ISOZYMES
OF RAT MUSCLE
PYRUVATE
KINASE
S. W. N. Wu, S. S. C. WING and D. C. Y. YEUNG Department of Biochemistry, University of Hong Kong, Hong Kong (Received IO May 1979) Abstract--i. Four different isozymes from rat muscle were resolved by hydroxyapatite chromatography. 2. Though all the isozymes exhibited hyperbolic kinetics, they possessed dissimilar atBnities for PEP. The phenylalanine inhibited enzyme responded differently to reactivation by fructose-l,&diphosphate. 3. The pattern of inhibition or activation by t_-alanine, t_-phenylalanine and ATP was common to all four isozymes. 4. The isozymes could not be separated by isoelectrofocusing nor interconverted from one to another by phosphorylating agents such as ATP and cyclic AMP dependent protein kinase. 5. It is postulated that they did not represent M,, M2 hybrids.
INTRODUCTION
Four isozymes of pyruv~te kinase (EC 2.7.1.40) have been detected in various vertebrate tissues. The L-type pyruvate kinase is the major isozyme in the liver and is immunologically similar to the R-type present in the erythrocytes (Imamura et al., 1973). The Mz-type is the most widely distributed isozyme and is found in fetal and many adult tissues. The fourth isozyme, the M,-type, exists in adult skeletal muscle, cardiac muscle and brain. The M, isozymes have been purified from many vertebrate muscles such as the ox (Cardenas et nl., 1973) the rat (Tanaka et al., 1967) the rabbit (Cottam et al., 1969) the chicken (Eigenbrodt & Schoner, 1977) and the human (Harkins et al., 1977). The physical, chemical and kinetic properties of the various M, ismymes have been extensively studied; different observations were obtained as to whether there is only one single Mi isozyme in skeletal muscle. Many workers purported that vertebrate muscles contain only one single type of Mr isozyme (Cardenas et al., 1973; Eigenbrodt & Schoner, 1977; Harkins et al., 1977; Harda et al., 1978) and there are no other variants such as those found in the kidney (Ibsen & Trippet, 1972; Ibsen et ai., 1975). On the other hand, the enzyme from rabbit muscle could be resolved into four peaks with enzymatic activity by means of isoelectrofocusing (Susor et al., 1969). Another similar observation was reported on chicken muscle pyruvate kinase (Cardenas et al., 1975). As the literature on rat muscle pyruvate kmase is still scanty as compared to that of other vertebrate muscles, this report attempts to investigate the possibility of the co-existence of more than one molecular form of pyruvate kinase in rat muscle. MATERIALS AND METHODS Chemicals
The sodium salt of adenosine-5’-diphosphate (ADP), adenosine-5’-triphosphate (ATP), tricyclohexylamine salt of phosphoenolpyruvate (PEP), reduced nicotinamide adenine dinucleotide (NADH), fructose-l, Gdiphosphate 1013
(FDP), t-phenylalanine, r.-alanine, bovine muscle protein kinase and rabbit muscle lactic dehydrogenase, Type II were obtained from Sigma Chemical Co. Sephadex G-25 used for desalting was a product from Pharmacia (Uppsala, Sweden). Ampholine with pH range of 5-9 was purchased from LKB Instruments, Inc. Hydroxyapatite was prepared according to the method of Tiselius (1956). All other chemicals were of standard analytical grade obtained from Merck. Enzyme
assay
Pyruvate kinase activity was assayed by coupling the pyruvate kinase reaction to that of lactic dehydrogenase according to the method of Bucher & Pfleiderer (1955). The assays were carried out at 37°C. in I ml of incubation medium containing 65 mM triethanolamine-HCI buffer pH 7.5, 75 mM KCI, 8 mM MgSO,, 1 mM PEP, 0.6 mM ADP, 0.26mM NADH and three units of lactic dehydrogenase. Partial
pur$cation
of rat muscle pyruvate
kinase
All purification steps were conducted at 0 to 4°C. unless otherwise stated. Step I: Extraction-Muscle was removed from the thighs of rats fed on normal rat chow and passed to a pre-cooled meat grinder. The minced muscle was then homogenized in 4~01 of 40mM potassium phosphate buffer pH 7.0, 0.25 M sucrose, 0.15 M KCI, 10 mM /?-mercaptoethanol using a Sorvall homogenizer at maximum speed for 1 min. The mixture was stirred for 1Omin and centrifuged at 13,OOOgfor 4.5min. The supernatant was strained through four layers of cheese cloth and the precipitate was discarded. Step 2: Heat treatment-The supernatant was heated rapidly to 60°C in a thermostated water bath with constant swirling and this temperature was maintained for 4 min. The suspension was immediately cooled in an ice bath to 5°C with constant agitation and the heat denatured protein was removed by centrifugation for 30 mm at 13,000 g. Step 3: Ammonium suiphate fractionation-The supernatant thus recovered was brought to 4504 ammonium sulphate saturation by slowly adding solid ammonium sutphate. The suspension was allowed to equilibrate for 20min and the precipitate was removed by centrifugation as before. The supernatant was then brought to 67.5% ammonium sulphate saturation and the precipitate obtained after centrifugation was dissolved in a minimum
S. W. N. Wu, S. S. C.
1014
WONG
and D. C. Y.
