....._..__...-...,—.._,.m‘ a-AMYLASE FROM GIBBERELLIN-INDUCED BARLEY ALEURONE CELLS: PARTIAL CHARACTERIZATION AND EFFECTS OF ‘5-FLUOROURACIL Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY SHIRLEY I. RODAWAY . 1977 T“ “I a! 31293 007051018 L [b’ R A R Y Michigan State University This is to certify that the thesis entitled a-Amylase from Gibberellin-Induced Barley Aleurone Cells: Partial Characterization and Effects of S-Fluorouracil presented by Shirley J. Rodaway has been accepted towards fulfillment of the requirements for Ph.D. degreein Botany and Plant Pathology LLO~§ 3- (RM Major professor Date February 22, 1977 0-7639 my. 0 4 2:22; . r‘ n) r" 3-11. ‘; ac”;- PART: ABSTRACT a-AMYLASE FROM GIBBERELLIN-INDUCED BARLEY ALEURONE CELLS: PARTIAL CHARACTERIZATION AND EFFECTS OF S-FLUOROURACIL BY Shirley J. Rodaway a-Amylase has been purified from the combined incubation medium and homogenate of de-embryonate grains ("half-seeds") of barley (Hordeum vulgare L. cv. Betzes) which have been treated with l u§_gibberellic acid (6A3) to induce a-amylase synthesis. The purified enzyme contained all the six isozymes of o-amylase found in the original crude extract. Based on sodium dodecyl sulfate- polyacrylamide gel electrophoresis there was only one component by molecular weight, a 41,400 dalton polypeptide. Free calcium, which stabilized the enzyme to heat at pH 8, and free carbohydrate could be removed by chromatographic procedures. Initially an investigation of the precise time course of the synthesis of a-amylase mRNA and a determination of the half-life of that mRNA were intended. Carlson had reported (Nature New Biol. 237: 39, 1972) that imbibition of aleurone tissue in H O for 1 day, 2 followed by incubation in 10“4 §_S-fluorouracil (S-FU) for 2 days and then by GA plus S—FU for one more day led to the synthesis 3 a-amylase which was more thermolabile in the absence of calcium than was d-amylase obtained from aleurone tissue incubated according to the same schedule but without S-FU. Presence of S-FU together unha onth 3 twc pogul tiers sore and less t Carlson conclué ‘55 more heat 1 With SA . Car 3 355A during tr 1mm 90.5315 In fitter; :43... d ., ' efitere :--,.. y at 10-2 ‘2‘ «Extent k“ .‘ 5N. .~'. b “\ . V. ~59 1.3: a“; ‘ ‘d 1% ‘ §Bek‘on S‘- ’ ‘z ‘1“ ‘ :‘z'Pn \‘J’laSe F ,- E‘s“: Shirley J. Rodaway withGA3 on the last day of incubation only,led to the synthesis of two populations of a-amylase molecules, apparently representing the more and less thermolabile populations of the first two treatments. Carlson concluded that the population of a-amylase molecules which was more heat stable must have been synthesized prior to treatment with GAB' Carlson proposed that S-FU was incorporated into a-amylase mRNA during transcription, thus leading to the altered (more thermo- labile) population of a—amylase molecules. In attempting to extend those observations and to justify some of the assumptions which were made, I collected the following data which were consistent with Carlson's conclusions. Metabolites of S-FU entered the pool of RNA precursors in aleurone tissue because S-FU at 10“2 §_inhibited synthesis of rRNA and 4 to SS RNA by as much as 90%. At the same S-FU concentration, a-amylase synthesis was only inhibited 10 to 20%. The a-amylase synthesized after S-FU treatment did not differ from normal a-amylase in isozyme composi- tion and molecular weight. However, some findings were not in agreement with those of Carlson. In my experiments a-amylase could not be purified by the method which he had employed because all of the a-amylase activity was lost during one of the intermediate puri- fication steps. Instead, an alternate, more gentle method was used. a-Amylase prepared by this procedure exhibited thermal denaturation kinetics which were independent of the previous incubation of the aleurone with S-FU. The rate of thermal denaturation was independent of a-amylase concentration, and no degradation of a-amylase occurred as a result of the heat treatment. These results have established that, contrary to the report by Carlson, one cannot use the thermal denaturation 0 measure th ti In Sectic 9r paring sna' Covalently be 901‘! Cryianié Ethniques of talysis of z Shirley J. Rodaway denaturation of a-amylase prepared from S-FU—treated half-seeds to measure the timing of the synthesis of a-amylase mRNA. In Section II of this dissertation, a method is described for preparing small amounts (<1 mg) of protein for analysis of its covalently bound sugar content. This method combined SDS- polyacrylamide gel electrophoresis and either aqueous or non-aqueous techniques of carbohydrate hydrolysis followed by gas-chromatographic analysis of the sugars. Using this method, the a-amylase secreted by whole grains of the Himalaya cultivar of barley was found to con- tain on the average up to 0.5 residues of mannose, glucose, and glucosamine. The amino-acid composition of o-amylase from Himalaya barley grains showed that it contained proportionately more histidine and tyrosine and less glutamate/glutamine than the average protein molecule. A second protein is described which was not synthesized during GA -treatment but which had many properties similar to 3 a-amylase, including partial glycosylation. . h \ A‘s. a-A- a-AMYLASE FROM GIBBERELLIN—INDUCED BARLEY ALEURONE CELLS: PARTIAL CHARACTERIZATION AND EFFECTS OF S-FLUOROURACIL BY Shirley J: Rodaway A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1977 - § - 9 HQ V n h. ESEEI‘ fir“ One 0 ertise i J: L G .‘l x ‘1‘ ‘ .tcn fro; 6 ideas i now to !9:I:;l A‘c e E . ‘ Ofiied: I 1‘ (h a~‘\‘- “0 acid h. ACKNOWLEDGEMENTS One of the major advantages of doing research at the Plant Research Laboratory is that an informal interchange of ideas and expertise is highly encouraged and we are continually learning as much from each other as we are from our own experiments. For this reason, I find that I must thank the whole of the PRL for philosophies and ideas which no doubt have incorporated themselves into my own attitudes and research. Hans Kende is especially acknowledged for setting a fine example of how to approach and solve basic problems in plant development, while encouraging independent thinking by his students. He is also acknowledged for teaching us the fine art of not taking ourselves too seriously, while taking our research very seriously. I thank Andrew Mort for teaching me techniques of sugar and amino acid analysis and Derek Lamport for the use of equipment main- tained within his laboratory. In addition, I thank J. E. Varner and P. Filner for valuable discussions concerning work reported in Section I of this dissertation. Finally, I would like to express my continuing gratitude to L. M. Blakely for encouraging and supporting my early interest in plant physiology which ultimately has led to the completion of this dissertation. ii The rese Administrati: The research reported here was supported by the 0.8. Atomic Energy Commission and the U.S. Energy Research and Development Administration under Contract B(ll-l)-l338. iii 532113“ 1 \‘Rsn ‘ ~ “ 4 .w, SECTION I TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . Control of Gene Expression in Eukaryotes. . . . . Induction of a-Amylase Synthesis by Gibberellic Acid in Barley Aleurone Layers . . . . . . . . Use of S-Fluorouracil to Study the Transcrip- tion of aA-mRNA. . . . . . . . . . . . . . . . MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . Preparation and Incubation of Barley Half-Seeds . Homogenization of Tissue. . . . . . . . . . . . . Purification of Calcium-Free a-Amylase (Method I) Partial Purification of o-Amylase (Method II) . . a-Amylase Assay . . . . . . . . . . . . . . . . . Protein Assays. . . . . . . . . . . . . . . . . . Carbohydrate Assays . . . . . . . . . . . . . . . Calcium Assay . . . . . . . . . . . . . . . . . . Thermal Denaturation. . . . . . . . . . . . . . . Separation of Proteins by Polyacrylamide Gel Electrophoresis. . . . . . . . . . . . . . . . Fairbanks (et a1.) Method. . . . . . . . . Laemmli Method . . . . . . . . . . . . . . Ingle Method . . . . . . . . . . . . . . . Isozyme Separation by Agar Gel Electrophoresis. . 32P-Labeling and Isolation of Ribonucleic Acid. . RESULTS 0 O O O O O O O O O O O O O O O O O O I O O O O 0 Response of Half-Seeds of Betzes Barley to GA3. . Effect of S-FU on o-Amylase Synthesis . . . . . . Effect of S-FU on RNA Synthesis . . . . . . . . . Purification of a-Amylase . . . . . . . . . . . . Removal of B-Amylase . . . . . . . . . . . Electrophoretic Homogeneity on SDS- Polyacrylamide Gels . . . . . . . . . . Removal of Carbohydrate. . . . . . . . . . The Loyter and Schramm Purification Procedure . . . . . . . . . . . . . . . Yield of c-Amylase During Purification . . Isozyme Recovery . . . . . . . . . . . . . iv Page 10 10 ll 14 25 3O 36 36 39 39 4O 4O 42 42 43 44 47 47 52 52 SS 64 65 75 76 79 83 (’1 U H (I) (I) (I) '5 \g‘ ~~“ ZEN 11 P‘~ . C“‘ U“ (r) {W ‘11 r‘ 3 Effects of 5-FU on Molecular Weight and Iso- zyme Content of o-Amylase. . . . . . . . . . . Molecular Weight . . . . . . . . . . . . . Isozyme Composition. . . . . . . . . . . . Recovery of Activity and Specific Activity of a-Amylase During Purification. . . . The Thermal Denaturation Process: General Aspects. . . . . . . . . . . . . . . . . . . . Effect of Calcium. . . . . . . . . . . . . Effect of Protein Concentration. . . . . . Test for Protease Activity . . . . . . . . Effect of S-FU on the Thermal Stability of d-Amylase. . . . . . . . . . . . . . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . Suitability of the a—Amylase Preparation for Studies of Thermal Inactivation Kinetics . . . Use of S—FU to Time the Synthesis of a-Amylase mRNA 0 O O O O O O O O O O O O O O O O O O O 0 SECTION II 0 O O O O O C O O O O O O O O O O O O O O O O O Q C O INTRODUCT ION O O O O O O O I O O O O O O O O O O O O C 0 MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . Incubation of Grains for a-Amylase Purification . Purification of a-Amylase . . . . . . . . . . . . Preparation of l4C-Labeled o-Amylase from Aleurone Layers. . . . . . . . . . . . . . . . Incubation of Grains on Agar. . . . . . . . . . . Polyacrylamide Gel Electrophoresis. . . . . . . . Staining of Gels for Carbohydrate . . . . . . . . Isoelectric Focusing. . . . . . . . . . . . . . . Ammonium-Sulfate Fractionation. . . . . . . . . . Amino Acid Analysis . . . . . . . . . . . . . . . Carbohydrate Analysis . . . . . . . . . . . . . . Glassware and Dialysis Tubing . . . . . . . . . . Source of Chemicals . . . . . . . . . . . . . . . RESULTS 0 C O O O O O O O O O O O O O O O O O O O O O O 0 Synthesis of o-Amylase by Barley Grains . . . . . Partial Purification of a-Amylase . . . . . . . . Purity of the a-Amylase Solution. . . . . . . . . Attempts to Remove the 20,500 Dalton Protein (Band-2) . . . . . . . . . . . . . . . . . . . Amino-Acid Composition of a—Amylase and the Band-2 Protein . . . . . . . . . . . . . . . . Glycosylation of a-Amylase and the Band-2 Protein. . . . . . . . . . . . . . . . . . . . Reactions with Carbohydrate Stains. . . . . . . . Origin of the Band-2 Protein. . . . . . . . . . . Page 86 86 86 87 92 92 95 95 97 103 103 106 110 110 112 112 112 114 114 115 117 117 118 118 120 122 122 124 124 124 129 129 138 140 149 155 VA.~..— LDCVI ‘7'-.." :- b . g),\'hv: "‘ Ufimn\\,_ . Page DISCUSSION . . . . . . . . . . . . . . . . . . . . 156 Amino-Acid Composition. . . . . . . . . . . . . . 156 Glycosylation . . . . . . . . . . . . . . . . . . 157 The Band-2 Protein. . . . . . . . . . . . . . . 162 REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . 165 vi £4) (17‘ LL“) LIST OF TABLES Table Page . 32 . l Incorporation of P into RNA by aleurone layers of Betzes barley . . . . . . . . . . . . . . . . . . . . 58 2 Effect of protein concentration on the thermal denaturation of o-amylase. . . . . . . . . . . . . . . . 96 3 Ammonium-sulfate precipitation of purified o-amylase . . 137 4 Amino acid composition of barley a-amylase . . . . . . . 139 5 Amino acid composition of the band-2 protein . . . . . . 141 6 Comparison of amino acids in barley and wheat o-amylases . . . . . . . . . . . . . . . . . . . . . . . 142 7 Residues of sugar per molecule of a-amylase. . . . . . . 144 8 Residues of sugar per molecule of band—2 protein . . . . 146 9 Comparison of a-amylase to an "average protein" molecule . . . . . . . . . . . . . . . . . . . . . . . . 158 vii ‘1! E . I I t T. C .1 a. S E a. . Q .l C S O u. C 5. e .D. E U L e I .C r 2 Z t c E no 1 2 C; e a": 0 ‘5‘ RN 5-4 i h: F: ‘5 C§ and hlq F‘ .91 ”.4 e oV¢f ~ \ S E L H D. H 5 B S O .3 5 .K... 6 7 «C 9 OJ 1 \ s ~n Q 3‘ O N v. 1.74 Ea e S r n o a QC Ru «1 xx 1 I. ‘4 2‘ :1 E f l O l r \\H ‘1‘ AM P rV. C S 2 Lu \ xi 2 s e S A r. .1 a? as \Q s a a t QC «a QC .1 «1.. In? 4: Q. ‘ V l‘ VI 1‘ Figure 10 11 12 13 14 15 16 LIST OF FIGURES Incubation of barley half—seeds. . . . . . . . . . . . . Dilution effect on o—amylase recovery during puri- fication O O O O O O O O O O O O O O O O O O O O O O O 0 Effect of increasing temperatures on the amylase activity of the crude homogenate . . . . . . . . . . . . DEAE-cellulose chromatography of a-amylase from Betzes barley . . . . . . . . . . . . . . . . . . . . . . . . . Stability of a-amylase at pH 10.0. . . . . . . . . . . . Effect of starch concentration on the development of the starch-iodine complex. . . . . . . . . . . . . . . . Hydrolysis of starch by a-amylase: reaction of the products with iodine . . . . . . . . . . . . . . . . . . Hydrolysis of starch by sweet potato B-amylase . . . . . Synthesis and release of amylase by half-seeds of Betzes barley in response to GA3 . . . . . . . . . . . . Synthesis and release of a-amylase from aleurone layers of Betzes barley in response to GA3: effect of 10‘3 M S-FU o o o o o o o o o a o o o o o o o o o o o o o o o o S-Fluorouracil concentration and a-amylase synthesis . . SDS-Polyacrylamide gel electrophoresis of RNA from barley aleurone layers . . . . . . . . . . . . . . . . . . 32 . . Incorporation of P into RNA spec1es from aleurone tissue of Betzes barley: Effect of S-FU . . . . . . . . Specific activities of the RNA species after treatment of half-seeds with 5-FU. . . . . . . . . . . . . . . . . SDS-Polyacrylamide gel electrophoresis of purified a-amylase. . . . . . . . . . . . . . . . . . . . . . . . Mobilities of protein standards in polyacrylamide gels prepared as in Figure 15 . . . . . . . . . . . . . . . . viii Page 13 16 19 24 29 33 35 38 49 51 54 57 6O 62 67 7O 163:1 Etfec inert E E;gure 17 n: F\~ hai; xi it O a to t ”I 5.‘ Fur F igure Page 17 Electrophoresis of a-amylase using the method of Laemmli (1970) . . . . . . . . . . . . . . . . . . . . . 72 18 Polyacrylamide gel electrophoresis of o-amylase without SDS. . . . . . . . . . . . . . . . . . . . . . . . . . . 74 19 Flow diagram for the purification of a-amylase . . . . . 81 20 Separation of a-amylase on agar slab gels. . . . . . . . 85 21 Effect of S-FU treatment on the recovery of a-amylase activity during purification . . . . . . . . . . 89 22 Specific activities of o-amylase at each stage of purification . . . . . . . . . . . . . . . . . . . . . . 91 23 Effect of calcium on the rate of thermal denaturation of purified a-amylase. . . . . . . . . . . . . . . . . . 94 24 Thermal denaturation of o—amylase from 5—FU-treated half-seeds . . . . . . . . . . . . . . . . . . . . . . . 99 25 Thermal denaturation of a-amylase: continuous incuba- tion in 10"4 M 5-FU. . . . . . . . . . . . . . . . . . . 102 26 Synthesis and release of o-amylase by germinating barley grains. . . . . . . . . . . . . . . . . . . . . . 126 27 Solubilization of o-amylase after glycogen precipi- tation O O O O O O I O O O O O O O O O O O 0 O I O O O O 128 28 Precipitation of the solubilized glycogen-amylase complex by ethanol . . . . . . . . . . . . . . . . . . . 131 29 Purity of the a-amylase after precipitation by glycogen. 133 30 Isoelectric focusing of a-amylase. . . . . . . . . . . . 136 31 Periodate-Schiff (PAS) staining of a-amylase in poly- acrylamide gels. . . . . . . . . . . . . . . . . . . . . 151 32 Periodate-dansyl hydrazine staining of a-amylase in polyacrylamide gels. . . . . . . . . . . . . . . . . . . 154 ix ”A n-rENA ACCd in. r.“ sf 3,. vs C .3- s . A L . . . . 