176
KINASES
[29]
[29] Pyruvate Kinase from Yeast
(Saccharomyces cerevisiae) B y ANN ACST, SHYUN-LoNG YVN, and C. H. SVELTEg H + + phosphoenolpyruvate + ADP
Mg~+,K +
~ ATP + pyruvate
Assay Method Principle. The reaction catalyzed by pyruvate kinase may be continuously followed by measuring uptake of protons with a pH star, 1 decrease in absorption at 230 nm due to loss of phosphoenolpyruvate (PEP) ~ or by coupling with lactic dehydrogenase to measure reduction of pyruvate with the oxidation of N A D H ? The latter method, because of its most widespread use, is described in detail. Procedure. The reaction mixture contains, per milliliter: (CH3)~N cacodylate pH 6.2, 100 ~moles; MgC12, 24 ~moles; KC1, 100 tLmoles; tricyclohexylammonium P E P , 10 nmoles 4; Tris ADP, 10 ~moles ; tetracyclohexylammonium F D P , 1 tLmole; N A D H , 0.16 ~mole; and lactic dehydrogenase, 33 ~g. After the reaction mixture is equilibrated at the desired temperature of 30 °, a blank rate arising from pyruvate kinase contamination of the coupling lactic dehydrogenase is determined. The reaction is initiated by addition of 5-10 ~l of pyruvate kinase solution containing 10-20 ~g/ml. The initial rate of reaction is corrected for the blank rate. Definition of Unit and Specific Activity. One unit of p y r u v a t e kinase activity is the amount of enzyme t h a t catalyzes the transformation of 1 umole of substrate per minute under the conditions specified. Throughout the purification procedure and until the first cellulose phosphate column at p H 6.5, protein is determined by the biuret 5 method. After the cellulose phosphate column at p H 6.5, the extinction coefficient determined for yeast p y r u v a t e kinase o~0.1~ 0.51 is used. 6 ~280 Purification Procedure The purification procedure is a modification of that previously describedJ All operations except for the lysis are completed at room temF. J. Kayne and C. H. Suelter, J. Amer. Chem. Soc. 87, 897 (1965). 2N. G. Pon and R. J. L. Bondar, Anal. Biochem. 19, 272 (1967). T. Biicher and G. Pfleiderer, this series, Vol. 1, p. 435 (1953). 4Two micromoles of PEP per milliliter may be used for general routine assays. 5E. Layne, this series, Vol. 3, 450 (1957). A. Aust, S. L. Yun, and C. H. Suelter, unpublished observation. 7j. R. Hunsley anti C. H. Suelter, J. Biol. Chem. 244, 4815 (1969).
[29]
177
YEAST PYRUVATE KINASE PURIFICATION OF PYRUVATE KINASE FROM
Fraction Initial lysate, fraction I ~ (NH4)~S04 fraction 4O-55 % Phosphocellulose chromatography, pH 6.5 DEAE-phosphocellulose chromatography, pH 7.5 Phosphocellulose chromatography, pH 6.5 Sephadex G-100
Saccharomyces cerevisiae
Volume (ml)
Total protein (rag)
Total units
Specific activity (units/mg)
1070 195
24,055 12,135
245,000 226,400
10 18.6
500
2,485
173,929
70
71
520
529
132,260
250
54
80
127
43,200
340
18
25
110
37,400
340
15
Yield (%) 100 92
This represents the yield from 1 pound of yeast. perature. The results of a typical procedure are summarized in the table. Buffers and Strategy. The following buffers are used: pH 6.5 buffer consists of 10 m M sodium phosphate, 5 m M E D T A , 5 m M 2-mereaptoethanol, and 25% glycerol adjusted to pH 6.5; p H 7.5 buffer is identical in composition to the pH 6.5 buffer but adjusted to pH 7.5; and (NH,)~S04 pH 6.5 buffer consists of 10 mM sodium phosphate, 5 m M E D T A , 5 m M 2-mercaptoethanol, 25% glycerol, and (NH4)2SO~ at the appropriate concentrations adjusted to pH 6.5. The purification .once initiated should be completed as soon as possible to prevent proteolytie degradation. If it is necessary to stop at some step, it is recommended that the enzyme be precipitated with 55% (NH~)2SO~ and stored as an (NH4)._,SO~ suspension, at room temperature. Lysis of Yeast. One pound of yeast (Budweiser bakers' yeast) is crumbled into 800 ml of distilled-deionized water at 4°C and stirred until suspension is complete. The resulting suspension is made 1 m M in diisopropylfluorophosphate ( D F P ) , ~ and passed twice through a precooled s One gram of DFP is dissolved in 49 ml of dried isopropanol (dried over Fisher type 5 A molecular sieve or sodium sulfate), giving an approximate 0.1 M solution. This stock solution was stored at --20 ° . Condensation of water vapor in the stock solution should be avoided. Whenever DFP is added, it is to a final concentration of 1 raM. To avoid denaturation of protein, the DFP stock solution (0.1 M) is diluted 10-fold in the protein solvent before addition to rapidly stirring protein solutions. Because of the toxic effects of DFP, the user is advised to take cognizance of the following precautions : a. All operations with the pure liquid and concentrated solutions (greater than 1 raM) are to be done in a hood with good air flow. The user must wear gloves
178
KINASES
[29]
