THE PURKFEATION AND PROPERTIES (2F PYRWATE KiNASE FROM BAKER'S YEAST Thesis fer the Degree: cf M. S. MBCHlGAE‘é STATE UNWERSWY James R. Hunsley 1x956 wags V i Mighigan Stats Il1 Univc: it, 6”" LIBRARY _‘ _ w «57;? “0117-; Z "5"— 2 2.41142 mg) '?9: RUE? USE. Lu .5. . lardqflvhuml. .4 . ’z‘IIIWiI_ null ‘ .‘I HIJU v.54, ..I r " é . I it. lidIhWII‘ {1* .. .i. . if . I111: ABSTRACT THE PURIFICATION AND PROPERTIES OF PYRUVATE KINASE FROM BAKER'S YEAST by James R. Hunsley Washio and Mano (1960) published a 17-fold puri- fication of pyruvate kinase from yeast, but the pre- paration was stable only a few days. In order to study monovalent cation and substrate requirements for this enzyme,”the primary goal of this work was, therefore, to purify and stabilize yeast pyruvate kinase. Polyanions and polyhydroxy compounds were found to stabilize the enzyme. Using the techniques of toluene autolysis, ammonium sulfate fractionation, treatment with DEAE cellulose, and chromatography on cellulose phosphate in 50 per cent glycerol, the enzyme was purified 28-fold to about 95 per cent purity. The LDH linked assay of Bficher and Pfleiderer (1955) was adapted for this work. The purified enzyme, as found by Washio and Mano (1960), had an absolute requirement for potassium ions which could be replaced by rubidium and ammonium ions to about 50 per cent of activity. The KA's for potassium ion at constant and varying ionic strength were 0.173 and 0.029 M_respectively. The pH Optimum of the reaction was shown to be 6.1 to 6.4 in cacodylate, imidazole, and maleate buffers with cacodylate giving the highest observable rates. Linear kinetics were obtained with the substrate ADP yielding a Km of 3.6 x 10‘“ 21- Kinetics toward PEP showed a cooperative effect with a large Hill slope of 4.2 and an apparent Km of 1.1 X 10"3 fl, FDP was shown to activate the enzyme at low concentrations of PEP. As previously predicted by Pye and Eddy (1965), Hommes (196A), and Hess and Brand (1965), the enzyme appears to be an important control point in yeast glycolysis. The SQO’W of the enzyme was calculated to be 8.23 S from sedimentation velocity data. THE PURIFICATION AND PROPERTIES OF PYRUVATE KINASE FROM BAKER'S YEAST BY James R. Hunsley A THESIS Submitted to Michigan State University in partial fulfillment of the requiremtnts for the degree of MASTER OF SCIENCE Department of Biochemistry 1966 ACKNOWLEDGEMENT The author wishes to express appreciation to Dr. Clarence Suelter under whose critical guidance this research was conducted. 11 TABLE OF CONTENTS Acknowledgement Table of Contents List of Tables List of Figures List of Abbreviations Introduction Materials and Methods Experimental Discussion Summary References 111 Page ii iii iv vi 10 39 45 47 LIST OF TABLES Table Page I The Yeast Pyruvate Kinase Preparation of Washio and Mano (1960) 11 II Results of Stabilization Experiments 12 III Summary of Yeast Pyruvate Kinase Preparation 19 IV Univalent Cation Requirement of Yeast PK (Washio and Mano, 1960) 21 V Relative Univalent Cation Requirement of Yeast PK 21 VI FDP Activation of Yeast PK 35 iv LIST OF FIGURES Figure Page I Elution Profile of Yeast PK from Cellulose Phosphate 17 II A/v versus A Plot of Potassium Activation of Yeast PK at Constant Ionic Strength 22 III A/v versus A Plot of Potassium Activation of Yeast PK at Increasing Ionic Strength 24 IV Optimum pH for Yeast Pyruvate Kinase Reaction 27 V v versus v/S Plot of ADP Kinetics of Yeast PK 29 VI Lineweaver-Burke Plot of PEP Kinetics of Yeast PK 31 VII Hill Plot of PEP Kinetics of Yeast PK 33 VIII Sedimentation Velocity Frame of Yeast PK 37 IX Polyacrylamide Gel Disc ElectrOphoresis of Yeast PK Purification Fractions 37 ADP ATP DEAE FDP LDH NAD(H) NADP(H) OD PEP PFK PK TMA tris LIST OF ABBREVIATIONS adenosine-5'-diphosphate adenosine—5'—mon0phOSphate adenosine—5'-triphosphate diethylaminoethyl cellulose fructose-1,6-diphosphate lactic acid dehydrogenase nicotinamide adenine dinucleotide (reduced) nicotinamide adenine dinucleotide phosphate (reduced) Optical density phospho(enol)pyruvate phosphofructokinase pyruvate kinase tetramethylammonium tris(hydroxylmethyl)aminomethane vi INTRODUCTION The sole previously published preparation (Washio and Mano, 1960) of pyruvate kinase (EC 2.7.1.40) from yeast was unsuccessful although the enzyme was early shown to exist by several laboratories (Parnas, §t_ El”, 1935; Muntz, 1947; Seits, 1949). Washio and Mano (1960) were unable to obtain stability, high purity, or a kinetic assay, but did manage to accumulate useful kinetic data using an end point assay for pyruvate. The yeast enzyme was shown by them to have requirements for potassium and magnesium ions like the rabbit muscle enzyme (Kachmar and Boyer, 1953) but differed in re- quiring higher Optimal concentrations and also in having a lower pH Optimum. These workers also demon— strated that the stoichiometry of the reaction, shown‘ below, was uniequivalent by chromatography of the reaction products. K‘ , Ms‘: ADP 4- PEP—e “"ATP +- pyruvate Interest in this laboratory arose in the yeast en- zyme as