Comp. Biochem. Physiol., 1976, Vol. 53B, pp. 489 to 493.
Pergamon Press.
Printed in Great Britain
COMPARATIVE KINETIC AND ELECTROPHORETIC PROPERTIES OF ERYTHROCYTE PYRUVATE KINASES MARY A. BEESON AND JOHN A. BLACK Department of Biochemistry and Division of Medical Genetics, University of Oregon Medical School, Portland, OR 97201, U.S.A.
(Received 28 February 1975) Abstract--1. We have determined the effect of variation of phosphoenolpyruvate ADP and ATP
concentrations on the kinetics of erythrocyte pyruvate kinase from man, rhesus monkey, rabbit, dog, phalanger, beef, and opossum. 2. The enzymes show a wide range of kinetic and electrophoretic properties indicating the incorporation of genetically determined structural alterations in the various molecules during evolution. 3. We propose that the functional consequences of these alterations can be interpreted on the basis of a molecular model for erythrocyte pyruvate kinase similar to that of hemoglobin.
Preparation of erythrocyte pyruvate kinase White cells were removed by passing the blood through a cotton column (Busch & Pelz, 1966). The red cells were washed three times with 0.15 M KC1 and then lysed with an equal vol of distilled water for 10 min. Three vol of 0.15 M KCI were added and the hemolysate fractionated by addition of solid ammonium sulfate. Erythrocyte pyruvate kinases precipitated between 20 and 40% saturation of ammonium sulfate for all animals except the rhesus monkey, in which case the enzyme was obtained in the fraction precipitating between 10 and 30% saturation. The precipitates were dissolved in a small vol of 0-1 M triethanolamine-HCl (pH 7.4) and used for the kinetic experiments.
INTRODUCTION
PYRUVATE kinase (ATP: P y r u v a t e phosphotransferase, E.C. 2.7.1.40) is one of three rate-controlling enzymes of human erythrocyte glycolysis (Minakami & Yoshikawa, 1966; Hamasaki et al., 1970). The h u m a n e r y t h r o c y t e e n z y m e has allosteric kinetic properties which are consistent with its ~ate-controlling function. The e n z y m e is homotropically regulated by phosphoenolpyruvate, shows positive heterotropic modulation by f r u c t o s e - l , 6-diphosphate and glucose-6-phosphate and negative heterotropic modulation by A T P (Koler & Vanbellinghen, 1968; Black & H e n d e r s o n , 1972). Genetic alterations in the activity of erythrocyte p y r u v a t e kinase are the most c o m m o n enzymatic cause of hemolytic anemia in man (Beutler, 1969). The activity of the e n z y m e in other mammalian erythrocytes varies in a manner which is fairly well correlated with the overall rate of glycolysis (Jacobasch et al., 1974). In this paper we c o m p a r e the kinetic and electrophoretic properties of pyruvate kinase f r o m h u m a n erythrocytes and the erythrocytes of a n u m b e r of other mammals. W e have previously shown that all of the e r y t h r o c y t e p y r u v a t e kinase molecules studied in this paper are immunologically related (Rittenberg et al., 1975). This suggests that they have e v o l v e d f r o m a c o m m o n ancestor and that any functional differences o b s e r v e d will be due to genetically determined structural alterations.
Pyruvate kinase assay Assays were performed in a 1 ml reaction vol at 25°C and pH 7.4 in a Gilford 2400 recording spectrophotometer using the coupled assay with lactic dehydrogenase as described by Bucher & Pfleiderer (1955). Each cuvette contained a final buffer concentration of 0.1 M triethanolamine-HC1 (pH 7.4), 75 mM KCI and 8 mM MgSO,. The exact substrate concentrations used in each case are given in the figure legends. The absorbance change at 340 nm due to oxidation of NADH was followed for 10min. The results presented are averages of • duplicate determinations for each species with the exception of the monkey were only one determination was possible. All substrates were obtained from Sigma Chemical Co., St. Louis. Rabbit muscle lactic dehydrogenase was purchased as a crystalline suspension in 2.2 M ammonium sulfate from Calbiochem, Los Angeles.