5. ‘1 ‘P. nine in \\u ~a \Q" Acca AMPS aA-mRNA DEAE-cellulose EDTA EGTA 5-FO 5-FU PAS pI r-preRNA SDS TCA TEMED TFA Tris Trisca ABBREVIATIONS a buffer of 1 mM_acetate, 100 mM_CaC1 ammonium persulfate o-amylase messenger RNA diethylaminoethyl—cellulose ethylenediaminetetraacetic acid 2' pH 4.8 ethyleneglycol-bis-(B-aminoethyl ether)N,N'- tetraacetic acid 5-f1uoroorotic acid S-fluorouracil gibberellic acid milliamperes periodic acid-Schiff reaction isoelectric point precursor to rRNA sodium dodecylsulfate trichloroacetic acid N,N,N'N'-tetramethylenediamine trifluoroacetic acid tris(hydroxymethyl)aminomethane a buffer of 5 mM Tris-HCl, 5 mM.CaCl 2 , pH 8.0 SECTION I INTRODUCTION Control of Gene Expression in Eukaryotes In eukaryotes the events which occur between transcription and appearance of a functional protein are separated both in space and time. This is unlike the situation in prokaryotes where the nascent RNA transcript may be engaged in translation even before its transcription is completed. In prokaryotes, gene expression has been found to be regulated at the transcriptional or post—translational levels. In eukaryotes, however, the supply of a specific protein may be controlled either at the level of transcription or at several possible post-transcriptional steps. Because development is a dynamic process, it is easy to see that by varying the rates at which each reaction occurs, or by varying the time spent between reactions, very fine controls can be exerted over the amounts and kinds of proteins present in the cell at any one time. For this reason, the striking effects of hormones in those systems where the amounts and species of proteins change must ultimately result from hormonally induced changes in the rates of such key reactions which lead to the synthesis or activation of protein molecules. Four levels at which gene expression may be controlled are generally distinguished: the transcriptional, post-transcriptional, translational, and post—translational levels of control. A transcrip— tional mechanism of control implies that a functional protein is synthesized as soon as the gene is transcribed. Cases of 1 past-trans: molecules 1 nucleus , tr. removal 0 f post-transcriptional control include processing of the pre-mRNA molecules into potentially active mRNA, transport of mRNA out of the nucleus, unmasking of the mRNA from an inactive to an active form by removal of protein, binding of the mRNA to the appropriate class of ribosomes, and regulating the degradation rate for that particular mRNA. At the transcriptional level, the rates of initiation, elonga- tion, and release of the polypeptide and/or mRNA may be limiting, and even after the encoded amino acids are joined together, that polypeptide may need to be covalently modified or activated by a cofactor as post-translational control mechanisms are effected. It is necessary when studying hormone-induced enzyme synthesis to suspect that any one of these events may be the rate—limiting reaction which is affected by the hormone. Induction of o-Amylase Synthesis by Gibberellic Acid in Barley Aleurone Layers The control of gene expression in eukaryotes has been most extensively studied in metazoan systems (Maclean, 1976) and rela- tively little in higher plants. I have been concerned with the mechanism by which the plant hormone, gibberellic acid (GA3), induces the synthesis of d-amylase in aleurone cells of mature barley grains. The aleurone cells constitute a homogeneous layer of non-dividing, triploid cells which are committed to the synthesis and secretion of hydrolytic enzymes and to the hydrolysis and release of most of their cell contents to provide substrates for the growth of the germinating embryo (reviewed by Varner and Ho, 1976). The induction of enzyme synthesis and secretion and the release of inorganic ions and carbo- hydrate is controlled by GA However, the rates of general protein 3. synthesis, of respiration (Varner et a1., 1965), and of total RNA rthesls Y A: lated I Q aqu- 1 9.. Y. e c-k L 2%0 ,1 ‘1 1‘. u. 3 to 4 u- Ru A a. :‘huul’ la , - -83 :31 It vvh \- A“ i. he. bu;- V AL -Cs‘-AA an e rate 0: ‘ Q sv;10w' C § 'e is i no U‘ a-‘h 4 . CC; 1 «Ecu‘ Etta] .3 CO 3 synthesis (Jacobsen and Zwar, 1974a) are unaffected by the hormone. a-Amylase is the most prominent among the hydrolases whose synthesis is regulated by 6A3; at its maximum rate of synthesis o-amylase represents 40% of the total protein being synthesized by the cell (Varner and Ho, 1976). Before GA3 is added to isolated aleurone layers, as few as l or 2 units of a—amylase may be present within the tissue, while after an 8-hour lag period, the GA3-treated layers may synthesize and secrete up to 40 units per layer over a period of 16 hr. An obvious major question with regard to the hormonally- controlled synthesis of o-amylase concerns the level of control at which GA3 exerts its effect. More specifically, does GA3 induce the transcription of the o-amylase mRNA (aA-mRNA)? The possibility that GA3 might exert its control over o-amylase synthesis by affecting the rate of transcription of aA-mRNA must be considered for the following reasons. First, the 8—hour lag period before the appearance of active a-amylase is of sufficient duration to allow time for transcription, processing and translation to occur. Second, it has now been proven that the complete polypeptide portion of the a-amylase molecule is synthesized at the time when the increase in enzyme activity is first detected, thus eliminating the possibility that GA3 controls a-amylase synthesis at the post-translational level. This conclusion can be drawn from work of Ho (1976), who showed that the buoyant density of a-amylase increased when l3C-amino acids were fed to aleurone layers after the lag phase, when the activity of a-amylase was increasing. Conversely, the addition of 13C-amino acids during the period of the lag phase alone had no effect on the buoyant density of the o-amylase. Earlier, Varner and Chandra (1964) had shown that nearly all of the tryptic peptides prepared from a-anylase i tare usly I a-anylase ‘ density of beer. incub E:asel the 1 AC ‘k “ B 0. 5 , s A . 'a: 1r.h“\ i. v «.1 “he in V. ‘ arEear ;‘ H bi (n 4 o-amylase were labeled when radioactive amino acids were added simul- taneously with GA3, and Filner and Varner (1967) had shown that a-amylase was synthesized after 6A3 was added, since the buoyant density of all of the o-amylase was increased when the layers had been incubated in medium containing GA3 and H2180. In this latter case, the endogenous amino acids became labeled with 18O as they were released from storage proteins by proteolysis, and the 18O-amino acids were incorporated into the nascent a-amylase molecules. Recent reports indicate that GA3 affects o-amylase synthesis at the transcriptional level. Higgins et a1. (1976) showed that there was an increase in the capacity of total poly(A)-RNA from aleurone layers to direct in vitro synthesis of a-amylase following treatment of the tissue with GA3 for increasing periods of time. Poly(A)-RNA was incubated with 35S-methionine in the wheat germ protein— synthesizing system, and labeled a-amylase was identified by immuno— precipitation. The amount of radioactivity which could be precipitated in the in vitro system increased with precisely the same kinetics as the appearance of a-amylase activity in Vivo. GA3-treatment of aleurone layers also increased the rate of polyadenylation of RNA, beginning half-way through the lag period (at 4 to 5 hr) and reaching a maximum (a 60% increase) at the end of the lag period (Ho and Varner, 1974; 1975). Within the first 16 hr of GA3 treatment, the total amount of new poly(A)-RNA species increased 70% over that of the control tissue (Jacobsen and Zwar, 1974a,b). Furthermore, a number of early observations indicated that some critical species of RNA molecule were synthesized during the lag period in response to GA although some of these conclusions have 3! been subsequently challenged (Jacobsen and Zwar, 1974a,b). These rem P ‘ V‘FWI -A‘ l . v- / A\A‘~ n R‘ , H‘ “(1. in ~ L4 ~ow‘ v V ‘ L ‘h e do R' ‘ {a v -4 erth overa + a \ y U cl: rigs a 6 ext: rm 3 thI‘ea ‘, LA)~§:' «- \ m! V ‘ '5 g e'# (floservations were based on the use of various inhibitors of RNA synthesis (Varner and Chandra, 1964; Varner et a1., 1965; Chandra and Varner, 1965; Varner and Johri, 1967), the analysis of the overall base composition of RNA molecules labeled before and after the hormone was added (Varner et a1., 1965), and the increase in the specific activity of the RNA labeled in the presence of the hormone (Chandra and Varner, 1965). Certain inhibitors of RNA synthesis, notably cordycepin (Ho and Varner, 1974) and Actinomycin D (Varner and Chandra, 1964; Goodwin and Carr, 1972), only exerted their inhibitory effects during the lag period before a-amylase synthesis had begun, again suggesting that a—amylase synthesis is regulated at the level of transcription or processing of aA-mRNA. From all of these data it is tempting to conclude that GA3 exerts a direct effect on the synthesis of the active aA-mRNA molecules. However, no direct proof was presented to show that GA3 increased the rate of aA-mRNA synthesis. Even though the rate (Ho and Varner, 1974) and amount (Jacobsen and Zwar, 1974) of total poly(A)-RNA synthesis increased in response to GA this did not 3, imply that the specific mRNA for o-amylase was polyadenylated or transcribed during that time. Even the demonstration that some of the poly(A)-RNA contained the a-amylase message, and that the in vitro activity of the a-amylase message appeared to increase after 6A3 treatment, did not constitute proof that either the transcription or the processing of aA-mRNA was dependent upon the presence of 6A3 (Higgins et a1., 1976). The experiments performed with inhibitors of RNA synthesis may provide "food for thought", but it should be pointed out that cordycepin inhibits total RNA synthesis as well as polyadenylation (Ho and Varner, 1974) and Actinomycin D interferes Mk 9 326 t I .J‘. . ‘ H E I y k IO f A new “WV-J . . s "\ .~‘ \— ‘5 V :18 nu 1:28 ecuiv ‘ ME (2:956 . ~' ¢. , 9'- 4 6.821139 ‘I H '1“. w v R I o vu‘v‘ ‘Jfl s 'E'n : 6 ‘with.the morphology of the endoplasmic reticulum (Vigil and Ruddat, 1972). In no case has it been shown that the primary effect of any RNA synthesis inhibitor involved the transcription or processing of (iAemRNA and not some other mRNA species required for the previously synthesized a—amylase message to be expressed. The following problem still remains to be solved: How can one determine which of the numerous events involving aA-mRNA synthesis and utilization may be affected by GA ? One approach which would 3 unequivocally show whether GA3 controls the de-repression of the a-amylase gene would be that used by Axel et al. (1973). In this classic experiment, DNA complementary to hemoglobin mRNA was synthe- sized using reverse transcriptase, and this DNA was used to test for the presence of a hemoglobin mRNA among the RNA molecules transcribed in vitro from isolated reticulocyte chromatin. Use of S-Fluorouracil to Studygthe Transcription of aA-mRNA A new technique to determine the timing of aA-mRNA synthesis was used by Carlson (1972) in experiments with barley aleurone layers. Barley aleurone tissue was exposed to the base analog S-fluorouracil (S-FU) either during GA -treatment or both prior to 3 and during GAB-treatment. In some treatments, no S-FU was given, while in others S-FU was applied as a "pulse" which was chased by an excess of exogenous uracil. Carlson hypothesized that S-FU would be incorporated into aA-mRNA if the transcript was being synthesized at the time when S-FU was in the medium. He also postulated that the presence of S-FU in the dA-mRNA would manifest itself through an altered amino acid sequence of the a-amylase molecules which were later synthesized in response to GA After the S-FU and 3. GA.-trea : obtained exhibits bxa Labsue W Nsets 0; lie .~ LL.“ ‘ ‘H L tie is r in, “9 th. ‘s 8‘ =12 y fin. . 4‘ ‘ "(J‘re D. "as. 7 GA3-treatments, Carlson determined the thermal stability of a-amylase obtained at the end of each GA3 treatment and found that the o-amylase exhibited much greater thermal lability when extracted from aleurone tissue which had been incubated with S-FU during the period before GA3 addition. The alteration of thermal stability had been taken as indication for changes in the primary structure of a-amylase. From a series of such experiments, Carlson concluded that much of the dA-mRNA must have been synthesized prior to addition of GA and 3. that transcription of the o-amylase gene therefore occurred inde- pendently of the action of GA3. It was upon Carlson’s experiments that I had wished to base the first section of this dissertation. I had proposed to use his technique of short-time treatment with S-FU followed by chase with uracil to obtain a precise time course for the transcription of the a-amylase gene, beginning at the time when the dry tissue was first imbibed in H20. The technique would also have allowed me to estimate the functional lifetime of the oA-mRNA. However, the data which Carlson had published were considered controversial because he had not established the rate at which S-FU entered the pool of RNA pre- cursors, nor the rate at which added uracil was able to reverse the effects of 5—FU. In addition, he had not established the purity of the a-amylase preparations which he had obtained, nor had he shown that the isozyme compositions of the various a—amylase preparations were the same. Studies of thermal decay kinetics of enzymes may require pure enzyme solutions, and constancy of isozyme composition was necessary because isozymes of a-amylase differ substantially in their stability characteristics (Jacobsen et a1., 1970; Frydenberg and Nielsen, 1965). Additional support for Carlson's hypothesis y. La § Vx ‘ e:r liie: ex 3 ex u as .13 i‘ “ .0“ b b ‘« + 5‘ Sizes in t'~.- r326a8e, ‘ --:r ‘ .A “0&3 I U in m; g T the e a: wk :5“ u would ha‘ \m h! A..nm me N» em 2: E 8 vmnald have been provided by showing directly that S—FU was able to cause random amino acid substitutions in o—amylase and/or that S—FU was incorporated into poly(A)—RNA. In the course of my investigations, it became evident that Carlson's conclusions may have been premature. When o—amylase was purified by a method which led to the recovery of all of the o-amylase isozymes at a high level of purity, it was found that incubation of the aleurone tissue with 5—FU had no effect on the thermal stability of the enzyme. This is in contrast to Carlson's observations, according to which the thermal stability of the enzyme could be altered by S-FU treatment. To examine further the feasibility of this technique, I proceeded to investigate the possibility that the a-amylase which I had purified was unlike the a-amylase originally synthesized and secreted by the aleurone tissue, or that the d-amylase I studied was contaminated by protein, carbohydrate, calcium, protease, etc., or that S-FU was not utilized by the aleurone tissue in my experiments. In the end, however, none of these possibilities could explain the lack of a S-FU effect. Based on my data, there appears to be no reason to believe that altered d-amylase is synthe- sized as a result of treating aleurone layers with S-FU. At least in this instance, use of this technique appears to be without any * practical benefit. * Because of the nature of the following report, it will be necessary to summarize the methods and results of Carlson (1972) so that a comparison may be made to methods which I have employed. Carlson incubated de-embryonated seeds of barley (Hordeum vulgare cv. Betzes) in H20 for one day; following this, he removed (most of) the starchy endosperm and incubated the aleurone tissue in buffer (Chrispeels and Varner, 1967) for two more days. At that point 1 uM_GA3 was added for 24 hr, and after the incubation was terminated, the tissue was homogenized, combined with the medium, and the enzyme purified by a method develOped for pancreatic a-amylase (Loyter and Schramm, 1962). S-Fluorouracil was added for different periods throughout the incubation period, up to a maximum of 48 hr before and 24 hr after GA3-addition, and it was implied that S-FU treatment had no effect on the amount of a—amylase which was synthesized. If S-FU was removed before the end of the incubation period, it was replaced with 2.5 x 10"4 M_uracil. The purity and isozyme composi- tion of the a-amylase preparation were not described. It is possible that B-amylase may have contaminated the final enzyme preparation. Other proteins and carbohydrates were removed from a-amylase by binding the a-amylase to glycogen, passing the o-amylase and glycogen hydrolysis products over a charcoal column (pH 10.0) to remove carbohydrate, precipitating the enzyme with acetone, ether and ethanol. Calcium was removed by dialysis against 1 mM_EDTA in the presence of l mM_hexametaphosphate. Without 5-FU treatment, the half—life of the enzyme at 72 C was 12 min and with the longest S-FU treatment the half-life was 5 min [these half-lives were calculated by Chandra (1974) in his re-interpretation of Carlson's results]. o-Amylase from other treatments exhibited a mixture of both elements, leading to biphasic decay kinetics. Carlson's conclusions about the timing of a-amylase mRNA synthesis were based on the shapes of these thermal decay curves. 55b v grains we the E; L." 'V s. - ‘ d I I . . We” mete I H i t .1 .Q A S g .3 «b QC . S a n... «4 .l . h a t I. .Q a I A . d S C a \ E x- a Q I my. e c r E a .1 0 f E E E 3.. as .n I. d C. s 0 4 n .D ‘Q a < §~ MATERIALS AND METHODS Preparation and Incubation of Barley Half—Seeds Barley grains (Hordeum vulgare L., cv. Betzes) were harvested at Michigan State University in 1968 and were stored at 7 C until 1974, then at 20 C during the course of these experiments. The grains were dehusked by stirring the seeds slowly in 50% H2804 at room temperature for 35 min. Following this, the seeds were immedi- ately rinsed several times with distilled H20, then with double- distilled H O, and were then blotted and dried on paper towels. The 2 dehusked seeds were de-embryonated by transsecting the seeds behind the embryo with a scalpel in a plane perpendicular to the seed axis. Seeds prepared in this manner will be called "half-seeds." The half—seeds were also nicked at the distal ends to aid H20 uptake during imbibition. Half-seeds were surface sterilized in 1% NaOCl for 25 min at room temperature and rinsed 5 times with 4 volumes of distilled H20. The half—seeds were incubated for a total of 4.5 days. During the first 24 hr, the half-seeds were incubated in one batch in 100 to 200 ml of bathing solution. The solution was well aerated with air passed first through a cotton filter, then through 2% KMnO4 (as a sterilizing agent) into a gas dispersion tube, the end of which was immersed in the incubation solution. After the first 24 hr, groups of 50 half-seeds were transferred to 125-ml flasks containing 8 ml of l mM_sodium acetate, pH 4.8, and 100 mg. CaCl2 ("Acca" buffer). Chloramphenicol (0.001%) was added to 10 ”~V' \‘AL- . onare F11! mm . V analog ‘ad. ,6“ ‘EJS'iSed Q; Q.» : a .. mm. M. a V m» u. s a: A. y A ‘1 .. I .w I t . S t I. at E \,AN 5 1“ nasty t 11 inhibit bacterial growth. The flasks were incubated for 48 hr more on a reciprocating shaker operating at 125 to 150 oscillations per min. Filter-sterilized GA3 was added to the incubation medium to give a final concentration of 1 uM, and hormone treatment was con- tinued for 36 hr during which time the tissue had synthesized a maximal amount of a-amylase. In treatments with 5-FU, the base analog was solubilized by briefly warming it in Acca buffer at 30-40 C. The solution was filter-sterilized and then added to the incubation medium. Typical incubation regimes are shown in Figure 1A and 1B. When aleurone layers were used, they were prepared by removing the starchy endosperm with thin spatulas under sterile conditions. It was necessary to incubate half-seeds for a minimum of 2 days (3 days was best) to effectively remove the starchy endosperm and to avoid damaging the aleurone layer in this process. No amylase was released into the medium before the start of the GA3 treatment. Homogenization of Tissue The medium was decanted from flasks containing half-seeds or aleurone layers and set aside. Aleurone layers or half-seeds were homogenized in one of two ways. Large batches of material were ground with one-half of their weight of acid—washed sea sand in an electric mortar (Model MG—2, Torsion Balance Company, Clifton, NJ, USA). After the tissue was homogenized, the mortar was rinsed 3 times with a minimum of Acca buffer. Small batches of 10 to 25 layers or 10 half—seeds were homogenized by hand with a small porcelain mortar and pestle using about 1.4 g of sea sand and 1 m1 of Acca buffer. The mortar was then rinsed 3 times with 1 m1 of buffer. When the 12 COZEDO NQO £339.95 mo mane m 33m emcee mm; 30m 333336 .PTm mad 8&3 CH Ho .ocoam HoMmsn uoauwm ca mannedcwucoo woumnsocfi ones mommmrmamm .m .cowuanflnfiw mo mmmo m H39... 825 magmas: : 38 oflagmnflo .Dhim uconuw3 Ho Sufi: .Hommsn wood cw coaumnsocw mwomon Hz vm How 0 m ca ooxmom mums mommmimamm .¢ .mowmmlmamn moawmn mo cofiumnsocH .H ousmwm H own—mum 25 w .22. mm me me A to d:- d- l3 OH I' .24 . 4a... Q... ~34... 5.... .p..so¢os.o...sos~ .skooskksévss... s... ' c<<. o. oo. Q. Q - o .111. .1 1 L z 1 1 q u... 8:68 mqo e: dTZI in . e‘eas V enzf-fie ' of the ' ~1 s; d‘ «-U-- v ~\~ect ( fixing : at: u... a K .. v . 