YEUNG
vol of 50 mM potassium phosphate buffer pH 6.9, 3 mM @mercaptoethanoi. Step 4: Hydroxyapatite chromatography--The enzyme was desalted by passing through a Sephadex G-25 column equilibrated with the same buffer used in dissolving the enzyme precipitate. The desalted enzyme was then applied to a hydroxyapatite column (2.5 x 50 cm) which was chromatographed using a linear gradient of 50-400 mM potassium phosphate buffer pH 6.9. The linear gradient was developed with a LKB Ultrograd Gradient Former. Fractions (10 ml) were collected. A typical elution pattern is shown in Fig. I. Kinetic
studies and effect of moduiaiors
The K, values for PEP were determined for each of the enzyme peaks obtained from hydroxyapatite chromatography. The PEP concentration varied from 32-400 PM whereas the ADP concentration was kept constant at 2 mM. For the investigation of the activating and inhibitory effects of the various modulators, the concentrations of FDP and ATP were 1mM and those of L-phenyIa~anine and L-alanine were 3 mM respectively. Phosphorylation
of the enzyme
Fractions containing 20 fig protein, corresponding to the first and second enzyme peaks from the hydroxyapatite column were incubated for 30min at 30°C. with 3Opg of bovine muscle protein kinase and 1mM ATP in the presence of 25 mM potassium phosphate buffer pH 7.0, 5 mM magnesium acetate, 7.5% glycerol and 0.1 mM cyclic 3’,5’-AMP in a total volume of 200~1. Control without ATP was run in parallel. The incubation was stopped by diluting the samples with 400~1 of cold 50 mM imidazoleHCl buffer pH 7.5, containing 50 mM KCI. 5 mM MgSO* and 0.1 mM dithiothreitol. After the termination of the phosphorylation reaction, the K, with respect to PEP for the treated samples were re-determined. Isoelectrofocusing
of partially
purified
enzyme
The partially purified enzyme obtained from hydroxyapatite ~hromato~aphy was subjected to isoelectrofocusing using LKB amphoiine of pH range 5-9 according to the procedure recommended by the LKB practical manual. The voltage was kept constant at 900 V and after 90 hr the gradient was withdrawn from the column using a peristaltic pump and 1ml fractions were collected. The fractions were assayed for pyruvate kinase activity as described previously.
I
i
J -04
Fig. 2. Hill’s plot for the various M, isozymes. (c+---0) Peak 1; (M) Peak 2; (HA) Peak 3; (A---A) Peak 4. RESULTS Purt~l
pur~cari~n
qf
muscle
pyruvate
kinase
on
hy-
~ro~ya~uii~e
The elution pattern of the enzyme is shown in Fig. 1. In contrast to the observation made by other workers that there is only one molecular form of pyruvate kinase in muscle, it was possible to resolve the partially purified extract into four distinct enzyme peaks by hy~oxyapatite chromato~aphy. This observation was not very likely to be due to artefacts as the same elution pattern was obtained each time when a fresh batch of rat muscle was processed. The first and second peaks seemed to be the major components as they presented much higher activities than the 3rd and 4th peaks and they were better separated from each other. K, (PEP)fir
the various MI isozymes The K, values with respect to PEP were determined and summarized in Table 1. Individual isozyme possesses non-identical K, values for PEP. The isozyme corresponding to Peak 2 was shown to have
Enzyme Activity W/ml)
“Hp4
conc.(Mf lo4
FRACTION No.
Fig. 1. A typical elution profile of rat muscle pyruvate kinase from hydroxyapatite Pyruvate kinase activity; (---) KH,PO, concentration.
column. (--)
1015
Isozymes of rat muscle pyntvate kinase Tabie 1. Table showing the K,‘s for PEP of the various forms of rat muscle pyruvate kinase Sample Peak Peak Peak Peak
f 2 3 4
K, (PEP) (mM)
Hill’s coefficient
5.5S68 5.03 I9 5.161 0.128
5.978 5.933
I .05 0.947
The ADP concentration was kept at 2 mM. Assay conditions were as described in Methods.
the highest afinity for its substrate, that is 3.19 x IO-* M, and the lowest at&&y for the substrate was observed for Peak 3 whose K, reached as high as 1.61 x 10e4M. However all peaks showed hyperbolic kinetics with respect to PEP, with their Hill’s coefficients equal to 1.