MATERIALS AND METHODS
Sources of blood Blood samples were collected in heparin by venapuncture from Homo sapiens (human), Macaca mulatta (rhesus monkey), Oryctolagus cuniculus (rabbit), Canis familiaris (dog), and Trichosurus vulpecula (brush tailed phalanger). Blood from Bos taurus (bovine) was obtained at a local slaughter house using ethylene-dinitrilo tetraacetic acid (EDTA) as anticoagulant. Blood from Didelphis marsupialis virginianus (opossum) was collected in heparin by cardiac puncture. 489
Electrophoresis Starch gel electrophoresis was carried out at pH 5.0 in formic acid/NaOH buffer (Giblett, 1969) containing 1 mM EDTA, 0.25mM fructose-l,6-diphosphate, 10raM /3mercaptoethanol. Samples were applied to 1 x 0.25 cm strips of Whatman 3MM filter paper which were then inserted in the gel slots. The gel was run for 18 hr at 40 mA in the cold. After electrophoresis pyruvate kinase bands were detected by a modification of the method of Susor & Rutter (1971). The overlay medium contained 0.17M Tris-HC1 (pH 8.0), 2 mM MgCI2, 3 mM KC1, 0.5 mM PEP,
M. A. BEESON AND J. A. BLACK
490
0.8 mM ADP, 0.2 mM NADH with 5 mg Nobel agar and 4.8 units lactic dehydrogenase/ml. Results were recorded by Polaroid photography with u.v. illumination of the gel in which pyruvate kinase appears as a dark band on a fluorescent background. Polyacrylamide gel electrophoresis was carried out at pH 8.2 by a modification of the method of Imamura & Tanaka (1972). The gel buffer contained 10 mM Tris-HCl (pH 8.2), 5 mM MgSO4, 0.5 mM fructose-l,6-diphosphate and 10mM /3-mercaptoethanol. The samples were dialyzed for 2 hr against the gel buffer and 10 txl aliquants were put directly into the slots and covered with warm Vaseline. The electrophoretic buffer was that used in the gel containing 100 mM Tris-HC1 (pH 8.2). The gel was run for 3-5 hr at 400V in the cold. After electrophoresis, pyruvate kinase bands were detected by the same overlay method described for the starch gel electrophoresis. RESULTS
The effect of variation of p h o s p h o e n o l p y r u v a t e c o n c e n t r a t i o n at constant A D P c o n c e n t r a t i o n on the v e l o c i t y of the reaction c a t a l y z e d by pyruvate kinase from the e r y t h r o c y t e s of o p o s s u m , human and m o n k e y is s h o w n in Fig. 1. T h e p h o s p h o e n o l pyruvate c o n c e n t r a t i o n n e c e s s a r y for half maximal activity is listed for all s p e c i e s studied in Table I. The Hill's n values (Hill, 1910) obtained by analysis of the individual substrate c o n c e n t r a t i o n vs v e l o c ity curves are also given in Table 1. The effect of variation of A D P c o n c e n t r a t i o n at constant p h o s p h o e n o l p y r u v a t e concentration on the v e l o c i t y of the reaction c a t a l y z e d by the o p o s s u m , human and m o n k e y e n z y m e s is s h o w n in Fig. 2. The p h o s p h o e n o l p y r u v a t e c o n c e n t r a t i o n used in these e x p e r i m e n t s w a s 0-3 mM w h i c h is c o n s i d e r a b l y l o w e r than the saturating concentration as determined in Fig. 1. This modification of the more c o n v e n t i o n a l t e c h n i q u e o f studying the effect of one substrate at saturating c o n c e n t r a t i o n s o f the other w a s undertaken in an effort to a p p r o x i m a t e the reality of intracellular conditions. In the human red cell at least the p h o s p h o e n o l p y r u vate c o n c e n t r a t i o n is around 17 ~aM ( J a c o b a s c h et al., 1974). The use of 0.3 m M rather than the p h y s i o l o g i c a l c o n c e n t r a t i o n w a s dictated by technic a l considerations. It does, h o w e v e r , represent a part of the p h o s p h o e n o l p y r u v a t e curve w h e r e allosteric modulation is likely to be readily observed. The A D P c o n c e n t r a t i o n s n e c e s s a r y for half
,ooF 90 80!
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4
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Fig. 1. The effect of phosphoenolpyruvate concentration on the velocity of the reaction catalyzed by opossum, human and monkey erythrocyte pyruvate kinase. The ADP concentration was 0.4mM throughout. Reaction velocities on the ordinate are expressed as a % of the maximum velocity observed. e
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:
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L _ _ 1Calculated by a l e a s t squares analysis
at pH 0.2.
/Z.
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maximal activity are g i v e n for all s p e c i e s studied in Table 1. The effect of A T P c o n c e n t r a t i o n at constant p h o s p h o e n o l p y r u v a t e c o n c e n t r a t i o n on the v e l o c i t y of the reaction c a t a l y z e d b y the e n z y m e s from o p o s s u m , human and m o n k e y is s h o w n in Fig. 3. The c o n c e n t r a t i o n s of A T P w h i c h g a v e 75% of the
Human
2Ca] c u l a t e d r e l a t i v e
t 1'3
Fig. 2. The effect of ADP concentration on the velocity of the reaction catalyzed by opossum, human and monkey erythrocyte pyruvate kinase. The phosphoenolpyruvate concentration was 0.3 mM. Reaction velocities on the ordinate are expressed as a % of the maximum velocity observed.
Opossum
,
•
/
Table I. Kinetic and electrophoretic properties of erythrocyte pyruvate kinase Species
--5
o f a p l o t o f log ~ E ~ a g a i n s t log v/(V max-v).
to the m o b i l i t y of the human enzyme at pll 5.0 and the slower m i g r a t i n 0 human band
The value f o r m~nkey is t h a t of the slower band.
Comparative kinetic and electrophoretic properties of erythrocyte pyruvate kinases ~
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i 1.5
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Fig. 3. The effect of ATP on the velocity of the reaction catalyzed by opossum, human and monkey erythrocyte pyruvate kinase. The phosphoenolpyruvate concentration was 0.3 mM and the ADP concentration 0.4mM. The velocities on the ordinate are expressed as a % of the velocity of a control reaction mixture containing no ATP. activity of a control reaction containing no ATP are listed in Table 1 for these enzymes and the others studied. We have used two different systems to study the electrophoretic variability of mammalian erythrocyte pyruvate kinase. The behavior of four of the enzymes on starch gel electrophoresis at pH 5-0 is shown in Fig. 4. In this system the human enzyme migrates as a single band. As has been reported by Nakashima et al. (1974), the human enzyme gives two activity bands on thin layer polyacrylamide gel electrophoresis at p H S . 2 (Fig. 5). The rhesus monkey also gives two bands under these conditions. We have not observed multiple activity bands for the erythrocyte enzyme from any of the other species. This may, however, only reflect the inadequacy of the two separation methods used, rather than a homogeneous activity in these species. The canine enzyme migrates in the same direction as the human enzyme in starch gel at pH 5-0 with a mobility of 1.2 relative to the human enzyme. It migrates towards the anode on thin layer polyacrylamide gel electrophoresis at pH 8-2 with a mobility of 0.8 relative to the slower of the two human bands. Colhode
8eel
Rabbit OpossumHumor
Origin
pH 5.0
Fig. 4. Starch gel electrophoresis of erythrocyte pyruvate kinases at pH 5.0. The origin is detectable as a line across the center of the photograph. The cathode was at the top, the anode at the bottom. From left to right the slots contained the enzyme from beef, rabbit, opossum and human.
