 .00 V .0 0. o'o'o‘o' ofofofof 2:203:30: O O O 0 0 - I o'o' otot v 0 C 0.0. ’3’ 0 . 0.0.0... 00 Phosphatase activity, umoles Pi 06.0.0.0 ... O O O .... I 2 3 4 5 Treatments 221 .emoeass.co case one use Hm comm 0H .z.m Hmum antm, at oooma om .z.m amum mmim Nm oomoa 0N .z.m Noua mlw mu coma 00H .294 maaaa omtw mm comes 0 .z.4 mesoa Him we comm om .z.4 mmum mum om .2.4 mean .watm a m wadnwsn Hopmm moauaao poom Hohoo vaoao pmo>aa£ unmadhoawo com um soapsaao mo made no open season apaaandpm awsoaeaosoo names meawwem an soapswaaapdem one as Hosasao s NH mHDwB 222 stability value for the phOSphatase in the extract from the leaves harvested at 2:31 P.M. does not fit the pat- tern. The two diurnals were quantitatively different. The time dependent stabilization (under aerobic condi- tions) of the phoSphatase in extracts from leaves har- vested at night was essentially 0% of the time dependent stabilization of the phOSphatase in extracts from leaves harvested during the day. The stabilization by buzzing of the phoSphatase in extracts from night harvested leaves was about 2/3 of the maximum stabilization by buzz- ing of the phoSphatase in extracts from day harvested leaves. Effect of Sephadex G-25 Chromatography Rapid passage of extracts from tobacco leaves through a Sephadex G—25 column stabilized the phospha- tase toward dilution at 30° (Figure 23). The time of the day at which the leaves were harvested had no effect on the results, i.e. the phOSphatase in extracts from leaves harvested during daylight hours was also completely stabilized by the same treatment. Furthermore, Signifi- cant activation was noted when extracts from leaves har- vested during the day or during the night were passed through the Sephadex c-25 column (Figure 23).1 1The actual activation probably was greater than indicated by Figure 23,Since in none of the eXperiments was an effort made to be sure that the fraction from the column which was saved contained all of the phoSphatase. 223 Figure 23 Effect of Sephadex G-25 Chromatography A Sephadex G-25 (medium grade) column 1.0 cm.x 8 MD high was prepared in a 0-40 room using gel which had been hydrated in 0.02M cacodylate, pH 6.3, for h hours, and was washed with 100 ml of the same buffer. The leaves were harvested from the field at 2:00 AJL on 8-17-1966. The leaf blades were homogenized in a mortar and pestle with sand with two weights of cold 0.02M cacodylate' pH 6.3. The pH of the extract, which had been centrifuged at 15.000 g for 8 minutes, was 6.3. 0.75 ml of the extract was added to the column and eluted with 0.02M cacodylate, pH 6.3, The elution was started at 2’45 A.M. and was completed in about 10 minutes. The Ph°&' phatase emerged with the leading color peak, and 1.5 m1 of the peak was collected. The "no treatment" preparation was made by diluting 0.75 ml of the extract to 1.5 ml with the buffer. Open bars: controls. Closed bars: activity after 10-fold dilution at 30°- 221+ 0. as _E ..oa 5. o. 5 o. 5. 2 2 I.“ I. 0 .56 dog _a 86E: 53:8 omoaocamocd Treatments No treahnent G-25 Sephadex 225 Stabilization by Acetone Precipitation When the phoSphatase in a fresh extract was pre- cipitated by acetone, the rate of the stabilization of the phosphatase was increased (Figures 24 and 42). Even from an extract in which the phoSphatase did not stabil- ize with time, the enzyme which had been precipitated by acetone became stable (Figure 24), although not as . rapidly as did the acetone precipitated enzyme which came from an extract in which the phoSphatase did stabil- ize with time (Figure 42). Effect of Dilution AS discussed later, the stabilization of the phos- phatase by Sephadex G-25 column chromatography or by pre- cipitation by acetone could have.been by decreasing the concentration of endogenous reductants and inhibitors of the oxidation of the enzyme, and by supplying 02 dissolved in the buffer. For the same reasons, it was anticipated that dilution of extracts with buffer might have had a stabilizing effect on the phoSphatase. About half of the activity of the enzyme was des- troyed by the first hour of dilution at 0°. The remaining activity was not stable toward dilution at 30°, for 90% of the remaining activity was destroyed by the next hour at 30° (Table 14). The data suggest that the dilution itself may prevent any possible stabilization to be 226 Figure 24 Stabilization of the PhOSphatase by Acetone Precipitation The leaves were harvested from the field at 5:201LM. on 7-14-56 (driZZling. 100% overcast, low visibility). 13w pH of the extract, which had been centrifuged at 15,000 8 for 10 minutes, was 6.4. A 10.0 ml aliquot of the extract was fractionated by adding ten 1.0 ml aliquots of acetone sequentially. After each addition, the precipitate was removed by centrifugation, rinsed with cold water, and reSuSPended in 2.0 ml of cold 0.02M cacodylate, pH 6.3- The most active phoSphatase fraction was precipitated by the 7th ml of acetone. For the rest of the experiment.1flw remainder of the extract and the most active phOSphatase fraction were held as the extract had been held before the fractionation, near 0° in glass containers cpen to the air. D--£] Controls. o-—-o Activity after 40-fold dilution at 30°. 6.0 E L a) ‘1 4.0 2.0 Phosphatase activity, umoles Pi per min. P 9’ 90 O O O N o 227 I l l i I l WU Extract After dilution at 30° A “34 Um Control Acetone precipitated fraction After dilution at 30° l L L l l l 3 e 9 l2 l5 l8 2: Post homogenization time, hours 228 00H m.H N.N we on m.o o.c om om m.m o.: as on m.m H.m om m.o a.o em pace. 4 . a u _ a musosnuoapowa as a .oom some "as a .oo as a .oom as H..oo one maaasb poaapxo asp no soapsadm mHoapaoo mo R .aoapaaao hound hpasapod g campanamosm as» no apdadnapm on» so aoapsaam mo poomafl as cases 229 expected from decreasing the concentration of endogenous reductants and inhibitors of oxidation, and from supplying O2 dissolved in the buffer. Apparently, something which was decreased in concentration by the dilution is needed to permit the stabilization of the phoSphatase by 02. Effect of Metals Calcium: In a single eXperiment, a fresh extract from greenhouse grown tobacco leaves was divided into two aliquots. To one aliquot was added an equal volume of 0.02M CaClz. The phosphatase became stable toward dilu- tion at 30° much more rapidly in the extract containing CaClz than in the extract containing no calcium. The preincubation with CaCl2 also partially inhibited the phoSphatase reaction. Magnesium sulfate (and sodium cacodylate buffer): In the assay for activity after dilution at 30°, the MgSOu and Na cacodylate concentrations during the 1 hour of dilution were 3 x 10'3M and 5 x 10’2M reSpectively. It was found that at the usual temperature of 30°, the M880“ and Na cacodylate did not protect the enzyme com- Pared to a control containing only water and enzyme dur- ing the 1 hour of dilution. However, when the dilution was at 0° rather than 30°, only 25% of the activity sur- vived when the dilution mixture included only water besides the enzyme, while 50% of the phoSphatase activity 230 survived when the dilution mixture contained the standard amounts of MgSOu and cacodylate. It was also found that the MgsolP alone at 3 x 10‘3M and the buffer alone at 5 x 10'2M were about equally effective in giving partial pro- tection against inactivation by dilution at 0°, but neither was quite as effective as the two together. The two did not act synergistically in conferring this limited stabilization. EDTA and orthophenanthroline: Preincubation of extracts with low concentrations of orthophenanthroline made the phoSphatase more stable toward dilution at 30°, but the magnitude of the stabilization varied. EDTA was not as effective as orthophenanthroline and EDTA was less effective as the concentration was increased (Table 15). Endogenous metal(s): The phoSphatase in an extract from tobacco leaves had 43% as much activity when no metal was included in the reaction mixture as when the standard amount of MgSOu was included. When a control included enough EDTA to make the reaction concentration 0.1M, the phOSphatase had zero‘activity, which is consis— tent with earlier findings (202). The initial stability toward dilution at 30° of the phoSphatase in the extract was 6%. When the extract was held near 00 under air for 3 days, the activity with MgSOu in the reaction mixture had dropped to 82% of its original value. The surviving activity had a stability toward dilution at 30° of 98%, 231 .maoHmeSOCaona Mao: H on» weaasu msoapaapnooaoot HN MUCH Om MIOH N WoN +~ NlOd as .13 so at: w a 2. TS m o a: o o .o u z a z m 2 com um Codpfiddfi *mQHHOHSp com #6 SoapSHaU *mzHHOHSD com um Soapfiflav *flBDW season apaaanupm IamzonaoSpao Samson hpaadnapm tamaosao£pao l N paoaaaoawm H paoaaaoaxm I llwll cameo» apaaaadpm csaaoaspsdsosaospao no seam co pooacm we oases 232 while the activity with no metal added to the reaction mixture was 47% of the 82% of the activity which survived. Thus,as the stability toward dilution at 30° of the phos- phatase changed from 6% to 98%, there was no significant change in the phosphatase activity which depended an endogenous metal. These results suggest that the time dependent change in the stability toward dilution at 300 was not because of a time dependent binding or release of endogenous metal by the active site of the enzyme. Effect of OHPMS and of Glycolate A ten minute preincubation with glycolate made the phoSphatase a little more stable toward dilution at 30°, but the preincubation with glycolate partially inhibited the stabilization of the enzyme by oxygen (Figure 25). Glycolate also inhibited the stabilization of the phoSpha- tase by post homogenization time (Figure 26). The small decrease in phoSphatase activity which always accompanied the stabilization by oxygen was also partially prevented by the ten minute preincubation with glycolate. A ten minute preincubation with OHPMS under aerobic or anaerobic conditions was even more effective in stabilizing the phos- phatase than was the oxygen treatment which was used (Figure 25). When an aliquot of fresh extract was made 10‘2M in OHPMS and then buzzed under air, the intense color change 233 Figure 25 Effect of OHPMS and of Glycolate The leaves were harvested from the field at 10:50 A.M. on 9-23-67 (light intensity = 10,600 ft-c). 