3 l4 ennzyme was to be purified, the homogenate was combined with the medium. For quantitation of the amount of enzyme synthesized and released, the layers were first washed with 200 mMCaCl2 for 10 min, and this rinse was combined with the medium for an estimate of the "released" amylase. If half-seeds were used, the starchy endosperm, medium and the 200 mMCaCl2 wash of the stripped aleurone layers were designated as total o-amylase released. Residual enzyme activity in the cell walls and periplasmic space were included as "tissue" amylase along with that actually retained within the cells. Purification of Calcium-Free d-Amylase (Method I) Step 1. The combined medium and homogenate from 300 to 400 barley half-seeds was diluted to 200 ml with Acca buffer. This dilution step was necessary to maximize the recovery of o-amylase during steps 2 and 3 (Figure 2), although dilution alone had no effect on enzyme activity at the end of step 1. The Acca buffer was superior to the following buffers for extracting the enzyme: 20 mg succinate, pH 4.8, with 0.1 MCaCl2 and 0.3 M_NaCl; 50 mM Tris [tris(hydroxymethyl)aminomethane], PH 8.0, with 0.1 M_CaCl and 23 50 WE Tris, pH 8.0, with 0.1 M_CaCl2 and 0.3 M_NaCl. In all experi- ments to be reported, the pH values are those determined at 22 C. The diluted homogenate was incubated at 25 C for 2 hr with gentle shaking to release most of the a-amylase from its native substrate. The solution, which still contained cell debris, starch grains and sand, was centrifuged at room temperature in an Inter- national Clinical Centrifuge at 1300 g_for 10 min. 15 .ucemeum ec ou ucmsocu >HHechHHo mm3 cecu oeue>ooeu ec ou muH>Huom eE>Nce eHccHOm euoe oeBOHHe mscu ocHumec ocm emceco mm ecu euomec eE>Nce ecu mo COHusHHQ .oeucemeumeu muceEumeuu coom Heume qu>Huom eE>Nce ecu uecu HemmHomcev mcHueec one emceco mm ecu euowen qu>Huoe oe>mmme >HHechHuo ecu mo uceouem ecu mm oemmeumxe eH AeuecHoHov wuH>Huoe emMH>Eeso m0 xue>ooeu ece .cHE mH you O on um oeumec ecoHusHOm ecu one o.m ou oemHeu mp3 mc0HusHom oeusHHo ecu mo mm eca .meHuH>Huoe eE>Nce oeuoemxe ecu oeoHeH> ec0He coHusHHQ .HE Hem muHc: omH one mm ceezuec ou oeusHHo euez ecoHusHom esawce ecB .mcoHusHom efihuce eoduo ecu m0 coHusHHo euowec o\o\c\e How 0. H\o. H\HI H\m. H eueB meHuH>Huom eHceuomHuxe Heuou ecu mo moHueH ecB .xllllx .o.m mm .Hoez.mE oom .NHOeOI :8 00H .mHuu SE om coy Ho DIIIIO o. m mm .NHOMOI SE 00H .mHuu :5 om on N¢III< .m.m mm .Huez SE oom .NHOmul SE 00H .uewmzc. euMCHUUSm SE om Acv “Ollllo w. v mm .NHonI XE 00H .eueueoe ESHUOm 28 H Amy .uemmsc uceueMMHo m CH peNHcemOEoc mm3 msoum comm .mmsoum usow oucH peuscHHumHoeu cecu .mxmeHm HesoH>HocH Eoum oeHoom euez moeemIMHmc one EdHer eca .Dmnm usocuH3 .«H eusmHm CH me oeuecsocH eue3 moeeminem .coHueoHMstm mCHnso >ue>ooeu emeHweelo co uoemme coHusHHQ .N eusmHm 16 ISO 200 I50- lOO» NOIlVOIJItInd ‘IVlltIVd BBL-JV All/\llOV BSV‘IAWV-D :IO AUBAOOEH IOO a-AMYLASE, UNITS PER ML BEFORE PARTIAL PURIFICA TION Figure 2 17 Step 2. The supernatant solution was carefully decanted, and the pH of the solution was changed to 8.0 by dropwise addition of unbuffered 0.1 M Tris base. The final concentration of Tris required was about 18 mM. Step 3. The solution was poured into a lSOO-ml Erlenmeyer flask equipped with a thermometer. The flask was incubated in a water bath set at 80 to 85 C while the temperature of the solution in the flask was maintained at 70 C. Five minutes were required to heat the solution in the flask to 70 C. For the next 10 min, the solution was maintained at 70 C‘: l C by either removing the flask to cool it or by returning it to the bath to warm it. This heating procedure was employed to inactivate B—amylases (Kneen et a1., 1943; Schwimmer, 1947; Schwimmer and Balls, 1949). During this heat treatment, a-amylase retained its activity since the presence of calcium at pH 8.0 prevented the denaturation of the enzyme. At pH 4.8 (the original pH of the medium), both o- and B-amylase activities were eliminated at higher temperatures (Figure 3). After the solution was heated, it was rapidly cooled by immersing the flask into an ice bath. The heat-precipitated proteins and carbohydrates were removed by centrifugation at room temperature. The crude preparation contained both o-amylase and B-amylase; however, no simple method was available to determine how much of the total amylolytic activity was contributed by either of those enzymes. Step 4. The clear supernatant solution was cooled to 7 C and ice-cold absolute ethanol was added dropwise to give a final concen- tration of 40%. This step was preparatory to the precipitation of 18 .o .o 8 9810 .o om “o .0 pm “ecHH oecmmo .0 mm ewes meusueuemfieu eca .m.v mm um menusc mood cH qu>Huoe emeHaem mo HE Hem muHc: oom usoce oechucoo mcoHusHOm HMCHmHHo ecB .meusueuemseu oeueoHocH ecu ou oeueec we: coHusHom HeeHo ecu .coHummstuuceo meHuc ueum< .cH eusmHm cH me oeuecsocH :eec oec cOch moeemIMHec Eoum oeuoeuuxe mez emeHwecio .euecemoaoc eoouo ecu Ho wuH>Huoe emeHmse ecu co meusueuemEeu mCHmeeuocH mo uoemmm .m eusmHm 19 m eusmHh 2:2 .ZOCdeUZ. m0 m2; Om Og mm H H HP Q wow I. 000 ONINIVWEH All/\IIOV BSV'IAWV-D .:IO 1N3083d 'pa, ‘ ~\ ’5 a lie V :al gl “0 IO l I e i the .dt V '11: F: ‘1 E t P?“ .A-ML {j ml?” .33 9 . mixture n3 ‘ H ‘t ‘ 1 G we... O a t t.& .Ni «man it P. .C\ 20 Hm eue eueuo>cocueo pee wuH>Huoe .x .AxeGCH e>HuoeumeH any coHueuuceocou HOez 0cm “0 .Aumeu .emeereno mo coHueoHMHusm ecu mo .v eusmHm emeH>5e18 .emeo chu CH “O .muH>Huoe emeH>Ecv eueuowcocueo .weHuec mewuem Eoum emeH>EeIo mo xcmeumoueEouco emoHsHHeolmdmo e eeum 24 W‘IODN :IO NOIthIlNBONOO 0. ‘0. V. '9. N O o o o o I I I I I BIVHCIAHOBHVO :IO 'llNV BAIIV'IBB IO N T T «OJ 1 cv. Betzes I II II [I II [I II II II II II II II II II II II II II I I \ 1 l C) 8 9 I") All/\IIOV BSV'IAINV-D BALLV'ISH 200 ~ 125 25 FRACTION NUMBER Figure 4 detect Fracti a rate vy pas 1: 3f the 2-7 a? ’ w .rcm t: . :sing I Q~, he mobilit S 25 detectability (5 x 10_8 M) could be recovered from the column. Fractions (0.73 ml) were collected from the 1.6 x 40 cm column at a rate of 0.7 ml/min. In some experiments, the calcium was removed by passing the sample over a Bio-Gel P-30 column equilibrated in 5 mM Tris, pH 8.0 with 1 mM EGTA. This step replaced the Sephadex G—25 column chromatography. The column volume was 1.6 x 40 cm, and the flow rate was 0.2 ml per min. Because of the decreased stability of the a-amylase upon removal of calcium, the enzyme was maintained at 2—7 C during this and subsequent steps. The active fractions from the void volume of the column were pooled and concentrated using Aquacide II or Sephadex G-200 placed on the outside of a dialysis bag containing the enzyme solution. The presence of either Aquacide II or Sephadex G-200 changed the pH of the solution inside of the bag. Aquacide II had the greatest effect, changing the pH from pH 8.0 to as low as pH 6.6, presumably by differential mobilities or ion exchange of EDTA and Tris across the membrane. Sephadex had a lesser effect, decreasing the pH from pH 8.0 to pH 7.6. Most experiments were performed using Aquacide II, but in later experiments the Sephadex material was used. Partial Purification of o-Amylase (Method II) a-Amylase was also partially purified using methods which closely approximated those of Loyter and Schramm (1962) for purifying pancreatic amylase. This purification procedure had been used by Carlson without modifications (1972). Because of the nature of barley a-amylase and the medium into which it was released, certain modifications of the Loyter and Schramm method were necessary. The it- been int“ . cell debri tion of O. of calcium solution w: cold, abso. EiXture Nd: 20,003 g f: ‘ GlYCor 26 The medium and homogenate from barley aleurone layers, which had been incubated in Acca buffer, were combined and centrifuged to remove cell debris. The supernatant fraction was brought to pH 6.9 by the addi- tion of 0.5 M_K2HPO4. A voluminous precipitate formed composed primarily of calcium phosphate salts which precipitated at pH 5.6 to 5.8. The solution was chilled at 4 C and centrifuged at 1300 g_for 8 min. Ice- cold, absolute ethanol was added until its concentration was 40%. The mixture was stirred for an additional 60 min and then centrifuged at 20,000 g_for 20 min. Glycogen was added in three steps. First, glycogen was added to the solution at pH 6.9 using 1 ul of a 1.6% solution per 7 units of a- amylase (units are defined later). After 15 min, 0.2 M_phosphate buffer, pH 8.0, was added to give a final buffer concentration of 10 mg, Ethanol was immediately added to bring the ethanol concentration back to 40%. A second volume of glycogen was added, equivalent to the first. After 15 min, the precipitate was pelleted by centrifugation. The pellet was drained and was washed with 5 ml of 10 mM_phosphate buffer, pH 8.0, in 40% ethanol. After a second centrifugation at 4000 g_for 5 min, the pellet was resuspended in 1 m1 of 20 mM_phosphate buffer, pH 6.9, con- taining 3 mM_CaC12. This latter solution contained a fine precipitate of calcium phosphate. The centrifuge vial was rinsed a second time with 1 m1 of the pH 6.9 buffer. The solutions were combined and incubated for 2.5 hr at 37 C. After the glycogen and u-amylase were solubilized, NH OH (0.2 N) was 4 used to increase the pH of the solution to 8.4 i 0.1. The solutions were centrifuged at 6000 g_for 5 min at room temperature to remove the insoluble carbohydrate. 27 The method of Loyter and Schramm (1962) requires a rapid change of buffer after the preceding steps, because the sample must be at pH 10.0 during subsequent chromatography on a charcoal column. a—Amylase was somewhat unstable at pH 10.0 (Figure 5). After 3 hr at room temperature, 40% of the activity was lost. Loyter and Schramm apparently precipi- tated pancreatic amylase at its isoelectric point, pH 7.0, then dissolved the precipitate in pH 10.0 buffer made of 10 mM glycine and NaOH. This spontaneous precipitation did not occur with barley a—amylase at the final concentration of enzyme obtained prior to this step (about 320 enzyme units/m1). Preparative isoelectric focusing of barley o-amylase, in fact, segregated barley a-amylase into two peaks of activity, one with a pI of about 5.0 and the second with a pI of about 6.1 (data not shown). Thus, barley d-amylase would not be expected to precipitate at pH 7.0, and it did not. However, in order to approximate the conditions to which Carlson must have exposed his a—amylase solutions by following the procedure of Loyter and Schramm, the following adaptation was made. Two samples of o-amylase were processed differently at this step. Sample A: The pH of the first sample was adjusted from pH 8.4 to pH 7.0 by the addition of 0.2 M_acetic acid. No precipitation resulted after a 60-min incu- bation at 2 C. This meant that the buffer could not quickly be exchanged with one at pH 10.0 by dissolving the precipitate in the new buffer. Instead, 0.1 M glycine-NaOH, pH 10.0, was added to the solution to give a concentration of 10 mg, and the pH of the solution was adjusted to 10.0 with NH4OH. Sample B: After first changing the pH of the solution to 7.0 and chilling it for 60 min, the second sample was dialyzed at room temperature against 10 mM_glycinenNaOH, pH 10.0, containing 5 mM CaClZ. The 2 liters of external buffer were changed every 20 min. 28 Figure 5. Stability of a—amylase at pH 10.0. a-Amylase was purified by Method II (Loyter and Schramm procedure). The enzyme was precipitated by the addition of glycogen, then solubilized by incubation of the glycogen/amylase complex at 37 C for 2.5 hr. The pH of the solution was adjusted by the addition of glycine-NaOH buffer (pH 10) until a concentration of 10 mM_was reached, followed by titration of the solution to pH 10.0 by the addition of 1N NH4OH. The activity was determined at intervals thereafter. The initial a-amylase concentration was 69.1 units per ml. The incuba- tion was performed at room temperature. PER CENT ACTIVITY REMAINING I00 PER CENT ACTIVITY REMAINING IOO 75 O 60 Iéo Iéo TIME(M|N) Figure 5 After th: 8.5. H01 was remo‘ solution The coal (act 30 After three changes (total time 1 hr), the pH of the dialysate was only 8.5. However, much of the phosphate had been removed. Calcium phosphate was removed from the dialysate by centrifugation and the supernatant solution was titrated to pH 10.0 with NH4OH. The charcoal-celite columns were prepared from a mixture of char- coal (activated Darco G-60, Matheson, Coleman, and Bell, Cincinnati, OH, USA) and celite 545 as suggested by Loyter and Schramm (1962), except that instead of washing the particles on a Buchler funnel, the particles were suspended and left to settle so that the fine particles could be removed. The final buffer contained 10 mM_glycine-NaOH, pH 10.0. The column material was equilibrated in this solution over a period of 3 weeks. The charcoal used was kindly supplied by Dr. P. Carlson. o-Amylase Assay a-Amylase was measured by the method of Varner and Mense (1972). Starch substrate was prepared by suspending 150 mg of potato starch (Nutritional Biochemical Corporation) in 100 ml of a solution con- taining 50 mM_KH PO and 10 mMyCaCl 2 4 This solution was boiled for 2. 1 min, cooled, and centrifuged at 12,000 g for 15 min at 0—5 C. The supernatant solution was separated from the gelatinous pellet and diluted with 1.5 volumes of H20. The iodine solution was prepared by diluting 0.3 ml of iodine stock solution (6 g KI and 0.6 g 12 in 100 m1 H20) with 100 ml of 0.05 N HCl. This acidified solution was sensitive to air and light and had to be freshly prepared every 2 hr. The a-amylase was diluted to 1.0 ml with H20, and 0.5 ml of starch solution was added at room temperature. After a suitable time interval, 1.0 ml of the acidified iodine reagent was added, thus 3'3“: ere 31 stopping the hydrolysis reaction. The absorbance of the blue reac— tion product was measured spectrophotometrically at 620 nm. Certain features of the assay are worthy of note. First, the reaction of iodine with starch was only linear at from zero to 5620 0.950 absorbancy units (Figure 6). Thus, the starch should be diluted enough to yield a starch-iodine product which has an absorbancy below this value. Second, the enzyme assay is not linear, especially when more than 70% of the starch has been hydrolyzed (Figure 7). For this latter reason, the enzyme concentration or incubation time was varied so that the final starch—iodine complex would exhibit between 30 and 70% of the possible color development. One enzyme unit is defined as that activity of enzyme which elicits a change of one unit in absorbance at 620 nm in one minute. A convenient formula for calculation is the following: AA umb f V -620 n “0 (1-3)( )= . enzyme units — b v x t min where a absorbance of the starch-iodine complex after hydrolysis b = absorbance of the starch-iodine complex before hydrolysis (a and b are different samples, of course) V = total volume of enzyme (m1) v = sample volume of enzyme assayed (ml) t = time allowed for hydrolysis in minutes. o-Amylase was assayed in four replicates, except that the fractions resulting from column chromatography were usually assayed only once. The presence of B-amylase would cause an over-estimate of the supposed o-amylase activity. However, the starch substrate contained a substantial amount of polymer which was inaccessible to B-amylase alone. Pure B—amylase from sweet potato (Sigma type IB with a 32 Figure 6. Effect of starch concentration on the development of the starch—iodine complex. The starch was boiled, centrifuged, then diluted to various concentrations. The standard iodine solu- tion was added and the absorbance at 620 nm was determined. IOO 0.75( 620 0.500 0.250 oIr l.000 0.750 A620 0.500 0.250 I I 1 l 0.2 0.4 0.6 0.8 RELATIVE STARCH CONCENTRATION Figure 6 I.O 34 Figure 7. Hydrolysis of starch by a-amylase: reaction of the products with iodine. a-Amylase was incubated with the starch solution for increasing amounts of time, whereupon the iodine solution was added. The amount of a-amylase used in the assay was 0.107 units. The dotted area designates the region of A§620 within which the a-amylase assays were considered for calculation. 4 4620 o 0.75 0.25 35 h 5 I0 I5 TIME, MINUTES Figure 7 4 4 L SC‘eCl Il9Sl} etic etc. a: V‘H. -.o q T. #03 “:1 “~11 Ll of $‘uy. y e 36 specific activity of 785 units per ml by the maltose assay) could only hydrolyze the starch solution to a A of 0.350 (Figure 8). 35620 Protein Assays Protein was assayed according to the procedure of Lowry et a1. (1951) as described by Layne (1957). In some cases, samples contained a high concentration of amino acids and other interfering substances. In these instances, the following procedure was adopted. Portions of each sample (10 to 20 ul) were spotted onto cellulose thin-layer plates of 0.1 mm thick cellulose MN300 (Brinkman Instruments, Westbury, NY, USA), and thin-layer chromatography was performed in n-butanol, acetic acid, and H20 (4:1:1 v/v). The protein remaining at the origin was assayed by scraping 0.5 by 1.0 cm sections from the plate. NaOH (0.1 N, 100 pl) was added to the powder, and after 10 min, 900 pl of a solution containing 2% NaCO3, 0.02% sodium potassium tartrate, and 0.01% CuSO4-5H20 was added with rapid mixing. The usual pro- cedure (Layne, 1957) was then followed, and the samples were assayed spectrophotometrically at 750 nm. Bovine serum albumin (BSA, Sigma type VI) was used as a standard. It was assumed that all proteins in these crude mixtures were solubilized after chromatography and processing. However, pure BSA was not easily released from the cellulose. Instead, scrapings of the cellulose MN300 plates from areas devoid of protein or ninhydrin—positive material were used as blanks and soluble BSA was assayed in the presence of the cellulose after the scrapings had been suspended in the first reaction mixture. Carbohydrate Assays The presence of carbohydrate was determined using the anthrone reagent (Sigma Chemical Company, St. Louis, MO, USA). This reagent 37 .pepuooeu mes emceco uceouem ecu one oeusmeea mes 5: 0mm um eocecuomce ece .coHueASUCH no meEHu eueHumoumme ecu Heume AHE Hv coHusHom ecHoOH ecu ou oeooe eue3 “HE m.Hv muosvHHe oce coHusHOm coueum ecu mo HE OOH cuH3 oexHE me3 Amfi m.ov emeH>Ee|m eEHu omen ua .emeH>Eelm oueuom uee3m >n coueum mo mHmmHOHo>m .m ensmHh I 19 r «e I -O ID -8 n o O O O 0 LG “I” 039 1V BONVBHOSBV 3AI1V'IBH TIMEIMIN) Figure 8 (zoo mg) ‘ solution thoroughl absorban: sugars we chromatog 39 (200 mg) was dissolved in 100 m1 of concentrated H SO The test 2 4' solution (0.5 ml) and the dissolved reagent (1.0 ml) were mixed thoroughly. The mixture was boiled for 10 min, cooled, and the absorbance measured spectrophotometrically at 620 nm. Individual sugars were identified as their alditol acetate derivatives by gas chromatography (Jones and Albersheim, 1972). Calcium Assay Free calcium was determined by complexing the ion with glyoxal— bis(Z-hydroxyanil). The method for qualitative identification of calcium (Feigl, 1958) was modified in the following manner to permit quantitative measurements. To a 0.5 to 1.0 m1 test solution were added sequentially, with mixing at each addition, 50 ul of 1 N HCl (or sufficient HCl to drop the pH to equal to or less than pH 7.0), 200 pl of a saturated solution (up to 1%) of glyoxal-bis(2-hydroxyanil) in ethanol, 100 pl of 10% NaOH, and 100 ul of 10% Na2C03. The aqueous solution was extracted with 3 m1 of chloroform, and the absorbance of the chloroform solution was determined at 560 nm. At this wave- length, a calcium-free solution showed very little absorbance. The limit of detection for the spectrophotometric assay was 5 ug (in 3 ml) or about 1.6 x 10-6 M.of calcium. Thermal Denaturation Calcium-free a-amylase was diluted to between 200 and 500 units per ml with 5 mM_Tris, pH 8.0, containing 1 mM_EGTA. The volume of enzyme was usually between 600 and 800 ul. The zero—time sample was usually assayed six times to determine the initial a-amylase concen- tration. The sample, placed in a tightly sealed, 3.5-ml vial, was heated in a small waterbath (Aloe Scientific, St. Louis, MO, USA), and after vex could be rap: which had pre Each heet-tre arount 0f u-e Secara:- K Protein' gelsbl’ one [J 11L: SOdiu: I 13er :CHNQ‘ .