Fig. 5. Thin layer polyacrylamide gel electrophoresis of erythrocyte pyruvate kinases at pH8.2. The origin is detectable as a thin line close to the bottom of the photograph. The anode was at the top, the cathode at the bottom. From left to right the slots contained the enzyme from opossum, phalanger, monkey and human. The position of the phalanger activity with a mobility between that of the opossum and the faster of the monkey bands was confirmed in a subsequent experiment. DISCUSSION Cohen et al. (1973) have proposed that the central role of the glycolytic pathway in the survival of the mature red cell has restricted the evolutionary process acting on erythrocyte glycolytic enzymes. They were unable to detect any electrophoretic polymorphism in a survey of human erythrocyte pyruvate kinase from a large number of individuals. The studies reported here show that a wide range of kinetic and electrophoretic properties exist in erythrocyte pyruvate kinase from different species. The molecule has, therefore, changed in structure and function during evolutionary time. It is not clear, however, whether these changes represent independent, random events or alterations dictated by other changes in the red cells of the various species. For instance, there are considerable species variations in the overall rate of red cell glycolysis, in intracellular 2, 3 diphosphoglycerate concentrations and in the relative proportions of the monovalent cations (Jacobasch et al., 1974). The answer to this question must await further insight into these differences and the responsible molecular interrelationships within the red cell. The North American oppossum has been considered to be an example of a "living fossil" (Simpson, 1949) in which case the opossum proteins might represent molecules that have been unchanged through 100 million years of evolution. Current thought favors a continuing evolutionary history for the opossum (Tyndale-Biscoe, 1973) and the recent sequence determination of opossum hemoglobin (Stenzel, 1974) supports this view. We, therefore, attach no special evolutionary significance to the lack of cooperative properties shown by opossum erythrocyte pyruvate kinase. The molecular basis of the allosteric properties of erythrocyte pyruvate kinase has yet to be elucidated. The human enzyme has been proposed (Staal et al., 1971; Jacobson & Black, 1971) to conform to the R ~ T model of Monod et al. (1965). Studies of genetically altered human erythrocyte pyruvate kinase show a number of possible kinetic consequences (Nakashima et al., 1974; Nakashima, 1974; Van Eys & Garms, 1971) which are similar to the functional variations observed in human
492
M. A. BEESON AND J. A. BLACK
hemoglobin mutants (Perutz & Lehmann, 1968). We propose that the hemoglobin model can be usefully applied to erythrocyte pyruvate kinase in interpreting functional variation on a structural basis. From the intensive study of hemoglobin it is clear that the functional effect of a genetically determined structural alteration will depend on the nature and location of the alteration. The effects fall within three major groups (a) alterations which influence all allosteric properties by changing the relative stabilities of the oxy or deoxy forms of the molecule (b) alterations which affect the interaction between the heine group and the globin subunit or the region of the "active site" of hemoglobin and (c) those alterations which can be detected by electrophoretic differences but appear to cause no functional change. If the hemoglobin model is appropriate, we would then expect genetic alterations in erythrocyte pyruvate kinase to affect (a) all allosteric properties (b) the active site at the region of phosphoenolpyruvate and/or ADP binding or the allosteric sites at the region of ATP or fructose-l, 6-diphosphate binding or (c) electrophoretic properties only. In the comparison of pyruvate kinase from erythrocytes of different species we are, no doubt, detecting the effects of multiple alterations incorporated during the evolutionary divergence from common ancestors. Nevertheless, consideration of the diverse kinetic properties in terms of the above model is instructive. Hill's n value is taken to be the best measure of cooperative, allosteric properties and the kinetic data are arranged in Table 1 in order of increasing n value. If the n value is a function of the relative stabilities of the R or T forms of the enzyme, a number of other kinetic parameters should also change as the n value changes. The K0~s value for phosphoenolpyruvate should be directly related while the inhibitory effect of ATP, as measured by the concentration required to give 75% of control activity, should be inversely related to the n value, in the absence of any other alterations. We have shown, in a comparison of normal canine erythrocyte pyruvate kinase and an abnormal enzyme present in Basenji dogs, that these relationships hold and in addition that the K0.ss for ADP is inversely related to the n value (Standerfer et al., 1975). The relationships of these kinetic parameters are shown in Figs. 1-3 for opossum, human and monkey erythrocyte pyruvate kinase for which the n values are 0.94, 1.11 and 1.49 respectively. The data are consistent with the incorporation of mutations, during the separate evolution of opossum, man and monkey, which changed the relative stabilities of the allosteric forms of the pyruvate kinase molecules. The kinetic parameters for all species studied are presented in Table 1. The K05s values for phosphoenolpyruvate for the rabbit and beef enzymes are lower than expected from the above interpretation of the n values and we suggest that this is due to additional mutations at the active sites in these enzymes which have a specific effect on phosphoenolpyruvate binding. In like fashion the K0~s value for ADP is lower than expected for dog and higher than expected for rabbit and phalanger, suggesting alterations at the ADP binding site. ATP
is a more effective inhibitor of the dog enzyme and a less effective inhibitor of the phalanger enzyme than would be expected from the n value alone. We postulate mutations in these enzymes at the ATP binding site. It is not known whether ATP binds at part of the active site or at an entirely separate allosteric site. The correlation of an increased affinity of the dog enzyme and a decreased affinity of the phalanger enzyme for both ADP and ATP may indicate an overlap in the ADP and ATP binding sites. There is no obvious correlation between electrophoretic mobility and the various kinetic parameters which is consistent with the proposed model, We conclude that genetically determined alterations have been introduced into erythrocyte pyruvate kinase molecules in the course of divergent evolution and that the functional consequences of these alterations are consistent with a molecular model of pyruvate kinase similar to that of hemoglobin.