Manton-Gaulin homogenizer at 8000-8500 psi. The homogenized suspension is immediately adjusted to p H 7.0 with 4 N K O H and treated with D F P as before. The extract is then made 40 m M in CaC1._, (4.4 g/liter), and stirred thoroughly2 Immediately after dissolution of CaC12, 1.0 M potassium phosphate (pH 7.0) is added to a final concentration of 0.1 M and the solution allowed to stand for 1 hr at room temperature with occasional stirring. The clear yellow supernatant obtained after centrifugation at 14,000 g for 30 min is fraction I. All remaining steps in the procedure are accomplished at room temperature. A m m o n i u m Sul]ate Fractionation. To fraction I is added 243 g/liter solid ammonium sulfate, slowly, with constant stirring. After dissolution of ammonium sulfate, the mixture is adjusted to pH 6.2 with 4 N K O H and allowed to remain for 1 hr with constant stirring. The supernatant obtained after centrifugation at 14,000 g for 30 min is fraction II. To fraction I I is added 97 g/liter of solid ammonium sulfate, slowly, with constant stirring. After dissolution, the mixture is adjusted to p H 6.2 with 4 N KOH, allowed to remain for 1 hr with constant stirring and centrifuged at 14,000 g for 30 min. The pellet is suspended in pH 6.5 buffer to give a final volume of near 200 ml and dialyzed overnight against 2 liters of pH 6.5 buffer containing 1 m M D F P . A precipitate at this stage must be removed by centrifugation. The dialyzate was diluted with p H 6.5 buffer to a final volume of 1500 ml to give fraction III. This dilution reduces the (NH~)2SO4 concentration to near 10 m M and protein concentration to near 10 mg/ml and is required to achieve adsorption of enzyme to the cellulese phosphate pH 6.5 column described in the next step. Fraction I I I has a hazy white appearance which precipitates slowly on standing. This requires that the fraction be applied to the cellulose phosphate p H 6.5 column immediately after dilution. The precipitation is markedly enhanced if the precipitated fraction II is dialyzed by gel at all times and take precautions not to contaminate clothing. Polyvinyl glovea are recommended. b. All contaminated glassware is placed in 0.5 N NaOtt for a period of at least 24 hr for complete hydrolysis. c. One-millimolar solutions are not allowed to come in contact with skin. d. The following accounts of the chemistry and toxicity of fluorophosphate are available : L. S. Goodman and A. Gelman, "The Pharmacological Basis of Therapeutics," 3rd ed., p. 441. Macmillan, New York, 1969. B. C. Saunders, "Some Aspects of the Chemistry and Toxic Action of Organic Compounds Containing Phosphorus and Fluorine." Cambridge Univ. Press, London and New York, 1957. ~J. A. Illingworth, Biochem. J. 129, 1119 (1972).
[29]
YEAST PYRUVATE KINASE
179
permeation chromatography. The nature of the precipitate is not known. Cellulose Phosphate Column pH 6.5. Cellulose phosphate (1.1 m E q / g 1°) is washed as previously described. 7 The cellulose is suspended in p H 6.5 buffer, and the slurry is adjusted to p H 6.5 directly, using a p H meter. The cellulose is suction-filtered dry on a Biichner funnel, resuspended in the p H 6.5 buffer, and degassed. A column (5 X 20 cm) is poured and prior to use washed with at least 1 column volume of buffer. The p H of the eluate must be 6.5; if not, wash with additional buffer. After application of fraction I I I at an optimum flow rate of 500 ml/hour, the column is washed with p H 6.5 buffer to remove excess protein. P y r u v a t e kinase is eluted with the 0.1 M (NH4)~SO4 p H 6.5 buffer. All fractions containing eluted protein are pooled to give fraction IV, and dialyzed overnight against 10 volumes of p H 7.5 buffer. 11 DEAE-Cellulose Phosphate Column pH 7.5. Cellulose phosphate (1.1 meq/g) is prepared as described earlier except p H 7.5 buffer is used. A column, 4 by 45 cm, is poured to a height of 20 cm. DEAE-cellulose (0.8 meq/g) is washed as previously described 7 and suspended in the p H 7.5 buffer. The slurry was adjusted to p H 7.5, then suction-filtered dry, resuspended in the p H 7.5 buffer, and degassed. The DEAE-cellulose was poured directly onto the p H 7.5 cellulose phosphate to form a combined DEAE-cellulose-cellulose phosphate column with a total height of 40 cm. Prior to use, the column is washed with 1 column volume of p H 7.5 buffer. I f the p H of the eluate is not 7.5, wash with an additional volume of buffer. Fraction IV after adjustment to p H 7.5, is applied to the combined DEAE-cellulose-cellulose phosphate column followed by at least 1 column volume of p H 7.5 buffer. P y r u v a t e kinase is not adsorbed to this 1, Cellulose phosphate with a lower binding capacity will not function satisfactorily for this procedure using the column sizes as prescribed. To maintain binding capacity, the washing should be completed rapidly and the resin stored at pH 6~. Cellulose phosphate which has been subjected 6 or more times to the wash cycle appears to lose a significant percentage of its binding capacity. Please be aware that as this manuscript is being prepared, we were advised that the cellulose phosphate (Schwarz-Mann Mannex-P High Capacity) used for the development of this procedure, is no longer available. However, we have found that Brown Co. makes ~ cellulose phosphate of high capacity (greater than 1.1 meq/g) which may be satisfactorily substituted. 11Dialysis of fraction IV may be accomplished by passage over Sephadex G-25 or G-100. P~ssage over G-100 would serve an additional purpose of removing a contaminating protease activity which copurifies with the enzyme. To proceed, the enzyme is first precipitated by addition of 662 g of ammonium sulfate per liter. The precipitate obtained after centrifugation at 14,000 g for 30 rain is dissolved in a minimum volume of pH 7.5 buffer and applied to the Sephadex column equilibrated with the pH 7.5 buffer. To achieve separation of the contaminating protease, no more than 10 ml of solution are applied to a 5 X 90 cm G-100 column.