4 r ll Preincubation conditions Preincubation mixture Addition Atmosphere Buzzing 1 H20 aerobic not buzzed 2 H O aerobic buzzed 3 Glycolate aerobic 1’1013 buzzed it Glycolate aerobic buzzed 5 OHPMS aerobic r10t buzzed 6 OHPMS anaerobic not buzzed .4..— Each preincubation mixture contained 9 parts by volume of extract and 1 part by volume of additive. The stock 613” colate and OHPMS solutions were both 0.10M, pH 7, so that the Preincubation concentration for bath was 10'2M. The preincu- bationS With SlYColate and OHPMS were for 10.0 min. be“re the start of the dilution at 30°. The buzzing of the SHOO- late preincubation mixture was for 2 min. starting 2 min. after the addition of the glycolate, The anaerobic preincuba" tion was in a Thu-T113138 tube. After the system was gassed with N2, the preincubation was started by dumping the OHPMS from the side arm into the extract. The Thunburg was opened Just before the start of the dilution at 30°. Open bars: controls. Cl°sed bars: activity after 20-fold dilution at 30°: ll 7'5“ A «i run» ’49-. iv" tcl‘ l" per ml i per min. Phosphatase activity. umoles P 234 4.0 l— 9‘ O V N o 'o 0 .0 1:302:91 O O O O O 0 0 0 0 0 0 .0 0 0 0 0 0 0 00 0 0 .0. o'o'o'o'o'o'o'o' 0.030303030303’ A. 00 00. o o o ’0’... o . . 0 '0...- oo oo- 00 00. co co: 00 or so . co: . 00 o oo« . co . oo- . co . ... . ow . co . co. . 00 o oo- o oo o 60' . co . ’ o 0.1 0.. 0.4 0.1 9.4 0.4 ’0‘ 0‘ o 0.0 0.0 9.: 9.4 o Preincubation mixtures 000’ . 0 o 0 0 0 0 0.0 0 0.0 °:°:°:° . 0 . 0 0 O 0.0... .0 0 0 0 00 .:.;.:. C . 0 0'0' .0.0 0 o o . 0.0.9 o‘o'o‘o'o 9‘0 0 9.0 0.0 o o 0 0 0 .0 0 0 0 0 0 0‘0 0 .0 0.0 C A O O 0 0 0 0 0. 0 235 Figure 26 Effect of Glycolate and Time After Hemogenization The four preincubation mixtures, each.containingi%60 ml of extract, plus either 0.40 ml of H O or 0.40 ml of 0.10M 2 glycolate, pH 7.0, were held near 0° in 1.6 cm diam. x 15 cm high test tubes open to the air during the time of this eXperiment. TOP flaure: Leaves harvested from the field atffizo P.M.. 9-15-1967 (light intensity = 4000 ft-c). The two pre- incubation mixtures were made up 35 min. after homogeniza- tion. Bottom figure: Leaves harvested from the field at 3:40 A-M-. 9-16-1967. These leaves were kept in complete darkness before and during the homogenization. The tWOIfle' incubation mixtures were made up about 30 min. after homo- genization. D-——{J Without glycolate; controls. D~--EJ With glycolate; controls. C>--C> :étgggt glycolate: activity after 20-fold dilution O---O Wit 30°h glycolate; a°t1Vity after 20-fold dilution at 236 J! j! 4.0r- Harvested during light _ 3.0— "‘~~'~s‘—, E /—==.—_-g \— at 0. 2.0—— E E ,_ l.Or- a) O. O.— m 0 % Post homogenization time, hours E 1 >2 :‘S‘ 4.0—— Harvested during night 2 o o a, 3.0—— m o .0— 0 €- 20 a) ' _ O .c O. l.O>——- O————_-_- a l “W O 20 4O 60 Post homogenization time, hours 23'? to dark orange brown, which always accompanied such buzz- ing, was completely prevented. OHPMS is an inhibitor of glycolate oxidase. Inhibition of the oxidase could have prevented the formation of H202 during the buzzing of the extracts. In this way,the oxidation of the colorless compounds to their brown form might have been prevented. Furthermore, under certain conditions, which seem to include the presence of light plus FMN or leaf extracts, OHPMS itself seems to be oxidized (personal communication from Dr. N. E. Talbert). Any oxidation of OHPMS, rather than the colorless compounds in the extract, could also have decreased the oxidation to their brown form. The prevention of the formation of H202 Would not eXplain the stabilization of the phoSphatase by OHPMS. Likewise.the possibility that OHPMS might serve as a reductant would not eXplain the stabilization of the enzyme. Both the prevention of the formation of H202 and the possibility that OHPMS might serve as a reductant would be expected to keep the phOSphatase in the reduced, unstable form. An eXplanation for the stabilization of the phOSphatase by OHPMS is proposed in the discussion which follows later. Protection of the PhoSphatase by cis-aconitate Cis-aconitate at 6 x 10'3M gave partial stabiliza- tion against inactivation by dilution at 30° (Figure 27), 238 Figure 27 Protection of the PhoSphatase by Cis-aconitate The extract used for this eXperiment was the same as the one described in Figure 6. Control: no cis-aconitate. Control: plus cis-aconitate. o—o Act1v1ty after 20-fold dilution at 30°; no cis- aconitate. O---o Activity after 20-fold dilution at 30°; plus cis- aconitate. Cis-aconitate was at 6 x 10'3M during the dilutimiat 30°, and at 4 x 10'3M during the reactions. per ml Phosphatase activity, umoles Pi per min. l.20 l.00 0.80 0.6 0 0.40 0.20 0.00 239 — I 0 Controls “-0 ________ .0 After dilution at 30° with cis-aconitate without cis-aconitate \ l l l l L L l 2 3 4 5 6 7 Post homogenization time, hours 240 The small percent inhibition by the cis-aconitate is con- sistent with the saturating level of substrate which was used (see footnote, p. 202). Effect of Arsenite and Cd++ Preincubation with either arsenite or Cd++, even at low concentrations, Slightly activated the phoSphatase (Table 16). At high concentrations, Cd++ caused much precipitation to occur in the preincubation mixtures,sug- gesting that its inhibition at higher concentrations was nonSpecific. Preincubation with arsenite or Cd++ at the concen- trations Shown in Table 16 did not stabilize the phoSpha- tase toward dilution at 30°. Neither 10'3M arsenite nor 10‘3M arsenite + 10'3M EAL had any significant effect (inhibitory of stimulatory) on the stabilization by oxygen of the phOSphatase in the preincubation mixtures. The arsenite-EAL combination in the preincubation mixtures also did not stabilize the phOSphatase toward dilution at 30°. Preincubation with the arsenite-EAL combination did not give as much activation as preincubation with arsenite alone (data not shown). P1, at a concentration of 5 x 10‘“M during the reaction, resulted in a Slight (1.5%) inhibition of the phOSphatase reaction. 241 I I ma aloe w N Nice I I I i an mica w m mica I I I I cos mica w m alas I moa I I was mica w m mica NGoo mos moa mos I owe sues w N miss I i I Has mma mica w m mica mos mos moH I was mica w m nice I I I I mas aloe w m mica onasomws ooa ooa ooa ooa ooa I I Hopaz a a a a a a 2 pl m .pamm : .pawm m .paNm N .paxm a .paxm manna waaadd acapaapaooaoo abapaoow obapaoom mo nodpaapsooaoo mpdbdpom soapansondonm .moaspxaa :oapaQSoaHoaa on» so may once masons musessamosa opuaooaawua cascades .oo no soaecpsosaoaa co soon a paced hoses .obapaopm ho oasaob me name one one pomnpwo among mo menace an enema 0 mac: moaapHHa Goaumn:oaaohm .uoma .pmsws< ad nooanopma can ad emphases: one: massed one ++po can opdaomh< an ommpmnamosm as» no soapabdpow on canoe 242: Reversal of the Stabilized PhoSphatase to an Unstable Enzyme Ten minutes of preincubation of buzzed extracts with glycolate partially reversed the oxygen stabilized phosphatase to a less stable but just as active enzyme. A combination of glycolate, and of fresh extract (not buzzed) which had been heated to 1000 for 15 min., gave more reversal than either additive alone. No reversal was noted when OHPMS was included with the glycolate and the heated extract during the preincubations. More reversal was obtained under anaerobic than under aerobic preincubation conditions (Figure 28, Table 17). Glyco- late was also effective in decreasing the stability toward dilution at 300 of the phOSphatase which had been stabilized by post homogenization time under aerobic con- ditions. In preliminary exPeriments, the following compounds, when preincubated at the indicated concentrations with buzzed extracts, were