Acknowledgements--This work was supported by US PHS Grant AM-13173. We wish to acknowledge the essential support of Drs. W. Montagna, P. Ogilvie and M. B. Rittenberg in the collection of blood samples. Field trips in pursuit of Didelphis marsupialis virginianis were sustained by the James Joyce Fund for Special Studies.
REFERENCES
BEUTLER E. (1969) Genetic disorders of red cell metabolism. Med. Clin. N. Am. 53, 813-826. BLACKJ. A. & HENDERSONM. H. (1972) Activation and inhibition of human erythrocyte pyruvate kinase by organic phosphates, amino acids, dipeptides and anions. Biochim. biophys. Acta 284, 115-127. BUCHERT. & PFLEIDERERG. (1955) Pyruvate kinase from muscle. Meth. Enzymol. 1,435-440. BUSCH D. & PELZ K. (1966) Erythrocytenisoloierung aut Blut mit Baumwolle. Klin. Wschr. 44, 983-984. COHENP. T. W., OMENNG. S., MOTULSKYA. G., CHEN, S. H. & GIBLETT E. R. (1973) Restricted variation in glycolytic enzymes of human brain and erythrocytes. Nature New Biol. 241, 229-233. GIBLETr E. R. (1969) Genetic Markers in Human Blood, p. 439. Davis, Philadelphia. HAMASAKIN., ASAKURAT. & MINAKAMIS. (1970) Effect of oxygen tension of glycolysis in human erythrocytes. J. Biochem. 68, 157-161. HILL A. V. (1910) The possible effects of the aggregation of the molecules of hemoglobin on its dissociation curves. J. Physiol., Lond. 40, 4--7. [MAMURAK. & TANAKAT. (1972) Multimolecular forms of pyruvate kinase from rat and other mammalian tissues 1. Electrophoretic studies. J. Biochem. 71, 1043-1051. JACOBASCH G., MINAKAMIS. d~. RAPOPORTS. M. (t974) In Cellular and Molecular Biology of Erythrocytes. (Edited by YOSHIKAWA H. & RAPOI'ORT S. M.) University Park Press, Baltimore. JACOBSON K. W. & BLACKJ. A. (1971) Conformational differences in the active sites of muscle and erythrocyte pyruvate kinase J. biol. Chem. 246, 5504-5509. KOLER R. D. & VANBELUNGHENP. (1968) The mechanism of precursor modulation of human pyruvate kinase I by fructose diphosphate. Advan. Enzyme Regul. 6, 127-142. MINAKAMI S. & YOSHIKAWA H. (1966) Studies on
Comparative kinetic and electrophoretic properties of erythrocyte pyruvate kinases erythrocyte glycolysis--II. Free energy changes and rate limiting steps in erythrocyte glycolysis. J. BMchem. 59, 139-144. MONOD J., WYMAN J. • CHANGEUX J. P. (1965) On the nature of allosteric transitions. A plausible model. J. molec. Biol. 12, 88-118. NAKASHIMA K., MIWA S., ODA S., TANAKA T., IMAMURA K., & NISHINA T. (1974) Electrophoretic and kinetic studies of mutant erythrocyte pyruvate kinases. Blood 43, 537-548. NAKASHIMA K. (1974) Further evidence of molecular alteration and aberration of erythrocyte pyruvate kinase. Clinica chim Acta 55, 245-254. PERUTZ M. F. t~ LEHMANN H. (1968) Molecular pathology of human hemoglobin. Nature, Lond. 219, 902-909. RITrENBERG M. B., CHERN C. J., LINCOLN n . R. & BLACK J. A. (1975) The immunological properties of pyruvate kinase---I Mammalian erythrocyte enzymes. Im munochemistry 12, 491-494. SIMPSON G. G. (1949) The Meaning of Evolution. Yale University Press, New Haven, Connecticut.
493
STAAL G. E. J., KOSTER J. F., KAMP H., VAN MILLIGENBOERSMA L. & VEEGER C. (1971) Human erythrocyte pyruvate kinase. Its purification and some properties Biochim. biophys. Acta 227, 86-96. STANDERFER R. J., RITTENBERG M. B., CHERN C. J., TEMPLETON J. W. & BLACK J. A. (1975) Canine erythrocyte pyruvate kinase--II Properties of the abnormal enzymes associated with hemolytic anemia in the Basenji dog Blochem. Genet. 13, 341-351. STENZEL P. (1974) Opossum Hb chain sequence and neutral mutation theory. Nature, Lond. 252, 62-63. SUSOR W. A. & RUTrER W. J. (1971) Method for the detection of pyruvate kinase, aldolase and other pyridine nucleotide linked enzyme activities after electrophoresis. Analyt. Biochem. 43, 147-155. TYNDALE-B1SCOE H. (1973) Life of Marsupials. Elsevier, New York. VAN EYS J. & GARMS P. (1971) Pyruvate kinase hemolytic anemia, a model for correlation of clinical syndrome and biochemical anomalies. Adv. Pediat. 15, 203-229.
Pergamon Press.
Printed in Great Britain
COMPARATIVE KINETIC AND ELECTROPHORETIC PROPERTIES OF ERYTHROCYTE PYRUVATE KINASES MARY A. BEESON AND JOHN A. BLACK Department of Biochemistry and Division of Medical Genetics, University of Oregon Medical School, Portland, OR 97201, U.S.A.