180
KINASES
[29]
column, and thus the eluate is collected fractionally until all protein is eluted. Those fractions having a specific activity greater than 200 are pooled to give fraction V. Cellulose Phosphate Column pH 6.5. The cellulose phosphate resin (1.1 mEq/g) is prepared in the pH 6.5 buffer as described above and poured into a 2.5 cm column to a height of 28 cm. Prior to use, the column is washed with an additional volume of pH 6.5 buffer. If the pH of the eluate is not 6.5, wash with additional buffer. Fraction V, after adjusting to pH 6.5 with 3 N acetic acid, is applied to the cellulose phosphate column. After application of sample, the column is washed with pH 6.5 buffer to remove unadsorbed protein. Pyruvate kinase is eluted with a 600-ml linear (NH4)2S04 gradient (0.01 to 0.2 M) in pH 6.5 buffer, and fractions are collected fractionally. The peak of the pyruvate kinase activity elutes at 70 mM (NH~)2SO4. Those fractions having pyruvate kinase with specific activities greater than 340 are pooled (fraction VI).12 The pooled fraction is dialyzed against 90% saturated ammonium sulfate prepared in 10 mM sodium phosphate, pH 6.5, 5 mM EDTA, 5 mM 2-mercaptoethanol, to precipitate all enzyme. The enzyme obtained at this stage migrates as a single band during sedimentation by analytical ultracentrifugation and electrophoresis on polyacrylamide gels. However, a solution of the protein in 0.1% sodium dodecyl sulfate containing 10 mM 2-mercaptoethanol incubated for 12 hr at room temperature gives several bands on SDS polyacrylamide gels after electrophoresis, consistent with a protease contamination. The contaminating protease was removed by the Sephadex G-100 column. Sephadex G-IO0 Column. The precipitated enzyme from fraction VI is sedimented by centrifugation at 27,000 g for 20 min and dissolved in a minimum volume of pH 6.5 buffer. One milliliter is applied to a Sephadex G-100 column, 1.6 X 70 cm, and eluted with the pH 6.5 buffer. The fractions containing pyruvate kinase with specific activities greater than 340, and free of protease contamination as determined by SDS polyacrylamide gel electrophoresis, are pooled and dialyzed against 90% saturated ammonium sulfate prepared in 10 mM sodium phosphate, pH 6.5, 5 mM EDTA, 5 mM 2-mercaptoethanol to precipitate enzyme. Yeast pyruvate kinase is stored as a suspension at 2-4 ° in 90% saturated ammonium sulfate and retains nearly full activity for at least 1 month. The specific activity of the purified enzyme is 320-360 units/mg at 30 °. The ratio of the specific activity determined with 10 mM P E P in the presence and in the absence of fructose 1,6-diphosphate should be 1. 12If most of enzyme obtained at this stage has a specific activity less t h a n 340 units/rag, a failure of the combined DEAE-cellulose phosphate p i t 7.5 column is indicated.