an alternative and comparative model to the rabbit muscle enzyme for monovalent cation activation. The possibility Of Obtaining mutant enzymes altered in catalytic prOperties toward monovalent cations is espe- -1- -2- cially attractive in the yeast system. In addition, the essentially irreversible reaction catalyzed by pyruvate kinase makes the enzyme a candidate for glycolytic control. Hommes (1964) applying the theory of crossover points (Chance, 33 a1}, 1958) to glycolyzing yeast extracts pointed out the inhibition and activation phenomena of glycolysis are between g1ucose-6-phOSphate and fructose-6—phosphate, triose phosphate and 3—phospho- glycerate, and PEP and pyruvate. Pye and Eddy (1965) claimed the behavior of glycolytic intermediates in yeast could by explained by 4 control points: PK, 3—phospho- glycerate kinase, phosphofructokinase, and sugar entry. Hess and Brand (1965) suggested that the points of control were at the same three kinases. Hess (1965) earlier in the same colloquium gave the first data available for the possible control of yeast PK by FDP activation. Hommes (1966a) again identified the control point in the conver— sion of PEP to pyruvate but stated that exhaustive attempts to demonstrate allosteric effects in pyruvate kinase had failed. Hommes (1966b) also has shown that the PK content of yeast grown in media with glucose concentrations varying from 0.6 to 1.2 per cent increases about twenty fold while phOSphOfructokinase remains almost constant, suggesting the induction of pyruvate kinase and thus another level of control. No control has yet been found for phospho- glycerate kinase (Hess and Brand, 1965). In this thesis the stabilization and purification Of -3- pyruvate kinase from yeast (Saccharomyces cerevisiae) is described, and an introduction given to the kinetic and allosteric properties of the enzyme. MATERIALS AND METHODS A. Enzymatic Activity and Protein Determinations l. Assay for pyruvate kinase Because of equilibrium considerations the pyruvate kinase reaction can only conveniently be measured in the direction of ATP synthesis. The pyruvate formed was con- tinuously followed spectrophotometrically by employing the lactic acid dehydrogenase reaction as modified from the assay or Bficher and Pfleiderer (1955) for rabbit muscle pyruvate kinase. For these assays a modified Beck- man DU ultraviolet spectrophotometer with a Gilford Model 2000 multiple sample absorbance recording attachment was used to record the decrease in Optical density at 340 my, the absorption maximum of NADH, according to the following scheme: ADP + PEP=ATP + pyruvate NADH’\\-> lactate NAD The assay temperature was 30°C., maintained by a circulating water bath. The stoichiometry for the assay is one mole of NAD formed for each mole of ATP. Conditions for routine assays in this study are as follows: -5- Assay component pmoleszml cacodylate (Na) 110 KCl 230 Mg012 24 NADH 0.15 ADP 0.80 PEP 3.0 LDH (33 ps/ml) The final volume of the assay mixture was 1.00 ml at pH 6.00, the reaction being initiated by addition of enzyme and recorded against a water blank. A11 dilutions of this enzyme for assay must be made in cold 50 per cent (v/y) aqueous glycerol solutions containing 0.010 M_phosphate (Na), pH 6.50. The best commercial lactic dehydrogenase sometimes contains traces Of PK, and small residual rates with this determination are due both to contaminant enzyme and hydrolysis of PEP to pyruvate at acidic (pH 6.00) assay conditions. Measured rates were corrected for the usual residual rate of about 0.0015 AOD/min. 2. Definition of a unit of activity and specific activity A unit of pyruvate kinase activity is defined as that amount of enzyme which catalyzes the production of one pmole of pyruvate per minute under the conditions described in the assay procedure. The molar absorbance 6 2 of NADH at 340 my was taken as 6.22 X 10 cm per mole (Horecker and Kornberg, 1948); therefore, an optical density change corresponding to the production of one Pmole of pyruvate is 6.22. The specific activity is defined as units of enzy- -5- matic activity per mg Of protein. 3. Determination of protein concentration The spectrOphotometric method of Warburg and Christian (1942) was used to estimate protein concentration. 4. Substrates and assay conponents a. PEP. Phospho(enol)pyruvic acid, tri- cyclohexylammonium salt, was purchased from the Sigma Chemical Company. The reagent was assayed by two methods: 1) according to Bficher (1955), based on the molar ab- sorbancy of PEP at pH 7.4 in the presence of 2.7 X 10‘3 M MgClg, and 2) a determination based on addition of a limiting amount of this substrate to a standard assay system containing an excess of muscle pyruvate kinase (Tietz and Ochoa, 1958) and calculating from the absolute 0D change of the reaction. b. ADP. Adenosine diphOSphate, sodium salt, was dissolved in water and adjusted to pH 7.0 with NaOH. ADP was estimated by two methods, 1) the procedure of Bock, gt_a1, (1956), based on the absorption of the adenine moiety at 259 mp, pH 7, and 2) the second method employed