(Received 28 February 1975) Abstract--1. We have determined the effect of variation of phosphoenolpyruvate ADP and ATP
concentrations on the kinetics of erythrocyte pyruvate kinase from man, rhesus monkey, rabbit, dog, phalanger, beef, and opossum. 2. The enzymes show a wide range of kinetic and electrophoretic properties indicating the incorporation of genetically determined structural alterations in the various molecules during evolution. 3. We propose that the functional consequences of these alterations can be interpreted on the basis of a molecular model for erythrocyte pyruvate kinase similar to that of hemoglobin.
Preparation of erythrocyte pyruvate kinase White cells were removed by passing the blood through a cotton column (Busch & Pelz, 1966). The red cells were washed three times with 0.15 M KC1 and then lysed with an equal vol of distilled water for 10 min. Three vol of 0.15 M KCI were added and the hemolysate fractionated by addition of solid ammonium sulfate. Erythrocyte pyruvate kinases precipitated between 20 and 40% saturation of ammonium sulfate for all animals except the rhesus monkey, in which case the enzyme was obtained in the fraction precipitating between 10 and 30% saturation. The precipitates were dissolved in a small vol of 0-1 M triethanolamine-HCl (pH 7.4) and used for the kinetic experiments.
INTRODUCTION
PYRUVATE kinase (ATP: P y r u v a t e phosphotransferase, E.C. 2.7.1.40) is one of three rate-controlling enzymes of human erythrocyte glycolysis (Minakami & Yoshikawa, 1966; Hamasaki et al., 1970). The h u m a n e r y t h r o c y t e e n z y m e has allosteric kinetic properties which are consistent with its ~ate-controlling function. The e n z y m e is homotropically regulated by phosphoenolpyruvate, shows positive heterotropic modulation by f r u c t o s e - l , 6-diphosphate and glucose-6-phosphate and negative heterotropic modulation by A T P (Koler & Vanbellinghen, 1968; Black & H e n d e r s o n , 1972). Genetic alterations in the activity of erythrocyte p y r u v a t e kinase are the most c o m m o n enzymatic cause of hemolytic anemia in man (Beutler, 1969). The activity of the e n z y m e in other mammalian erythrocytes varies in a manner which is fairly well correlated with the overall rate of glycolysis (Jacobasch et al., 1974). In this paper we c o m p a r e the kinetic and electrophoretic properties of pyruvate kinase f r o m h u m a n erythrocytes and the erythrocytes of a n u m b e r of other mammals. W e have previously shown that all of the e r y t h r o c y t e p y r u v a t e kinase molecules studied in this paper are immunologically related (Rittenberg et al., 1975). This suggests that they have e v o l v e d f r o m a c o m m o n ancestor and that any functional differences o b s e r v e d will be due to genetically determined structural alterations.
Pyruvate kinase assay Assays were performed in a 1 ml reaction vol at 25°C and pH 7.4 in a Gilford 2400 recording spectrophotometer using the coupled assay with lactic dehydrogenase as described by Bucher & Pfleiderer (1955). Each cuvette contained a final buffer concentration of 0.1 M triethanolamine-HC1 (pH 7.4), 75 mM KCI and 8 mM MgSO,. The exact substrate concentrations used in each case are given in the figure legends. The absorbance change at 340 nm due to oxidation of NADH was followed for 10min. The results presented are averages of • duplicate determinations for each species with the exception of the monkey were only one determination was possible. All substrates were obtained from Sigma Chemical Co., St. Louis. Rabbit muscle lactic dehydrogenase was purchased as a crystalline suspension in 2.2 M ammonium sulfate from Calbiochem, Los Angeles.
MATERIALS AND METHODS
Sources of blood Blood samples were collected in heparin by venapuncture from Homo sapiens (human), Macaca mulatta (rhesus monkey), Oryctolagus cuniculus (rabbit), Canis familiaris (dog), and Trichosurus vulpecula (brush tailed phalanger). Blood from Bos taurus (bovine) was obtained at a local slaughter house using ethylene-dinitrilo tetraacetic acid (EDTA) as anticoagulant. Blood from Didelphis marsupialis virginianus (opossum) was collected in heparin by cardiac puncture. 489
Electrophoresis Starch gel electrophoresis was carried out at pH 5.0 in formic acid/NaOH buffer (Giblett, 1969) containing 1 mM EDTA, 0.25mM fructose-l,6-diphosphate, 10raM /3mercaptoethanol. Samples were applied to 1 x 0.25 cm strips of Whatman 3MM filter paper which were then inserted in the gel slots. The gel was run for 18 hr at 40 mA in the cold. After electrophoresis pyruvate kinase bands were detected by a modification of the method of Susor & Rutter (1971). The overlay medium contained 0.17M Tris-HC1 (pH 8.0), 2 mM MgCI2, 3 mM KC1, 0.5 mM PEP,
M. A. BEESON AND J. A. BLACK
490
0.8 mM ADP, 0.2 mM NADH with 5 mg Nobel agar and 4.8 units lactic dehydrogenase/ml. Results were recorded by Polaroid photography with u.v. illumination of the gel in which pyruvate kinase appears as a dark band on a fluorescent background. Polyacrylamide gel electrophoresis was carried out at pH 8.2 by a modification of the method of Imamura & Tanaka (1972). The gel buffer contained 10 mM Tris-HCl (pH 8.2), 5 mM MgSO4, 0.5 mM fructose-l,6-diphosphate and 10mM /3-mercaptoethanol. The samples were dialyzed for 2 hr against the gel buffer and 10 txl aliquants were put directly into the slots and covered with warm Vaseline. The electrophoretic buffer was that used in the gel containing 100 mM Tris-HC1 (pH 8.2). The gel was run for 3-5 hr at 400V in the cold. After electrophoresis, pyruvate kinase bands were detected by the same overlay method described for the starch gel electrophoresis. RESULTS