completely ineffective in decreas- ing the stability toward dilution at 30° of the oxygen stabilized phosphatase: NADPH, NADH, or Fe++ at io-3M. citrate, isocitrate, or cis-aconitate at 10'2M, cur a combination of cysteine at iO'ZM and Fe++ at 10‘ M. The effect of preincubation with ascorbate on the activity and stability of P-glycolate phoSphatase was tested using an enzyme with the following history: it had originated 243 Figure 28 Reversal of the Stabilized PhoSphatase to an Unstable Enzyme The leaves were harvested from the field at 11:45 A.M. on 9-14-1967 (light intensity = 11,000 ft-c). The pH aflflw extract was 5.6. Preincubation mixture 1 contained 0.175nfl of extract (not buzzed) plus 0.075 ml of H20. The other six preincubation mixtures contained the following: _- Extract,. Extract, Mixture buzzed H20 boiled and OHPMS Glycolate clarified ml ml ml ml ml 2 0.175 00075 - " '- 3 0.175 0.050 0.025 - - 4 0.175 0.050 - - 0.025 5 0.175 0.025 0.025 — 0.025 6 2.800 0.400 0.400 - 0.400 needed. Extract, buzzed: Extract, boiled and clarified: OHPMS and glycola preincubation concentration for both was 1 6.0 ml of extract buzzed under arr. 3 ml of the extract (not buzzed) was placed in a boiling water bath,with a marble over the test tube,for 15 minutes. was decanted and held at 0° in a stoppered test tube until te were both 0.10M 0-2 The clear supernatant pH 7.0. The M. The compo- nents of the preincubation mixtures were added in the order indicated in the above table, from left to right. Preincubation no. 6 was made anaerobically. The other six preincubations were aerobic. The aerobic preincu- bations, at 0°, were in 5 ml test tubes open to the air. The anaerobic preincubation. at 0°, was in a double Side arm Warburg flask. buzzed extract and H and clarified extrac This system was gassed with N tipped into the buzzed extrac added. The main part of the flask contained the %0. One side arm contained the boiled and the other contained the glycolate. of the dilution at 30°. t'y The boiled extract was ust before the glycolate was The Warburg flask was opened Just before the start The addition of glycolate started the 10 minute pre- incubations, and the start of the dilution at 300 ended them. .I' Afi‘ . ooooo H l" t it: i It ri' rt 244 During the 1 hour of dilution at 30° and during the 10. minute phOSphatase reactions, the assay tubes were open to the air. Open bars: controls. Closed bars: acgivity after 20-fold dilution at 30 . The stabilities toward dilution at 30° of the phoSphatase in preincubation mixtures 1 and 2 were 0.8% and 69.5% reSpectively. For any of the other preincuba- tion mixtures: stability of Reversal of stability = 63.5% - the phOSphatase x 100% toward dilution at 30° 69.5% - 0.8% . per ml per mm. Phosphatase activity, umoles Pi 245 3.0 Reversal of stability toward dilution at 30° (°/o) 0 5 23 37 7| o 0‘1 o o c 0’: , .o.‘ o 1 0 6% 0 . 0 0 0: 0‘ o 0’: 0 a 0 0 .. 04 0 0% 0 a 0 ., ... . co ’0‘ ... so ” .0.: ’o’e‘ ’5' ”' oo ” D0! "' 00 '° '0‘ ”' 00 ’° IO- "‘ 00 " ... D01 .. co ... po- .. co ,., b0! ,. co ... vo- .. co ... DO: .. o. ,. D0- ,... '9’- ., 00 00 co to co .0 00 b0 .0.. An 2 3 Preincubation mixtures 0 ' 0 9’0 0 a 0 0 0 0 9a 0 f0' ‘0 ‘0 O 0 o 0 0 0 '0 .0 0 ‘9 0 0 0 O 0 0 0 0 0.3 0;. o 0 O '0 0!. 0 O 0Wf0'0 Q‘Q‘Q‘D ..... o o a... 00 00 ‘0‘. o q. 0 05’30' " O 'o'ooo‘o‘c ' 0.0.0.0.... 0%,;0 O O O O 0 0 0 O O O 3%.: °' . . .nwo I as ho .pHo on» ad omapmnamona on» no mpaaanapm as» and poaapwo ocuuzn on» ma omapasamosa as» mo mpdaanmpm on» no owaaoba oopswdos on» ma oopaHSOHao was aamnosoa ohomofl com pa soapsaao cameo» mpaaaoopm asp ..pMo no .pwo I wz ooosaoaa scans moaspaaa soapaQSoaaona mo made on» aH Hmmaoboa ohomop pomnpwo oouusmzmo anaaascem I com as scansaao season Hmmhmsoh chowon pomhpxo I hpaflanapm mo Hamhobom i causes no apaaandpm Abounsp pocv I . . .Iwodaewo ac abwaasmnm mooa a _Hamao>oh nopma poaapxo causes no apaflapmnm .mm oasmam an confluence he one: maoapaQSosaoha canoaoaa< .had on» on soao mops» pump as m Ca who: maoameSoaHoaa capoaow .oo on message ma no oH you one: can opaaoomam mo sodpdbba on» Spa: ompaapm one: maoameSosaoam . Ioa ma: nodpaapaooaoo madame Isosdoaa on» pan» on .o.m ma .zoa.o was opmaoomaw one .mohfipxaa nompa9502acaa on» mo oasaob one .oaauwda aofimeSoadoaa a no oasdob an Rea some opoaaaoo op .Uohaddon muons .bobpm was nova: macs .pxo I Nzfiznw.poaapxo .pocapxo oofidon .opaaoomaw .moaspwda aodpanaoaaoaa on» go Had 2H .pomhpxo acumen can on sodpaooa 2H poaapxo ooaaob one opmaobhflw poaampzoo monapxaa soapansoaaoha canoaomsa one .moazpwaa aoameSosaoaa on» mo oasaos an Rom was poaapxo mousse as» .mpnoauhoaxo Hosea one ad oH«£3.moH:pxaa soapaQSoaaoaa on» mo oedaob an Ron was poaapxo bonusn on» .5 and .m .m mpaoaaaoaxo aH .saa ma you paoapaoap Span omm msaaaon a above Abouusn posv poaawxo can scan pampmGaoQSm Macao on» u .pxo ooaaom .Nz spas oowmaw Apouusn poav poahpxo n .pMo I z .Abouusn ponv because u .me .uomalmmtm op mmeINNIm aohm bopoSpGoo macs mpaoadhoawo and: one soapcudaabmpm on» do vacuum on» one .oaanam cannons: cap on omapanamosm oonaaabmpm on» yo Homeosom on» no psopwm on» noospom soapaaonnoo a cases 6 ll». 2 247 .0 ma paonm Op m.m ma psonm thm madame adaaahoa mpodhpwa £05m ho heads» ma one .Uopmsnuaas whoa whoa mpomhpxo amp mo mosaab mm on» .mpcoaahoawo m Hanna on» GH .mom 2H.o Spas ©.m op wopmsndm was pomapxo as» mo mm as» .w unoadhomxo ho pampm an» p2 :2: > z 0 O 3,’ 0.70 D :2: 0.60 C 8 0.50 < 0.40 0.30 0.20 0.l0 L i IO’ZM . 5xIO'2M M9804 concentration 284 Figure 32 Activation of Aconitase by Sulfates or Chlorides The aconitase for this SXperiment was prepared as described in Figure 31,except the Swiss chard was grown in the greenhouse, the extract was not adjusted with reSpect to pH, and. 0.02M cacodylate, pH 6.3, rather than water, was used to resuspend the acetone precipitated enzyme. All reaction mixtures contained 0.050 ml of 0.20M cacodylate, pH 6.3. 0.008 ml of 0.01M sodium cis-aconitate, pH 7.5, salt as indicated below, 0.005 ml of enzyme, and. enough water to bring the reaction volume to 0.30 ml. The reactions were initiated. by the addition of cis-aconitate. o-——o M8804. o—o (NHMZSOLV A__.ANHL,01. (3......[3 NaCl. I-—I KCl. For NHLl'Cl, NaCl, and KCl, the ionic strength is equal to the concentration in moles/liter. For (NH4)ZS°4' the ionic strength may be converted to the concentration in moles/liter by dividing the ioniclstrength by 3, and for MgSOu, by dividing the ionic strength by 4. A A240 /I0 minutes Aconitase activity, 285 0.20 0.I2—- 0.08 0.0 6 0.04 — 0.02 —— J Sulfates Chlorides l l | l 0.0 0.04 0.08 0.I2 0.I6 0.20 Ionic strength 286 Figureg33 Activation with Citrate, Isocitrate, or CiS-aconitate as Substrate The acetone precipitated enzyme described in Figure 39 was used. All reaction mixtures contained 0.40 ml of 0.10M TES, pH 7.5. 0.020 ml of enzyme, substrate and (NH4)2804 as described below, and enough water to bring the reaction volumes to 1.20 ml. Reactions were initiated with substrate. Cis: reaction mixture contained 0.030 ml of 10'84 sodium cis-aconitate, H 7.5 (reaction con- centration = 2.5 x io'RM). Iso: reaction mixture contained 0.12 ml of 0.10M isogitrate,pH 7.5 (reaction concentration = 10- m) 1 Citrate: reaction mixture contained 0.36 ml of 0.10M citrate,pH 7.5 (reaction concentration a 3 x 10'2M). Closed bars: reaction mixtures contained.0.24 ml of 0.10M (NH ) SO“, pH 7.5 (reaction concen- tration = 0.820). Open bars: reaction mixtures contained no (NH4)éflhp Figure A: absolute rates. Figure B: normalized rates. 110‘2M was the total isocitrate concentration. The concentration of the naturally occuring isomer, t reo-Ds- isocitrate.is thus assumed to have been 0.5 x 10“ M. 287 L__l e t 0 N n AVA. H \\\\\\N NV. II \\N m S I.” INNN\\N\N\N\N\N\\\N\\N\\\\\\\\ . W \\N m Mlv Un4 m «NNNNNNNNN\NNNNNNNNNNN\\\\\\N\\\N\N\\ ..l. a N .6 ( 1| w 4. SNNNNNNNNNNNNNNNNNNNN\\\\\\\\\\\\N\\\N C 0 V. 3 _ f b T _ _ _ . O O 0 O m 2 O 8 6 33:8 525on do o\o / m /E .\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\.\\.\u sulfate sulfate plus Cis. lso. Citrate . \ \\\ \\hh.3“\\\\\\\\\\\hK\\\\\.