in assaying PEP. c. NADH. Nicotinamide adenine dinucleotide (reduced), di-sodium salt Obtained from Pabst Laboratories, was dissolved in 1.0 x 10"2 M tris (HCl) buffer, pH 7.5. d. LDH. Lactic acid dehydrogenase, obtained from Sigma, was the Type II crystalline rabbit muscle enzyme substantially free of pyruvate kinase. A stock -7- solution was prepared by diluting the ammonium sulfate suspension (10 mg/ml) to 330 pg per ml in water. B. Other Reagents and Materials "Budweiser" baker's yeast, Anheuser-Busch, Incor- porated, was obtained fresh from Michigan State University Food Stores. Analytical reagent grade chemicals were used throughout whenever possible. All solutions were prepared from deionized distilled water with a conductivity reading (Crystalab Deeminizer) below 1.0 ppm. Glycerol was the Fisher Certified Reagent, Fisher Scientific Company. Imidazole was recrystalized from chloroform. Fructose-1,6-diphosphate was the sodium salt from Sigma. Cacodylic acid, Sigma or Fisher, was titrated to the desired pH (Sargent pH Meter, Model LS) with NaOH or TMAOH. Tetramethylammonium hydroxide and chloride were purchased from Eastman Organic Chemicals and used without further purification. Special Enzyme Grade ammonium sulfate was obtained from Mann Research Laboratories, In- corporated and was used throughout these studies. Visking Corporation dialysis tubing was soaked at least one hour before using in several changes of water. Conductivity readings were taken with an Industrial Instruments Corporation conductivity bridge Model RC 16B2 in a platinum-glass flow cell of unknown cell constant. C. Preparation of Columns for Chromatography 1. DEAE cellulose (about 0.9 meq/g) was purchased either from Sigma or Gallard-Schlesinger Chemical Manu- -8- facturing Corporation. 150 g of medium mesh material was washed consecutively with two liters of the following solutions, filtering after each wash: 95 per cent ethanol, 0.5 N_Na0H, water, 0.1 §_HC1, and 0.1 N HCl again. The residue was then washed 5 to 7 times with water decanting fines and filtering until the wash was free of chloride ion by the silver nitrate test. The damp dry filtered material was then suspended in 50 per cent aqueous (v/y) glycerol containing 1.0 X 10’2 M NaHQPOM, adjusted directly to pH 7.50 with NaOH, and refiltered. The material was then suspended in phosphate buffered glycerol, pH 7.50, added to a glass column 8.0 cm in diameter with a sintered glass bottom, and gravity packed to a height of 18 cm. The column was then washed with one column volume (900 m1) of buffered glycerol, pH 7.50, and placed in the cold room at 4° c. 2. Cellulose phosphate, Sigma, (0.9 meq/g) was treated the same as the DEAE above except that the HCl and NaOH washes were reversed. The damp dry filtered derivative was equilibrated with 0.01 M_phosphate buffered 50 per cent glycerol adjusted to pH 6.50. This slurry was added to a glass column 5.0 cm in diameter with a sintered glass bottom and packed by gravity to a height of 18 cm. After washing the column with an additional column volume (400 ml) of buffered glycerol, pH 6.50, the column was placed in the cold room. D. Polyacrylamide Disc ElectrOphoresis -9- Disc electrOphoresis patterns of enzyme fractions of the purification scheme given later in this thesis were obtained by subjecting appropriate samples to analysis by modified techniques of Ornstein (1962) and Canalco (1963). All reagents were obtained from Canalco. To 7.5 per cent polymerized standard gels in glass tubes 4.5 mm inside diameter by 75 mm containing a Spacer gel were added apprOpriate samples of protein (less than 200 pg) in 50 per cent aqueous (v/v) glycerol containing 0.015 M phosphate buffer adjusted to pH 6.90 with tris. Samples were electrOphoresed at room temperature in an apparatus constructed in this laboratory at 5 ma constant current per tube in standard tris-glycine buffer, pH 8.3 using brom phenyl blue tracking dye and a Heath Company regulated power supply, Model IP-32. The runs were concluded at the time the tracking dye came within about 5 mm of the anodic end. The gels were removed within 10 minutes Of the end of the run, stained in 0.55 per cent Amido-Schwarz in 7.5 per cent acetic acid, then destained in 7.5 per cent acetic acid. E. Sedimentation Velocity Determination in the Ana- lytical Ultracentrifuge The Spinco Model E Analytical Ultracentrifuge with phase plate schlieren optics was used to determine the sedimentation constant for the enzyme. Calculations were made according to Schachman (1957) and corrected to the viscosity of water at 200 C. EXPERIMENTAL A. Stability Studies of Yeast Pyruvate Kinase The failure of the only previously published attempt to Obtain a stable pyruvate kinase from yeast was due to the extraordinary instability of the enzyme both in the unpurified and purified states (Washio and Mano, 1960). Washio and Mano achieved a 17 fold purification with poor yields as can be seen in Table I but the preparation lost all activity "within a few days." Extensive stability studies of various preliminary stages