‘htr . athp J: a ‘ 40 and after various times the vial was briefly Opened so that samples could be rapidly mixed with one-half of their volume of l MCaCl2 which had previously been pipetted into small vials placed on ice. Each heat-treated sample was assayed three or four times for the amount of o-amylase activity remaining. Separation of Proteins by Polyacrylamide Gel Electrophoresis Proteins were separated by electrophoresis on polyacrylamide gels by one of three methods. Fairbanks (et al.) Method Usually, the method of Fairbanks et a1. (1971) was used, with some modifications. The running buffer consisted of 40 mM Tris base, 20 mM_sodium acetate, 2 mM_EDTA, and 1% SDS. The pH of the running buffer was adjusted to pH 7.4 with acetic acid before SDS was added. The gels consisted of 5.6% acrylamide, 0.21% bis-acrylamide, 10% glycerol, and the running buffer. Polymerization required 24.2 ul of TEMED and 65 mg of ammonium persulfate (AMPS) per 100 ml of gel solution. The gel solution was distributed to glass tubes (6 x 150 mm) which had been silanized with 1% dichlorodimethyl silane (v/v) in toluene for 1 hr and rinsed thoroughly with methanol. Each gel tube contained about 3.5 m1 of gel solution, and the gels were about 10 cm long. When the polymerizing gels were layered with isobutanol, a very flat gel surface resulted. Polymerization was complete within 45 min, after which the isobutanol was washed with running buffer from the gel surfaces. The protein samples were dialyzed if necessary to reduce the salt concentration. Two volumes of protein were then added to one volume of a boiling solution of 5% SDS and 12.5% B-mercaptoethanol, and boiling we additional glycerol, PH of the HCl. The and buffe. The 1 Units of I Onto the . The POrti imm‘irSed the EleCt direction for 3 hr. tubes usi betweEn t The Blue R in Sari. Af remm,ed b 41 boiling was continued for 10 min. The samples were cooled and one additional volume of a buffer-tracker dye solution containing 40% glycerol, 40 mM Tris, 2 mM_EDTA, and 0.02% pyronin Y was added. The pH of the buffer-tracker solution had first been adjusted to 8.0 with HCl. The ratio of the volumes of the solution of protein, denaturants and buffer were thus 2:1:1, respectively. The protein samples, each of which contained a maximum of 10 units of a-amylase or 100 pg of a mixture of proteins, were layered onto the gel surface under the running buffer of the upper chamber. The portions of the gel tubes which contained the polyacrylamide were immersed in the buffer of the lower chamber to facilitate cooling since the electrophoretic runs were performed at room temperature. The direction of electrophoresis was toward the anode using 5 mA per gel for 3 hr. After electrophoresis, gels were removed from the glass tubes using a jet of H 0 from a 2-inch, 25-gauge needle inserted 2 between the gel and the glass tubing. The gels were stained for protein with 0.1% Coomassie Brilliant Blue R in 50% methanol and 7.5% acetic acid. Pre-fixing was unneces- sary. After 8—16 hr in the staining solution, excess stain was removed by several incubations in 5% methanol and 7.5% acetic acid at 60 C. The stained gels were scanned at 560 nm using a Beckman DUR monochrometer equipped with a Gilford linear transporter, or the mobilities relative to pyronin Y were determined with a ruler. India ink, injected with a needle, was used to mark the dye position before the gel was stained. Poor staining could be remedied by repeating the staining procedure or could be prevented by staining the gels in large volumes (50 ml per gel) of staining solution. glycera (25,703 theses by £20 1y. CYtochr: Obtains; a". Ili Lag The Of prOtg SDS, 0 o final C0 to the b 42 The molecular weight of o-amylase was determined by comparison to those of several marker proteins. These were bovine serum albumin (68,000), ovalbumin (43,000), horse liver alcohol dehydrogenase (41,000), aldolase (40,000), yeast alcohol dehydrogenase (37,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), chymotrypsinogen (25,700) and cytochrome c (12,400). The molecular weights in paren- theses were taken from Weber and Osborne (1969) and had been determined by polyacrylamide gel electrophoresis. The molecular weight of cytochrome c was given by the distributor. All marker proteins were obtained from Sigma. Laemmli Method The method of Laemmli (1970) was also used for electrophoresis of protein samples. Samples were prepared by adding crystalline urea to a boiling solution containing 0.125 M_Tris base, 0.119 N HCl, 4% SDS, 0.0002% bromophenol blue, and 10% mercaptoethanol to give a final concentration of 8 molal urea. One volume of protein was added to this with rapid mixing, and the solution was immediately returned to the boiling water bath for 10 min. Ingle Method A third method used a gel system based on that of Ingle (1968), using non-denaturing conditions. The running buffer was 5 mM_Tris and 5 mM_glycine at pH 8.9. The gels contained buffer, 7.5% acrylamide, and 0.15% bis—acrylamide, with 50 ul TEMED and 7.5 mg of aqueous AMPS per 100 m1 of gel solution. A H20 overlay was used to cover the gels during polymerization. After 1 hr of pre-electrophoresis at 7 C and 2.5 mA per gel, the enzyme samples, which contained 8% sucrose, and 0.008% bromophenol blue, were layered onto the gel surfaces. Electroph min. The Coomassie unstained (approxi: agar, 0.5 (pH 5.6), SliCes We IZKI solu cate the 43 Electrophoresis was continued in the cold at 2.5 mA per gel for 50 min. The gels were removed from the tubes and either stained with Coomassie blue or were examined for enzymatic activity. These unstained gels were sliced at room temperature into 2-mm sections (approximately) which were placed onto petri plates containing 1% agar, 0.5% soluble potato starch, 20 mM potassium phosphate buffer (pH 5.6), and l mM_CaC1 After 20 hr at 25 C in the dark, the gel 2. slices were removed, and the plates were flooded with acidified IZKI solution. The diameter of the clear circles was used to indi- cate the relative o-amylase activity. Isozyme Separation by_Agar Gel Electrophoresis Amylase isozymes were separated by electrophoresis in agar gel slabs (Jacobsen et a1., 1970). The gels were formed from 1% Difco purified agar dissolved in the running buffer, 4 mM_sodium phosphate, pH 7.55. The samples were each placed in one of eight individual, l-cm—long slits arranged across the plexiglass tray holding the gel. The dimensions of the tray were 17.5 cm x 19.5 cm x 0.3 cm. The slits were arranged parallel to and 6 cm away from the narrower (17.5 cm) side of the plate. Up to 8 ul of solution containing a total of 0.3 units of o-amylase were placed on 2.5 x 10 mm wicks of Whatman No. 2 filter paper and these were gently inserted into the slits. Thin sponges (duPont cellulose sponge cloth) were used as wicks for the buffer during the electrophoresis. The edge of one Sponge cloth was placed over the shorter end of the gel to within 2 mm of the samples. This was the anode end of the gel. The second cloth was placed 3.5 cm from the cathode end but was underlain with a single layer of dialysis membrane (4.0 cm wide) which helped prevent electroos with a 5: prevent c 3-Amylase After 20 the filte was ident at 37 c 1 Starch (; CaC12. I allowed t 44 electroosmotic H20 flow. The agar and exposed sponges were covered with a smooth piece of Dow Handi-WrapR (a thin plastic film) to prevent otherwise inevitable moisture loss from the surface. o-Amylase migrated toward the cathode at 25 mA and 7 C for 2 hr. After 20 min, the electrophoresis was briefly interrupted to remove the filter papers and the slits were gently pushed closed. Amylase was identified by incubating the gels (still in the trays) for 1 hr at 37 C in a boiled and cooled solution of 0.5% Lintner soluble starch (Fisher Scientific Company) containing 1% KB PO and 0.1% 2 4 CaClz. The agar gels were briefly rinsed in distilled H20 and were allowed to rest on a table for 15 min at room temperature. They were subsequently incubated for l to 2 min in a solution of 0.5% HCl, 0.6% KI and 0.06% 12. The gels were then briefly rinsed a final time with distilled H20. They were photographed using PolaroidR color print film, type 48, using diffused transmitted light from a fluorescent light source. The a-amylase bands appeared as clear zones in a dark blue background. 32P-Labeling and Isolation of Ribonucleic Acid One hundred half-seeds were incubated for 24 hr in aerated H20, followed by 36 hr in Acca buffer, pH 4.8. Inorganic 32F, at 70.3 uCi per ml or 560 uCi per flask of 50 half-seeds, was added to the medium for a further 12 hr. After labeling, the half-seeds were placed on paper towels and aleurone layers were prepared. Each layer was immediately rinsed in 50 mM_PO4, blotted, and dropped into a tube partially filled with liquid nitrogen. The frozen layers were 1yophilized overnight and ground to a powder in the presence of additional liquid nitrogen. Ribonucleic acid was extracted from the aleurone 1 Ralph (19 volumes 0 saturated 0.01% 8-} and the diSSOlve 5'5: con 0f 1% w for 30 n 10 min a three ti PH 5.5, PH 5.5, 45 aleurone powder by a method which was based on that of Bellamy and Ralph (1968). The powder was homogenized with sea sand using equal volumes of (a) 1% SDS with 200 ug/ml sodium heparin and (b) a H20- saturated solution containing 80 g phenol, 10 ml of m—cresol and 0.01% 8-hydroxyquinoline. The mixture was again stirred and then centrifuged in a clinical centrifuge to separate the phases. The phenol phase was extracted a second time with fresh SDS solution. The two resulting aqueous upper phases were combined, and 2 volumes of ice-cold ethanol were added. The solution was chilled for 1 hr, and the precipitate pelleted by centrifugation. The pellet was dissolved at room temperature in 5 ml of 0.1 M_sodium acetate, pH 5.5, containing 0.5% SDS and 0.1% sodium heparin. CTA-bromide (1 ml of 1% w/v) was added dropwise, and the solution was placed in ice for 30 min. The precipitate was collected by centrifugation for 10 min at 13,300 g, and the pellet was washed and recentrifuged three times with cold 70% ethanol containing 0.1 M_sodium acetate, pH 5.5. The washed pellet was resuspended in 0.33 M_sodium acetate, pH 5.5, and reprecipitated with two volumes of cold ethanol. The final pellet was dissolved in 0.3 ml of electrophoresis buffer. This method extracted rRNA and tRNA, but poly(A)-RNA species were probably left behind. The RNA was separated electrOphoretically using the method of Loening (1967). The electrophoresis buffer included 40 mM_Tris, 33 thsodium acetate, 2 mM_disodium EDTA-ZHZO and 0.4% SDS. The pH was adjusted to 7.8 with glacial acetic acid. The gels contained 2.5% acrylamide, 0.125% bis-acrylamide, 82 ul of TEMED per 100 ml of solution, and 0.8% ammonium persulfate. Plexiglass tubing (6 mm x 120 mm) was fitted at one end with a small piece of Tygon tubing so that t] the gel added t 4 mm of gels we at 5 ma 5. Lin 460 onto the for 105 from the “EStaine were the at the 1. sliCEd i1 PaCkard 1 used to c tained 0. 46 that the opening of the tube was reduced to l to 2 mm. This prevented the gels from sliding out of the gel tubes. The gel solution was added to a depth of 9—10 cm and was immediately over—layered with 4 mm of 820. Polymerization was complete within 30 to 45 min. The gels were pre-treated by electrophoresis without samples for 1 hr at 5 mA per gel. Afterwards, samples were prepared by mixing one 5260 unit of RNA with ribonuclease-free sucrose (8%) and were layered onto the tOps of the gels, and electrophoresis was allowed to proceed for 105 min at 5 mA per gel. Following this, the gels were removed from the tubes and were incubated in distilled H20 for 1 hr. The unstained gels were scanned spectrophotometrically at 260 nm. They were then lain at a slight angle in foil troughs with rubber plugs at the lower end and were frozen over dry ice. The frozen gels were sliced into 0.7 or 1.0 mm slices with a Mickle Gel Slicer. A Packard Tri-Carb liquid scintillation spectrometer, model 544, was used to determine the radioactivity. The scintillation fluid con- tained 0.5 g PBBO and 8 g of butyl-PBO per liter of toluene. RESULTS Response of Half—Seeds of Betzes Barley to GA 3 Half-seeds of Betzes barley contained no detectable a-amylase activity after a three—day imbibition period on moistened sand. When 10-6 §_GA was added and the half—seeds were shaken in a liquid 3 medium, a-amylase activity increased sharply after a lag period of about 8 hr (Figure 9). Ninety-five percent of the enzyme was released into the medium or the starchy endosperm. Only a low level of d-amylase was produced when no hormone was added. One half-seed of Betzes barley each synthesized between 10 and 15 units of amylase activity in a period of 40 hr. Aleurone layers prepared from half-seeds of Betzes barley synthe- sized 5.0 to 5.6 units of a-amylase, again after a lag period of about 8 hr (Figure 10). However, about 30% of this activity remained associated with the tissue, even after the layers were rinsed with 0.2 §_CaC12. S-Fluorouracil at a concentration of 10-3 §_did not affect the lag time for a-amylase synthesis and the amount of a-amylase synthesized by or released from the aleurone layer (Figure 10). Less than 0.12 units of enzyme were synthesized without the addition of GA3' Despite the fact that the Betzes barley seed had been stored (at 7 C) for five years before these studies were begun, the seeds were completely viable when placed on moist filter paper. The 47 48 £50 I Figure 9. Synthesis and release of amylase by half-seeds of Betzes barley in response to GA3. Amylase activity was measured in the medium plus starchy endosperm ("released") and in the homogenate of the aleurone layers ("tissue"). Solid lines, 1 u§_GA3; dashed lines, no GA3. Released, 0; tissue, 0. IOOL 50t c2-11A471_/15H§ lQCITV\/I7"Y’(LJPJITYS FDEH? IC)lflllLJ:-EHEEHDES) d: 49 .9.-- 24 48 36 l2 ISO IOO - 5 Amowmminar O. mum mtznv >._._>.._.0< mmdj>§<-c TIME (hr) Figure 9 50 Figure 10. Synthesis and release of a—amylase from aleurone layers of Betzes barley in response to GA3; effect of 10‘3 §_5—FU. The layers were prepared from half—seeds which had been pre- incubated for 3 days, with or without S-FU. Fresh medium (with or without S-FU) was added with l ug_GA3 at zero time. The aleurone layers were rinsed with 0.2 fl_CaC12 in l m§_sodium acetate, pH 4.8 before homogenization, and the rinse was added to the medium. No S-FU, solbdlines;1 m§_S—FU, dashed line. Released, open symbols; tissue, solid symbols. cr-AIA4Y1_AMSETaACITV\/FTVfl LHVITTS FWEF?IC)1_IPYEI?$; 40 IO. 4O p b p O 3 m m mmmhqn. O. mum mtz: .>._._>_._.o< mm<4>208mu mos Summmoocm .NH madman cfl mm owumnsocfi oumz mmanmn monumm mo momwmlwammm v.am was moa x H.a omum.m NIOH m.mm «mma mod x H.a amum.m euoa o.ooH vmmm moa x a.» palm on w Amzm m: mom Emuv Axmmam mom smov mucwfiumwua >ua>auo< cemeowmm m>flumamm sua>auo< oflmnommm ass m2 as a 8 mm xmaumn mmuumm mo mumme mcouzmam ma 42m oucfi m mo GOHUMHOQHOUGH .H dance mm 59 Figure 13. Incorporation of 32F into RNA species from aleurone tissue of Betzes barley: Effect of S-FU. One 5260 unit (about 41.7 ug) was applied per gel. After electrophoresis on polyacrylamide gels, each gel was sliced, and the radio- activity per slice was determined. A. No S-FU, 0.7 mm slices. B. 10-4 g S-FU, 1.0 mm slices. c. 10-2 g S-FU, 1.0 mm slices. Notice that the slices from (A) are thinner and thus have rela- tively fewer cpm per slice. The total radioactivity (cpm) in each peak is printed next to the peak. 32p, CPM PER SLICE 60 EKXD+ 3954 A. no 5- FU 400- 2104 200' 1634 GM - . 0 so IOO I50 200 lOOO» 3118 8. low 5-FU 800- 600- 400- 1716 200- 942 00 2'5 5'0 75 IOO 200 c. IO'ZMS-FU '00 -WW 0 1 o 25 50 75 I00 SLICE NUMBER Figure 13 61 Figure 14. Specific activities of the RNA species after treatment of half-seeds with 5-FU. The relative specific activity is the ratio of radioactivity (cpm) of 32P per 5260 peak, relative to the specific activity of the RNA species from control (no S-FU) tissue. 