[29]
YEAST PYRUVATE KINASE
181
Properties Homogeneity and Molecular Properties. The enzyme prepared by this procedure is homogeneous by the following criteria: the protein sediments as a single symmetrical peak during analytical ultracentrifugation, equilibrium sedimentation data indicates no heterogeneity, and a single band is obtained after electrophoresis on polyacrylamide gel prepared with 5 different concentrations of acrylamide2 The absorbancy ratio A2sonm/A2~onm of the pure enzyme is greater than 1.7. The molecular weight of the enzyme determined by the sedimentation equilibrium method is 209,400 ± 1700. Electrophoresis of the protein in sodium dodecyl sulfate on polyacrylamide gel and comparison of its migration rate with those of standard proteins according to the method of 0sborn and Weber 13 gives a subunit molecular weight of 50,000-52,000 consistent with a tetrameric subunit protein. The preparation from Saccharomyces carlsbergensis ~4,~5 has a molecular weight of 190,000 and also consists of 4 subunits. The previous preparation of yeast (S. cerevisiae) pyruvate kinase had a molecular weight of 167,000 which dissociated in guanidine HC1 to give subunits of 42,000-46,000 molecular weight. 16 These data in light of the present results suggest that the previous preparation had undergone partial proteolytic breakdown. The evidence available to date indicates that the preparation described in this chapter is native to yeast. 17 However, the techniques that have been used are not sufficient to eliminate a microheterogeneity. The enzyme is cold labile 6 as described for an earlier preparation. TM Fructose 1,6-diphosphate enhances the rate of inactivation both at 0 ° and at 23 °. Glycerol (25%) a n d / o r 0.1 M KC1 and 20 m M MgC12 protects against the FDP-induced and cold inactivation. 19 The enzyme 1~K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). ~P. Roschlau and B. Hess, Hoppe-Seyler's Z. Physiol. Chem. 335, 435 (1972). 15H. Bischofberger, B. Hess, and P. Roschlau, Hoppe-Seyler's Z. Physiol. Chem. 352, 1139 (1971). 1~R. T. Kuczenski and C. H. Suelter, Biochemistry 9, 4032 (1970). 1, The presence of a contaminating protease at each step of the purification suggests a possible proteolytic degradation. However, the following evidence does not support this degradation. (1) Attempts to inhibit the contaminating proteasc by DFP at the stage just prior to the Sephadex G-100 column have failed, suggesting that the protease does not react with DFP or that it exists as a zymogen or as an enzyme-inhibitor complex~'; SDS may activate the zymogen or disrupt the complex. (9) No protease activity can be detected in the absence of SDS. (3) The enzyme migrates es a single sharp band on polyacrylamide gels. (4) The enzyme in an initial extract of yeast has the same mobility as purified enzyme on polyacrylamide gels prepared from 5 different concentrations of acrylamide. ~'J. F. Lenney and J. M. Dalbec, Arch. Biochem. Biophys. 129, 407 (1969). ~9R. T. Kuczenski and C. H. Suelter, Biochemistry 9, 939 (1970).
182
KINASES
[30]
contains 20 sulfhydryl residues; 8 are buried in the interior of the folded native enzyme, and 12 are exposed to the solvent as determined by reaction of native enzyme with 5,5'-dithiobis (2-nitrobenzoic acid).6 Kinetic Properties. Yeast pyruvate kinase is an allosteric enzyme exhibiting sigmoid substrate velocity saturation curves for PEP which are hyperbolic in the presence of fructose 1,6-diphosphate. The Km values at pH 6.2 and 30 ° in the presence of 1 mM FDP for PEP, ADP, K +, and Mg + are 99 ~M, 0.16 mM, 50 mM, and 2 mM, respectively. Reaction of the 12 exposed sulfhydryl residues with 5,5'-dithiobis(2nitrobenzoic acid) results in a marked increase in the Km for PEP as noted by an increase in the ratio of the specific activity determined in the presence of FDP to that determined in the absence of FDP at 10 mM PEP. A ratio of 1 is found for native enzyme; ratios as high as 14 have been observed for modified enzyme.
[30] Human Erythrocyte Pyruvate Kinase By G. E. J. STAAL, J. F. KOSTER, and C. VEEGER Phosphoenolpyruvate + ADP --~ pyruvate + ATP
Pyruvate kinase (EC 2.7.1.40) is a key enzyme in glycolysis. Human erythrocytes rely most exclusively on glycolysis to fulfill their energy requirements. Pyruvate kinase deficiency is, after glucose-6-phosphate dehydrogenase deficiency, the most common erythrocyte metabolic error. The deficiency comprises a heterogeneous group of disorders characterized by both qualitative and quantitative enzymic abnormalities.1 It is well accepted that different forms of pyruvate kinase exist. Erythrocyte pyruvate kinase resembles very much the liver L type, but differences do exist. An important difference between these two enzymes is that the hepatic enzyme is under hormonal and dietary control, whereas the erythrocyte enzyme is not. -~
Assay Method Pyruvate kinase activity is measured at 25 ° by coupling the system with lactate dehydrogenase, according to the method of Biicher and Pfleiderer. 3 The oxidation of NADH is followed at 340 ~m. 1p. Boivin, "Enzymopathies," Vol. 1, p. 190. Masson, Paris, 1971. 2T. Tanaka, Y. Harano, F. Sue, and It. Morimura, J. Biochem. (Tokyo) 62, 71 (1967). T. Bficher and G. Pfleiderer, this series, Vol. I, p. 435.