of the purification scheme were conducted in an attempt to find a stabilizing agent which would be effective both at low and high protein concentrations and which Could be conveniently used throughout a protein puri- fication procedure. Table II shows the partial results of these studies. Polyhydroxy compounds are very effective in the desired stabilization, and glycerol in particular was chosen because Of lower viscosity and efficiency of stabilization both at low and high protein concentrations. A typical experiment consisted of incubating known amounts of fresh active enzyme in cold reagent solutions of wide concentration ranges, incubating at temperatures of 4 and 250 C. for periods of either 2 or 3 days, and assaying for activity after the incubation period. B. Isolation Of PK from Yeast A protein estimated to be about 95 per cent pure -10- .pcoEpmomp mmOHSHHOO m:a>co:QOLpficfipa:.m 0:» >9 dopammma mm wuscfie pom mpw>5pma no OHoEA moo mEpom seas: Campopa mo ucsoew wasp ma was: 0:0* -11- o.efi HA H.3H moa NH m - we m.m mom mm s 3.: am m.m sflm mm m m.m mm m.H ems es: m . ooH m.o onH emwfi H cofimeHmfipsm R .OHOHM AwENmuHCSV hpfi>fipo< Ampficsv mpfi>fipo< AMEV cHopOpm coapompm oeom oeeaooom Heooew Hoeoe Aommav one: new ofismwz no cofipmpmdmpm mmmCHm mpw>spmm endow 039 H OHQMB -12- 00 0H OOHlom om OOHlom OOHnom OOHnom mm OOHuom OH 8-3 oofinom mmoco>fipoommm R* m m «-3 x 64 Sm: .2. TB x 6;“ «Se: .8 OH.o m.© mm .ouwpppmuuA+vuO .8 OH.o m.m mm .mz .opmcompm .8 o.Humm.o 0.0 mm .mz .mpmnmmocaonppo .8 mica x o.H m.© ma aoz .mpmnamonmopza .8 MIOH x o.m m.w ma .mz .opmcamosmzaoafipp e8 I 66 mo £8.36 mumndmoca oz 2 OH.o cfi Hoomaw mCOHmcpm me HOPHQLOm .8 OH.o omoosam .8 o.mam.H I. 0.0 mm .Lmemzn mpmndmoza oz 2 OH.o CH omop05m A?\>v Rowuo: Homooham cofiumppcoocoo .mmmwmmm mucosapmdxm cofipmufiflfinmum no measmom HH OHQOB -13- .mpcoEpmopp paoo one use: new .mz< wouHmHSm ssfiOOm .opwndmonmnwuomoosaw .opmnamocananomoosfiw .mmm .opmuomH .mcfioummo .ocoanuMQSHw voodoop .Ofiom OHpoomwpuopmcfiEmHUOCOHmnpm .muduoowOOOH ammm .HocwnpmoudmopoEum .cfiesnam Edmom "nonnaocfi mCOHuHOcOo pom mesowmop o>apoommocH .mposumz new mamfipmuwz CH Omnapomoo no new: no: henna ppmpcmum one .uxmp one CH confipomop one maawpmo Hopewefipomxm .omopw pcsoem czocx co women OOALOO coapmn302a pound zufi>auom wchHMEop pcoo pom* me-om .8 m-oH x o.H moz em .8 m-oH x m.m m.m mo .aze .oeseeao 8H .8 m-oH x 6.: age ooH-om .8.m-oH x onm m m-oH x o H ape mafia How: mmoco>fipooMQm R: coauMchoocoo .mmmwmmm Aooseaocoov HH oases -14- exhibiting PK activity was isolated from fresh baker's yeast by a procedure involving autolysis with toluene, ammonium sulfate fractionation, treatment with DEAE, and chromatography on cellulose phOSphate. l. Autolysis with toluene and filtration Plasmolysis of yeast was accomplished by a method modified from that of Kunitz and McDonald (1946). Ten pounds of yeast was crumbled into a stainless steel pail and to it added 2.4 liters of reagent grade toluene at 450 C. The covered pail was incubated in a 450 C. water bath and stirred occasionally with a wooden paddle until the yeast reached about 370 C. and liquified. The mixture was allowed to stand at room temperature for one to 2 hours, then rapidly cooled to 10° c. in an ice bath. This mix- ture was added to 3400 ml or water at 4° c. in the cold room and stirred for one hour and allowed to stand over- night (about 16 hours) until two phases separated. All successive Operations were carried out at 0 to 40 C. The aqueous bottom layer was carefully siphoned off and centri- fuged at 8500 rpm for 20 minutes in a GSA head, Sorvall RC-2 refrigerated centrifuge, resulting in a three layer system. The relatively clear brownish middle layer was removed with a suction collecting apparatus. To this centrifugate was added 20 g per liter of Fisher infusorial earth and the mixture filtered over 2 sheets of Whatman #1 filter paper, yielding a crystal clear crude enzyme solution designated Fraction 1. -15- Immediately to Fraction I was slowly added 242 g per liter of solid ammonium sulfate with rapid stirring to 1.62 M, Upon dissolving, the pH Of the solution was adjusted to pH 6.2 by direct measurement with 0 to 5 m1 of 3'3 NHMOH. After allowing to stand between 3 and 12 hours, the suspension was Spun as before, discarding the precipitate. To the clear supernatant, Fraction II, was added 96 g per liter of ammonium sulfate to 2.22 M_stirring rapidly until dissolved. This was centrifuged as before after standing 2 to 4 hours, the supernatant was discarded, and the pre— cipitate was rapidly dissolved in a minimum (less than 100 m1) of 2.0 x 10’2 g phosphate buffer (Na), pH 7.5. To this was added an equal volume of glycerol, yielding Fraction III. Fraction III was then dialyzed 48 hours ver- sus 2 changes of 10 volumes Of 1.0 X 10'2 M_phosphate (Na), pH 6.5, in 50 per cent glycerol. The conductivity of the resultant dialysate (Fraction IV) should be near that of the glycerol buffer alone. Fraction IV is stable for at least 3 months if stored at -200 C. 2. DEAE treatment A volume of Fraction