\\\\\\\\\\\\\\\\\\\\\\N\\\\\\\u 0.30 r- V O 2 O moScE. o_\o¢~< 4 $3280 $2284 0.I0L- 288 present with the chlorides or sulfates did not seem to exert as pronounced an effect on the plant aconitase as did the anions (Figure 32). Sulfate was found to acti- vate aconitase from tobacco leaves with any of the three tricarboxylic acids as substrate. However, the amount of activation depended on which tricarboxylic acid was used as substrate and/or the concentration of the acid (Figure 33). Classically, when an endogenous activator is present with an enzyme, the velocity vs. enzyme concen- tration plot shows an upward curvature. However, includ- ing the activator in the reaction mixtures straightens out the plot (68). Thus,the linear plot of velocity vs. aconitase concentration which was found with 801+"2 present (Figure 5) is not unexpected. Peters (191) found that increasing amounts of NaCl dramatically increased the activity of aconitase from pig heart, while KCl at low concentrations increased the activity and at high concentrations decreased the activity. To my knowledge, the activating effect of anions other than substrate on aconitase from plants has not been previously reported. However, it has been reported that increased isocitrate concentrations acti- vate the aconitase from mustard (185). From a control standpoint, it may be of signifi- cance that passage of acetone precipitated aconitase 289 through a Sephadex G-10 (Figure 31) or G-25 column rever- sibly inactivated the enzyme,while passage of fresh extracts through a Sephadex G-25 column activated the phoSphatase (Figure 23). Relative Reaction Rates with Citrate or Isocitrate The rate of formation of cis-aconitate from iso- citrate was considerably greater than the rate of forma- tion of cis-aconitate from citrate, even though the con- centration of citrate was 6 times greater than the con- centration of threo-DS-isocitrate (Figure 33). Although the data of Figure 33 do not give Vmax values, they are consistent with the finding that for beef liver aconitase, the relative maximum.velocity for the disappearance of isocitrate was 1.7 times greater than for the disappear- ance of citrate (114). The Adeguagy of Aconitase In 5 experiments with tobacco leaves harvested from the field, the aconitase activity in the extracts varied from 0.40 to 1.2 umoles of cis-aconitate converted/ gram fresh weight of leaf blade tissue/minute. In these same experiments, the ratio of P-glycolate phoSphatase to aconitase activity, each eXpressed as umoles of substrate 290 converted/minute/ml of extract, varied from 13 to 23,1 with the average ratio being 19. The adequacy of P-gly- colate phoSphatase has already been discussed. From the activities present in the extracts of leaves, it is estimated that enough phoSphatase activity is present 22:3222.t° allow all the newly fixed C02 to be acted upon by this enzyme. The reSpiratory evolution of CO2 in plants in the dark has been estimated to be about 1/25 the rate of photosynthetic C02 fixation (120). For both P-glycolate phoSphatase and aconitase, the enzyme must catalyze one reaction per 2 C02 molecules fixed or respired. Therefore, the amount of aconitase activity which was measured in the extracts from tobacco leaves seems adequate to account for 12.3212 dark reSpiration rates. The Fractionation of Aconitase and P-glycolate PhoSphatase It was found that P-glycolate phosphatase 1P-glycolate phoSphatase assays were by the standard procedure. All aconitase reaction mixtures contai ed 0.033M TES, pH 7.5. 0.02M ammonium sulfate, and 2.5 x 10‘ cis- aconitate in a volume of 1.2 ml. The estimates of the aconitase activities must be considered approximations of the lg vivo activities at 30° for at least the following reasons. The activities were converted from those which were measured at 25° to the values given, which are the estimated activities at 30°, by multiplying by 1.41 (this was based on an estimated doubling of the rate for each 10° increase in the 25 to 30° range). The substrate for aconi- tase for dark reSpiration is citrate,and the rate with cit- rate is considerably less than with cis-aconitate (Figure 33) (114). .Although the inactivation rate of aconitase in the extracts was Significant (Figures 40 and 42), no allow- ance was made for the inactivation which occured before the aconitase assays were run. 291 and aconitase from tobacco leaves were fractionated in parallel by acetone. Significant recoveries were obtained for both enzymes (Figures 34 and 35). Although protein concentrations were not determined in the SXperiments shown in Figures 34 and 35, in every other similar frac- tionation of extract which had been prepared by mortar and pestle and in which protein concentrations were mea- sured, the purification of the phOSphatase was in excess of 20-fold. In a preliminary eXperiment similar to those described in Figures 34 and 35, aconitase and the phos— phatase from Swiss chard leaves were also fractionated in parallel by acetone (Figure 36). It seems significant that although mare than twice as much acetone was required to precipitate the two enzymes from Swiss chard leaves than from tobacco leaves, they fractionated together as they did from tobacco. In the eXperiment with Swiss chard, activities of glycolate oxidase and NADPH glyoxylate reduc- tase were also measured. Unlike P-glycolate phoSphatase and aconitase, most of the glycolate oxidase and of the reductase precipitated in the 7.50-8.75 fraction (data not shown). Under conditions which included at least some eXposure of tobacco leaves to light before homogenization, aconitase and the phoSphatase, after a preliminary purifi- cation by an acetone fractionation, also fractionated in parallel during DEAE-cellulose chromatography (Figures 37 292 and 38).1 When any eXposure of field grown tobacco leaves to light was completely avoided for 3 to 4 hours before har- vest and homogenization, after a preliminary purification by an acetone fractionation, aconitase and the phoSphatase did not fractionate in parallel during DEAE-cellulose chromatography (Figure 39). The slight shoulder on the aconitase peak of Figure 39 may reflect an affinity between the two enzymes, or it could be fortuitous,or because of possible inaccuracies in the assays. When aconitase and the phosphatase were separated, isoCitrate was no longer found to be associated with the phoSphatase. (Although isocitrate was with aconitase (Figure 39), the shapes of the curves do not strongly suggest binding between aconitase and isocitrate.) In contrast, the posi- tion of one of the isocitrate peaks was found to coincide with the phOSphatase peak during chromatography on DEAE- 1No significant amount of either enzyme from leaves which had been harvested in strong sunlight was retained by the column (Figure 37). (A second nearly identical eXperiment in which the leaves were harvested from the field when the light was at 11,000 ft-c gave the same results, i.e. after a preliminary purification by acetone fractionation, neither enzyme was retained by the DEAE- cellulose column. The column had been prepared as des- cribed for Figure 39.) In contrast, all of the aconitase and the phOSphatase, from leaves which were exposed to no. light for at least 3 to 4 hours before homogenization, was retained by an equivalent DEAE-cellulose column, even though a greater amount of protein was applied in the lat- ter case (Figure 39). Also, the enzymes from leaves which were exposed to only a small amount of light before homog- enization were retained by a DEAE-cellulose column (Figure 38). 293 odofiaahwpmd 904» was» 0009. 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Mynuoo wnngow 4o °/o 299 _ .oamnno UHoo no H1 om mo Soapaood on» up Umpwapaca who: occupOQon on» pamoxo .im ohswam Mom dopahommo mm ohms whammm one .mmmpanoQ4 A¥IILQ . mmwmemm onm opmHoohlem OIIIO .Umpooaaoo who; nowo Ha m.o poopm mo mnoapoanw muchlhpuoze .zom.o op 20o.o Bong psoaUMHm hmozaa m CH Huwz wcacdwpcoo .m.w mm .mpmamuoowo smo.o mo HE o.om and: nmpsao mm: ucm nasaoo m:oo mpwpasoomumdo mo moHoad omo.o and mahuno mo Ha\:«a\uommodon A no moaoan 5.0 «coma who; suds: wnoapaonoo momma on» nouns modpabapod wna3oaaom on» can ANV coapoahm mbapow pmoa one .Rooa mm Umpm: Imamou zodpomnm obapom pmoa on» :H mpa>dpom on» Spas .ooudamaho: who: .mahuso no Ha\:da\uopnobcoo opmnpwnsm mo moHoan mm.oommonmxm .mmapabdpow mahnnm spom 301 ”‘v ..\-...ne- ..., A la 1 IO0.0 — W Rumor) 50.0 — wnngow ;o % I2 l4 l6 I8 20 22 24 IO F rection 302 .oahuco no H1 «HE 0.N ho .omdpanoo¢A¥llLQ .oahuno Mo godpaoom on» an oopdapdnd who! mfloapodoh one 005 no oom gonna» can .m.u mo .opwpdoooaumao az nod x m.a oESHob a ad wnaxoaaom on» docampnoo mohfipxaa no podoh one .mwmmm Unmuzwpw on» an .owmpdsmmonn mpdaoomdmlmnvlllnv .Uopooaaoo who: zoom Ha m.a psonm mo mcoapomnw amopndse .zom.0 on 200.0 Bonn pnodvmhm Hammad d n« Homz wcacampzoo Mona: no as 0.0m no“: copsHm was and .qadHoo Mdmn on» ouno uohmmma max codpmpaaaoona onopoom on» Sony noapwhmmmna mamwco on» go He 0.: .omm go He coo psonm.npds umsmmz cam .m.u mm on Umpmsnuw .omm ad amazon imam soon was mon on» was» mopsnda m o» 0 no doanon on» mud .Apmobhms op noHyQ mndon m 90% Mpmc coon 0m: honamno npsonw envy mwma\m\m. 3959 .2.4 m pm Amnamno npsohw on» Scum Umpmmbhmz who: mmbmoa ooomnop one Q0 Axpwm on» mcdhsm honamno npzmmo 02p Bong topmobhmm mobmoqv mmmpacoofi find mmwpmnamozm opwHoomelm mo azmmhwopGEOHso omOHSHHoolmdmm animaswfim 303 .mom dad RmmH ohms omprnoom Mom monstm quunoamonnoo ose . mm was AmcoHpomhu MH Haw GHV nasaoo omoafiaaoolm¢mm msp Scum humboomh on» 0cm Rmn was :oHpmpHQHooha onopmoo on» How mmapmsamosm mo 0HmHh one .mahunm mo Ha\:Ha \uopymbnoo opmhpmpzm ho mmdoan mm commonaxm who: moHpHpHpom Ham .UoNHHmano: who: mos» ohommm .Aw QOHpomHm nHV msawb mmmpmnmmosm doNHHmaho: azaHNma oz» mo RMNH was H :oHpome :H mprHpom mmMpmsmmosa mpwfloomelm on» oHHsz.Am :oHpome QHV monb mmmanoom uouHHwaho: aoawaa on» no Room mm: H moHpomHu.:H mprHpom mmwp IHQoow one .RO0H mo conuanmmo was :oHpomnm dopsam oprom pmoa on» :H mprHpom one .umNHHaano: mums Howz anz dodeo who: 02w Mdmm on» on xospm SOHQB moHpH Iprow 0:» mafia .m cam H mnoHpomhm 2H mama :Hmpohm pmHHm on» Ssz umwnoao 02m mhms ohms mobmmH ooonou msa .mnoHmeoxm wszoaaom on» 29H; .mm oHSme Mom UoDHHommU mm umahomhmm was pnmaHthNo mnp .mmoasaaoosm_0-d 20:88-90 I o r 0 n 030 n .0-: 0:: Pro \ _ x I .