KINASES
[29]
[29] Pyruvate Kinase from Yeast
(Saccharomyces cerevisiae) B y ANN ACST, SHYUN-LoNG YVN, and C. H. SVELTEg H + + phosphoenolpyruvate + ADP
Mg~+,K +
~ ATP + pyruvate
Assay Method Principle. The reaction catalyzed by pyruvate kinase may be continuously followed by measuring uptake of protons with a pH star, 1 decrease in absorption at 230 nm due to loss of phosphoenolpyruvate (PEP) ~ or by coupling with lactic dehydrogenase to measure reduction of pyruvate with the oxidation of N A D H ? The latter method, because of its most widespread use, is described in detail. Procedure. The reaction mixture contains, per milliliter: (CH3)~N cacodylate pH 6.2, 100 ~moles; MgC12, 24 ~moles; KC1, 100 tLmoles; tricyclohexylammonium P E P , 10 nmoles 4; Tris ADP, 10 ~moles ; tetracyclohexylammonium F D P , 1 tLmole; N A D H , 0.16 ~mole; and lactic dehydrogenase, 33 ~g. After the reaction mixture is equilibrated at the desired temperature of 30 °, a blank rate arising from pyruvate kinase contamination of the coupling lactic dehydrogenase is determined. The reaction is initiated by addition of 5-10 ~l of pyruvate kinase solution containing 10-20 ~g/ml. The initial rate of reaction is corrected for the blank rate. Definition of Unit and Specific Activity. One unit of p y r u v a t e kinase activity is the amount of enzyme t h a t catalyzes the transformation of 1 umole of substrate per minute under the conditions specified. Throughout the purification procedure and until the first cellulose phosphate column at p H 6.5, protein is determined by the biuret 5 method. After the cellulose phosphate column at p H 6.5, the extinction coefficient determined for yeast p y r u v a t e kinase o~0.1~ 0.51 is used. 6 ~280 Purification Procedure The purification procedure is a modification of that previously describedJ All operations except for the lysis are completed at room temF. J. Kayne and C. H. Suelter, J. Amer. Chem. Soc. 87, 897 (1965). 2N. G. Pon and R. J. L. Bondar, Anal. Biochem. 19, 272 (1967). T. Biicher and G. Pfleiderer, this series, Vol. 1, p. 435 (1953). 4Two micromoles of PEP per milliliter may be used for general routine assays. 5E. Layne, this series, Vol. 3, 450 (1957). A. Aust, S. L. Yun, and C. H. Suelter, unpublished observation. 7j. R. Hunsley anti C. H. Suelter, J. Biol. Chem. 244, 4815 (1969).
[29]
177
YEAST PYRUVATE KINASE PURIFICATION OF PYRUVATE KINASE FROM
Fraction Initial lysate, fraction I ~ (NH4)~S04 fraction 4O-55 % Phosphocellulose chromatography, pH 6.5 DEAE-phosphocellulose chromatography, pH 7.5 Phosphocellulose chromatography, pH 6.5 Sephadex G-100
Saccharomyces cerevisiae
Volume (ml)
Total protein (rag)
Total units
Specific activity (units/mg)
1070 195
24,055 12,135
245,000 226,400
10 18.6
500
2,485
173,929
70
71
520
529
132,260
250
54
80
127
43,200
340
18
25
110
37,400
340
15
Yield (%) 100 92
This represents the yield from 1 pound of yeast. perature. The results of a typical procedure are summarized in the table. Buffers and Strategy. The following buffers are used: pH 6.5 buffer consists of 10 m M sodium phosphate, 5 m M E D T A , 5 m M 2-mereaptoethanol, and 25% glycerol adjusted to pH 6.5; p H 7.5 buffer is identical in composition to the pH 6.5 buffer but adjusted to pH 7.5; and (NH,)~S04 pH 6.5 buffer consists of 10 mM sodium phosphate, 5 m M E D T A , 5 m M 2-mercaptoethanol, 25% glycerol, and (NH4)2SO~ at the appropriate concentrations adjusted to pH 6.5. The purification .once initiated should be completed as soon as possible to prevent proteolytie degradation. If it is necessary to stop at some step, it is recommended that the enzyme be precipitated with 55% (NH~)2SO~ and stored as an (NH4)._,SO~ suspension, at room temperature. Lysis of Yeast. One pound of yeast (Budweiser bakers' yeast) is crumbled into 800 ml of distilled-deionized water at 4°C and stirred until suspension is complete. The resulting suspension is made 1 m M in diisopropylfluorophosphate ( D F P ) , ~ and passed twice through a precooled s One gram of DFP is dissolved in 49 ml of dried isopropanol (dried over Fisher type 5 A molecular sieve or sodium sulfate), giving an approximate 0.1 M solution. This stock solution was stored at --20 ° . Condensation of water vapor in the stock solution should be avoided. Whenever DFP is added, it is to a final concentration of 1 raM. To avoid denaturation of protein, the DFP stock solution (0.1 M) is diluted 10-fold in the protein solvent before addition to rapidly stirring protein solutions. Because of the toxic effects of DFP, the user is advised to take cognizance of the following precautions : a. All operations with the pure liquid and concentrated solutions (greater than 1 raM) are to be done in a hood with good air flow. The user must wear gloves
178
KINASES
[29]