The effects of the various modulators on the activity of the different MI isozymes were listed in Table 2, and the combined effects of two or more modulators presented in Table 3. The results revealed that none of the MI isozymes was activated by FDP, in contrast to the behaviour of the L and Mz iso-
zymes to the effector. However, ah the isozymes were inhibited by 3 mM L-phenylalanine. Peak 2 was most sensitive to phenylalanine inhibition whilst Peak 4 was least sensitive. L-Alanine, which is a potent inhibitor for the L and M, isozymes, did not exert any inhibitory effect on any of the forms of MI and in fact a slight activation by the amino acid was demonstrated. Similar observations were made when ATP was used as the modulator. When the enzyme was incubated with more than one modulator, a different pattern of inhibition was observed. FDP was shown to be capable of reversing the inhibition by L-phenytafanine by reducing the inhibition from 76% to only iI% or 14% for Peak f and Peak 2 isozymes, although FDP itself was not an inherent activator for the isozymes. However the reactivation of the inhibited Peak 3 and Peak 4 isozyme by the same concentration of FDP (I mM) was much less. Thus FDP appears to be a less efficient deinhibiting agent for the L-pheny~a~anine inhibited Peak 3 and Peak 4 isozymes. t.-Alanine, on the other hand, behaved differently from FDP in its manner of reversing t-phenylalanine inhibition. The difference lies in the fact that all 4 M, isozymes were reactivated to almost the same extent, No significant effect of ATP on L-pheny~a~anine inhibition was observed in any of the isozymes
Table 2. Effect of modulators on the activities of the different M, isozymes
Sample Peak Peak Peak Peak
1 2 3 4
7; Inhibition or activation by ~-phenyialan~oe L-alanine
FIX
AT-P
0 0
-25 -20 - 18.5 -7.8
76.0 19.2 73.3 69.6
0 -8.0 -10 -8.5
The concentration of FDP and ATP was I mM and the amino acids were kept at 3 mM. Negative values denote activation by the modulators.
Table 3. Combined effect of modulators on the activity of the various M r isozymes
L-E-%
y/, Inhibition or activation by I-Phe t-Phe L-Ala
+ Sample Peak Peak Peak Peak
1 2 3 4
r--Ala t
&P
A+TP
L-Ala
A+TP
FDP
6.15 16.7 63.3 53.7
86.2 76.7 83.3 79.0
13.8 11.0 13.3 fS.2
- 19.2 -38.3 0 13.6
-16.2 - 18.3 - 23.5 0
The concentrations of the modulators were as those listed under Table 2. Negative values denote activation by the modulators. Table 4. Effect of phosphorylation on the K, (PEP)‘s of Peak I and Peak 2
fL WW fmW Sample Peak 1 Peak 2
Before phosphoryiat~on 0.0568 0.5319
After phospborylation 0.0458 5.0388
1016
Efict of
S. W. N. Wu. S. S. C. WONG and D. C. Y. YEUNG
of
phosphorylation
the M,
isozymes
Treating the fractions corresponding to Peaks 1 and 2 with ATP and cyclic-3’,5’-AMP dependent protein kinase did not seem to affect the kinetic properties of the isozymes to any extent (Table 4). This can be gathered from the fact that the K,‘s for PEP were not even significantly altered and we also found that the V,,, of the isozymes were not changed. The isozymes, after phosphorylation, still demonstrated hyperbolic kinetics with the Hill’s coefficients close to 1. In other words, the isozymes retained their basic kinetic properties after the attempted phosphorylation. Isoelectric
points
of
the
M, isozymes
When the different M, isozymes were pooled and subjected to isoelectrocusing using ampholine (pH range 559), only one distinct enzyme peak was obtained. This would suggest that the different isozymes have very similar, if not identical isoelectric points. Ibsen (1975) also observed one single band in isoelectrofocusing of the muscle enzyme. However the possibility of inactivation during the lengthy period of isoelectrofocusing cannot be ruled out.
DISCUSSION It has been reported by many workers that purified pyruvate kinase from vertebrate muscle contains one single isozyme which consists of subunits of equal molecular weight. Their conclusion is deducted from the observation that the purified enzyme gives only one single protein band when subjected to polyacrylamide gel electrophoresis in the absence or presence of sodium dodecyl sulphate. However there have been reports suggesting that more than one molecular form of muscle pyruvate kinase may exist (Susor et al., 1969; Cardenas et al., 1975) but no extensive work has been carried out to support their claim. Recently, it is reported that some intermediate forms of the enzyme other than the parental isozymes-M,, M, and L types, have been demonstrated in rat tissues (Saheki et al., 1978) by means of thin-layer polyacrylamide gel electrophoresis which offers a better resolution of the different isozymes. The four enzyme peaks resolved by hydroxyapatite chromatography represented different isozymes of muscle pyruvate kinase as they possessed dissimilar kinetic properties. The isozymes have different affinities for their substrate PEP as revealed by their different K, values (Table 1) although they all have a Hill’s coefficient of nearly 1. This latter property is consistent with the well established observation that Mi type pyruvate kinase exhibits MichaelisMenten kinetics. The K, with respect to PEP determined by other workers is 0.075 mM (Tanaka et al., 1967) and this may be a resultant value presented by a mixture of the four isozymes. On the whole, all isozymes behaved similarly towards different modulators such as FDP, ATP, L-phenylalanine and L-alanine (Table 2). These include their sensitivity to inhibition by L-phenylalanine, insensitivity to inhibition by ATP (1 mM), L-alanine or activation by FDP. However the minor isozymes i.e. Peaks 3 and 4 behaved differently from the major isozymes i.e. Peaks 1 and 2 as to the reversal of L-phenylalanine