\ .\ i. . . at... +1 IVUAI +T_ IVWAI 0,, o o\-,o o--,o 340 the similarity between the substrates and the possible similarity between the reaction mechanisms of aconitase and P-glycolate phOSphatase. Other conformations could no doubt be found which would serve this same illustra- tive purpose. In any case, the conformations which are represented are imagined as those which might occur while the molecules are bound to the enzyme. For each of the _ RI molecules, there are of course an infinite number of conformations which could result from rotation about single bonds. At pH 6.:3(pH Optimum for the phOSphatase), most of the P-glycolate molecules should bind one proton at one of the phoSphate oxygens,while the other two ioniz- able groups, one on the phoSphate and the other being the carboxyl group, would be fully ionized. (The pKa values for phOSphate are 2.1, 7.2,and 12.2,and for glycolate, 3.8.)1 At pH 6.3 or higher (pH range for aconitase), most of any one of the tricarboxylic acids should be fully dissociated (the pKa values for citrate are 3.1, 4.7, and 5.4).1 The phoSphate moiety of P-glycolate would contain two non-bridging oxygen atoms,which would share a single negative charge between them. Both isocitrate and cis- aconitate would contain a carboxylate group at this 1Reference; footnote, p.102, 341 analogous position, in which a single negative charge would also be distributed between two oxygen atoms. If attention is focused on the longest chain of atoms which includes this negative charge, the main difference between P-glycolate and cis-aconitate or isocitrate, apart from a phoSphorous and an oxygen being substituted for two carbon . atoms, is that one more atom (a carboxylate carbon) is in w the tricarboxylic acid chain (Figure 47). Furthermore, "the chemistry of carbon and that of phOSphorous are very closely connected, as would be eXpected from the diagonal relationship of these elements in the Periodic Table" (247). In both the proposed phosphatase reaction mechanism and the reaction mechanism for the conversion of cis—aconi- tate to isocitrate by aconitase, a proton is added to the atom alpha to the carboxymethyl moiety and water is added to the atom which is beta to this moiety. The oxygen of P-glycolate which accepts the proton acts as a base by donating a pair of electrons. The two pi electrons of the double bond of cis-aconitate act in a similarly basic fashion. Using aconitase from pig heart, Rose and O'Connell have provided evidence that a proton from solution is passed by means of a basic group of the enzyme to cis- aconitate during its hydration to isocitrate or citrate (204). The structure of citrate would likewise be expected to be analogous to that of P-glycolate. The -OH group of 342 isocitrate is in a position analogous to the -OH (on the phOSphate) of P-glycolate, i.e. both are on atoms 6 to the carboxymethyl moiety (Figure 47). The position of the -OH of citrate is not as analogous. This difference may partly explain the observation that isocitrate was more effective in stabilizing the phOSphatase than was citrate (Figure 15). The increased stabilization by MgSOu in con- junction with citrate or isocitrate,but not in conjunction with cis-aconitate (Table 11% suggests that Mg++ may bridge between the enzyme and the -OH group of citrate or iso- citrate. An analogous link with the -OH of P-glycolate would be consistent with one of the known principles by which Mg++ catalyzes phOSphatase reactions, i.e. by polar- izing the phoSphate group making it more subject to nucleo- philic attack by water. Discussion of Similarities, Differences, and Relationships Between Aconitase and P-glycolate PhOSphatase Stabilization by Tricarboxylic Acids The substrates of aconitase have been found to be the most effective stabilizers of that enzyme (literature review). The substrates of.aconitase were also found to be effective, as well as Specific (to the extent tested), stabilizers of the phOSphatase (Figure 15).1 In the 1The order of effectiveness of the tricarboxylic acids in stabilizing the phOSphatase (cis-aconitate some- what more effective than isocitrate which was somewhat 343 literature review, 4 enZymeS are described which are acti- vated by at least one of the tricarboxylic acids,while 11 are described which are inhibited by one or more of the tricarboxylic acids. For none of these enzymes was it reported that any of the tricarboxylic acids stabilized the enzyme, nor to my knowledge is there a report that any enzyme other than the phOSphatase and aconitase is stabilized by the tricarboxylic acids. Furthermore, in some cases where all 3 tricarboxylic acids were tested for inhibition or activation, not all of the tricarboxylic acids were found to be effective. For example, although citrate was found to be the most effective activator of acetyl CoA carboxylase, cis-aconitate was found to be without effect. While citrate was found to be an effec- tive inhibitor of mitochondrial malate dehydrogenase, isocitrate and cis-aconitate were found to be ineffective. On the other hand, deoxyribose phOSphate aldolase was found to be activated by citrate, isocitrate, cis-aconitate, and trans-aconitate (literature review). Thus, even among those enzymes which are affected by the tricarboxylic acids, the phOSphatase and aconitase are somewhat unique in that more effective than citrate (Figure 15)) shows an apparent correlation with the order of the K values of aconitase for these acids (K for cis-aconitage <’Km for isocitrate.< Km for citrate (ligerature review)). The effect of trans- aconitate seems to be Similarly correlated, since it was about as effective as citrate in stabilizing the phOSpha- tase (Figure 15),while for aconitase the K for trans- aconitate was found to be about equal to the Km for citrate (literature review). 34L. they are Significantly affected by citrate, isocitrate, cis-aconitate, and trans-aconitate. Competitive Inhibition by CiS-aconitate The competitive inhibition of the P-glycolate phos- phatase reaction by cis-aconitate was not caused by chela— tion of the Mg++ which is required for the reaction (Figure 18). By contrast, the competitive inhibition of phoSphoglucomutase and of intestinal alkaline phOSphatase by citrate could be reversed by the addition of relatively high concentrations of Mg++. It was therefore hypothesized that citrate exerted its effect by complexing the Mg++ required by the latter two enzymes as an activator (79, 275). In the case of P—glycolate phOSphatase, the alterna- tive is that cis-aconitate competes with P-glycolate by binding with the phosphatase itself. Product inhibition is usually competitive inhibition (149). For the conver- sion of any one tricarboxylic acid, the other two tricar- boxylic acids should be competitive inhibitors of aconitase. Tomizawa (241) demonstrated that DL—isocitrate-Z-luc com- petitively inhibited the formation of unlabeled citrate from unlabeled cis-aconitate. Thus,aconitase and P-glyco- late phOSphatase are similar in that reactions of both enzymes can be competitively inhibited by cis-aconitate. The available evidence suggests that in both cases the site of inhibition is on the enzyme. 345 Substrates and Reaction Mechanisms The substrates of P-glycolate phOSphatase and acon- itase seem to be structurally analogous, and the phOSpha- tase reaction mechanism seems to be analogous to the mech- anism for the conversion of cis-aconitate to isocitrate (Figure 47). These Similarities are consistent with the observations on stability and competitive inhibition. They are also consistent with another property that the two enzymes have in common, i.e. each one is highly Specific for its substrate(s) (literature review). Effect of 02 Oxygen had a qualitatively similar effect on the activity and stability toward dilution at 30° of P-glyco- late phOSphatase and aconitase in fresh extracts from tobacco leaves (p. 317). An important practical consider- ation concerning the stability toward dilution at 30° of both enzymes is that,except when it is desired to study this stability itself, the reactions should be initiated by the addition of cold enzyme to the otherwise complete reaction mixtures (including substrate). Stability Toward Acetone Fractionation Both enzymes were very stable to fractionation by acetone. High phoSphatase recovery was obtained,and similarly high recovery of aconitase was obtained if 346 allowance was made for the rapid inactivation of the latter enzyme in the fresh extracts, by using the activity which prevailed at the