Manton-Gaulin homogenizer at 8000-8500 psi. The homogenized suspension is immediately adjusted to p H 7.0 with 4 N K O H and treated with D F P as before. The extract is then made 40 m M in CaC1._, (4.4 g/liter), and stirred thoroughly2 Immediately after dissolution of CaC12, 1.0 M potassium phosphate (pH 7.0) is added to a final concentration of 0.1 M and the solution allowed to stand for 1 hr at room temperature with occasional stirring. The clear yellow supernatant obtained after centrifugation at 14,000 g for 30 min is fraction I. All remaining steps in the procedure are accomplished at room temperature. A m m o n i u m Sul]ate Fractionation. To fraction I is added 243 g/liter solid ammonium sulfate, slowly, with constant stirring. After dissolution of ammonium sulfate, the mixture is adjusted to pH 6.2 with 4 N K O H and allowed to remain for 1 hr with constant stirring. The supernatant obtained after centrifugation at 14,000 g for 30 min is fraction II. To fraction I I is added 97 g/liter of solid ammonium sulfate, slowly, with constant stirring. After dissolution, the mixture is adjusted to p H 6.2 with 4 N KOH, allowed to remain for 1 hr with constant stirring and centrifuged at 14,000 g for 30 min. The pellet is suspended in pH 6.5 buffer to give a final volume of near 200 ml and dialyzed overnight against 2 liters of pH 6.5 buffer containing 1 m M D F P . A precipitate at this stage must be removed by centrifugation. The dialyzate was diluted with p H 6.5 buffer to a final volume of 1500 ml to give fraction III. This dilution reduces the (NH~)2SO4 concentration to near 10 m M and protein concentration to near 10 mg/ml and is required to achieve adsorption of enzyme to the cellulese phosphate pH 6.5 column described in the next step. Fraction I I I has a hazy white appearance which precipitates slowly on standing. This requires that the fraction be applied to the cellulose phosphate p H 6.5 column immediately after dilution. The precipitation is markedly enhanced if the precipitated fraction II is dialyzed by gel at all times and take precautions not to contaminate clothing. Polyvinyl glovea are recommended. b. All contaminated glassware is placed in 0.5 N NaOtt for a period of at least 24 hr for complete hydrolysis. c. One-millimolar solutions are not allowed to come in contact with skin. d. The following accounts of the chemistry and toxicity of fluorophosphate are available : L. S. Goodman and A. Gelman, "The Pharmacological Basis of Therapeutics," 3rd ed., p. 441. Macmillan, New York, 1969. B. C. Saunders, "Some Aspects of the Chemistry and Toxic Action of Organic Compounds Containing Phosphorus and Fluorine." Cambridge Univ. Press, London and New York, 1957. ~J. A. Illingworth, Biochem. J. 129, 1119 (1972).
[29]
YEAST PYRUVATE KINASE
179
permeation chromatography. The nature of the precipitate is not known. Cellulose Phosphate Column pH 6.5. Cellulose phosphate (1.1 m E q / g 1°) is washed as previously described. 7 The cellulose is suspended in p H 6.5 buffer, and the slurry is adjusted to p H 6.5 directly, using a p H meter. The cellulose is suction-filtered dry on a Biichner funnel, resuspended in the p H 6.5 buffer, and degassed. A column (5 X 20 cm) is poured and prior to use washed with at least 1 column volume of buffer. The p H of the eluate must be 6.5; if not, wash with additional buffer. After application of fraction I I I at an optimum flow rate of 500 ml/hour, the column is washed with p H 6.5 buffer to remove excess protein. P y r u v a t e kinase is eluted with the 0.1 M (NH4)~SO4 p H 6.5 buffer. All fractions containing eluted protein are pooled to give fraction IV, and dialyzed overnight against 10 volumes of p H 7.5 buffer. 11 DEAE-Cellulose Phosphate Column pH 7.5. Cellulose phosphate (1.1 meq/g) is prepared as described earlier except p H 7.5 buffer is used. A column, 4 by 45 cm, is poured to a height of 20 cm. DEAE-cellulose (0.8 meq/g) is washed as previously described 7 and suspended in the p H 7.5 buffer. The slurry was adjusted to p H 7.5, then suction-filtered dry, resuspended in the p H 7.5 buffer, and degassed. The DEAE-cellulose was poured directly onto the p H 7.5 cellulose phosphate to form a combined DEAE-cellulose-cellulose phosphate column with a total height of 40 cm. Prior to use, the column is washed with 1 column volume of p H 7.5 buffer. I f the p H of the eluate is not 7.5, wash with an additional volume of buffer. Fraction IV after adjustment to p H 7.5, is applied to the combined DEAE-cellulose-cellulose phosphate column followed by at least 1 column volume of p H 7.5 buffer. P y r u v a t e kinase is not adsorbed to this 1, Cellulose phosphate with a lower binding capacity will not function satisfactorily for this procedure using the column sizes as prescribed. To maintain binding capacity, the washing should be completed rapidly and the resin stored at pH 6~. Cellulose phosphate which has been subjected 6 or more times to the wash cycle appears to lose a significant percentage of its binding capacity. Please be aware that as this manuscript is being prepared, we were advised that the cellulose phosphate (Schwarz-Mann Mannex-P High Capacity) used for the development of this procedure, is no longer available. However, we have found that Brown Co. makes ~ cellulose phosphate of high capacity (greater than 1.1 meq/g) which may be satisfactorily substituted. 11Dialysis of fraction IV may be accomplished by passage over Sephadex G-25 or G-100. P~ssage over G-100 would serve an additional purpose of removing a contaminating protease activity which copurifies with the enzyme. To proceed, the enzyme is first precipitated by addition of 662 g of ammonium sulfate per liter. The precipitate obtained after centrifugation at 14,000 g for 30 rain is dissolved in a minimum volume of pH 7.5 buffer and applied to the Sephadex column equilibrated with the pH 7.5 buffer. To achieve separation of the contaminating protease, no more than 10 ml of solution are applied to a 5 X 90 cm G-100 column.