IV containing a maximum of 60,000 units was carefully adjusted to pH 7.50 with 3 N NHMOH. This was added to the DEAE column described on page7', washing after loading with at least one column volume (900 ml) Of glycerol buffer, pH 7.50 and collecting 15 ml fractions about every 15 minutes at the maximum flow rate. After assaying the fractions, those with a specific activity -16- greater than 22 were pooled yielding Fraction V which was stored in a freezer at —200 C. 3. Cellulose phosphate chromatography To a volume of the DEAE effluent (Fraction V) con- taining about 50,000 units was added 3 N acetic acid to pH 6.50. This was then added to the cellulose phosphate column described on page 83 and after loading was washed with one column volume (400 m1) of the phosphate buffered glycerol solution, pH 6.50. No activity washed through the column. The enzyme was eluted from the column with a 600 ml 0.010 to 0.20 M_linear ammonium sulfate gradient in 50 per cent aqueous (v/v) glycerol. Conductivities of 15 m1 fractions collected at the maximum flow rate of about one ml per minute were measured to insure linearity of the gradient. The active fraction eluted at about 0.07 M_ ammonium sulfate. Figure I shows the elution profile of the column. Tubes were pooled that contained enzyme of Specific activity above 120, yielding Fraction VIa, and above 60, yielding Fraction VIb. Both were stored at -200 C. The highest purity almost homogeneous Fraction VIa was used for all successive studies. A summary of the purification scheme is presented in Table III with calculated yields. Fractions of the purification III through VIb were subjected to polyacrylamide disc electrOphoresis to check for heterogeniety of protein species. Figure IX displays the patterns Obtained. -17- .moocpmz cam meHpopwz CH ponfisommo mm osmocwum mos momma one .mep 0:» no :OHpoom Hmucmefipomxm 02p Op wcHOLOoom Hopoomfiw pooo pom om CH pamfiowpw opHMHSm sea-tosses 8 omo op 8.6 some: s f:- ocefio as: omo mo .Amzv Lehman mumsamona.8 10H x O.H moacHMpcOo A?\>V Hommoham m pcoo pom om CH 8m ammo» mo mafia: oooqom hammeHXOpmd< OQBLQmocm omOHSHHoo Eopm Mm unmow no OHAMOLm Coapdam H opswam Figure I -18- (IW/5W) NIBJ. 03d 00. :r. 0. ‘9 N Q v: Q N N N ‘: _: 0 o o (scum) AIIAIlanaNOO o o O o o O o o :3 a. a 9 ” °° ‘7 o l\ - \\ .o \ I...” - 0’..‘§ 000.00.000.00...“ \ «- qpa::&3;::........ 0000......000000000000000000 '- ....., d ‘x \ . .- o——o Protein .............. Specific Activity .-——-5 Conductivity AJIAIJOV OIJIOBdS 60 70 80 90 IOO TUBE NUMBER 50 o .n o -N .5 ljllilll—IJI ll 2 0 0,0 0 o o o 0 _¢_ N',9 m (0 v- N -19- .mponpoz new mamfihopw: CH ponfiuomop mm cow: was mmmmm Ohmpcmpm .COHpomem msofi>mpd one EOLM copr means prOp no woman pamah pcoo pom* was Aoms.mv msfi Aooe.afiv 5.6m Aoos.msv m.mH ooo.mmo H.mH ooo.mmm om.s ooo.mmm sm.m ooo.ome Amofiv AmoHv Ammmv mam 8m: 0mm: omfls QH> MH> > >H HHH HH H one m.Hm m.mm m.mm* :HH :.wm w.wm* mmfi om.m m.ee* oeHH mm.m e.om omHH mm.m m.ms mma - m.sm see - ooH He\moae: coaeooseHeoa ofioaw & UHOm cofipmpmmopm ommcfix opm>sezm ammow mo hemEESm sesesooa mmeacsv Assesooe caesooom Hoeoe HHH OHQME AHEM mEDHo> coapompm -20- The enzyme was concentrated by dialyzing aliquots of Fraction VIa against 10 volumes of 2.0 M_ammonium sulfate for 12 hours, then adding 172 g per liter of solid ammonium sulfate with rapid stirring to 3.0 M, The pre- cipitated enzyme was centrifuged and dissolved in appro- priate buffers. Less than one per cent Of the activity remains in the supernatant. B. Catalytic PrOperties of the Enzyme 1. Univalent cation requirement of PK Yeast pyruvate kinase like the muscle enzyme (Kachmar and Boyer, 1953) has an absolute requirement for univalent cations as shown by Washio and Mano (1960) with partially purified enzyme (see Table IV). The univalent cation activation kinetics of this enzyme was investigated fur- ther by assaying the enzyme in the presence of varying concentrations of KCl, NH401, RbCl, LiCl, NaCl, and TMACl, replacing the usual sodium cacodylate buffer with TMA cacodylate. Assays with each cation were performed at the concentration Optimum for KCl, 0.23 M, Table V shows the relative activities given by these cations. The potassium requirement was pursued further in 2 experiments, varying the KCl concentration in each, ionic strength kept constant with added TMACl in one, and increasing ionic strength (no TMACl) in the other. A/v versus A plots (see Figures 11 and III) of the ac- tivation in both experiments lead to estimated KA's of 0.17 and 0.029 M_and Vmax's of 216 and 145 units per mg Cation K+ + NH“ Li+ Na+ -21- Table IV Univalent Cation Requirement of Yeast PK (Washio and Mano, 1960) Concentration (g2_ *Pyruvic Acid Formed (pmoles) 2.25 x 10‘1 1.05 " 0.51 " 0.06 H O *2,4-dinitr0phenylhydrazine assay of Friedman and Haugen (1943). Table V Relative Univalent Cation Requirement of Yeast PK Cation % Activity, Based on K+ K+ 100 NHu* 50.4 Rb* 49.5 TMA+ 1.6 Na+ 0.05 Li+ 0.005 The standard assay was used as described in Materials and Methods substituting given cations as chloride salts at 0.23 M for KCl as described in the Experimental section. TMA cacoaylate was used as the buffer, pH 6.00. The reaction was initiated with 0.19 pg of enzyme. -22- .mEhNCo mo mm ma.o spas pepmfipficfi mm; coapommp one a8 mm.o pm pcwpmcoo no: coapnpuCoocoo Hofipo< EDHmmwpom no poam < m5mpm> ?