inhibition by FDP (Table 3). FDP almost totally reactivated the L-phenylalanine inhibited major isozyme forms whereas the minor isozymes only showed a very small extent of reactivation. The isozymes seemed to be non-interconvertible by phosphorylating agents since they conserved their respective kinetic parameters after treatment with ATP, and cyclic 3’-5’-AMP dependent protein kinase (Table 4). This observation not only indicated that the different isozymes were not created by phosphorylation of one of the isozymes, but also gave support to the current idea that muscle pyruvate kinase is not regulated by a phosphorylation-dephosphorylation mechanism. Another possible explanation for the co-existence of these four isozymes is that they may be hybrids of the parental i.e. the Mi and M2 subunits. But the results on the kinetic behaviour of the isozymes revealed that the above proposal was not probable. The rationale for this was based on the fact that the isozymes were neither activated by FDP nor inhibited by L-alanine (3 mM), ATP (1 mM) and did not show sigmoidal kinetics with PEP. On the other hand the M, isozyme demonstrates sigmoidal kinetics with PEP, inhibited by L-alanine and ATP, and liable to FDP activation. If they were hybrids of MI and Mz subunits, the isozymes would have the following subunit composition: (M1)4r (Mi)sMz, (M&(M&, (M1)(MJ3. This means that the isozymes with a higher percentage composition of the Mz subunit should exhibit kinetic properties resembling more towards the M, isozyme. But we could not detect an isozyme which appeared to resemble the M2 type. Therefore the Mi isozymes are probably not M, , M, hybrids. Experiments are presently in progress to purify the individual M, isozyme so as to gain an understanding of their amino acid composition, physical and kinetic properties, and their immunological behaviour with respect to each other and to other pyruvate kinase isozymes.
REFERENCES
BUCHER T. & PFLEIDERERG. (1955) In Methods in Enzymology, Vol. 1 (Edited by COLOWICK S. P. & KAPLAN N. O.), pp. 435440. Academic Press, New York. CARDENAS J. M., DYSON R. D. & STRANDHOLM J. J. (1973) Bovine pyruvate kinases. I. Purification and characterization of the skeletal muscle isozyme. J. hiol. Chem. 248, 6931-6937. CARDENAS J. M., BALCHLY E. G., CE~XZOTT~ P. L. & Dusoh: R. D. (1975) Properties of chicken skeletal muscle pyruvate kinase and a proposal of its evolutionary relationship to the other avian and mammalian isozymes. Biochemistry
14, 2247-2252.
COTTAMG. L., HOLLENBERG P. F. & C&IN M. J. (1969) Subunit structure of rabbit muscle pyruvate kinase. J. hiol. Chem. 244, 1481-1486. EIGENBRODT E. & SCHONER W. (1977) Purification and properties of the pyruvate kinase isozymes type L and M2 from chicken liver. Hoppe-Seyler’s Z. physiol. Chem 358,
1033-1046.
HARKINS R. N., BLACK J. A. & RI~~ENBERG M. B. (1977) Purification and characterization of human muscle pyruvate kinase. Cnn. J. Biochem. 55, 301-307. HARDA K., SAHEKI S., WADA K. & TANAKA T. (1978) Puri-
fication of four pyruvate kinase isozymes of rats by affinity elution chromatography. Biochim. biophys. Actu 524,
327-339.
Isozymes IBSENK. H. & TRIPPET P. (1972) Interconvertible
of rat muscle
and noninterconvertible forms of rat pyruvate kinase. Biochemistry II, 44424450. IBSENK. H., TRIPPET P. & BASABE J. (1975) Properties of rat pyruvate kinase isozymes. In Isoz~~mes, Vol. 1, Molecular Structure (Edited by MARKERT C. L.) pp. 543-559. Academic Press, New York. IMAMURA K.. TANAKA T.. NISHINA T., NAKASHIMA K. & MIWA S. (1973) Electrophoretic, kinetic and immunological studies on pyruvate kinase of erythrocytes and other tissues. J. Biochem.. Tokyo 74, 1165-l 175. SAHEK~ S., HARDA K.. SANNO Y. & TANAKA T. (1978) Hybrid isozymes of rat pyruvate kinase. Their subunit
pyruvate
kinase
1017
and developmental changes in the liver. BioActa 526, 116-128. SUSORW. A.. KOCHMAN M. & RUTTER W. J. (1969) Heterogeneity of presumably homogeneous protein preparations. Science 165. 1260-I 262. TANAKA T.. HARANO Y. SUE F. & MORIMURA H. (1967) Crystallization, characterization and metabolic regulation of two types of pyruvate kinase isolated from rat tissues. J. Biochem., Tokyo 62, 71-91. TISELIUS A., HJERTEN S. & LEVIN 0. (1956) Protein chromatography on calcium phosphate columns. Archs Biochem. Biophys. 65, 132-155. structure chim.
hiophys.