The effect of variation of p h o s p h o e n o l p y r u v a t e c o n c e n t r a t i o n at constant A D P c o n c e n t r a t i o n on the v e l o c i t y of the reaction c a t a l y z e d by pyruvate kinase from the e r y t h r o c y t e s of o p o s s u m , human and m o n k e y is s h o w n in Fig. 1. T h e p h o s p h o e n o l pyruvate c o n c e n t r a t i o n n e c e s s a r y for half maximal activity is listed for all s p e c i e s studied in Table I. The Hill's n values (Hill, 1910) obtained by analysis of the individual substrate c o n c e n t r a t i o n vs v e l o c ity curves are also given in Table 1. The effect of variation of A D P c o n c e n t r a t i o n at constant p h o s p h o e n o l p y r u v a t e concentration on the v e l o c i t y of the reaction c a t a l y z e d by the o p o s s u m , human and m o n k e y e n z y m e s is s h o w n in Fig. 2. The p h o s p h o e n o l p y r u v a t e c o n c e n t r a t i o n used in these e x p e r i m e n t s w a s 0-3 mM w h i c h is c o n s i d e r a b l y l o w e r than the saturating concentration as determined in Fig. 1. This modification of the more c o n v e n t i o n a l t e c h n i q u e o f studying the effect of one substrate at saturating c o n c e n t r a t i o n s o f the other w a s undertaken in an effort to a p p r o x i m a t e the reality of intracellular conditions. In the human red cell at least the p h o s p h o e n o l p y r u vate c o n c e n t r a t i o n is around 17 ~aM ( J a c o b a s c h et al., 1974). The use of 0.3 m M rather than the p h y s i o l o g i c a l c o n c e n t r a t i o n w a s dictated by technic a l considerations. It does, h o w e v e r , represent a part of the p h o s p h o e n o l p y r u v a t e curve w h e r e allosteric modulation is likely to be readily observed. The A D P c o n c e n t r a t i o n s n e c e s s a r y for half
,ooF 90 80!
HJmon / / A/ / Monkey
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//o /
50
i
.
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30
,/
20
~ I
/ A/ /
2
,o!/ 2
3 mM PEP
4
5
Fig. 1. The effect of phosphoenolpyruvate concentration on the velocity of the reaction catalyzed by opossum, human and monkey erythrocyte pyruvate kinase. The ADP concentration was 0.4mM throughout. Reaction velocities on the ordinate are expressed as a % of the maximum velocity observed. e
U0nke
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t 8
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ATP I n h i b i t i o n 75% of Control w i t h no ADP
Electrophoretic Mobility 2 pH 5.0
pH 8,2 2,3
0.94
:
0.12
0.14
None
0.3
i. II
i!
0,44
0.09
0.53
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1,31
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1.49
Rabbit
1.53
1.08
0.05
0,19
0.46
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0.15
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1.85
1.01
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Beef
2.41
0.43
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0.10
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0,8
I
1.5
0.3 2.0 1.8
L _ _ 1Calculated by a l e a s t squares analysis
at pH 0.2.
/Z.
J 40
maximal activity are g i v e n for all s p e c i e s studied in Table 1. The effect of A T P c o n c e n t r a t i o n at constant p h o s p h o e n o l p y r u v a t e c o n c e n t r a t i o n on the v e l o c i t y of the reaction c a t a l y z e d b y the e n z y m e s from o p o s s u m , human and m o n k e y is s h o w n in Fig. 3. The c o n c e n t r a t i o n s of A T P w h i c h g a v e 75% of the
Human
2Ca] c u l a t e d r e l a t i v e
t 1'3
Fig. 2. The effect of ADP concentration on the velocity of the reaction catalyzed by opossum, human and monkey erythrocyte pyruvate kinase. The phosphoenolpyruvate concentration was 0.3 mM. Reaction velocities on the ordinate are expressed as a % of the maximum velocity observed.
Opossum
,
•
/
Table I. Kinetic and electrophoretic properties of erythrocyte pyruvate kinase Species
--5
o f a p l o t o f log ~ E ~ a g a i n s t log v/(V max-v).
to the m o b i l i t y of the human enzyme at pll 5.0 and the slower m i g r a t i n 0 human band
The value f o r m~nkey is t h a t of the slower band.
Comparative kinetic and electrophoretic properties of erythrocyte pyruvate kinases ~
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•
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01.5
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i 1.5
21.0
Fig. 3. The effect of ATP on the velocity of the reaction catalyzed by opossum, human and monkey erythrocyte pyruvate kinase. The phosphoenolpyruvate concentration was 0.3 mM and the ADP concentration 0.4mM. The velocities on the ordinate are expressed as a % of the velocity of a control reaction mixture containing no ATP. activity of a control reaction containing no ATP are listed in Table 1 for these enzymes and the others studied. We have used two different systems to study the electrophoretic variability of mammalian erythrocyte pyruvate kinase. The behavior of four of the enzymes on starch gel electrophoresis at pH 5-0 is shown in Fig. 4. In this system the human enzyme migrates as a single band. As has been reported by Nakashima et al. (1974), the human enzyme gives two activity bands on thin layer polyacrylamide gel electrophoresis at p H S . 2 (Fig. 5). The rhesus monkey also gives two bands under these conditions. We have not observed multiple activity bands for the erythrocyte enzyme from any of the other species. This may, however, only reflect the inadequacy of the two separation methods used, rather than a homogeneous activity in these species. The canine enzyme migrates in the same direction as the human enzyme in starch gel at pH 5-0 with a mobility of 1.2 relative to the human enzyme. It migrates towards the anode on thin layer polyacrylamide gel electrophoresis at pH 8-2 with a mobility of 0.8 relative to the slower of the two human bands. Colhode
8eel
Rabbit OpossumHumor
Origin
pH 5.0
Fig. 4. Starch gel electrophoresis of erythrocyte pyruvate kinases at pH 5.0. The origin is detectable as a line across the center of the photograph. The cathode was at the top, the anode at the bottom. From left to right the slots contained the enzyme from beef, rabbit, opossum and human.