62 5- FU CONCENTRATION, M Figure 14 .J 1 T 8 p— .. ZI00 o o u. o '— z m o a: m 0- . ,: I: 2 so - - '— o < 2 E o 33 a) 0 285 m o l8$ 2 x4m55 I— . <1 .1 g 0 1L 1 1 o " I0" IO‘Z 63 The reason for this is that S-FU treatment of the aleurone tissue may have affected the synthesis and/or release of compounds other than a-amylase which may have resulted in there being different kinds and levels of contaminants in the enzyme solutions at the time the thermal stability was determined. Certain of the contaminants may have either affected the stability of the a-amylase or have also utilized the starch substrate. Some examples of substances which could interfere with the determination of the thermal stability of d-amylase, and the levels of which may have been especially affected by S-FU treatment, are B—amylases, proteases, and relatively high concentrations of proteins or of polysaccharides. B-Amylase. The B-amylases become activated by proteases which are synthesized and released along with a-amylase (Jacobsen and Varner, 1967). B-Amylases are exo-hydrolases while a-amylases are endo-hydrolases. Since calcium stablizes d-amylase against thermal inactivation (Kneen et a1., 1943), all of the calcium must be removed from the enzyme. In the absence of calcium, both a-amylase and B-amylase are unstable to heating. Thus, B—amylases would definitely interfere with determinations of the heat stability of a-amylase. Proteases. Even trace amounts of protease could increase the apparent rate of enzyme inactivation during heating. If isozymes of a-amylase were differentially "inactivated" by proteases, multi- phasic inactivation kinetics could be produced. Other proteins. Carbohydrases, in addition to B-amylase, which utilize soluble starch as substrate, might also cause the appearance of altered heat stability of a-amylase which would manifest itself 64 through multi-phasic decay kinetics. Contamination by large amounts of protein (on the order of 1% w/v or more of the test solution) could decrease the activity of 820 in the solution and thus decrease the rate of thermal inactivation. Such proteins could also bind to either the a-amylase or the starch substrate, or they could interfere with the renaturation of the a—amylase during cooling of the enzyme solution. Polysaccharides. In the aleurone system, there are many sources of polysaccharide contamination, including the cell walls of the aleurone and starchy endosperm, the starch grains, and the glycogen used to precipitate the a-amylase. Because enzymes bound to their substrates are frequently more stable than when they are free, the presence of carbohydrate polymers could affect the stability of the a-amylase during heating. Removal of B-Amylase There are two ways to avoid the contamination of a-amylase by B-amylase. One would be to remove all of the starchy endosperm from the aleurone layer before placing the layer into medium which con- tained GA3. For the starchy endosperm to be efficiently removed, it must have been "softened" during a suitable hydration period of at least 2 days of incubation in an aqueous medium. Although Carlson (1972) indicated he had used aleurone layers for his studies, I found that I could not prepare clean aleurone layers by incubating half- seeds for only 24 hr in H20 as he had. Therefore, I used half-seeds, heavily contaminated with B-amylase, as a source of the a-amylase, but I employed a different method to eliminate that contaminant. B-Amylases are inactivated by heating the extract of the half-seeds 65 at 70 C for 15 min at pH 8.0, a-amylases are stable to this procedure, as long as the medium contains calcium. Therefore, a heat treatment was used to inactivate B—amylases. Following this, B-amylases should no longer bind to glycogen, permitting separation of the two amylases in the glycogen precipitation step. Electrophoretic Homogeneity on SDS-Polyacrylamide Gels 1. a-Amylase was purified using Method I and was subjected to polyacrylamide gel electrophoresis using the method of Fairbanks et al. (1971) with modifications as indicated. This method leads to the separation of proteins according to their molecular weight. When purified a-amylase was subjected to electrophoresis, a single band of protein appeared following staining with Coomassie Brilliant Blue R (Figure 15). When compared to proteins of known molecular weights, this protein was calculated to have a molecular weight of about 41,400 daltons (Figure 16). In some instances, prolonged storage (several months) of the stained gels in the de-staining solution led to the staining of a trace protein component with a molecular weight of about 21,000 daltons, or roughly one-half the molecular weight of a-amylase. The presence or absence of S—FU in the incubation medium of the half- seeds had no effect on the presence or absence of this minor component on the gels. Data obtained in the second part of this dissertation indicated that the minor protein component did not derive from a-amylase. This contaminant represented less than 3% of the protein on the gels where it eventually became visible. 66 Figure 15. SDS—Polyacrylamide gel electrophoresis of puri- fied a-amylase. The protein was applied to a polyacrylamide gel, and electrophoresis was performed according to the method of Fairbanks et al. (1971). The gel was scanned at 5560 after staining with Coomassie Brilliant Blue R. A560 0.4 67 l l l I 2 4 6 8 DISTANCE MIGRATED (CM) Figure 15 68 2. When the procedure of Laemmli (1970) was used for electro- phoresis of denatured a-amylase on polyacrylamide gels, two additional bands of protein were evident (Figure 17). By comparison, crude malt a-amylase from Sigma was low in a-amylolytic activity and contained very little of the band of a-amylase protein. In the latter case, two bands of protein predominated, which had mobilities similar to those of the "additional" bands of protein mentioned above (Figure 17). Since the electrophoresis of the purified a-amylase by Fairbanks' method had not led to the separation of these two additional proteins, and since the Fairbanks method uses strong denaturing conditions which are designed to severely inhibit proteolysis, it appeared that the two additional proteins may have been derived from hydrolysis of a-amylase. This is supported by the observation that no proteins from the purified a-amylase were retarded by a column of Bio-Gel P-30 which theoretically should have retarded these smaller proteins. The origin of these two additional protein bands was not investigated further. If these bands were produced as a result of proteolysis during the denaturation of protein prior to electrophoresis, this could have meant that a trace amount of protease was present in the final a-amylase preparations. However, as will be discussed later, no hydrolysis of the d-amylase occurred in the absence of SDS when calcium-free a-amylase was heated to 60 C in the thermal denaturation experiments. 3. If the purified protein was electrophoresed on polyacrylamide gels prepared by the method of Ingle (1968), five bands of protein could be separated (Figure 18). This method separates "native" pro- teins and leads one to conclude that the a-amylases, which have 69 Figure 16. Mobilities of protein standards in polyacrylamide gels prepared as in Figure 15. The mobility of a-amylase under these conditions was 0.556, relative to Pyronin Y. The standards used were: 1) bovine serum albumin, 2) ovalbumin, 3) horse liver alcohol dehydrogenase, 4) aldolase, 5) yeast alcohol dehydrogenase, 6) glyceraldehyde-3-phosphate dehydrogenase, 7) chymotrypsinogen, and 8) cytochrome c from horse heart. MOLECULAR WEIGHT I00,000 70 80,000 r 60,000 40,000 20,000 Pyronin Y dye I L l l I l #1 I0,000 0.4 0.6 0.8 I.O MOBILITY, RELATIVE TO PYRONIN Y Figure 16 71 Figure 17. Electrophoresis of d-amylase using the method of Laemmli (1970). Protein samples were prepared by adding first 8 fl urea and then the protein to a boiling solution of SDS, mercaptoethanol, and buffer. The solid line represents purified a—amylase from Betzes barley half-seeds, while the dashed line represents the soluble protein in barley malt amylase from Sigma. The gels were stained for protein and were scanned at 560 nm. 72 2.0 A560 I.0 -- --—-——-—-————— \2- DISTANCE, CM Figure 17 73 Figure 18. Polyacrylamide gel electrophoresis of a-amylase without SDS. Half—seeds were incubated as in Figure 18, then the a-amylase was purified and the protein components were separated by electrophoresis using non-denaturing conditions. of protein applied to each gel varied. A. S-FU. c. 10-2 gs-FU. The amount No S-FU. B. 10-4 91 74 I.O - 0.5 - top DISTANCE Figure 18 75 approximately the same molecular weight, differ in their net charges. The amylase in these polyacrylamide gels was still active after electrophoresis, as was shown by the following test. The gels were sliced with a razor blade, and each slice was placed in a petri plate containing a mixture of starch and agar. After 20 hr, the gel slices were removed, and the plates were flooded with an Iz/KI solution which turned the plates blue except for where a—amylase had diffused into the starch-agar and had hydrolyzed the starch. The region of the polyacrylamide gel which contained protein also contained a-amylase activity. However, the isozymes were not sufficiently separated to test each protein band individually for d-amylase activity. Removal of Carbohydrate After the a—amylase-glycogen complex was resuspended and incubated in Trisca buffer, it was no longer pelletable by centrifugation at 6000 g_for 10 min. However, the glycogen had probably not been completely hydrolyzed by the bound a-amylase. In later experiments with a-amylase from Himalaya barley seeds as little as 10% ethanol again precipitated the a-amylase from the supernatant solution as a carbohydrate-a-amylase complex, but 5% ethanol did not. The hydrolysis products of glycogen could not be removed from the solution by dialysis. Interestingly, glucose was not the only sugar residue present in this mixture. Gas-chromatographic analysis established the presence of arabinose residues, which may have originated from the aleurone cell walls (Jones and Albersheim, 1972), in addition to the glucose residues. The neutral carbohydrate polymers were removed by column chroma- tography using DEAF-cellulose. The carbohydrate was eluted from the 76 column in the first column volume of Trisca buffer. Following this, the enzyme was eluted with a gradient of sodium chloride in Trisca (Figure 4). The fractions with a-amylolytic activity did not react with the anthrone reagent. The Loyter and Schramm Purification Procedure Carlson (1972) purified barley d-amylase by the method of Loyter and Schramm (1962) designed for the purification of pancreatic a-amylase.* I attempted to use this procedure so that Carlson's and my methods of purification could be compared. A minor modification was necessary in the early stages of purification, because the barley a-amylase was secreted into a medium containing CaCl2 which led to precipitation of the phosphate buffer which Loyter and Schramm had used during homogenization of their tissue. The addition of 0.012 b_d_ phosphate buffer (pH 6.9), as required by Loyter and Schramm, was therefore insufficient to change the pH of the homogenate. Instead, dibasic potassium phosphate was used to titrate the homogenate to * An outline of the procedure of Loyter and Schramm (1962) is included here for comparison to the methods employed in this disser- tation. Commercial pancreatin (9 g) was homogenized in 40% ethanol containing 12 mg phosphate buffer, pH 7.9, 4 mg_NaCl, and 3 mg_ CaClZ, and the precipitate was removed. The pH was changed to 8.0, glycogen was added to bind the a-amylase, and the amylase-glycogen complex was pelleted by centrifugation. The pellet was resuspended in pH 6.9 buffer and incubated for 1 hr at 25 C. A small precipitate could be removed by centrifugation at pH 8.5 at 20 C. The a-amylase was precipitated at pH 7.0 by incubating the solution at 0 C for 30 min. The enzyme precipitate was resuspended in glycine-NaOH buffer (pH 10.0) and passed down a column of charcoal and celite which adsorbed contaminating carbohydrates. The eluted a-amylase was precipitated by acetone followed by ethanol-ether at -10 C. The enzyme was resuspended in distilled H20, the pH was changed to 6.5 with NH4OH, and a lipid precipitate was removed. After a second acetone and ethanol-ether precipitation, a phosphate-CaClz buffer (pH 6.8) was added and the enzyme, at a protein concentration of 3%, crystallized spontaneously within 2 days. 77 pH 6.9. This resulted in the formation of a large precipitate of calcium phosphate before pH 6.9 was attained. Certain problems arose later in the procedure for which I could find no solution, however. These problems involved (i) the difficulty of precipitating a-amylase from the resuspended glycogen-amylase mixture, (ii) the lability of the a-amylase in the pH-lO buffer used during chromatography on the charcoal-celite column, and (iii) the difficulty of removing a-amylase from the charcoal—celite column. With regard to point (i), the a-amylase which I had prepared could not be precipitated by adjusting the pH of the digested glycogen—amylase mixture to 7.0 and by placing the solution at 0 C for 30 min. Even when left at 0 C overnight, the a-amylase which I had prepared did not precipitate. This pre- sented certain difficulties in rapidly changing the buffering solution to the glycine-NaOH buffer (pH 10.0). As can be seen in Figure 6, d-amylase was quite unstable at pH 10.0. Forty percent of the enzyme activity was lost after 3 hr of incubation at that pH. The purified a-amylase was split into two samples, and the pH of each was adjusted to 10.0 by slightly different means. Sample A (see Materials and Methods) was applied to a short charcoal-celite column (1.8 cm wide x 1.4 cm long) immediately after its pH had been changed to 10.0. This sample contained 320 units of activity. An attempt to remove a-amylase from the charcoal column was made by the further addition of glycine-NaOH buffer (10 mg, pH 10.0) to the column. Fractions of 2 ml were collected at a flow rate of 1 m1 min-l. After five fractions were collected, the pH of each fraction was lowered to 7.0 by the addition of 0.2 §_acetic acid. Although Loyter and Schramm (1962) reported that 70% of the pancreatic a—amylase could be removed in the second and third column volumes of 78 buffer, the barley a—amylase was not removed by 50 column volumes. Ovalbumin was then added to the column in an attempt to displace competitively the a—amylase from the charcoal, but again no enzyme was eluted. Sample B (420 units) was applied to an even smaller column con- taining 0.5 ml of charcoal-celite. Again no a-amylase could be recovered, although 200 ml of buffer were used to wash the column. Increasing the ionic strength of the buffer lO-fold or adding Triton X-100 to the elution buffer were equally ineffective. These results precluded the possibility of a direct comparison between a-amylase as prepared by Carlson and a-amylase prepared by the method which I have reported (Method I). The a-amylase could not be precipitated at 0 C at pH 7.0, it was unstable at pH 10.0, the pH of the elution buffer from the charcoal column, and it could not be removed from the charcoal column. It is not altogether clear that the d-amylase purified by Carlson was a pure preparation of a-amylase. Although he indicated that aleurone layers were used for his studies, there is reason to believe that these layers were not completely free of starchy endo- sperm. It is likely that his final enzyme preparation was contaminated by proteins originating from the starchy endosperm (such as B-amylase) which would bind to glycogen. In one experiment, aleurone layers were isolated after 3 days of incubation on moist sea sand (Chrispeels and Varner, 1967). The layers were incubated for 24 hr on a shaker in Acca medium containing 1 uM GA Following this the a-amylase was purified by the Loyter 3. and Schramm method as far as the glycogen precipitation step and subsequent incubation to release the a-amylase. This preparation was 79 subjected to electrophoresis on polyacrylamide gels (Fairbanks method) and was found to contain only one protein band. Thus if Carlson was able to obtain very clean aleurone layers, it was possible that his enzyme preparation had been fairly pure. The purity of his final d-amylase preparation must therefore have depended on his ability to remove all of the starchy endosperm. Because I found it impossible to carry out the entire Loyter and Schramm purification procedure, I could not determine which of the a-amylase isozymes would have been released in the final steps of that method. Yield of a-Amylase During Purification The final recovery of d-amylase activity was 20-25% of the total activity in the original solution. A flow diagram is included to indicate the fate of the enzyme during the purification procedure (Figure 19). The major loss of activity occurred because of inacti- vation of some of the enzyme by the addition of ethanol. Some of the a-amylase was also not precipitated by glycogen, but much of that activity could be precipitated by addition of more glycogen. The two glycogen-amylase hydrolysates were combined, and the solu- tion was applied to a DEAE—cellulose column. Two peaks of activity were eluted, the first containing 22% and the second 10% of the original activity. In order to determine the effect of removing the calcium from the enzyme in each peak, the fractions constituting each peak were concentrated and chromatographed separately on a column of Sephadex G-25 equilibrated in l mM_EGTA and 5 mM_Tris at pH 8.0. The solutions were recovered with yields of 19% and 9%, respectively, and a combined yield of 28%. Normally, the a-amylase- containing fractions were combined after the DEAR-cellulose chroma- tography step. 80 Figure 19. Flow diagram for the purification of a-amylase. The a-amylase was purified by Method I. Some additional steps are included to test procedures during which the enzyme activity might be lost. The numbers under many of the entries refer to the percent of the original amylase activity which was repre- sented in that fraction. Footnotes are included under the diagram. 81 CRUDE HOMOGENATE AND MEDIUM 100 I Step 2, pH 8.0 .l . inactive 9.5 l pellet + inactive F ’1 pellet SUPERNATANT FRACTION 0.1 90.5 Step 3, 70 1 SUPERNATANT FRACTION 7.1 83.3 I Step 4. 40% EtOH I 1 pellet SUPERNATANT FRACTION inactive 1.4 55.3 28.0 I Step 5, glycogen precipitation I ' . I . SUPERNATANT FRACTION PELLET Inactive more glycogena re-precipitationb I l l PELLET supernatant supernatant PELLET fraction fraction 1.8 6.4 . . . b re-prec1pitation L l l PELLET supernatant fraction 0.8 Step 5, hydrolysis Step 5, hydrolysis L j I T I pellet SUPERNATANT SUPERNATANT pellet + FRACTION FRACTION + inactiveC 8.7 27.5 inactiveC .___P O O L E D___J 36.2 Figure 19 82 POOLE D (36.l2) Step 6, DEAE-Cellulose I ' I PEAK I (lost) PEAK II 22.4 3.5 10.3 Step 7, concentration Step 7, concentration 27.3 10.3 Step 8, Sephadex G-25 Step 8, Sephadex G—25 I ‘ . inactive VOID VOLUME VOID VOLUME inactive 3.0 19.3 9.1 1.2 FINAL YIELD—'— 2 8 . 4 % aAn equivalent amount of glycogen (as at Step 5) was added a second time. bThe glycogen/ amylase complex was suspended in Trisca for 1 to 2 min, then ethanol was added until it reached a concentration of 40% . CTogether, these must account for 10.0% of the total. quuacide II was used. Figure 19, continued 83 2E§pzyme Recovery It was important to establish whether any of the d-amylase iso- Srymes might have been lost during the purification of the enzyme. cx—Amylase was purified from Betzes barley, following Method I, and ‘the isozymes were separated by electrophoresis on agar slab gels (Figure 20). The presence of the various isozymes was monitored at each step during the purification. At each step the pattern of isozymes was essentially unchanged, except that electrophoresis of the original solution did not separate the isozymes as clearly as did electrophoresis of the less crude preparations. The preparations routinely examined were (a) the original, crude homogenate, (b) the solution after titration to pH 8.0, (c) the solution after heat treatment at 70 C for 15 min, (d) the solu- tion after DEAF—cellulose chromatography, and occasionally (e) the solution after the removal of calcium. The a-amylase activity on the agar gels separated into six bands. The active regions were those which remained clear after incubation of the slab with a starch solution prior to staining the gel with IZKI. The original solution also contained three or four additional bands which moved more rapidly toward the cathode and which were suspected to be B-amylases on the basis of their incomplete hydrolysis of the starch substrate, their disappearance after heat treatment at 70 C, and the fact that their mobilities were close to those of B-amylases from Himalaya barley (Jacobsen et a1., 1970). The a-amylases of peak I from the DEAE-cellulose chromatography migrated the farthest and those of Peak II migrated more slowly during the electrophoresis. 