\< HH enemas Figure II 2.0 - l “D 0.9;!!!" 6w W) 90/ x A/v‘ 0.0 O.l6 0.20 0.24 A (M ) OJ 2 0.08 0.00 0.04 -24- .oEmNco mo ml mH.o npfiz ooumfipficfi mm: cofipommh one .Hom poops spa: pommmLocH Cameoppm OHCOH one .oo.m ma pm ouwflhooomo <29 eefiz commas oesfisoooso soaooo masseusenoom moored: paw mamapmumz CH ponfipommp mm pom: mm; hmmmw pudendum one newcmepm eacOH mcfimmmuocH pm Mm pmmmw no coaum>au08 Edammmuom 80 uon ¢ m5mpo> >\¢ HHH mpswfim _25- 254 ¢N.0 0N0 0.0 «.0 00.0 ¢0.0 00.0 . HHH choose (,_s;!un 6w W) Go, x A/V -26- reSpectively. 2. pH activity profile The pH activity profile of the enzyme was determined with the standard assay substituting equimolar amounts of imidazole (HCl), cacodylate (TMA), or maleate (TMA) buffers. The pH of duplicate or triplicate reactions was measured directly. The results seen in Figure IV show that the enzyme has more absolute activity in caco- dylate than either imidazole or maleate buffers in the ratios of 1.0 to 0.65 to 0.73 respectively at the Optimal pH. 3. Kinetics Of ADP and PEP Kinetics for ADP and PEP were Obtained under standard assay conditions using varying amounts Of ADP or PEP. If nonlinear rates were observed only initial rates were measured. A v versus v/S plot of ADP kinetics is shown in Figure v yielding an estimated Km of 3.6 x 10"LL g, PEP kinetics are nonlinear; a Lineweaver-Burke plot and a Hill plot of the data can be seen in Figures VI and VII. 4. Effect of FDP on the kinetics Of the enzyme Hess (1965) in a terse note containing unpublished data and no methodology pointed out the FDP stimulation of PK in presumably crude yeast extracts. Under conditions of low catalytic rates due to the allosteric effect of PEP, low concentrations of FDP can fully activate the enzyme as can be seen in Table VI. -27- .oEmuco 00 ml 050.0 npfiz ompmeHcfi mm; :Ofipommp 0:3 .Umppoam ma was coauomop Hanuom one 00 ma one .m-mm mafizpm> pm Aaomv mHoumofiEH no .A¢zev mummfiwe .A30§m endow pom m0 Esafipao >H mpsmam -28_ . 00. 0/ \R 23.22 OIIIIO r . l \ L OIIOIIIIO\ 0.0NOBEH G .......... 0 ON. . 323360 o...ll.o . 0v. ___-._____.- - >H musmflm (,-5w suun) AIM/10V -29- .oesnco eo m1 mH.o eo coaeaops as ooosaeaea mm: cofipomop 028 .00000 m0< mo mangoes on» wcH>Lm> moocpmz 0cm mfimfimopmz CH umnfipomoc mm 00m: mm: zmmnm cpwpcwum one 80 unwow mo moameHm m0< mo poam m\> mSmpo> > > mpsmfim A7279: wees-PS x m\> ow m._ N.- 0.0 v.0 0.0 _ _ _ .14 14 . _ . ON 00 00 00. > mnsmflm (,-6w $110an -31- .oesseo eo ml 3.0 no soaps-ops so Accesses-:- mmz coapowmp one .Uoonm mmm no mpcsosm on» mcfizpw> moonpoz 02m mawfipopmz CH umnapomop ma now: was henna pesocmpm 059 Km pmwmw mo weapocfix 0mm 00 poam exasmupo>wmzoch H> magma-m 000m 000m 2.20 m\_ . 000. 000. H> enemas 00 mo N0 00 10 90 (1-51/00 5w) ll/l _33_ .oeznco eo ml mH.o eo coaeaooo an oopsaease mm: coapowmp 059 .00000 mmm mo mpcsosw esp wcfihmm> mpocumz 0cm mamfipmpmz CH omnfipommo no now: was momma unaccepm 029 mm pmmow .HO moapmcfim mmm mo poam Hafim HH> onsmHm 2): m 00.. no- _.m- mm. mm- on- 3. _.~- . . _ _ _ _ a _ J _ _ _ .. o u . O O lime-- ”in-3m \ \\ \ \ \\ \\ \\ I-Imaonm \\\\ \\ _ b _ _ _ _ _ _ . L _ _ HH> musmfim -35- Table VI FDP Activation of Yeast PK p§_ PEP (m) FDP (m) Activity (units/mg) 6.5 6 0 x 10'4 - 0 6.5 6 0 x 10"LL 2.0 x 10 5 36 6 5 6 0 x 10'“ 1.0 x 10'“ 106 5:5"21'532‘154 ------ I --------------- 37' ------- 6.0 6 0 x 10‘“ 2 0 x 10"5 83 6.0 6.0 x 10'” 1.0 x 10‘” 143 63"23'5633 ''''''' I """"""""""" 85 """"" 6.5 2 4 x 10'3 2.0 x 10"5 108 6.5 2.4 x 10'3 l 0 x 10’ 139 Reaction mix contained cacodylate (Na) 110 moles,I«fl.230 pmoles MgCl 24 pmoles, NADH 0.15 pmoles, ADP 0.8 ‘pmoles, LDH 33 pg, and FDP and PEP in amounts shown. Reaction volume was 1.00 ml. The reaction was initiated by addition of 0.19 pg of enzyme. -36- C. Physical PrOperties of the Enzyme One ml of concentrated enzyme in ammonium sulfate solution prepared according to the method on page 20 and containing 4.8 mg of protein was dialyzed against one liter of 0.050 M phosphate buffer (Na), pH 6.0, over- night. 0.50 ml of the resultant dialysate (4.5 mg/ml) was subjected to sedimentation analysis in the Model E analytical ultracentrifuge at 4.350 C. as described in the methods section. A frame from the resultant plate (Figure VIII) shows a single unskewed peak with a small low molecular weight contaminant of unknown origin. The specific activity of the enzyme used in this experiment was reduced by about 75 per cent during dialysis. The calculated s value corrected to water at 200 C. is 8.23 S. -37- Figure VIII Sedimentation Velocity Frame of Yeast PK Sedimentation is proceding from left to right in this picture. The conditions for the experiment are given in the Experimental section. Figure IX Polyacrylamide Gel Disc ElectrOphoresis Of Yeast PK Purification Fractions See