time of the precipitation by ace- tone. Furthermore, when this same allowance was made, the phOSphatase and aconitase were both purified many fold and about the same amount by acetone fractionation (p. 320). Stability toward precipitation from plant extracts by ace— tone is somewhat unique, Since most of the protein present seems to be irreversibly denatured by the treatment (p. 166). The phOSphatase from tobacco leaves was unstable toward further acetone fractionation after an ammonium sulfate fractionation (p. 178). It may also be that acon- itase from pig heart is stable to organic solvents before ammonium sulfate treatment, but not after (literature review, p. 62). Effect of pH Although the pH Optimum of the phOSphatase from Swiss chard has not been determined, the optimum of the enzyme from the leaves of the plants tested (wheat, Spinach, or tobacco), with Mgsou present at 10'3M, was found to be 6.3 (265). The pH Optimum of partially puri- fied aconitase from Swiss chard, with MgSOu present at 10‘2 M and with cis-aconitate as substrate, was found to be between 6.0 and 6.5 (p. 278). Thus, under the assay conditions which were used, aconitase from the leaves of 34? Swiss chard is similar to the phosphatase from the leaves of the other higher plants tested, with reSpect to the pH optimum. Marked loss of pig heart aconitase activity occured when the pH of the clarified crude extract was adjusted below pH 5.0 (162). The same observation was made for P-glycolate phosphatase in an extract from tobacco leaves (Figure 9). Effect of Freezing and Thawing During the early stages of purification, the phos- phatase from tobacco leaves was found to be unstable to freezing and thawing,while the enzyme after the fifth purification step was found to be stable (p, 175), Like the phOSphatase from tobacco leaves, aconitase from pig heart was unstable toward freezing at the earliest stage of purification, and the purified enzyme was unaffected by repeated freezing and thawing (162). How- ever, unlike the phOSphatase from tobacco leaves, highly purified aconitase from mustard leaves was totally inac- tivated by freezing (185). Association of the phOSphatase and aconitase from tobacco leaves during the early stages, but not the last stage of purification,might eXplain the behavior of the phOSphatase toward freezing, but this possibility has not been tested. 348 Effect of Sulfhydryl Reagents P-glycolate phOSphatase from tobacco was inhibited 90% by 10'3M p-chloromercuribenzoate in the reaction mix- ture,but was not inhibited at all by 3 x io-3M iodoacetate in the reaction mixture (202). peMercuribenzoate at 0.53 x 10‘6M inhibited the animal aconitase 50% while iodoace- tate of 10'2M or arsenite at 10"2 gave no inhibition. The animal enzyme had been activated by Fe++ and ascorbate, and the inhibitors were at the concentrations indicated during a 5 minute preincubation at 30° (63). Arsenite at 10‘2 M during a one hour preincubation at 0° resulted in a Slight stimulation of P-glycolate phOSphatase in fresh extracts from tobacco leaves (Table 16). Buchanan and Anfinsen (39) reported that cysteine alone, in the initial stages of the purification, Showed a stabilizing effect on pig heart aconitase,but as the purification of the enzyme continued, cysteine alone strongly inhibited the enzyme. P-glycolate phOSphatase from tobacco was inhibited 90% by 8 x 10‘3M cysteine in the reaction mixture,and was inhib- ited 84% by glutathione at 8 x 10'3M in the reaction mix- ture (202). Cysteine partially stabilized the phOSphatase from wheat, though not as effectively as did the tricar- boxylic acids (Tolbert and Yu, unpublished data). Thus, the aconitase from animal tissues and P-glycolate phospha- tase from higher plants are affected qualitatively in essentially the same way by the four sulfhydryl reagents 3H9 for which comparisons are available. Little such work has been done with aconitase from plants. However, Palmer (185) did find that preincubation of the aconitase from mustard with iodoacetate (final concentration 10'3M) for 10 minutes had no effect on the activity, but he also found that cysteine had no stabilizing effect on the purified aconitase from mustard. In the present work, it was found that cysteine (without iron) in the reaction mixture inhibited the aconitase from tobacco leaves (p. 323 ). Molecular Weight The molecular weight of neither aconitase nor P- glycolate phosphatase has been determined. However, the EEO value for purified aconitase from mustard leaves was found to be #.7 by Palmer (185). Using the $20,w value of 7.7 and M.w. of 150,000 for aldolase (58% and the s = (constant) Mz/3 relationship which is based on the assump- tion of Spherical shape, a rough approximation of the molecular weight of the mustard aconitase is 70,000. The Bf of 1.6 for the phoSphatase from tobacco leaves during Sephadex G-100 gel filtration chromatography (p. 173) allows a very rough approximation of its molecular weight of about 20,000 to 30,000. Although aconitaSe from plants and animals seems to be a single protein Species with a single active site 350 for both of the aconitase half reactions, there is some evidence from Saccharomyces that aconitase may comprise two polypeptide chains (literature review). Also, the phOSphatase reaction seems to be analogous only to the cis-aconitate to isocitrate half reaction (pp. 337-3h2). These observations lead to the hypothesis that the phos- phatase might have a considerably smaller molecular j 1 weight than aconitase. Effect of Cations and Anions P-glycolate phOSphatase from the leaves of a variety of plants seems to be totally dependent on di- valent cations in the reaction mixture for activity (202, 265), while aconitase from tobacco or Swiss chard leaves may be totally dependent on anions in the reaction mixa ture for activity (pp. 281-289). Parallel Fractionation of the Enzymes Concerning the parallel fractionation of aconitase and P-glycolate phOSphatase (Figures 34 through 38), at least three hypotheses are worth consideration: A: The two enzymes are not bound together, either directly or indirectly. i.e. they fractionated together by coincidence. The similarities in the properties of the two enzymes are consistent with this hypothesis. If this 5 hypothesis were true, the parallel fractionation of the two I 351 enzymes during acetone fractionation and during DEAE- cellulose chromatography would have to be considered as two additional similarities between the two enzymes. How- ever, the separation of the two enZymes during DEAE- cellulose chromatography under certain conditions (Figure 39) would then have to be considered an anomoly. B: The two enzymes are bound together indirectly, E by being bound to a third structure. Since both enzymes have an affinity for the tricarboxylic acids, one or more 1 Furthermore, of these could serve as the third structure. the possibility of an unidentified third structure has not been ruled out. C: The two enzymes are bound together directly. The similarities in the properties of the two enzymes are also consistent with this hypothesis.? 1Isocitrate (Figures 16, 17 and 39) and the tri- carboxylic acids in general (Figure 10) consistently showed either a double peak or a pronounced shoulder, while both enzymes consistently showed single peaks during fractionation (Figures 10, 11, 3h, 35, 36, 37, 38 and 39). These data suggest that the tricarboxylic acids were not tightly bound to either enzyme,but may have been in equi- librium with one or both of them. Nevertheless, with a» enough matching binding sites on both enzymes, the tri- carboxylic acids could have kept the enzymes bound together while the acid at any one pair of sites was in equilibrium with the medium. 2Studies with E. coli alkaline phOSphatase (212), fumarase (121), and muscle or heart lactate dehydrogenase (130) have demonstrated that they are composed of identi- cal or nearly identical subunits. From interallelic com- plementation studies with.Neur03pora (46), it is thought that a great number of enZymes are likewise composed of identical or nearly identical subunits. (See also comments by Dr. Lacks, Brookhaven Symp. Biol. l1, p. 17fi). Rever- sible dissociation into subunits of rabbit muscle aldolase 352 On the basis of present evidence, it is not possible to eliminate any of these three hypotheses. Furthermore, should hypothesis B or C be true, it would still be neces- sary to determine whether the binding is artifactual or of physiological significance. Some of the apparent similarities between aconitase and P-glycolate phOSphatase, eSpecially in the early stages of purification, could have been because the enzymes were bound together. Even for the enzymes in the later stages of purification, this possibility cannot be excluded. Thus, the stabilization of the phoSphatase by the tricarboxylic acids (Figures 10 and 15) could have been because of sta- bilization of aconitase which may have been bound to the phOSphatase.1 Also, competitive inhibition does not (58), E. coli alkaline phOSphatase (212), and fumarase (121) Has been demonstrated.and definitive evidence has been presented in the latter two cases that the subunits are held together by non covalent interactions (121, 212). 