OF RAT MUSCLE
PYRUVATE
KINASE
S. W. N. Wu, S. S. C. WING and D. C. Y. YEUNG Department of Biochemistry, University of Hong Kong, Hong Kong (Received IO May 1979) Abstract--i. Four different isozymes from rat muscle were resolved by hydroxyapatite chromatography. 2. Though all the isozymes exhibited hyperbolic kinetics, they possessed dissimilar atBnities for PEP. The phenylalanine inhibited enzyme responded differently to reactivation by fructose-l,&diphosphate. 3. The pattern of inhibition or activation by t_-alanine, t_-phenylalanine and ATP was common to all four isozymes. 4. The isozymes could not be separated by isoelectrofocusing nor interconverted from one to another by phosphorylating agents such as ATP and cyclic AMP dependent protein kinase. 5. It is postulated that they did not represent M,, M2 hybrids.
INTRODUCTION
Four isozymes of pyruv~te kinase (EC 2.7.1.40) have been detected in various vertebrate tissues. The L-type pyruvate kinase is the major isozyme in the liver and is immunologically similar to the R-type present in the erythrocytes (Imamura et al., 1973). The Mz-type is the most widely distributed isozyme and is found in fetal and many adult tissues. The fourth isozyme, the M,-type, exists in adult skeletal muscle, cardiac muscle and brain. The M, isozymes have been purified from many vertebrate muscles such as the ox (Cardenas et nl., 1973) the rat (Tanaka et al., 1967) the rabbit (Cottam et al., 1969) the chicken (Eigenbrodt & Schoner, 1977) and the human (Harkins et al., 1977). The physical, chemical and kinetic properties of the various M, ismymes have been extensively studied; different observations were obtained as to whether there is only one single Mi isozyme in skeletal muscle. Many workers purported that vertebrate muscles contain only one single type of Mr isozyme (Cardenas et al., 1973; Eigenbrodt & Schoner, 1977; Harkins et al., 1977; Harda et al., 1978) and there are no other variants such as those found in the kidney (Ibsen & Trippet, 1972; Ibsen et ai., 1975). On the other hand, the enzyme from rabbit muscle could be resolved into four peaks with enzymatic activity by means of isoelectrofocusing (Susor et al., 1969). Another similar observation was reported on chicken muscle pyruvate kinase (Cardenas et al., 1975). As the literature on rat muscle pyruvate kmase is still scanty as compared to that of other vertebrate muscles, this report attempts to investigate the possibility of the co-existence of more than one molecular form of pyruvate kinase in rat muscle. MATERIALS AND METHODS Chemicals
The sodium salt of adenosine-5’-diphosphate (ADP), adenosine-5’-triphosphate (ATP), tricyclohexylamine salt of phosphoenolpyruvate (PEP), reduced nicotinamide adenine dinucleotide (NADH), fructose-l, Gdiphosphate 1013
(FDP), t-phenylalanine, r.-alanine, bovine muscle protein kinase and rabbit muscle lactic dehydrogenase, Type II were obtained from Sigma Chemical Co. Sephadex G-25 used for desalting was a product from Pharmacia (Uppsala, Sweden). Ampholine with pH range of 5-9 was purchased from LKB Instruments, Inc. Hydroxyapatite was prepared according to the method of Tiselius (1956). All other chemicals were of standard analytical grade obtained from Merck. Enzyme
assay
Pyruvate kinase activity was assayed by coupling the pyruvate kinase reaction to that of lactic dehydrogenase according to the method of Bucher & Pfleiderer (1955). The assays were carried out at 37°C. in I ml of incubation medium containing 65 mM triethanolamine-HCI buffer pH 7.5, 75 mM KCI, 8 mM MgSO,, 1 mM PEP, 0.6 mM ADP, 0.26mM NADH and three units of lactic dehydrogenase. Partial
pur$cation
of rat muscle pyruvate
kinase
All purification steps were conducted at 0 to 4°C. unless otherwise stated. Step I: Extraction-Muscle was removed from the thighs of rats fed on normal rat chow and passed to a pre-cooled meat grinder. The minced muscle was then homogenized in 4~01 of 40mM potassium phosphate buffer pH 7.0, 0.25 M sucrose, 0.15 M KCI, 10 mM /?-mercaptoethanol using a Sorvall homogenizer at maximum speed for 1 min. The mixture was stirred for 1Omin and centrifuged at 13,OOOgfor 4.5min. The supernatant was strained through four layers of cheese cloth and the precipitate was discarded. Step 2: Heat treatment-The supernatant was heated rapidly to 60°C in a thermostated water bath with constant swirling and this temperature was maintained for 4 min. The suspension was immediately cooled in an ice bath to 5°C with constant agitation and the heat denatured protein was removed by centrifugation for 30 mm at 13,000 g. Step 3: Ammonium suiphate fractionation-The supernatant thus recovered was brought to 4504 ammonium sulphate saturation by slowly adding solid ammonium sutphate. The suspension was allowed to equilibrate for 20min and the precipitate was removed by centrifugation as before. The supernatant was then brought to 67.5% ammonium sulphate saturation and the precipitate obtained after centrifugation was dissolved in a minimum
S. W. N. Wu, S. S. C.
1014
WONG
and D. C. Y.