84 Figure 20. Separation of a-amylase on agar slab gels. After electrophoresis, the gels were stained for a-amylase activity. Top: diagrammatic sketch; zones 1-6 are a-amylases, zones 7-10 are believed to be B-amylases. (A) original mixture of homogenate plus medium, (B) purified a-amylase. Bottom: photographs of actual slab gels; I. The original mixture of medium and homogenate, left to right are alternating pairs of control and l mM_5-FU treat- ments at increasing protein concentrations; II. After pH 8.0 treatment, with better resolution of zones 7 to 10; left to right are control (at two concentrations) and 1 mg 5-FU (two concentrations); III. Purified a—amylase, left to right are four samples of control then four of l mM_5-FU. The control is without S-FU treatment. 85 r—'——‘| ORIGIN o o. .o u s. s n o . a. u n o o. . c n- o. n o- .o n a. u u o. 86 Effects of 5-FU on Molecular Weight and Isozyme Content of a-Amylase Inolecular Weight Half-seeds were imbibed with concentrations of S-FU between -5 -2 . -6 10 and 10 M, after Wthh 10 _IV_I_GA3 was added to the S-FU— containing solutions for 36 hr. The a-amylases which were subse- quently purified had the same molecular weights, and no enzyme with altered molecular weight was generated. Isozyme Composition Half—seeds were imbibed as in Figure 1A or 18 using zero, 10.-4 M, or 10-3 M_5-FU. The isozymes were separated in agar slab gels. Because two of the isozymes predominated in the mixture, several different dilutions of the a—amylase had to be examined for the real pattern of isozymes to be understood. S-Fluorouracil was found to have no effect on the pattern of isozymes present in the crude homogenate, in the solutions at subsequent purification steps, or in the final solutions after the removal of calcium (Figure 20). In addition the amylase was separated into isozymes and stained for protein with Coomassie Brilliant Blue R after electrophoresis on acrylamide gels according to Ingle's method (Figure 18). In one case the half-seeds were incubated without 5-FU and in the others in the presence of 10"4 M or 10_2 M_5-FU. The relative optical densities of the protein bands appeared to be identical despite the S-FU treat- ment. This is of some significance because the isozyme activities on the agar gels only present a qualitative measure of the relative enzyme activities, while staining with Coomassie Blue dye indicates in a more quantitative way that the amounts of protein in each band 87 xnaintained the same ratio, despite the S-FU treatment. This supports the conclusion that S-FU had no effect on the composition of isozymes which were recovered after purification of the a-amylase. Recovery of Activity_and Specific Activigy of d-Amylase During Purification Half-seeds were incubated as in Figure 18 in buffer without S-FU or with S-FU at concentrations between 10_5 and 10.2 M, These con- centrations of S-FU had had little effect on the amount of a-amylase which was synthesized by the half—seeds (Figure 118). The homogenate and medium from each of the S-FU treatments were pooled and the recovery and specific activity of the a-amylase was determined at each purification step. The amount of a-amylase activity recovered from each S-FU treatment at each purification step is shown in Figure 21. Thus, the percent recovery after glycogen precipitation, for example, has been calculated as the activity after precipitation divided by the activity before the precipitation step. S-Fluorouracil had little effect on the recovery of a-amylase during the purification of the enzyme. Changing the pH of the original homogenate to pH 8.0 may have the greatest differential effect on the a—amylase activities. However, because only one sample (of 75 half—seeds) was used for each determination, it is difficult to conclude whether these differences were significant. The effect of the 5-FU treatments on the specific activities of the enzyme is shown in Figure 22. It should be recalled that, except for the case of the most purified a-amylase, differences in specific activity may be based on differences in the amount of contaminating protein present rather than in any changes in the amount of a-amylase . . . —3 activ1ty recovered. At concentrations of S—FU greater than 10 M, 88 .maa musmflm CA omucmmmumon Hafiuwums onu Eoum pwuomaaoo wmonu mums mcofiudaom Hosamfluo one .ooa >n ooflamfluasa mmum m50H> Iona ozu mo cam mnu um >ua>fiuom mzu an poofi>flc mmum on» NO cam wnu um >ua>wuum man no touMHSUHmo mm3 Amumcflouov coflumoflwfiusm wnu ca ommum Hmzofl>flocfi comm um mmmamsolo mo >Hw>oomu ucmowmm mze .coflumoflwflusm mcfluso >ue>fluom mmdaxfimIo mo >um>oomw on» so ucoeummuu DmIm mo uommmm .Hm musmflm 89 Hm musmsm .2. .3th ~50. «YO. v-0. VOICJ 0 53520685 32230 930 .O . 00 o\o o o J 00. O O .2. ARE .0_ .0. .0_ T0 0 N . m . s . I.) . om axe o o O O O 011 . 00. .EE 9 .0 .0» .m 5.3.73 N-0_ m0. 70. may 0 oIOIOIIIIOIOIILI O O . OD o\o . 00. 8:25.85 50320 .0 2.31-2 ~.0_ m.0_ v.0. 90.1 0 d d 4 4 NJ .. On oxo 9|? In 0 . 8. 0.0 In 3 0955 Ia .< 90 Figure 22. Specific activities of a-amylase at each stage of purification. The d-amylase collected from the half-seeds repre- sented in Figure 118 was purified and the specific activity of the d-amylase was determined as a function of the stage of puri- fication. BSA was used as a protein standard. For the first three curves, the protein sample was chromatographed on a cellulose thin layer plate before the protein assay was performed. Crude homogenate and medium, A; after pH change to pH 8.0, x; after 70 C treatment, 0; after glycogen precipitation and hydrolysis, C]; after DEAF-cellulose chromatography, 0. 91 so. x 56E 3 Es €22 omit/$2-8 Lo >t>po< omomam o. O n .m nu nu ..s o n .m 4 s .o o 6 O D 1m o. s. Tlv E is LIV - _ PI! - by — p p O m. m m m w m 4 s 2 .. o S-FU CONCENTRATION, M Figure 22 92 the final specific activity was less than that of the control a-amylase. Again, this may or may not be significant, but it may correlate with the lower recovery of a-amylase after the change of pH to 8.0 as seen in Figure 21, or the small decrease in the amount of a-amylase synthesized at the higher concentrations of S-FU. By the methods used to separate and identify the a—amylase isozymes, however, there was no indication that such differences, if real, were caused by the loss of a particular isozyme. The Thermal Denaturation Process: General Aspects Effect of Calcium When calcium was present in the crude homogenate, the enzyme was not inactivated by heating it at 70 C for 15 min if the pH of the solution had been adjusted to 8.0. The purified enzyme was also stabilized by the presence of calcium in the solution (Figure 23). If calcium was removed from the solution by passing the solution over a Bio-Gel P-30 column equilibrated in 1 mM EGTA and 5 mM Tris (pH 8.0), the a-amylase lost 99% of its activity after incubation for 20 min at 54 C. However, addition of calcium to a final concen- tration of 0.33 M prevented the inactivation of the enzyme during the 40—min test period. For this reason, the thermal denaturation process could be stopped by the addition of the heated sample to one-half of that volume of l M CaCl in an ice-chilled vial. The 2 high concentration of CaCl did not itself inactivate the a-amylase 2 but had the added advantage of rapidly binding to any EGTA present in the final solution. Since the thermal inactivation process exhibited log-linear decay kinetics over as much as four decades, I 93 Figure 23. Effect of calcium on the rate of thermal denatura- tion of purified a—amylase. a—Amylase was heated at 54 C in the presence or absence of 0.33 M CaC12. The a-amylase was purified by Method I. Calcium was removed with the aid of a Bio-Gel P-30 column equilibrated in 1 mM EGTA with 5 mM Tris, pH 8.0, and the sample was concentrated using Aquacide II. Control (no calcium), at 224 units per ml, 0; 0.33 M CaClz, at 335 units per m1, 0. 94 o—lfi—o O 02_2_F_>_._.U< wm_2<-d .rzmo mum b O O H J 40 25’ TIME (MIN) Figure 23 95 have assumed that calcium was effectively removed from the a-amylase by the EGTA de-salting column. Effect of Protein Concentration The rate of thermal inactivation of a-amylase should be inde- pendent of the concentration of the enzyme during the heat treatment at low concentrations of enzyme. If the decay rate was dependent on the concentration of enzyme, one would suspect that a second, "non-amylase" component was affecting the denaturation process. Table 2 records the effect of diluting the enzyme solution with buffer on the amount of a-amylase activity remaining after 5 min of heating at 56 C. In the range of 50 to 500 enzyme units per m1, no significant effect of protein concentration on the degree of thermal inactivation was observed. Test for Protease Activity The removal of calcium from the a-amylase also makes the enzyme highly susceptible to hydrolysis by protease. Thus, it was necessary to examine whether a-amylase was hydrolyzed by traces of proteases present during the thermal denaturation treatment. If proteolysis had occurred, the apparent inactivation rate would be dependent on the activity of the protease and not the structure of the a-amylase. Samples of calcium-free a—amylase were subjected to SDS-polyacrylamide gel electrophoresis (Fairbanks' method). The samples were examined before heating, or after 99% of the enzyme activity had been destroyed. The electrophoretic properties of the protein remained unchanged, indicating that no hydrolysis of the a—amylase had occurred during heat inactivation, and that the inactivation rate must have been 96 Table 2. Effect of protein concentration on the thermal denaturation of d-amylase Initial Concentration, Activity Remaining, Treatmenta units/ ml % no S-FU 503 9.01 402 8.31 302 9.07 201 8.66 101 8.89 50 8.76 10'4 g S-FU 493 8.97 394 8.71 296 9.14 197 8.93 98.6 9.16 49.3 8.63 aHalf—seeds were incubated as in Figure 1A. After calcium'was removed from the purified a-amylase, the enzyme was heated at 56 C for 5 min, and the remaining activity was determined. 97 dependent on heat-induced structural changes in the a—amylase molecules themselves. Effect of S—FU on the Thermal Stability of a-Amylase The kinetics of thermal inactivation of a-amylase from tissues incubated either in the presence or in the absence of S-FU are shown in Figure 24. For Figure 24A, half—seeds were incubated as in Figure 1A using 10"4 M_5-FU. These conditions were the same as those under which Carlson obtained a-amylase with the maximum decrease in thermal stability. It is clear that S—FU had no effect on the thermal sta- bility of the a-amylase. A second such experiment is shown in Figure 24B. This experiment differs from that of 24A in that a higher denaturation temperature (60 C as compared to 54 C) was used. In Figure 24C, a similar experiment is shown, except that the concentra- tion of 5-FU had been raised to 10—2 M, In none of these experiments had the treatment of the half-seeds with 5-FU affected the thermal stability of the a-amylase. These results disagree with those reported by Carlson. Closer examination of Figure 24C shows that the denaturation rates of both the control and the 5-FU samples was more rapid than those of a comparable experiment shown in Figure 24B. The Aquacide II which was used to concentrate the samples was later found to have decreased the pH of the enzyme solutions, resulting in more rapid rates of thermal decay. Thermal stability determinations were performed six times, using six separate preparations of a-amylases from S-FU—treated half-seeds. Along with each a-amylase sample from S-FU-treated tissue, a sample of a-amylase from control tissue was purified in parallel. I‘ 98 .0 .UmumoHuIDhIm “0 .ADmIm ocv Houucoo am NIOH mm3 ucoafiummxm was» won poms coflumuucoocoo Dmum one .0 oo um ooEHOMHmm mm: cowumusumcwo Hashmca .o>onm Adv 2H mm omcfimuno mm3 mmMaasdua .0 am vIoa mos coHumuucmocoo DmIm on» one 0 cm mm3 wusumummfimu coflumnsumcmo one .wmmnu Eoum omaoom mm3 mmmH>EMId on» can .omEHonmm mama mmmum cofiumufimflomum cmmooxam ozu umnu ammoxm .m>onm Adv cw mm mommmumamn Scum omummmum mm3 mmmaafimlo .m um VIOH mm3 coflumuu Icoocoo Dmum map 0cm 0 em mm3 ounumummsmu cowumuswmcoo one .Umuwafius mm3 HH mcfiumswm .mmoum coflumuucmocoo on» cH ._mm >.:>:.0< mm_>_<-0 H2 0. m0 mud Figure 25 DISCUSSION Suitability of the a-Amylase Preparation for Studies of Thermal Inactivation Kinetics The report by Carlson that a-amylase was rendered heat labile by the inclusion of S-FU in the incubation medium for barley aleurone tissue (Carlson, 1972) has not been supported by this study. If S—FU was able to affect the synthesis of proteins other than or in addition to d—amylase, artifacts could have been introduced which could have affected the analysis of the thermal stability character- istics of this isozymic enzyme system. Certain isozymes which may have been altered as a result of S—FU treatment could have been lost during the purification of the a-amylase. This could have produced artificial differences in the kinetics of thermal inactivation if the isozymes had had different thermal stability properties. Alterna- tively, the co-purification of a second factor, which was present in different amounts in each solution of "purified" a—amylase, could have influenced the kinetics of thermal inactivation. It was there- fore necessary to determine as closely as possible that isozymes of a-amylase had not been lost during the purification and that no factors had co-purified with the enzyme which would have affected its thermal stability. The a-amylase synthesized by Betzes barley aleurone tissue was found to be heterogeneous, six isozyme species having been separated. During electrophoresis on SDS-polyacrylamide gels, the isozymes 103 104 co-migrated as one band of protein corresponding to a molecular weight of 41,400 daltons. A faint trace of a second component of 21,000 daltons was sometimes visible after the stained SDS-gels were stored for a long time. In the absence of calcium at least 99% of the purified enzyme was heat labile while in the presence of calcium the enzyme was stable to the same heat treatment (Figure 23). The isozyme composition of the a-amylase was monitored through- out the purification process and was found to be unaltered as a result of purification or removal of calcium from the enzyme. The base analog S-FU, which had little or no effect on a-amylase synthesis or secretion by the aleurone tissue, had no effect on the isozyme composition of the a-amylase, its molecular weight, or its purity. Thus, the purification method which I selected for obtaining a-amylase must be considered to be at least as effective as that which was employed by Carlson (1972), although Carlson did not char- acterize the a-amylase purified by his procedures, and a direct comparison of the properties of our preparations cannot be made. I was unable to purify a-amylase using the method which Carlson had employed (Loyter and Schramm, 1962). The a-amylase did not pre- cipitate spontaneously in the cold at pH 7.0, it was unstable at pH 10.0, the pH of the charcoal column chromatography, and it bound irreversibly to or was inactivated by the charcoal. Although the a—amylase had to be purified by an alternative procedure, attempts were made to simulate Carlson's incubation conditions. Thus, the half-seeds were soaked by a batch method for one day before they were incubated in flasks on a shaker, and Betzes barley seeds were used instead of the more commonly studied Himalaya variety. In addition, 105 the aleurone tissue was homogenized and the homogenate was added to the medium (plus the extract of the starchy endosperm) before puri- fication was begun. Because Carlson did not heat-treat his original extract in the presence of calcium, it is probable that other proteins such as B-amylases were present in his preparations of purified a-amylase. This suggestion is made because aleurone layers cannot be scraped clean of starchy endosperm after one day of incubation in H20 or buffer. Such proteins originating from the endosperm may have appeared to represent a second population of molecules with the capability of hydrolyzing starch, or they may have affected the rate of inactivation of the calcium-free a-amylase during the thermal denaturation experiments. The identification of the isozymes of a-amylase which retained their activity after purification and removal of calcium was not a trivial requirement. Jacobsen et al. (1970) have shown that there are two types of a—amylases synthesized by Himalaya barley. One type was strongly affected by GA3-treatment (a 12.1-fold increase), was unstable at pH 3.7 and bound calcium only weakly. The second type of a-amylase also produced amylose from starch, but it was stable at pH 3.7 and it tightly bound calcium. Unlike the first group, the second group of isozymes was less affected by 6A3, being increased only 2.6-fold by GAB-treatment. The a-amylases of Betzes barley undoubtedly exhibit such poly- morphic behavior as well, leading Chandra (1974) to presume that Carlson (1972) may have only been able to remove the calcium from the isozymes which weakly bound the ion. The incomplete removal of calcium may have generated biphasic decay curves. Alternatively, 106 the calcium—free a-amylases in Carlson's solutions may have been partially inactivated before the thermal denaturation experiments were begun, or the charcoal-binding step may have selected for certain isozymes, again giving rise to a series of biphasic decay curves. It is easy to see that the thermal decay kinetics could depend on the relative proportions of the active isozymes which were present in each solution if traces of calcium were present. To establish further that the a-amylases which I had purified were of sufficient purity for the thermal denaturation experiments, the thermal inactivation reaction was shown to occur as if it was a monomolecular reaction since the concentration of a—amylase, at concentrations of protein below 50 ug (BSA equivalents) per ml, had no effect upon the rate of the inactivation. In addition, the inac- tivation of a-amylase during heating was not due to proteolysis. From the evidence described in this study no conclusion can be drawn concerning the timing of a-amylase mRNA synthesis. This is contrary to the conclusion of Carlson (1972) who, based on similar experiments with S—FU, proposed that the<1-amylase in RNA was pro- duced both in the presence and absence of 6A3. Use of S-FU to Time the Synthesis of a-Amylase mRNA Since treatment of the half-seeds with 5-FU did not cause the formation of a more thermolabile population of a-amylase molecules, one cannot make any conclusions concerning the timing of a—amylase mRNA synthesis in barley aleurone layers from the data which have been presented here. a-Amylase mRNA may have been produced before imbibition or, equally as likely, 5-FU may have been incorporated into nascent mRNA during the course of these experiments; but the 107 presence of substitutions of S—FU in the mRNA may not have led to amino acid substitutions in the