'The very rapid ig_vitro association of E. coli alkaline phoSphatase subunits, even in crude cell extracts, points to the very high degree of recognition between the monomers (212). It seems likely that for multichain enzymes, the amino acid sequence in the polypeptide chains uniquely determines the chain conformation as well as the Spatial relationships of the subunits in the native enzyme mole- cules (58, 211, 212). Thus,there exists ample precedence for peptide chains with identical or nearly identical structure having a highly Specific affinity. These iden- tical or nearly identical peptide chains should have identical or nearly identical prOperties. 1The phOSphatase used for the experiment of Figure 10 had a Specific activity of 161, or half of 333, which is the highest Specific activity attained for the phospha- tase. The additional protein could have included aconi- tase. However, in the experiment of Figure 15, the Rf of the phosphatase on the G—100 column which was used was 353 require that the substrate and the inhibitor bind at the same site. Conceivably. if aconitase were bound to the phOSphatase, the binding of cis-aconitate at the aconitase substrate site could competitively inhibit the phOSphatase reaction (Figure 18).1 However, I know of no precedent in which the binding site of the competitive inhibitor is on an enzyme different from the one with the substrate binding site. Furthermore, the structural Similarity between P-glycolate and cis-aconitate suggests that the site of inhibition by cis-aconitate is the P-glycolate binding site. Also, before the competitive inhibition eXperiment of Figure 18, the phOSphatase preparation was tested for aconitase activity and was found to be essen- tially inactive, and aconitase reactivation attempts yielded negative results. Still, the enzyme might have been preSent in a conformation in which it could bind cis-aconitate but not catalyze the reaction. Parallel Fractionation of the PhOSphatase and the Tricarboxylic Acids Because citrate, cis-aconitate, and isocitrate are the substrates of aconitase, the fractionation of large 1.6. This Rf value on G-100 suggests that aconitase and the phOSphatase were separated before or during the experie ment. Still.the phosphatase was stabilized by the tricar- boxylic acids (Figure 15). 1The enzyme used for the experiment of Figure 18 had a Specific activity of 161, or half of 333, which is the highest Specific activity attained for the phOSphatase. The additional protein could have included aconitase. 35h quantities of these acids with the phosphatase under cer- tain conditions of harvest and purification (PP. 137-147, 181-191, and.195-201) would seem to be closely related to the parallel fractionation of aconitase and the.phOSphatase under similar conditions of harvest and purification (Figures 3# through 38). The concept that the fractiona- tion of the tricarbOXylic acids with the phOSphatase is related to the fractionation of aconitase with the phos- phatase is strengthened by.the evidence that isocitrate no longer fractionated with the phOSphatase when aconitase was separated from the phOSphatase (Figure 39). There is some evidence that aconitase possesses multiple binding Sites for the tricarboxylic acids. For most enzymes, the effect of anions at ordinary concentra- tions is negligible (71). However, enzymes such as fumar- ase (158), soluble malate dehydrogenase from pea seeds (253), and glyoxylate reductase from tobacco leaves (266), which are activated by anions, have each been found to be activated by many different anions. Thus, a role for tri- carboxylic acid anions as activators of the plant aconitase would be consistent with the finding that chloride or sul- fate activates aconitase from tobacco or Swiss chard leaves (Figures 31, 32, and 33). Isocitrate has in fact been found to activate the aconitase from mustard leaves,and an argument is presented that the activating effect by iso- citrate is by combination with groups other than at the 355 active center (literature review). The activation of the tobacco aconitase by so“? supports the concept that the enzyme may possess groups, other than at the active center, capable of binding the tricarboxylic acids. Furthermore, Benson and Cleland (114) reported that citrate, which was added during purification for stability purposes, remained bound to beef liver aconitase even after passage through a Sephadex G-25 column. The amount of citrate that was bound to the enzyme was sufficient to require removal before kinetic experiments could be performed. Since the phoSphatase is stabilized by the tricar- boxylic acids and is competitively inhibited by cis-aconi- tate, and since there is some evidence that the phOSphatase might be activated by the tricarboxylic acids under certain conditions (footnote, p. 202), the phosphatase itself must also possess binding sites for the acids. Therefore, the fractionation of endogenous tricarboxylic acids with the phOSphatase may also be partly due to properties of the phOSphatase itself. The two enzymes fractionating in parallel would offer more binding sites for the tricarboxylic acids than would the phOSphatase alone. The ratio of 1 isocitrate per 10 to 15 amino acid residues of the phOSphatase (pp. 195-201) is misleading if some of the isocitrate were bound to aconitase.1 1Although the Specific activity of the phOSphatase depicted in Figures 16 and 17 was not measured, the Specific 356 From the available data, it is not possible to know the nature of the relationship between aconitase, the tri- carboxylic acids, and the phOSphatase when they fraction- ate together. The finding that isocitrate was with aconi- tase and was not with the phOSphatase when the two enzymes . were separated (Figure 39) suggests as one possibility _fi that the tricarboxylic acids might have fractionated with (I the phosphatase because they were bound to aconitase which 1 fractionated with the phOSphatase. However, although isocitrate was with aconitase, the shapes of the curves do not strongly suggest binding between aconitase and iso- citrate (Figure 39). (The curves of Figures 16 and 17 are more suggestive of binding.) Therefore, as a second possi- bility, it might be that under some conditions isocitrate was bound to both enzymes, and that under other conditions it was bound to neither. Other possibilities also exist. Effect of Light Although the effect of light before harvest on the time dependent stabilization of P-glycolate phOSphatase in tobacco extracts (Figure 20) could be related to the pos- sible role of light as a factor in the determination of activity in a nearly identical preparation. (both prepara- tions had come from the same preparation of 3 times acetone purified phosphatase and were further fractionated on nearly identical DEAE-cellulose columns) was 84.7 (Table 3), or % of the highest Specific activity of 333 which was obtained. Thus, the phosphatase fractions of Figures 16 and 17 con- ’ tained enough other protein to include aconitase. 357 whether aconitase and P-glycolate phOSphatase fractionate together (pp. 291-308), no direct evidence for such a relationship has been sought or obtained. However, these effects of light are of further interest in view of some evidence that aconitase may be inhibited in some plants in the light (pp. 334-337) and in view of the requirement for light for the synthesis of glycolate (literature review). The Possibility of an Evolutionary Relationship Butter (207) has pointed out that the study of enzymes with related mechanisms may suggest meaningful evolutionary relationships. Thus,the similarity between the mechanism for the hydration 0f cis-aconitate to iso- citrate and the proposed mechanism for the enzymatic hydrolysis of P-glycolate (Figure 47) leads to the Specu- lation that an evolutionary relationship may exist between the two enzymes. Furthermore, the phOSphatase and aconi- tase should contain homologous regions if they evolved from a Single prototype gene (207). Many of the observa- tions reported in this thesis concerning these two enzymes would be more or less eXpected with such an evolutionary relationship. Also, a possible evolutionary relationship between aconitase and the phOSphatase might be of signifi- cance in view of the requirement for Mn++ for the develop— ment of aconitase activity in AsEergillus gigs; and in Chlorella vulgaris, and in view of the Mn++ requirement 358 for glycolate synthesis (literature review). It would be of interest to see if P-glycolate phOSphatase has a similar Mn'"+ requirement for the development of its activity. In Vivo Correlation Between the hOSphatase and Aconitase The localization of P-glycolate phosphatase in or on the chloroplasts (literature reviewx together with the