YEUNG
vol of 50 mM potassium phosphate buffer pH 6.9, 3 mM @mercaptoethanoi. Step 4: Hydroxyapatite chromatography--The enzyme was desalted by passing through a Sephadex G-25 column equilibrated with the same buffer used in dissolving the enzyme precipitate. The desalted enzyme was then applied to a hydroxyapatite column (2.5 x 50 cm) which was chromatographed using a linear gradient of 50-400 mM potassium phosphate buffer pH 6.9. The linear gradient was developed with a LKB Ultrograd Gradient Former. Fractions (10 ml) were collected. A typical elution pattern is shown in Fig. I. Kinetic
studies and effect of moduiaiors
The K, values for PEP were determined for each of the enzyme peaks obtained from hydroxyapatite chromatography. The PEP concentration varied from 32-400 PM whereas the ADP concentration was kept constant at 2 mM. For the investigation of the activating and inhibitory effects of the various modulators, the concentrations of FDP and ATP were 1mM and those of L-phenyIa~anine and L-alanine were 3 mM respectively. Phosphorylation
of the enzyme
Fractions containing 20 fig protein, corresponding to the first and second enzyme peaks from the hydroxyapatite column were incubated for 30min at 30°C. with 3Opg of bovine muscle protein kinase and 1mM ATP in the presence of 25 mM potassium phosphate buffer pH 7.0, 5 mM magnesium acetate, 7.5% glycerol and 0.1 mM cyclic 3’,5’-AMP in a total volume of 200~1. Control without ATP was run in parallel. The incubation was stopped by diluting the samples with 400~1 of cold 50 mM imidazoleHCl buffer pH 7.5, containing 50 mM KCI. 5 mM MgSO* and 0.1 mM dithiothreitol. After the termination of the phosphorylation reaction, the K, with respect to PEP for the treated samples were re-determined. Isoelectrofocusing
of partially
purified
enzyme
The partially purified enzyme obtained from hydroxyapatite ~hromato~aphy was subjected to isoelectrofocusing using LKB amphoiine of pH range 5-9 according to the procedure recommended by the LKB practical manual. The voltage was kept constant at 900 V and after 90 hr the gradient was withdrawn from the column using a peristaltic pump and 1ml fractions were collected. The fractions were assayed for pyruvate kinase activity as described previously.
I
i
J -04
Fig. 2. Hill’s plot for the various M, isozymes. (c+---0) Peak 1; (M) Peak 2; (HA) Peak 3; (A---A) Peak 4. RESULTS Purt~l
pur~cari~n
qf
muscle
pyruvate
kinase
on
hy-
~ro~ya~uii~e
The elution pattern of the enzyme is shown in Fig. 1. In contrast to the observation made by other workers that there is only one molecular form of pyruvate kinase in muscle, it was possible to resolve the partially purified extract into four distinct enzyme peaks by hy~oxyapatite chromato~aphy. This observation was not very likely to be due to artefacts as the same elution pattern was obtained each time when a fresh batch of rat muscle was processed. The first and second peaks seemed to be the major components as they presented much higher activities than the 3rd and 4th peaks and they were better separated from each other. K, (PEP)fir
the various MI isozymes The K, values with respect to PEP were determined and summarized in Table 1. Individual isozyme possesses non-identical K, values for PEP. The isozyme corresponding to Peak 2 was shown to have
Enzyme Activity W/ml)
“Hp4
conc.(Mf lo4
FRACTION No.
Fig. 1. A typical elution profile of rat muscle pyruvate kinase from hydroxyapatite Pyruvate kinase activity; (---) KH,PO, concentration.
column. (--)
1015
Isozymes of rat muscle pyntvate kinase Tabie 1. Table showing the K,‘s for PEP of the various forms of rat muscle pyruvate kinase Sample Peak Peak Peak Peak
f 2 3 4
K, (PEP) (mM)
Hill’s coefficient
5.5S68 5.03 I9 5.161 0.128
5.978 5.933
I .05 0.947
The ADP concentration was kept at 2 mM. Assay conditions were as described in Methods.
the highest afinity for its substrate, that is 3.19 x IO-* M, and the lowest at&&y for the substrate was observed for Peak 3 whose K, reached as high as 1.61 x 10e4M. However all peaks showed hyperbolic kinetics with respect to PEP, with their Hill’s coefficients equal to 1.