Materials and Methods for experimental procedures. Anodic ends are shown down. Fractions III, IV, V, VIa, and VIb as shown in order contained 200, 100, 100, 20, and 100 pg reSpectively. -38- Figure VIII Figure IX DISCUSSION A. Pyruvate Kinase from Yeast The per cent yield column in Table III summarizing the enzyme purification demonstrates that somewhat variable results are obtained in the assay of crude enzyme. Due to the allosteric nature of this enzyme a measured rate is highly dependent on the concentration of PEP in the assay. Early Fractions (I, II, III) can be shown to contain an interfering enzyme which hydrolyzes PEP to pyruvate and does not require ADP. This tends to lower the effective PEP concentration and the rate, resulting in misleading lower activities. The highest purity enzyme was obtained in high yield, about 29 per cent. Figure IX shows the polyacrylamide disc electrOphoresis patterns of Fractions III through VIb. The almost homogeneous Fraction VIa contains a 4 to 6 per cent impurity which can be removed if desired by rechromatography on cellulose phosphate. This contami- nant was demonstrated to arise from the higher ionic strength side of the active peak by disc electrOphoresis. The highest Specific activity obtained of about 150 in the absence of FDP compares favorably to the crystalline muscle PK specific activity of from 130 to 250 (Kayne and Suelter, 1965). It is not known whether yeast PK acts as a fluorokinase or hydroxylamine kinase as described for the muscle enzyme (Tietz and Ochoa, -39- -40- 1958; Kupiecki and Coon, 1960). While the stabilization of enzymes through the use of polyhydroxy compounds has been increasingly used, a common mechanism explaining the increased stability has as yet not been elucidated (Jarabek, §t_a1,, 1966). Of interest to this problem are the findings of Chilson, g£_§1, (1965) who showed that high concentrations of glycerol, sucrose, ethylene glycol, glucose, and pro- pylene glycol protected LDH from hybridization during freezing and thawing. Jarabek, g£_§1, (1966) discovered that 20 per cent glycerol solutions as well as high concentrations of phOSphate protected human placental 17Q-hydroxysteroid dehydrogenase from an inactivating aggregation phenomenon due to cold. These workers also pointed out the accumulated evidence that polyanions and glycerol might have a common protective mechanism. Macromolecular polyanions, as suggested by Bernfield, 33 a1, (1965), have a tendency to support the dissociation of several enzymes at high dilution. The catalytic pro- perties of mammary g1ucose-6-phOSphate dehydrogenase could be explained, Levy, gt_§1, (1966) found, by an Xa#=Y equilibrium in which formation of Y was promoted by NADP or NADPH and X by 40 per cent glycerol and NAD. Allosteric kinetics were seen in aqueous solutions but not in 40 per cent glycerol. These data all suggest that polyanions and glycerol in particular prevent pro- tein-protein interaction. -41- Table 11 lists some stabilizing reagents discovered for yeast pyruvate kinase. The most effective agents are polyhydroxy compounds and polyanions. The action of Mg“ , Mn“ , and ADP can probably be explained through substrate or cofactor protection, with fluoride and ATP plus Mg+I effects remaining unaccounted for. The gross effects of these stabilizing agents on molecular con- figuration are as yet unknown but undoubtedly some changes occur. Polyanions were not used in the purification as stabilizers because of the high concentrations required and because of a tendency to ineffectiveness at high protein concentrations. Cellulose derivative chromatography is also sensitive to high ionic strength. B. Yeast Catalytic and Physical PrOperties The indication of Washio and Mano (1960) (see Table IV) that the Optimal concentrations of activating mono- valent cation is 0.225 M has been substantiated as shown in Table V. In addition, rubidium ion is as effective an activator as ammonium ion. The 1.6 per cent remaining activity with TMA ion, a nonactivating and noninhi- bitory species, is probably due to contaminating ammonium ion from the LDH suSpension used in the assay. The KA for KCl (see Figures II and III) activation is higher in the presence of TMA ion at constant ionic strength than in the absence (0.17 and 0.029 M2 respectively). A possible explanation is that TMA+ competes for MgEI but -42- this seems unlikely. The higher Vmax observed in the first case possibly points to an ionic strength effect. The A/v versus A plots under both conditions were linear and the KA's obtained were both higher than the K of 2 A M found for the rabbit muscle enzyme (Kach- l.15 X 10- mar and Boyer, 1953). As mentioned before, yeast PK has a significantly lower pH Optimum of 6.1 to 6.4 (see Figure IV) than the muscle enzyme of 7.5 (Boyer, 1962) with a shift upwards of about 0.2 pH units observed with maleate buffer. This is considerably higher than the Optimum of pH 5.7 found by Washio and Mano (1960). The kinetics of yeast PK toward ADP are self-explanatory (Figure V) yielding a Km of 3.6 X 10'“ M, The PEP