classical concept that aconitase is a mitochondrial enzyme, would seem to rule out the possibility of a physiologically significant binding between P-glycolate phosphatase and aconitase. However, the localization of aconitase in animal as well as in plant cells actually is controversial (literature review). Thus,it would be premature to consider an in zizg association of P-glycolate phOSphatase and acon- itase as being ruled out. Further Observations on the Stability of P-glygolate PhOSphatase As discussed previously, both aconitase and isocit- rate (and presumably the other tricarboxylic acids) were separated from the phOSphatase under the conditions described for Figure 39. Yet, when the stability of the phOSphatase in the 6 most active fractions (10 through 15) was tested, it was found that only 4 to 18% of the activity was lost by storage at 0-4° for 6 days. The average loss of activity was 8%. Thus, at this stage of purification, ( I 359 the phosphatase was stable to storage at 0-4° even without the tricarboxylic acids. In contrast, when the tricar- boxylic acids were removed from the phosphatase, which had been purified by 3 successive acetone fractionations, by passage through a Sephadex G-100 gel filtration column (p. 193), 66% of the activity was lost by storage at 0-40 for 4 days. These findings suggest that the loss of stability during the Sephadex G-100 gel filtration chroma- tography, after 3 acetone purification steps, may not have been only because of the separation of the tricarboxylic acids from the phOSphatase. Many of the observations on the stability of the phOSphatase (pp. 175-178) might be eXplained by hypothe- sizing an unknown factor(s) which interacts with the enzyme independently from the tricarboxylic acids and from 02. It could have been an unknown factor which made the enzyme stable even without the tricarbOXylic acids, stable to acetone fractionation, and unstable to freezing. The fac- tor was apparently not removed by acetone or DEAE frac- tionation, or by dialysis, but it apparently was removed by (NH#)2804 fractionation,or when the history of the enzyme was that of the Bio-Gel P-60 preparation of Table 3. Removal of the unknown factor may have been the reason why the (NH4)230u fractionated enzyme was stable to freezing, unstable to acetone fractionation, and unstable to dialysis, and why the Bio-Gel P-60 enzyme of Table 3 was stable to 360 freezing with or without citrate, but was unstable without added tricarboxylic acids. Likewise, the presence of the unknown factor may have been the reason why the acetone purified enzyme was stable to repeated acetone fractiona— tion, unstable to freezing, stable to and after dialysis, and stable without the tricarboxylic acids even after further purification by DEAE-cellulose chromatography. Furthermore, gradual loss of the unknown factor during ;L purification could have been the reason for the decreas- ing stability toward dilution at 30° of the phosphatase in the later stages of the purification (p. 261). It is worth noting that every preparation that behaved as though the hypothetical factor were present was colored (orange-brown),and every one that did not was not colored (or, in the case of the (NHu)2804 purified enzyme, was relatively pale in color). As pointed out previously (p. 174), the colored compound(s) absorbed strongly at 254 mu. Thus,the coincidence of a 254 mu peak with the phosphatase peak (Figure 16) could possibly. mean that a colored compound was bound to the phOSphatase after 3 acetone fractionations and DEAE-cellulose chroma— tography. SUMMARY A.purification procedure for P-glycolate phoSphatase from tobacco leaves, which included three successive ace- ‘tone fractionations, DEAEacellulose chromatography, and *! gel filtration chromatography, was develOped. The enzyme was purified 1000-fold to a Specific activity of 333 umoles of substrate hydrolyzed per minute per mg of protein. During the last purification step, the tobacco phOSphatase became unstable, as determined by heating the enzyme to 45° for 1 hour, but could be restabilized with fractions which emerged after the phOSphatase from the gel filtration column. The endogenous stabilizing factors in these fractions were identified as citrate, isocitrate, and cis-aconitate. Commercial citrate, isocitrate, and cis-aconitate, as well as trans-aconitate, also stabilized the enzyme, but mono and dicarboxylic acids which were tested did.not. Ole-aconitate was found to be an inhibitor, competi- tive with reSpect to P-glycolate, of the phosphatase. The apparent K for cis—aconitate was 5.0 x 10‘3M when the I Mg+2 was 2 x 10'3M. Increasing the Mg+2 to 2 x 10'2M. ‘ZM. did not significantly affect with cis-aconitate at 10 the apparent KI. The data suggest that cis-aconitate did not cause inhibition by complexing the mg+2 required as an activator of the phosphatase. When the Mg+2 was 2 x 10'3M, 361 362 the apparent Km for P-glycolate was 6.7 x 10'5M. Increas- ing the Mg*2 to 2 x 10‘2M did not significantly affect the apparent Km. The apparent EA for mg+2 was 3.5 x 10'”M. When isocitrate and the phOSphatase were assayed in fractions from the DEAE-cellulose chromatographic step, one of the two isocitrate peaks approximately coincided with the phOSphatase peak. At this DEAE stage of purifi- cation, approximately 1 mole of isocitrate per 10 to 15 moles of amino acid of the phOSphatase fractionated with the enzyme. Furthermore, comparison of the sizes of the spots on paper ohromatograms suggests that more citrate than isocitrate fractionated with the phoSphatase. In most of the eXperiments, aconitase and P-glyco- late phOSphatase from tobacco leaves fractionated in parallel during acetone fractionation and during subse- quent DEAE-cellulose chromatography. However, when field grown tobacco leaves were kept in darkness for 3 to 4 hours before harvest and homogenization, the phOSphatase and aconitase, after a preliminary purification by acetone fractionation, were separated during DEAE-cellulose chromatography. In the latter case, isocitrate no longer fractionated with the phOSphatase. Although in all the eXperiments in which the phosphatase and aconitase frac- tionated together and in which isocitrate fractionated with the phOSphatase, the tobacco leaves had been exPosed to at least some light in the period before harvest and/or homog- 363 enization, eXperiments controlled so as to determine the effect of light on the parallel fractionation of the two enzymes, or of the phOSphatase and the tricarboxylic acids, were not performed. Nevertheless, the evidence suggests that the fractionation of the endogenous tricarboxylic acids with the phOSphatase was closely related to the fractionation of aconitase with the phOSphatase. . Although it is not known whether the parallel frac- tionation of the two enzymes was artifactual or of physio- logical significance, it is of interest that the tricar- boxylic acids are also effective stabilizers of aconitase, and that any one tricarboxylic acid would be expected to competitively inhibit the interconversion of the other two. Thus, aconitase and.P-glycolate phOSphatase seem to possess at least two similarities, i.e. stabilization by the tricarboxylic acids and competitive inhibition by cis- aconitate. Other apparent similarities between the two enzymes are discussed. Partially purified aconitase from tobacco or Swiss chard leaves could be deactivated by Sephadex G-10 or G-25 gel filtration chromatography and could be reactivated by fractions which emerged after aconitase from the column, or by sulfates or chlorides. 'The activation of aconitase by sulfates or chlorides is consistent with the concept that the enzyme may possess groups, other than at the active center, capable of binding citrate, isocitrate, or 364 cis-aconitate. In fresh extracts from tobacco leaves, P-glycolate phoSphatase was unstable toward dilution of 20 to 30-fold at 30° for 1 hour, but under aerobic conditions, the enzyme in the extracts slowly became stable toward this dilution at 30°. A variety of treatments of the extracts including mixing under air or 02 (but not N2 or 002), a 10 minute preincubation with 10’2M OHPMS, or rapid passage through a Sephadex G-25 column, quickly and completely stabilized the phosphatase toward dilution at 30°. The stabilized enzyme could be partially and rapidly recon- verted to the unstable but just as active enzyme by pre- incubation of the oxygenated extracts with glycolate. OHPMS completely inhibited, while anaerobic conditions enhanced, this glycolate dependent conversion. Glycolate also inhibited the stabilization toward dilution at 30° of the phOSphatase by 02. Other aSpects of the stability toward dilution at 30° of the phosphatase are discussed. The data suggest that the phOSphatase can exist in an oxidized or in a reduced state and that in the extracts, glycolate oxidase could be involved in the interconversion between the two states. 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