Fig. 5. Thin layer polyacrylamide gel electrophoresis of erythrocyte pyruvate kinases at pH8.2. The origin is detectable as a thin line close to the bottom of the photograph. The anode was at the top, the cathode at the bottom. From left to right the slots contained the enzyme from opossum, phalanger, monkey and human. The position of the phalanger activity with a mobility between that of the opossum and the faster of the monkey bands was confirmed in a subsequent experiment. DISCUSSION Cohen et al. (1973) have proposed that the central role of the glycolytic pathway in the survival of the mature red cell has restricted the evolutionary process acting on erythrocyte glycolytic enzymes. They were unable to detect any electrophoretic polymorphism in a survey of human erythrocyte pyruvate kinase from a large number of individuals. The studies reported here show that a wide range of kinetic and electrophoretic properties exist in erythrocyte pyruvate kinase from different species. The molecule has, therefore, changed in structure and function during evolutionary time. It is not clear, however, whether these changes represent independent, random events or alterations dictated by other changes in the red cells of the various species. For instance, there are considerable species variations in the overall rate of red cell glycolysis, in intracellular 2, 3 diphosphoglycerate concentrations and in the relative proportions of the monovalent cations (Jacobasch et al., 1974). The answer to this question must await further insight into these differences and the responsible molecular interrelationships within the red cell. The North American oppossum has been considered to be an example of a "living fossil" (Simpson, 1949) in which case the opossum proteins might represent molecules that have been unchanged through 100 million years of evolution. Current thought favors a continuing evolutionary history for the opossum (Tyndale-Biscoe, 1973) and the recent sequence determination of opossum hemoglobin (Stenzel, 1974) supports this view. We, therefore, attach no special evolutionary significance to the lack of cooperative properties shown by opossum erythrocyte pyruvate kinase. The molecular basis of the allosteric properties of erythrocyte pyruvate kinase has yet to be elucidated. The human enzyme has been proposed (Staal et al., 1971; Jacobson & Black, 1971) to conform to the R ~ T model of Monod et al. (1965). Studies of genetically altered human erythrocyte pyruvate kinase show a number of possible kinetic consequences (Nakashima et al., 1974; Nakashima, 1974; Van Eys & Garms, 1971) which are similar to the functional variations observed in human
492
M. A. BEESON AND J. A. BLACK
hemoglobin mutants (Perutz & Lehmann, 1968). We propose that the hemoglobin model can be usefully applied to erythrocyte pyruvate kinase in interpreting functional variation on a structural basis. From the intensive study of hemoglobin it is clear that the functional effect of a genetically determined structural alteration will depend on the nature and location of the alteration. The effects fall within three major groups (a) alterations which influence all allosteric properties by changing the relative stabilities of the oxy or deoxy forms of the molecule (b) alterations which affect the interaction between the heine group and the globin subunit or the region of the "active site" of hemoglobin and (c) those alterations which can be detected by electrophoretic differences but appear to cause no functional change. If the hemoglobin model is appropriate, we would then expect genetic alterations in erythrocyte pyruvate kinase to affect (a) all allosteric properties (b) the active site at the region of phosphoenolpyruvate and/or ADP binding or the allosteric sites at the region of ATP or fructose-l, 6-diphosphate binding or (c) electrophoretic properties only. In the comparison of pyruvate kinase from erythrocytes of different species we are, no doubt, detecting the effects of multiple alterations incorporated during the evolutionary divergence from common ancestors. Nevertheless, consideration of the diverse kinetic properties in terms of the above model is instructive. Hill's n value is taken to be the best measure of cooperative, allosteric properties and the kinetic data are arranged in Table 1 in order of increasing n value. If the n value is a function of the relative stabilities of the R or T forms of the enzyme, a number of other kinetic parameters should also change as the n value changes. The K0~s value for phosphoenolpyruvate should be directly related while the inhibitory effect of ATP, as measured by the concentration required to give 75% of control activity, should be inversely related to the n value, in the absence of any other alterations. We have shown, in a comparison of normal canine erythrocyte pyruvate kinase and an abnormal enzyme present in Basenji dogs, that these relationships hold and in addition that the K0.ss for ADP is inversely related to the n value (Standerfer et al., 1975). The relationships of these kinetic parameters are shown in Figs. 1-3 for opossum, human and monkey erythrocyte pyruvate kinase for which the n values are 0.94, 1.11 and 1.49 respectively. The data are consistent with the incorporation of mutations, during the separate evolution of opossum, man and monkey, which changed the relative stabilities of the allosteric forms of the pyruvate kinase molecules. The kinetic parameters for all species studied are presented in Table 1. The K05s values for phosphoenolpyruvate for the rabbit and beef enzymes are lower than expected from the above interpretation of the n values and we suggest that this is due to additional mutations at the active sites in these enzymes which have a specific effect on phosphoenolpyruvate binding. In like fashion the K0~s value for ADP is lower than expected for dog and higher than expected for rabbit and phalanger, suggesting alterations at the ADP binding site. ATP
is a more effective inhibitor of the dog enzyme and a less effective inhibitor of the phalanger enzyme than would be expected from the n value alone. We postulate mutations in these enzymes at the ATP binding site. It is not known whether ATP binds at part of the active site or at an entirely separate allosteric site. The correlation of an increased affinity of the dog enzyme and a decreased affinity of the phalanger enzyme for both ADP and ATP may indicate an overlap in the ADP and ATP binding sites. There is no obvious correlation between electrophoretic mobility and the various kinetic parameters which is consistent with the proposed model, We conclude that genetically determined alterations have been introduced into erythrocyte pyruvate kinase molecules in the course of divergent evolution and that the functional consequences of these alterations are consistent with a molecular model of pyruvate kinase similar to that of hemoglobin.