a—amylase. S-Fluorouracil was also found to inhibit the synthesis of rRNA and tRNA in barley aleurone tissue without affecting the synthesis or release of a-amylase in response to GA3. The synthesis of new species of rRNA and tRNA is therefore not required for the synthesis or release of a-amylase. These results agree with those of Jacobsen and Zwar (1974) that 15 mM S-FU inhibited the synthesis of rRNA (90%) and tRNA (over 50%) by barley aleurone tissue (cv. Himalaya) during the first 16 hr of GA3-treatment with no concomitant inhibition of a-amylase synthesis. In a rat liver-system l4C-S- fluoroorotate (5-FO) was used as a more efficient precursor of S—fluorouracil ribosylmonophosphate in RNA than S-FU. The 14C-S-FO was incorporated into 458 RNA (r-preRNA) but not into 288 or 185 RNA (Wilkinson et al., 1971). In addition, S—FU was shown to have no effect on the transcription or methylation of 45S RNA in rat liver (Wilkinson and Pitot, 1973). Therefore, S-FU apparently affects the last steps of rRNA processing rather than inhibiting the synthesis of the original r—preRNA transcript. Radioactively labeled S-FU is also incorporated into polysomal mRNA of HeLa cells (Gleason and Fraenkel—Conrat, 1976), TMV-RNA in tobacco leaves (Mandel, 1969), and D-RNA from soybean (Key, 1966). In fact, the use of 5-FO has been proposed as a method to label mammalian mRNA specifically (Wilkinson et al., 1971). In barley aleurone tissue, 1 mM_S—FU had no effect on the labeling of 58 to 14S RNA by 3H-adenosine (Zwar and Jacobsen, 1972), indicating that S-FU does not inhibit the synthesis of mRNA in this system. ._-__—-—g 108 No conclusive evidence has been obtained from studies of eukaryotes which would prove that the incorporation of S-FU is responsible for the substitution of amino acids in the polypeptides of specific proteins. It has been hypothesized that the substitution of fluorine in the 5-position of uracil affects the ratio of the keto and enol forms of the pyrmidine by decreasing the pK of the pyrimidine in RNA in viva, since poly (U) has a pK of 9.8 while poly(S-FU) has a pK of 8.1 (Mandel, 1969) . The protein-synthesizing capacity of poly (U) and poly(S-FU) has been examined in vitro with a protein-synthesizing 1 system from wheat germ. Poly(S-FU) was translated less efficiently than poly(U) , but the S-FU polymer was read exclusively as if it were a poly (U) message (Mandel, 1969). In contrast, the substitution of S-FC in poly (C) elicited some unexpected changes in the amino- acid composition of the resultant polypeptides, indicating that S-FC may have occasionally been read as U, and it was hypothesized that the infrequent converstion of 5-FU to S-FC in vivo causes the muta- genic effects currently attributed to S-FU (Gleason and Fraenkel- Conrat, 1976) . In some instances, treatment of bacterial cultures with S-FU has Produced phenocopies of the bacteria or of the bacteriophage infecting them. Such cases have provided the only unambiguous demon- Stra.tions that 5-FU can alter the phenotype of an organism by being incorp<>rated into messenger or messenger-like RNA. Growth of Escherichia coli on S-FU, for example, was effective in reversing the Phenotypes of 17 out of 339 mutants of T bacteriophage, the 4 original mutations of which had arisen by the change of a single base in the viral RNA (Champe and Benzer, 1962). S-Fluorouracil was also able to induce amber (nonsense) mutations in the RNA coding for 109 the coat protein of R17 bacteriophage, and these mutations could be suppressed if the S—FU-mutants were subsequently grown on suppressor strains of the host (Tooze and Weber, 1967). Thus, a precedent for 'the induction of phenocopies by S-FU treatment has been established .in bacterial viruses, and the absence of such examples in eukaryotes rnay simply derive from the lack of well-defined eukaryotic systems 143 which the hypothesis can be tested. The results summarized here iaudicate that the induction of d—amylase synthesis by GA3 in barley aileurone tissue does not provide a suitable eukaryotic system for I such studies. SECTION II I NTRODUCT ION At least two carbohydrases are involved in the hydrolysis of starch reserves in the endosperm of barley grains. These are the a-amylases (E.C. 3.2.1.1) and the B-amylases (E.C. 3.2.1.2). The a—amylases randomly cleave the a-l,4-glucosyl linkages and are thus "endo"-enzymes. The reducing ends of their hydrolysis products are in the alpha configuration. a-Amylases are synthesized by the living aleurone layer in response to gibberellin, which originates in the germinating embryo (McLeod and Palmer, 1967) . The embryo can be replaced by exogenous gibberellic acid (GA3) which stimulates the synthesis and/or release of a number of hydrolases, including d—amylase, from isolated aleurone tissue (Yomo and Varner, 1966) . d-Amylase constitutes the major protein synthesized in response to GA3. The B-amylases, on the other hand, cleave maltose (disaccharide) units sequentially from the non-reducing ends of a-l,4-glucans. They are thus "exo"-enzymes, yielding maltose units which are in the beta configuration. In wheat grains, B-amylases are synthesized during the maturation of the grain, but remain inactive until the embryo germine-ates. The B—amylases can be activated prematurely by incuba- tion of the starchy endosperm with proteolytic enzymes or sulfhydryl reagents (Rowsell and Goad, 1962); in the germinating grain it seems likely that the proteases, which are synthesized by the aleurone tissue, are responsible for activation of B—amylases. 110 111 The barley aleurone system has been chosen as a model system for studying the action of gibberellic acid (Varner, 1964 and references therein). Since the synthesis and secretion of a-amylase are major events which are controlled by GA3, it would be useful to know the primary structure of the a-amylases from barley grains. Because many glycoproteins are destined for secretion or are sequestered from the ground cytoplasm of the cell (Winterburn and Phelps, 1972) , I was particularly interested in determining whether the a-amylases svere also glycoproteins. Perhaps the presence of a carbohydrate moiety could partially account for the remarkable stability of a-amylases to protease (Jacobsen and Varner, 1967) and heat (Kneen et al., 1943) in the presence of calcium. Such a study is complicated try the fact that the a-amylases from different varieties of barley ginains are electrophoretically heterogeneous (Jacobsen et al., 1970; lanrdenberg and Nielsen, 1965; Bdg-Hansen and Daussant, 1974; Tanaka and Akayawa, 1970) and that at least two antigenically different tYpes of (II-amylase are produced in those systems where antibody sPecificity has been studied (Jacobsen and Knox, 1973; Daussant et a].-,, 1974). With this underlying heterogeneity in mind, the average aHULruo acid and carbohydrate composition of the a-amylase secreted by barley grains has been analyzed and is described in the following. MATERIALS AND METHODS Incubation of Grains for a-Amylase Purification Barley seeds (Hordeum vulgare L. cv. Himalaya) were obtained from the Agronomy Club at Washington State University in Pullman, Vflashington, USA. One hundred sixty grams of the grains were surface sterilized by partially evacuating them for 30 min in 1% sodium luypochlorite in a stoppered flask which was attached to a water easpirator. The grains were rinsed several times with sterile, (distilled H20 until 2 liters of H20 had been used. The grains were (listributed into five l-liter flasks containing 200 ml of incubation Ineuiium each, and were incubated for 4.5 days in the dark. The huaisley grains were aerated by shaking the flasks on a rotary shaker. Tile: incubation medium contained 10 mM succinate buffer (pH 5.3), 0.1% Chloramphenicol, and 10 11M GA At the end of the 20 mM CaCl 3. 2’ incubation period, some roots had emerged from the seed, and the maximum length of these was about 6 mm. Purification of d-Amylase After incubation of the grains, the medium was collected by deC’I'Su'itation. The grains were further incubated for 30 min with an additional 100 ml per flask of 1 mM sodium acetate buffer, pH 4.8, The high concentration of CaCl was used to wash With 200 mM CaCl 2 2. adsorbed (II-amylase from the starchy endosperm and cell walls of the aleurone layer (Varner and Mense, 1972) . This wash was added to the 112 113 original medium. The grains were subsequently pressed with a pestle in a mortar to remove the starchy endosperm from the aleurone layer. The aleurone and extruded starch were then combined with the medium and wash, and l M CaCl2 was added to yield a final concentration of 200 mM. The aleurone, starch, medium, and wash were incubated for another 2 hr at room temperature on a reciprocating shaker. At this point, the extract contained intact aleurone tissue, whole embryos, and the diluted components of the starchy endosperm. The extract was filtered through cheesecloth and then through glass wool, and the filtrate was centrifuged at 5900 g for 20 min. The supernatant solution contained about 200,000 units of a-amylase activity (see Materials and Methods of Part I of this dissertation for a descrip- tion of the assay of a-amylase and the definition of III-amylase units). The cloudy supernatant solution was adjusted to pH 8.0 by the addition of 0.1 M tris base, and was centrifuged at 5900 g for 30 min in a refrigerated centrifuge (0 to 4 C). The resultant supernatant fraCtion was heated in two 2-liter flasks to 70 C. The total heating time was 20 min, about 7 min being required to reach 70 C. At 53 C, a Precipitate of denatured protein began to form. After heating, the precipitate was removed by centrifugation at 5900 g for 30 min. F01-lowing this treatment, about 150,000 units of a-amylase remained in the supernatant solution. Cold ethanol was added to the soluble fraction to give a final ConCentration of 40%, and this solution was stored in the cold over- night- The mixture was subsequently centrifuged at 6900 g for 30 min, and the pellet was discarded. Deproteinized glycogen (1.6%) was added to the cold supernatant fraction in the proportion of 1 I11 per 7 units of (II-amylase. At the end of 18 min of constant stirring, 114 the solution was centrifuged at 5900 g for 30 min. The supernatant solution was removed, and the pellets were suspended in a buffer containing 5 mM tris, pH 8.0, and 5 mM CaClz. A total of 12 ml were used to collect the d-amylase-glycogen complex. . 4 Preparation of l C—Labeled a-Agylase from Aleurone Layers Aleurone layers (200) were separated from barley grains which had been de-embryonated and imbibed under sterile conditions on sand for 3 days (Chrispeels and Varner, 1967). The aleurone layers were transferred to four 125-ml flasks containing 8 ml each of 20 mM succinate buffer (pH 5.3), 0.2 M CaClz, 0.1% Chloramphenicol, l 1.1M 6A3, and 50 uCi l4C-(U)L-leucine (286 mCi/mmole). The aleurone layers were incubated at 25 C for 24 hr on a reciprocating shaker. The medium was decanted, and diluted to 50 ml with 0.2 M CaClZ. The pH was adjusted to 8.0 with 0.1 M tris base, and the solution was heated to 70 C for 20 min. The solution was centrifuged at 13: 000 g for 5 min, and the precipitate was discarded. Cold ethanol was added to a final concentration of 35%, and after 1 hr of incuba- tiOri in the cold the solution was centrifuged at 13,000 g for 10 min. One milliliter of 1.6% glycogen was added, and the glycogen-amylase complex was removed by a second centrifugation. The pellet was resu3pended in 1 ml of 5 mM tris-HCl buffer (pH 8.0) with 5 mM CaC12. Fj-fteen hundred units of (II-amylase were recovered. Incubation of Grains on Agar In order to estimate the time required for barley grains to Sy“thesize and release a maximal amount of a-amylase, the grains were incubated on agar for different periods of time, and the distribution 0 - O I I I O I f a amylases in the tissues was determined. Grains were nicked with 115 a scalpel blade at the end distal to the embryo, surface sterilized with 1% sodium hypochlorite, and rinsed with sterile distilled H20. The grains were placed on 2% agar and were incubated at 27 C in the dark. At the designated times, grains were slit open and the starchy endosperm tissue was removed and ground in 1 m1 of 0.2 M CaClZ. To this homogenate was added four l-ml H20 washes of the aleurone layers and two l-ml H O washes of the portions of the embryo still in the seeds. All of the amylase collected by this method was termed "released" amylase. The agar was not assayed for a-amylase activity. The HZO-washed aleurone layers were briefly rinsed three times in 10 m1 of distilled H20 and were incubated twice successively for 5 min in 2-m1 portions of 0.2 M CaClz. The (II—amylase thus released was termed "cell-wall" amylase. The aleurone layers were further incu- bated for 10 min in 1 mM HCl to inactivate the remaining a-amylase, were rinsed three times in 10 m1 of distilled H20, and the layers Were homogenized with a mortar and pestle and acid-washed sea sand in 1 m1 of 1 mM sodium acetate buffer, pH 4.8, containing 0.2 M CaClZ. Three l-ml washes (same buffer solution) of the mortar and PeStle were added to the homogenate. This amylase activity was termed "tissue" amylase. Polyacrylamide Gel Electrophoresis Proteins were separated electrophoretically by SDS—polyacrylamide gel electrophoresis. The method of Fairbanks et al. (1971) was used With the slight modifications described in Part I of this disserta- tion- For analytical separations, 8 to 10 units of (II—amylase activity were used per gel. For preparative separations, about 100 units of aTamYlase or up to 400 pg of total proteins were added per gel. The .—_ 116 large amount of protein present in the preparative gels caused the proteins to have increased mobility. Thus, attempts to localize proteins in unstained gels on the basis of the relative mobility of the proteins after analytical separation was not reliable. Instead, the proteins in preparative gels were localized directly by a careful visual examination made possible by the refractile nature of the protein in the gel at the leading edge of the protein band. Protein was eluted from the gels in the following way. Portions of the gels which contained the protein in question were excised and all gel pieces containing the same protein band were placed in line in an empty gel tube. Up to three segments were placed in each tube. In order to exclude bubbles, a drop of electrOphoresis buffer was placed between the gel pieces. The gel pieces were pushed to the very bottom of the gel tube. A lO-cm piece of dialysis tubing was closed at one end with a dialysis bag clip (National Scientific, Cleveland, OH, USA), 1 ml of electrophoresis buffer was pipetted into it, and the gel tube was gently slid to the bottom of the dialysis bag. A rubber band was used to fasten the upper portion of the bag to the gel tube. The rubber band was wrapped around the bag and tube about 3 cm above the closed end of the dialysis bag. Buffer was added to the lower reservoir of the electrophoresis apparatus to a height equal to that of the buffer in the dialysis bag when the tubes were in place. Next, buffer was added to the upper reservoir, and 50 ul of the buffer-tracker solution was placed on top of each gel. The tracker dye was pyronin Y. Up to 100 volts were applied to the total system for twice the time required for the tracker to be eluted from the end of the gel. (The mobility of a-amylase relative to the tracker dye is greater than 0.5). 117 Gels were stained for protein using Coomassie Brilliant Blue R250 and were scanned at 560 nm. Radioactivity in the gels was determined by cutting the gels into l-cm pieces, drying the pieces under a heat lamp, and combusting them using a Packard Tri-Carb Sample Oxidizer (Model 306). StainingMof Gels for Carbohydrate The SDS-polyacrylamide gels were stained for carbohydrate by the method of Fairbanks (1971) using a periodate-Schiff reaction or by the method of Eckhardt et al. (1976) which is based on the transfer of a dansyl group from dansyl hydrazine to periodate-oxidized carbo- hydrates. The gels which were stained with the Schiff reagent were scanned spectrophotometrically at 560 nm. Gels which were exposed to the dansyl hydrazine were photographed using a 350 nm light source, a Whatman 2D filter and Kodak "Kodalith" film. The light source (four black-light tubes) was held 12 to 14 inches from the gels and the film was exposed for 10 min at f/5.6. Isoelectric Focusing Isoelectric focusing was accomplished in a preparative column containing 110 m1 of liquid. A gradient was formed in the column by mixing 48.5 g glycerol, 14.25 9 H20 and 2.25 ml of a 40% stock of Ampholines (LKB) with a solution of 0.75 ml of the Ampholine stock plus the sample in a total volume of 55 ml (H 0 was used to adjust 2 the volume). The sample initially contained 22,000 units of a-amylase activity; 21% of that activity was recovered after focusing. The column was cooled at 8 C, the initial voltage was 600 volts, and 2.5 days were allowed for focusing to be completed. The pH of each fraction subsequently collected was measured by equilibrating the 118 tubes in an ice-bath and measuring the pH after 1 min of electrode immersion in each sample. Ammonium-Sulfate Fractionation Protein was fractionated by precipitation with ammonium sulfate (Mann Research Laboratories, special enzyme grade) at O C. After a suitable incubation period, precipitates were removed by centrifuga- tion at 0 to 2 C for 30 min at 29,000 2, Before determination of a-amylase activity or protein content, the pellets were dissolved in 1 ml of Trisca buffer (5 mM_tris buffer, pH 8.0, and 5 mM_CaClz) and dialyzed for 40 hr against cold (about 7 C) Trisca. Amino Acid Analysis Protein was eluted electrophoretically from preparative SDS- polyacrylamide gels. The protein solution was carefully removed from the dialysis bag with a Pasteur pipette and transferred to a pre- weighed test tube (Pyrex, 13 x 100 mm). The dialysis bag was rinsed once with a minimum volume (100 pl) of distilled H20, and the rinse was added to the test tube. Trichloroacetic acid (TCA) was added from a 100% (w/v) stock solution (i.e., 100 9 TCA made up to 100 ml with H20) to a final concentration of 20%. At 15% TCA, a precipitate formed but 20% TCA was used to insure that all of the protein was precipitated. After 60 min at room temperature, the solution was centrifuged using an International Clinical Centrifuge at low speed (a setting of '2' indicating 500 rpm). The supernatant solution was removed with a Pasteur pipette, but not completely, and 1 ml of 95% ethanol (re-distilled) was added to the precipitate. After 15 min at room temperature, the centrifugation was repeated. The pellet was not suspended after addition of ethanol, but was washed once more 119 with 1 ml of 95% ethanol and then three times with 4 ml each of 95% ethanol. After each rinse, the sample was centrifuged. This pro- cedure removed SDS, the electrophoresis buffer, and TCA. The final pellet was carefully dried under