The effects of the various modulators on the activity of the different MI isozymes were listed in Table 2, and the combined effects of two or more modulators presented in Table 3. The results revealed that none of the MI isozymes was activated by FDP, in contrast to the behaviour of the L and Mz iso-
zymes to the effector. However, ah the isozymes were inhibited by 3 mM L-phenylalanine. Peak 2 was most sensitive to phenylalanine inhibition whilst Peak 4 was least sensitive. L-Alanine, which is a potent inhibitor for the L and M, isozymes, did not exert any inhibitory effect on any of the forms of MI and in fact a slight activation by the amino acid was demonstrated. Similar observations were made when ATP was used as the modulator. When the enzyme was incubated with more than one modulator, a different pattern of inhibition was observed. FDP was shown to be capable of reversing the inhibition by L-phenytafanine by reducing the inhibition from 76% to only iI% or 14% for Peak f and Peak 2 isozymes, although FDP itself was not an inherent activator for the isozymes. However the reactivation of the inhibited Peak 3 and Peak 4 isozyme by the same concentration of FDP (I mM) was much less. Thus FDP appears to be a less efficient deinhibiting agent for the L-pheny~a~anine inhibited Peak 3 and Peak 4 isozymes. t.-Alanine, on the other hand, behaved differently from FDP in its manner of reversing t-phenylalanine inhibition. The difference lies in the fact that all 4 M, isozymes were reactivated to almost the same extent, No significant effect of ATP on L-pheny~a~anine inhibition was observed in any of the isozymes
Table 2. Effect of modulators on the activities of the different M, isozymes
Sample Peak Peak Peak Peak
1 2 3 4
7; Inhibition or activation by ~-phenyialan~oe L-alanine
FIX
AT-P
0 0
-25 -20 - 18.5 -7.8
76.0 19.2 73.3 69.6
0 -8.0 -10 -8.5
The concentration of FDP and ATP was I mM and the amino acids were kept at 3 mM. Negative values denote activation by the modulators.
Table 3. Combined effect of modulators on the activity of the various M r isozymes
L-E-%
y/, Inhibition or activation by I-Phe t-Phe L-Ala
+ Sample Peak Peak Peak Peak
1 2 3 4
r--Ala t
&P
A+TP
L-Ala
A+TP
FDP
6.15 16.7 63.3 53.7
86.2 76.7 83.3 79.0
13.8 11.0 13.3 fS.2
- 19.2 -38.3 0 13.6
-16.2 - 18.3 - 23.5 0
The concentrations of the modulators were as those listed under Table 2. Negative values denote activation by the modulators. Table 4. Effect of phosphorylation on the K, (PEP)‘s of Peak I and Peak 2
fL WW fmW Sample Peak 1 Peak 2
Before phosphoryiat~on 0.0568 0.5319
After phospborylation 0.0458 5.0388
1016
Efict of
S. W. N. Wu. S. S. C. WONG and D. C. Y. YEUNG
of
phosphorylation
the M,
isozymes
Treating the fractions corresponding to Peaks 1 and 2 with ATP and cyclic-3’,5’-AMP dependent protein kinase did not seem to affect the kinetic properties of the isozymes to any extent (Table 4). This can be gathered from the fact that the K,‘s for PEP were not even significantly altered and we also found that the V,,, of the isozymes were not changed. The isozymes, after phosphorylation, still demonstrated hyperbolic kinetics with the Hill’s coefficients close to 1. In other words, the isozymes retained their basic kinetic properties after the attempted phosphorylation. Isoelectric
points
of
the
M, isozymes
When the different M, isozymes were pooled and subjected to isoelectrocusing using ampholine (pH range 559), only one distinct enzyme peak was obtained. This would suggest that the different isozymes have very similar, if not identical isoelectric points. Ibsen (1975) also observed one single band in isoelectrofocusing of the muscle enzyme. However the possibility of inactivation during the lengthy period of isoelectrofocusing cannot be ruled out.
DISCUSSION It has been reported by many workers that purified pyruvate kinase from vertebrate muscle contains one single isozyme which consists of subunits of equal molecular weight. Their conclusion is deducted from the observation that the purified enzyme gives only one single protein band when subjected to polyacrylamide gel electrophoresis in the absence or presence of sodium dodecyl sulphate. However there have been reports suggesting that more than one molecular form of muscle pyruvate kinase may exist (Susor et al., 1969; Cardenas et al., 1975) but no extensive work has been carried out to support their claim. Recently, it is reported that some intermediate forms of the enzyme other than the parental isozymes-M,, M, and L types, have been demonstrated in rat tissues (Saheki et al., 1978) by means of thin-layer polyacrylamide gel electrophoresis which offers a better resolution of the different isozymes. The four enzyme peaks resolved by hydroxyapatite chromatography represented different isozymes of muscle pyruvate kinase as they possessed dissimilar kinetic properties. The isozymes have different affinities for their substrate PEP as revealed by their different K, values (Table 1) although they all have a Hill’s coefficient of nearly 1. This latter property is consistent with the well established observation that Mi type pyruvate kinase exhibits MichaelisMenten kinetics. The K, with respect to PEP determined by other workers is 0.075 mM (Tanaka et al., 1967) and this may be a resultant value presented by a mixture of the four isozymes. On the whole, all isozymes behaved similarly towards different modulators such as FDP, ATP, L-phenylalanine and L-alanine (Table 2). These include their sensitivity to inhibition by L-phenylalanine, insensitivity to inhibition by ATP (1 mM), L-alanine or activation by FDP. However the minor isozymes i.e. Peaks 3 and 4 behaved differently from the major isozymes i.e. Peaks 1 and 2 as to the reversal of L-phenylalanine