kinetics, however, in con— trast to the muscle enzyme, are nonlinear (Figure VI) and yield a limiting Hill s10pe of 4.2 with an apparent 3 Km of 1.1 X 10- M, For comparison, a line with lepe equal to l is drawn on the graph which would correspond to normal linear kinetics. Of particular interest is the nonlinearity of the Hill plot. At very high PEP con- centrations (greater than 5 X 10"3 M) substrate inacti- vation appears to occur, but may be due to the companion salt of cyclohexylamine. Intermediate PEP concentrations show kinetics with normal lepe. The muscle enzyme in contrast exhibits normal kinetics toward ADP and PEP in both crude and purified states (Dr. Karl Smiley, Jr., _u3_ unpublished results). Hommes' (1966a) failure to obtain cOOperative effects with this enzyme cannot be accounted for at this time. The accumulated evidence reviewed in the introduction that pyruvate kinase in yeast is a control point in glycolysis can now be explained not only by the PEP OOOperative effect but by the effect of FDP as shown in Table VI. PK exhibiting low catalytic rates with low concentrations of PEP can be fully activated by FDP. The rates Observed at pH 6.5 with PEP concentrations of 6.0 x 10‘“ M_are particularly striking; the enzyme was observed to be completely inactive in the absence of FDP but in the presence of 1.0 X 10"4 M FDP gave a rate of 106 units per mg. The combined work of Vinuela, gt-al, (1963, 1964) and Ramaiah, g£_§1. (1964) indicated that PFK of yeast is inhibited by ATP and stimulated by AMP, and Moore, §£_§1, (1965) showed that the PFK inhibition by ATP can be decreased by FDP. Gancedo, gt_31, (1965) demonstrated fructose diphOSphatase inhibition in yeast by AMP. These data added to the PK data give a fine control for glycolysis as shown in the scheme below: MN") 0 glucose—5 fructose-6-phosphate—Ib FDP-OPEP—D yruvate 3'?‘ 6 ATP i ADP ADP "27‘ ATP I FDP(+) FDP + ATP t AMP — -uu_ As previously mentioned, no control has been found for the other likely regulatory point, 3-phOSphoglycerate kinase. Other effectors have as yet not been found for yeast PK. A rough estimate of the molecular weight of the enzyme can be made from the sedimentation velocity run (Figure VIII), assuming a spherical molecule of 0'73Q’ of about 110,000 to 140,000. A probably asymetric con- figuration would raise this estimate considerably. The small contaminant seen in the sedimentation velocity plate is possibly the 4 to 6 per cent contaminant of the best purity Fraction VIa used for the experiment, but this remains to be proven. The molecular weight in the pre- sence of cofactors and substrates is not known, and determinations in the presence of FDP are especially needed. SUMMARY 1. Pyruvate kinase from yeast was shown to be stabilized by a variety Of reagents including polyanions and polyhydroxy compounds at high concentrations. 2. The enzyme was purified almost to homogeniety at high yields by toluene autolysis, ammonium sulfate fractionation, dialysis versus 50 per cent glycerol, treatment with DEAE cellulose, and chromatography on cellulose phosphate. 3. The purified enzyme was shown to have an absolute requirement for monovalent cations. Rubidium ion and ammonium ion could replace potassium ion with about one half efficiency. The K for potassium ion was A measured and was found to be higher in the presence of TMA ion than in the absence, yielding KA's of 0.17 and 0.029 respectively. 4. The pH activity profile was shown to have a maximum in the range of 6.1 to 6.4 in maleate, cacodylate, and imidazole buffers. 5. The kinetics of the enzyme toward ADP were shown to be linear with a Km of 3.6 X 10’“ M, PEP kinetics showed a c00perative effect with a limiting Hill shOpe of 4.2 and an apparent Km of 1.1 X 10"3 M, The allosteric nature of the enzyme was shown to be in agreement with the previous predictions that pyruvate kinase is a control -45- 4 —46- point in yeast glycolysis. 6. Full activation of the enzyme at low PEP con- centrations could be brought about by low concentrations of FDP. 7. The 320, w Of the enzyme by sedimentation velocity analysis was demonstrated to be 8.23 S. REFERENCES Bernfield, P., Berkeley, B., and Bieber, R. (1965), Arch. Biochem. BiOphys. 111, 31. Bock, H., Ling, N., Morell, s., and Lipton, s. (1956), $219, ég, 253. Boyer, P. (1962), in The Enzymes, V01. 6, Boyer, P., Lardy, H., and Myrback, K., eds., New York, N. Y., Academic Press, p. 95. Bficher, T. (1955), in Methods in Enzymology, Vol. 1, Colowick, S., and Kaplan, N., eds., New York, N. Y., Academic Press, p. 427. Bucher, T. and Pfleiderer, G. (1955), 1213,, V01 1, p. 435. Canalco (1963), Disc ElectrOphoresis Trial Kit Instruc- tions, Bethesda, Md. Chance, B., Holmes, w., Higgings, J., and Connelley, E. (1958), Nature 182, 1190. Chilson, 0., Costello, L., and Kaplan, N. (1965), Fed. Proc. Suppl. N0. 15, 24, S-55. Friedman, T. and Haugen, G. (1943), g, Biol. Chem. 147, 415. Gancedo, 0., Salas, M. L., Giner, A., and 8018, A. (1965), Biochem. BiOphys. Res. Commun. 20, 15. Hess, B. 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