180
KINASES
[29]
column, and thus the eluate is collected fractionally until all protein is eluted. Those fractions having a specific activity greater than 200 are pooled to give fraction V. Cellulose Phosphate Column pH 6.5. The cellulose phosphate resin (1.1 mEq/g) is prepared in the pH 6.5 buffer as described above and poured into a 2.5 cm column to a height of 28 cm. Prior to use, the column is washed with an additional volume of pH 6.5 buffer. If the pH of the eluate is not 6.5, wash with additional buffer. Fraction V, after adjusting to pH 6.5 with 3 N acetic acid, is applied to the cellulose phosphate column. After application of sample, the column is washed with pH 6.5 buffer to remove unadsorbed protein. Pyruvate kinase is eluted with a 600-ml linear (NH4)2S04 gradient (0.01 to 0.2 M) in pH 6.5 buffer, and fractions are collected fractionally. The peak of the pyruvate kinase activity elutes at 70 mM (NH~)2SO4. Those fractions having pyruvate kinase with specific activities greater than 340 are pooled (fraction VI).12 The pooled fraction is dialyzed against 90% saturated ammonium sulfate prepared in 10 mM sodium phosphate, pH 6.5, 5 mM EDTA, 5 mM 2-mercaptoethanol, to precipitate all enzyme. The enzyme obtained at this stage migrates as a single band during sedimentation by analytical ultracentrifugation and electrophoresis on polyacrylamide gels. However, a solution of the protein in 0.1% sodium dodecyl sulfate containing 10 mM 2-mercaptoethanol incubated for 12 hr at room temperature gives several bands on SDS polyacrylamide gels after electrophoresis, consistent with a protease contamination. The contaminating protease was removed by the Sephadex G-100 column. Sephadex G-IO0 Column. The precipitated enzyme from fraction VI is sedimented by centrifugation at 27,000 g for 20 min and dissolved in a minimum volume of pH 6.5 buffer. One milliliter is applied to a Sephadex G-100 column, 1.6 X 70 cm, and eluted with the pH 6.5 buffer. The fractions containing pyruvate kinase with specific activities greater than 340, and free of protease contamination as determined by SDS polyacrylamide gel electrophoresis, are pooled and dialyzed against 90% saturated ammonium sulfate prepared in 10 mM sodium phosphate, pH 6.5, 5 mM EDTA, 5 mM 2-mercaptoethanol to precipitate enzyme. Yeast pyruvate kinase is stored as a suspension at 2-4 ° in 90% saturated ammonium sulfate and retains nearly full activity for at least 1 month. The specific activity of the purified enzyme is 320-360 units/mg at 30 °. The ratio of the specific activity determined with 10 mM P E P in the presence and in the absence of fructose 1,6-diphosphate should be 1. 12If most of enzyme obtained at this stage has a specific activity less t h a n 340 units/rag, a failure of the combined DEAE-cellulose phosphate p i t 7.5 column is indicated.