inhibition by FDP (Table 3). FDP almost totally reactivated the L-phenylalanine inhibited major isozyme forms whereas the minor isozymes only showed a very small extent of reactivation. The isozymes seemed to be non-interconvertible by phosphorylating agents since they conserved their respective kinetic parameters after treatment with ATP, and cyclic 3’-5’-AMP dependent protein kinase (Table 4). This observation not only indicated that the different isozymes were not created by phosphorylation of one of the isozymes, but also gave support to the current idea that muscle pyruvate kinase is not regulated by a phosphorylation-dephosphorylation mechanism. Another possible explanation for the co-existence of these four isozymes is that they may be hybrids of the parental i.e. the Mi and M2 subunits. But the results on the kinetic behaviour of the isozymes revealed that the above proposal was not probable. The rationale for this was based on the fact that the isozymes were neither activated by FDP nor inhibited by L-alanine (3 mM), ATP (1 mM) and did not show sigmoidal kinetics with PEP. On the other hand the M, isozyme demonstrates sigmoidal kinetics with PEP, inhibited by L-alanine and ATP, and liable to FDP activation. If they were hybrids of MI and Mz subunits, the isozymes would have the following subunit composition: (M1)4r (Mi)sMz, (M&(M&, (M1)(MJ3. This means that the isozymes with a higher percentage composition of the Mz subunit should exhibit kinetic properties resembling more towards the M, isozyme. But we could not detect an isozyme which appeared to resemble the M2 type. Therefore the Mi isozymes are probably not M, , M, hybrids. Experiments are presently in progress to purify the individual M, isozyme so as to gain an understanding of their amino acid composition, physical and kinetic properties, and their immunological behaviour with respect to each other and to other pyruvate kinase isozymes.
REFERENCES
BUCHER T. & PFLEIDERERG. (1955) In Methods in Enzymology, Vol. 1 (Edited by COLOWICK S. P. & KAPLAN N. O.), pp. 435440. Academic Press, New York. CARDENAS J. M., DYSON R. D. & STRANDHOLM J. J. (1973) Bovine pyruvate kinases. I. Purification and characterization of the skeletal muscle isozyme. J. hiol. Chem. 248, 6931-6937. CARDENAS J. M., BALCHLY E. G., CE~XZOTT~ P. L. & Dusoh: R. D. (1975) Properties of chicken skeletal muscle pyruvate kinase and a proposal of its evolutionary relationship to the other avian and mammalian isozymes. Biochemistry
14, 2247-2252.
COTTAMG. L., HOLLENBERG P. F. & C&IN M. J. (1969) Subunit structure of rabbit muscle pyruvate kinase. J. hiol. Chem. 244, 1481-1486. EIGENBRODT E. & SCHONER W. (1977) Purification and properties of the pyruvate kinase isozymes type L and M2 from chicken liver. Hoppe-Seyler’s Z. physiol. Chem 358,
1033-1046.
HARKINS R. N., BLACK J. A. & RI~~ENBERG M. B. (1977) Purification and characterization of human muscle pyruvate kinase. Cnn. J. Biochem. 55, 301-307. HARDA K., SAHEKI S., WADA K. & TANAKA T. (1978) Puri-
fication of four pyruvate kinase isozymes of rats by affinity elution chromatography. Biochim. biophys. Actu 524,
327-339.
Isozymes IBSENK. H. & TRIPPET P. (1972) Interconvertible
of rat muscle
and noninterconvertible forms of rat pyruvate kinase. Biochemistry II, 44424450. IBSENK. H., TRIPPET P. & BASABE J. (1975) Properties of rat pyruvate kinase isozymes. In Isoz~~mes, Vol. 1, Molecular Structure (Edited by MARKERT C. L.) pp. 543-559. Academic Press, New York. IMAMURA K.. TANAKA T.. NISHINA T., NAKASHIMA K. & MIWA S. (1973) Electrophoretic, kinetic and immunological studies on pyruvate kinase of erythrocytes and other tissues. J. Biochem.. Tokyo 74, 1165-l 175. SAHEK~ S., HARDA K.. SANNO Y. & TANAKA T. (1978) Hybrid isozymes of rat pyruvate kinase. Their subunit
pyruvate
kinase
1017
and developmental changes in the liver. BioActa 526, 116-128. SUSORW. A.. KOCHMAN M. & RUTTER W. J. (1969) Heterogeneity of presumably homogeneous protein preparations. Science 165. 1260-I 262. TANAKA T.. HARANO Y. SUE F. & MORIMURA H. (1967) Crystallization, characterization and metabolic regulation of two types of pyruvate kinase isolated from rat tissues. J. Biochem., Tokyo 62, 71-91. TISELIUS A., HJERTEN S. & LEVIN 0. (1956) Protein chromatography on calcium phosphate columns. Archs Biochem. Biophys. 65, 132-155. structure chim.
hiophys.