a stream of nitrogen. The pellet, upon drying, separated from the test tube and could be weighed on a sensitive balance to give an estimate of the maximum weight of protein present in the pellet. The pellet was carefully returned to the test tube to which 0.6 ml of constant boiling HCl was added. The tube was flushed with nitrogen for l min without placing the needle attached to the nitrogen line directly into the acid solution. The tube was quickly sealed with the aid of a gas-oxygen torch and incubated in a heating block at 107 C for 18 hr. Thereafter, the tube was cooled to room temperature and cracked open. The constant boiling HCl was evaporated to dryness at 50 C under a stream of nitrogen. The residue was resuspended in 200 to 500 ul of 0.01 N HCl, and was frozen or was used directly for amino acid analysis. Amino acid analysis was performed using a Technicon system (Technicon Corporation, Tarrytown, NY, USA) as modified by Lamport (1969). The Chromo-Beads ion exchange column (Type C-2, 0.6 x 70 cm) was eluted under pressure with buffer which was applied as a gradient of increasing pH and ionic strenth. The column effluent was monitored at 570 nm and 440 nm, after it was mixed with ninhydrin reagent. For best results 60 to 100 ug of protein hydrolysate were applied to each column per determination. Three or four determinations were made for each protein analyzed. The amounts of each amino acid were determined relative to 100 nmoles of norleucine. The values were corrected by comparison to the color intensities of known amounts of standard 120 amino acids recovered from the columns. Tryptophan was destroyed by this procedure and yields of cysteine were not reliable. Glutamine was converted to glutamate and asparagine to aspartate. Carbohydrate Analysis Sugars in glyc0proteins were identified and quantitated by gas chromatography. A glass column packed with a support of Gas Chrom-Q which was coated with SE 30 (Applied Science Laboratories, Inc., State College, PA, USA) was used to separate sugars as their methyl- glycosides after trimethylsilylation (Bhatti et al., 1970). The following sugars were used as standards: arabinose, rhamnose, fucose, xylose, mannose, galactose, glucose, N-acetyl glucosamine, N-acetyl galactosamine, and sialic acid. The internal standard was mannitol at 100 nmoles per sample. The detection limit was about 1 nmole of sugar per sample in 25 ul of the trimethylsilylation reaction mix- ture. About 1 mg of a—amylase and 0.24 mg of band-2 protein (the weight being based on the amino acid composition) were used per sample in these carbohydrate determinations. Two methods were used to cleave the carbohydrate moiety of the glycoproteins into monosaccharides. The first method was non-aqueous, using dry methanol containing 1.5 M HCl (Chambers and Clamp, 1971). Mannitol (100 nmole) was added to the test sample, and the sample was dried in vacuo for 12 hr over P O 2 5. One milliliter of dry methanolic HCl was added to the sample, and the vial or test tube was flushed with N2 for l min. The vial or tube was sealed, and the mixture was heated at 80 C for 12 hr. If vials were used, the paper liners of the caps were replaced by teflon liners. After methanolysis, the solu- tion was cooled, the container opened, and the HCl neutralized by .h. _. 121 cautious addition of solid silver carbonate. When gas was no longer evolved upon further addition of silver carbonate, 100 pl of dry acetic anhydride was added, the vial was closed again and incubated for 6 hr. The liquid was removed and placed in a lS-ml centrifuge tube. The solid was resuspended two times in 1 ml of dry methanol and was removed by low-speed centrifugation. The rinses were added to the centrifuge tube which was centrifuged at 13,300 g_for 20 min. 1 The supernatant solution was subsequently evaporated at 45 C under a In stream of nitrogen. The sample was dried under vacuum over P205 for 6 to 10 hr. The trimethylsilylating reagent (25 p1) was added, and after 45 min, the samples were analyzed on a Perkin-Elmer 900 gas chromatograph. The silylation reagent, containing pyridine, trimethyl- chlorosilane, and hexamethyldisilazane (5:1:1, v/v/v), was stored up to 72 hr and was centrifuged before use. After derivatization, the sample was diluted with dry ethyl acetate, if required. The second method was designed to allow the cleavage of the carbohydrate into monosaccharide units to occur while the glycopro- tein was still in the polyacrylamide gel. The gels were fixed for 48 hr in 50% methanol containing 5% acetic acid. Several changes of about 500 ml of fixing solution per gel were used. Portions of the gel which contained the precipitated protein were excised with a razor blade, and up to 1.25 cm3 of gel was placed in a test tube (Pyrex, 13 x 100 mm). The gel pieces were subsequently minced up using a dissecting tool with an arrow-shaped tip. Two volumes of 3 N trifluoroacetic acid (TFA) were added to one volume of gel so that the concentration of TFA in the total aqueous phase was about 2 N. Mannitol was added as an internal standard and the tubes were sealed with a gas—oxygen torch. The sealed tubes were incubated at 122 3.211 C for 60 min. The tubes were cooled, the aqueous phase was t:1:ansferred to a new tube, and the H20 and TFA were removed by a ssizream of nitrogen while the tube was maintained at 45 C. The gel theaterial, which had been left behind, was meanwhile rinsed with a srnall volume of H20 which was subsequently combined with the first enqueous phase which was being evaporated. The residues were dried .in vacuo for 7 to 10 hr over P205. The monosaccharides released by 'this aqueous procedure were then processed as in the first method, Ibeginning at the step where dry methanolic—HCl was added to the sample. Glassware and Dialysis Tubing Glassware used for carbohydrate and amino—acid analyses were cleaned with Dichrol (Scientific Products, McGaw Park, IL, USA) followed by detergent, acetone, and methanol. Every attempt was made to avoid contamination of the samples by dust. Dialysis tubing (Dialyapor A, molecular weight cutoff 6000 to 8000, National Scientific Company) was boiled once in 2% sodium bicarbonate, three times in 2% sodium bicarbonate plus 0.03% EDTA, and once in dis- tilled HZO. The dialysis tubing was rinsed and stored in 50% glycerol with 10% ethanol. Source of Chemicals Chemicals used for carbohydrate analysis were obtained from sources suggested by Bhatti et al. (1970) or Chambers and Clamp (1971). Those for the dansyl hydrazine reaction were as suggested by Eckhardt (1976). Reagents for electrophoresis included SDS from Pierce (Rockford, IL), acrylamide from Ames (Miles Laboratories, Elkhart, IN), bis—acrylamide, TEMED, and ammonium persulfate from 123 Bio-Rad (Richmond, CA). Basic fuchsin was obtained from Allied Chemical Corporation (New York, NY). Ovalbumin, BSA, Coomassie Brilliant Blue R, and chymotrypsinogen were obtained from Sigma Chemical Company (St. Louis, MO), while lysozyme (muramidase) was obtained from Worthington Biochemicals (Freehold, NJ). Isoelectric focusing was performed using ampholines from LKB (Rockville, MD). RESULTS Synthesis of d-Amylase byfiBarley Grains A time course of the synthesis and release of amylolytic activity by germinating Himalaya barley grains is shown in Figure 26. The amylase activity increased for 5 days, after which time the starchy endosperm was completely solubilized. After one day of imbibition, 87% of the a-amylase was released from the aleurone cells, and after 5 days, 99% of the a-amylase which had been synthe- sized had been released from the cells. Partial Purification of a-Amylase Based on the results of the time course experiment, grains were incubated for 4.5 days, and the secreted a-amylase was collected .and.purified. The grains were continuously incubated in buffer and 110 pM_GA to insure that the maximum amount of a-amylase was synthe- 3 ssized. a-Amylase was bound to glycogen in the presence of 40% esthanol and was precipitated as a glycogen-amylase complex. The Iprecipitate was subsequently solubilized in Trisca buffer at room ‘temperature. Most of the a-amylase and glycogen were solubilized VVithin the time required to collect the precipitates from the centri- fuge bottles (Figure 27). The a-amylase solubilized during zero to 45 min of incubation in Trisca were pooled. The solubilized a-amylase 'Was precipitated a second time by 30% ethanol, and the pellet was SOlubilized again in Trisca. This d-amylase was then precipitated 124 125 .o .Hmuou a. .oommmaou “ I .263 See a D .osmmau ”as.” mmmefiTo .580 9.3 ca Homo co mumcflgmm ou omBOHHm who», mcamuo .mcflmno >maumb mafiumcflsuwm >b ommaxsoId mo mmmmamu tam mwmobucmm .om musmflm 126 on assess m>2EoIa obu oocfloucoo nuanz How mo mcofiuoom EUIH bufl3 oouoHUOmmo >ua>fluooofloou onu mucom Ioumou Eonmoumflb one .ocHH oouuoo .osmmflu oconsoam touoaomfl Eoum moGHH owHOm .mcfloum woauob >Q oououoom mcflouonm .Ec oom um ooccooo ouo3 0cm ommm osam NCMHHHHHm ofimmmsooo nua3 oocflmum ouoB maom ooflsoa>uommaomImom .comoo>am sh ceaumuamflooum uoumo omoassolo onu mo aufludm 132 .mm ousmflm 133 099V DJ 0.0. unit I_\ , N — (WVHDOlSII-I)Q-OI 3: VHS 'BNIOI'IB'I'OM P band-2 fl 5 DISTANCE/ SEGMENT NUMBER Figure 29 134 two proteins. Only the sample taken from the trailing edge of the peak was pure a-amylase. This suggested that in its native state the protein of band-2 contained two of the 20,500 dalton peptides and thus had a molecular weight about equal to that of a-amylase. As very little pure a-amylase was obtained by this method, use of this method as a preparative technique was not feasible. Since the native proteins could not be separated on the basis of their molecular weights, an attempt was made to separate the two by preparative isoelectric focusing (Figure 30). Two components were resolved, one with a pI of 5.0 and the second with a pI of 6.8, based on the regions of a-amylase activity in the column. The first com- ponent (pI 5.0) was pure a-amylase. The second (pI 6.8) included both a-amylase and the second protein in fraction IIc and IId. [In actuality, the true pI of the second component lay between pH 5.5 and pH 6.8 based on the amounts of protein (assayed after dialysis of the fractions) present in each region. Some of the a-amylase had been inactivated at the region of its true pI.] From this it appeared that the a-amylase and the band-2 protein could not be separated by differences in net charge of the molecules. In addition, the contaminating protein could not be removed by differential ammonium-sulfate precipitation. Most of the a-amylase (95%) was precipitated by ammonium sulfate at 20 to 40% of saturation (Table 3). A small amount of a-amylase activity (5%) precipitated when the ammonium sulfate concentration was at 50 to 60% of saturation. The a—amylase from the first precipitate contained the band-2 con- taminant. That from the second precipitation (50 to 60%) was very nearly pure a-amylase, with only a faint trace of the second protein being present. The first precipitate (20 to 40% saturation of 135 Figure 30. Isoelectric focusing of d-amylase. A mixture of d-amylase and band-2 protein was focused at 8 C using a gradient of pH 5 to 8 in glycerol. After focusing, the indicated fractions were pooled, dialyzed, concentrated (Millipore immersible mole- cular separation) and analyzed on SDS-polyacrylamide gels. Fraction IIb contained the most protein. Fractions I and IIa contained pure a-amylase. The gels are not shown. a-AMYLASE, UNITS 500- 10 11b c 11d 40 8O FRACTION NUMBER Figure 30 I20 I I2 .IO Table 3. 137 Ammonium-sulfate precipitation of purified a-amylase (NH4)ZSO4,a Time a-Amylase, Protein, % of saturation at 0 C units of activity pgb 0 to 20% 1 hr 0 -- 20 to 40% 1C 1 hr 23,402 15,360 20 to 40% IId 3 hr 1533 1290 40 to 50% 10 hr 17.4 -- 50 to 60% 1 hr 1341 1703 60 to 80%c 72 hr <1 —- At 0 C. CD‘S” d Measured as bovine serum albumin equivalents. Some non-proteinaceous crystals formed during dialysis. Incubation of the supernatant fraction from the first ammonium-sulfate precipitation was continued for a further 3 hr. 138 ammonium sulfate) was thereafter designated as aA-4OI, and was kept separate from the second precipitate (50 to 60% saturation), which was designated as aA-60. The band-2 protein had roughly the same molecular weight as a-amylase (in its native state) and the same net charge. It also precipitated with the bulk of the a-amylase during ammonium sulfate precipitation. Furthermore, the protein also bound to glycogen and was not precipitated by the 70 C heat treatment which was a part of the purification procedure. The band-2 protein was also rendered soluble to the same extent as was a-amylase during the solubiliza- tion of the glycogen-amylase pellet (Figure 27), and it precipitated in a constant ratio to a-amylase during the repeated ethanol precipi- tations at different ethanol concentrations (Figure 28). However, it did not appear that the presence of this peptide was necessary for a-amylase to be active since fractions which contained only the 41,000 to 42,000 dalton protein were fully active, based on the expected specific activity of a-amylase. No method for the separation of active a-amylase isozymes from the band-2 protein was found. Therefore, the two proteins were separated by preparative SDS-polyacrylamide gel electrophoresis. Amino-Acid Composition of a-Amylase and the Band-2 Protein The two proteins, a-amylase and band—2, were characterized on the basis of their amino-acid compositions. The a-amylase, it should be recalled, was composed of four a-amylase isozymes. The average amino-acid composition of this mixture of a-amylases is shown in Table 4. For reference, the ratio of each of the amino acids to isoleucine was determined. The amino acid composition of the 20,500 139 Table 4. Amino acid composition of barley a-amylase Number of Residues, Amino Acid normalized to 1.0 of isoleucine ala 1.93 : 0.015a 91y 2.69 i 0.044 val 1.30 i 0.021 thr 0.93 i 0.006 ser 0.81 i 0.010 leu 1.47 i 0.006 ilu 1.00 pro 1.14 i 0.044 met 0.38 i 0.010 asp 2.86 t 0.051 phe 0.98 t 0.026 glu 1.54 i 0.012 lys 1.29 i 0.010 tyr 0.83 t 0.021 arg 0.97 i 0.023 his 0.94 i 0.104 a . . . Standard deViation, based on four replicates. 140 dalton protein is shown in Table 5. The relative ratio of the indi- vidual amino acids in a-amylase and the band-2 protein was also determined. If the two proteins had the same amino acid composition, the values determined in the last column of Table 5 would have been identical. The variation in the ratios suggests that the band-2 protein is different from the average d-amylase molecule. Cysteine was detected in both proteins, but the amount was not quantitated. The amino acid composition of barley d-amylase was compared to that of wheat a-amylase (Tkachuk and Kruger, 1974) in Table 6. The percent of the weight of d-amylase which was represented by each amino acid was determined, assuming that the tryptophan and cysteine compositionscnfthe barley enzyme were the same as those of wheat. The average number of residues of each amino acid present in barley a-amylase was determined using this same assumption. Barley a-amylase differed from that of wheat a-amylase by being noticeably poorer in glutamate/glutamine, serine and proline, while being somewhat richer in aspartate/asparagine, lysine, and phenylalanine. Glycosylation of a—Amylase and the Band-2 Protein Mitchell (1972) reported that barley a-amylase was a glycopro- tein, although the mole percent of each sugar residue had not been determined. Preliminary experiments indicated that my barley a-amylase preparations, which still were contaminated by the 20,500 dalton protein, contained a variety of sugars including arabinose, xylose, glucose, and small amounts of mannose. These samples had been prepared after the solubilized glycogen-amylase complexes had been incubated at room temperature, then passed over a column of DEAE-cellulose to remove the neutral sugars and polysaccharides. 141 Table 5. Amino acid composition of the band-2 protein Number of Residues, Ratio of Amino Acid Normalized to 1.0 of ilu Band-2 to (Ir-Amylasea ala 3.08 i 0.078b 1.60 gly 3.85 i 0.014 1.43 val 1.89 i 0.014 1.45 thr 1.70 i 0.035 1.83 ser 2.10 i 0.092 2.59 leu 2.32 t 0.049 1.58 ilu 1.00 1.00 pro 2.96 i 0.113 2.36 met 0.30 i 0.071 0.79 asp 3.47 i 0.085 1.21 phe 1.46 i 0.212 1.49 glu 2.62 i 0.156 1.70 lys 1.48 i 0.071 1.14 tyr 1.44 i 0.495 1.73 arg 2.36 i 0.071 2.43 his 1.94 i 0.134 2.06 aNormalized to a ratio of 1.0 for ilu. Standard deviation, based on three replicates. 142 Table 6. Comparison of amino acids in barley and wheat d—amylases Amino Weight Percent Ratio, Number of Residues Acid barleya wheat barley: wheatb barleyc wheatd ala 5.68 5.42 1.05 33.2 33.0 gly 6.27 5.55 1.13 46.2 42.0 val 5.27 5.68 0.93 22.3 24.8 thr 4.10 4.76 0.86 16.0 20.4 ser 2.89 4.11 0.70 13.9 20.4 1eu 6.94 7.16 0.97 25.3 27.4 ilu 4.22 5.21 0.81 17.2 19.9 pro 4.43 5.86 0.76 19.6 26.1 met 2.12 1.88 1.13 6.5 6.2 asp 13.42 11.00 1.22 49.1 41.5 phe 6.08 4.87 1.25 16.8 14.3 glu 8.16 12.20 0.67 26.5 41.0 lys 6.97 5.56 1.25 22.2 18.8 tyr 5.70 6.05 0.94 14.3 12.7 arg 6.35 6.25 1.02 16.7 17.3 his 4.66 4.06 1.15 16.2 12.8 cys __e 1.04 -- -- 2.2 trp __e 4.75 -- -- 11.0 IAdjusted, as if barley a-amylase contained the same weight percent of cys and trp as did wheat a-amylase. bCalculated from weight percent. CPer molecule of 41,400 daltons. dPer molecule of 43,300 daltons. eNot determined. 143 Even when the a-amylase was fully inactivated by incubation at pH 3 and then dialyzed extensively, the dialysate still contained these sugars, although their amounts in different samples varied. Because a—amylase is known to bind tenaciously to its substrate (Fischer and Stein, 1960), even during its crystallization, I had concluded that the a-amylase would have to be denatured before it was analyzed for the presence of sugars. However, while the dialysate of the pH 3-denatured a-amylase contained sugars, there was no proof that these were covalently bound to the protein. Perhaps the sugars were not removed by dialysis, although they should have been released from the protein during denaturation. The two proteins, a-amylase and band-2, could only be separated by SDS-polyacrylamide gel electrophoresis. This procedure was also well suited to the removal of carbohydrate polymers which were not covalently attached to the a-amylase molecule. Since SDS treatment converts proteins into more or less linear molecules, it would be nearly impossible for any non-covalent association of carbohydrate and protein to be maintained during electrophoresis. Unattached carbohydrate would be left behind at the top of the gel. a-Amylase from the first precipitation by ammonium sulfate (20 to 40%) was denatured and subjected to SDS-polyacrylamide electrophoresis, and the number of sugar residues per molecule of a-amylase was determined by gas chromatography (Table 7). The sugars were identified as their trimethylsilylated methyl glycosides. 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