[29]
YEAST PYRUVATE KINASE
181
Properties Homogeneity and Molecular Properties. The enzyme prepared by this procedure is homogeneous by the following criteria: the protein sediments as a single symmetrical peak during analytical ultracentrifugation, equilibrium sedimentation data indicates no heterogeneity, and a single band is obtained after electrophoresis on polyacrylamide gel prepared with 5 different concentrations of acrylamide2 The absorbancy ratio A2sonm/A2~onm of the pure enzyme is greater than 1.7. The molecular weight of the enzyme determined by the sedimentation equilibrium method is 209,400 ± 1700. Electrophoresis of the protein in sodium dodecyl sulfate on polyacrylamide gel and comparison of its migration rate with those of standard proteins according to the method of 0sborn and Weber 13 gives a subunit molecular weight of 50,000-52,000 consistent with a tetrameric subunit protein. The preparation from Saccharomyces carlsbergensis ~4,~5 has a molecular weight of 190,000 and also consists of 4 subunits. The previous preparation of yeast (S. cerevisiae) pyruvate kinase had a molecular weight of 167,000 which dissociated in guanidine HC1 to give subunits of 42,000-46,000 molecular weight. 16 These data in light of the present results suggest that the previous preparation had undergone partial proteolytic breakdown. The evidence available to date indicates that the preparation described in this chapter is native to yeast. 17 However, the techniques that have been used are not sufficient to eliminate a microheterogeneity. The enzyme is cold labile 6 as described for an earlier preparation. TM Fructose 1,6-diphosphate enhances the rate of inactivation both at 0 ° and at 23 °. Glycerol (25%) a n d / o r 0.1 M KC1 and 20 m M MgC12 protects against the FDP-induced and cold inactivation. 19 The enzyme 1~K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). ~P. Roschlau and B. Hess, Hoppe-Seyler's Z. Physiol. Chem. 335, 435 (1972). 15H. Bischofberger, B. Hess, and P. Roschlau, Hoppe-Seyler's Z. Physiol. Chem. 352, 1139 (1971). 1~R. T. Kuczenski and C. H. Suelter, Biochemistry 9, 4032 (1970). 1, The presence of a contaminating protease at each step of the purification suggests a possible proteolytic degradation. However, the following evidence does not support this degradation. (1) Attempts to inhibit the contaminating proteasc by DFP at the stage just prior to the Sephadex G-100 column have failed, suggesting that the protease does not react with DFP or that it exists as a zymogen or as an enzyme-inhibitor complex~'; SDS may activate the zymogen or disrupt the complex. (9) No protease activity can be detected in the absence of SDS. (3) The enzyme migrates es a single sharp band on polyacrylamide gels. (4) The enzyme in an initial extract of yeast has the same mobility as purified enzyme on polyacrylamide gels prepared from 5 different concentrations of acrylamide. ~'J. F. Lenney and J. M. Dalbec, Arch. Biochem. Biophys. 129, 407 (1969). ~9R. T. Kuczenski and C. H. Suelter, Biochemistry 9, 939 (1970).
182
KINASES
[30]
contains 20 sulfhydryl residues; 8 are buried in the interior of the folded native enzyme, and 12 are exposed to the solvent as determined by reaction of native enzyme with 5,5'-dithiobis (2-nitrobenzoic acid).6 Kinetic Properties. Yeast pyruvate kinase is an allosteric enzyme exhibiting sigmoid substrate velocity saturation curves for PEP which are hyperbolic in the presence of fructose 1,6-diphosphate. The Km values at pH 6.2 and 30 ° in the presence of 1 mM FDP for PEP, ADP, K +, and Mg + are 99 ~M, 0.16 mM, 50 mM, and 2 mM, respectively. Reaction of the 12 exposed sulfhydryl residues with 5,5'-dithiobis(2nitrobenzoic acid) results in a marked increase in the Km for PEP as noted by an increase in the ratio of the specific activity determined in the presence of FDP to that determined in the absence of FDP at 10 mM PEP. A ratio of 1 is found for native enzyme; ratios as high as 14 have been observed for modified enzyme.
[30] Human Erythrocyte Pyruvate Kinase By G. E. J. STAAL, J. F. KOSTER, and C. VEEGER Phosphoenolpyruvate + ADP --~ pyruvate + ATP
Pyruvate kinase (EC 2.7.1.40) is a key enzyme in glycolysis. Human erythrocytes rely most exclusively on glycolysis to fulfill their energy requirements. Pyruvate kinase deficiency is, after glucose-6-phosphate dehydrogenase deficiency, the most common erythrocyte metabolic error. The deficiency comprises a heterogeneous group of disorders characterized by both qualitative and quantitative enzymic abnormalities.1 It is well accepted that different forms of pyruvate kinase exist. Erythrocyte pyruvate kinase resembles very much the liver L type, but differences do exist. An important difference between these two enzymes is that the hepatic enzyme is under hormonal and dietary control, whereas the erythrocyte enzyme is not. -~
Assay Method Pyruvate kinase activity is measured at 25 ° by coupling the system with lactate dehydrogenase, according to the method of Biicher and Pfleiderer. 3 The oxidation of NADH is followed at 340 ~m. 1p. Boivin, "Enzymopathies," Vol. 1, p. 190. Masson, Paris, 1971. 2T. Tanaka, Y. Harano, F. Sue, and It. Morimura, J. Biochem. (Tokyo) 62, 71 (1967). T. Bficher and G. Pfleiderer, this series, Vol. I, p. 435.