Acknowledgements--This work was supported by US PHS Grant AM-13173. We wish to acknowledge the essential support of Drs. W. Montagna, P. Ogilvie and M. B. Rittenberg in the collection of blood samples. Field trips in pursuit of Didelphis marsupialis virginianis were sustained by the James Joyce Fund for Special Studies.
REFERENCES
BEUTLER E. (1969) Genetic disorders of red cell metabolism. Med. Clin. N. Am. 53, 813-826. BLACKJ. A. & HENDERSONM. H. (1972) Activation and inhibition of human erythrocyte pyruvate kinase by organic phosphates, amino acids, dipeptides and anions. Biochim. biophys. Acta 284, 115-127. BUCHERT. & PFLEIDERERG. (1955) Pyruvate kinase from muscle. Meth. Enzymol. 1,435-440. BUSCH D. & PELZ K. (1966) Erythrocytenisoloierung aut Blut mit Baumwolle. Klin. Wschr. 44, 983-984. COHENP. T. W., OMENNG. S., MOTULSKYA. G., CHEN, S. H. & GIBLETT E. R. (1973) Restricted variation in glycolytic enzymes of human brain and erythrocytes. Nature New Biol. 241, 229-233. GIBLETr E. R. (1969) Genetic Markers in Human Blood, p. 439. Davis, Philadelphia. HAMASAKIN., ASAKURAT. & MINAKAMIS. (1970) Effect of oxygen tension of glycolysis in human erythrocytes. J. Biochem. 68, 157-161. HILL A. V. (1910) The possible effects of the aggregation of the molecules of hemoglobin on its dissociation curves. J. Physiol., Lond. 40, 4--7. [MAMURAK. & TANAKAT. (1972) Multimolecular forms of pyruvate kinase from rat and other mammalian tissues 1. Electrophoretic studies. J. Biochem. 71, 1043-1051. JACOBASCH G., MINAKAMIS. d~. RAPOPORTS. M. (t974) In Cellular and Molecular Biology of Erythrocytes. (Edited by YOSHIKAWA H. & RAPOI'ORT S. M.) University Park Press, Baltimore. JACOBSON K. W. & BLACKJ. A. (1971) Conformational differences in the active sites of muscle and erythrocyte pyruvate kinase J. biol. Chem. 246, 5504-5509. KOLER R. D. & VANBELUNGHENP. (1968) The mechanism of precursor modulation of human pyruvate kinase I by fructose diphosphate. Advan. Enzyme Regul. 6, 127-142. MINAKAMI S. & YOSHIKAWA H. (1966) Studies on
Comparative kinetic and electrophoretic properties of erythrocyte pyruvate kinases erythrocyte glycolysis--II. Free energy changes and rate limiting steps in erythrocyte glycolysis. J. BMchem. 59, 139-144. MONOD J., WYMAN J. • CHANGEUX J. P. (1965) On the nature of allosteric transitions. A plausible model. J. molec. Biol. 12, 88-118. NAKASHIMA K., MIWA S., ODA S., TANAKA T., IMAMURA K., & NISHINA T. (1974) Electrophoretic and kinetic studies of mutant erythrocyte pyruvate kinases. Blood 43, 537-548. NAKASHIMA K. (1974) Further evidence of molecular alteration and aberration of erythrocyte pyruvate kinase. Clinica chim Acta 55, 245-254. PERUTZ M. F. t~ LEHMANN H. (1968) Molecular pathology of human hemoglobin. Nature, Lond. 219, 902-909. RITrENBERG M. B., CHERN C. J., LINCOLN n . R. & BLACK J. A. (1975) The immunological properties of pyruvate kinase---I Mammalian erythrocyte enzymes. Im munochemistry 12, 491-494. SIMPSON G. G. (1949) The Meaning of Evolution. Yale University Press, New Haven, Connecticut.
493
STAAL G. E. J., KOSTER J. F., KAMP H., VAN MILLIGENBOERSMA L. & VEEGER C. (1971) Human erythrocyte pyruvate kinase. Its purification and some properties Biochim. biophys. Acta 227, 86-96. STANDERFER R. J., RITTENBERG M. B., CHERN C. J., TEMPLETON J. W. & BLACK J. A. (1975) Canine erythrocyte pyruvate kinase--II Properties of the abnormal enzymes associated with hemolytic anemia in the Basenji dog Blochem. Genet. 13, 341-351. STENZEL P. (1974) Opossum Hb chain sequence and neutral mutation theory. Nature, Lond. 252, 62-63. SUSOR W. A. & RUTrER W. J. (1971) Method for the detection of pyruvate kinase, aldolase and other pyridine nucleotide linked enzyme activities after electrophoresis. Analyt. Biochem. 43, 147-155. TYNDALE-B1SCOE H. (1973) Life of Marsupials. Elsevier, New York. VAN EYS J. & GARMS P. (1971) Pyruvate kinase hemolytic anemia, a model for correlation of clinical syndrome and biochemical anomalies. Adv. Pediat. 15, 203-229.
