Sep 25, 1981 - Hochachka 1968) the Coneholepas-coneholepas pyru- vate kinase also .... Acknowledgements--We thank Dr Peter Ward for his help in the ...
Comp. Biochem. Physiol. Vol. 72B, pp. 65 to 69, 1982 Printed in Great Britain.
0305.0491/82/050065-05803.00/0 © 1982 Pergamon Press Ltd
PURIFICATION AND CHARACTERIZATION OF PYRUVATE KINASE F R O M MUSCLE O F THE SEA MOLLUSC CONCHOLEPAS CONCHOLEPAS OSCAR LEON, ARSENIOMOR~.N and RUBYGONZALEZ Departamento de Biologia Molecular, Facultad de Ciencias Biol6gicas y de Recursos Naturales, Universidad de Concepci6n, Concepci6n, Chile S.A. (Received 25 September 1981) Abstract--1. Muscle pyruvate kinase from the mollusc Concholepas concholepas was purified by DEAEcellulose chromatography, affinity chromatography on Blue Dextran-Sepharose and gel filtration on Sephadex G-200. 2. The final specific activity of the enzyme was 177 U/mg of protein with a 20% yield. 3. The molecular weights of the native enzyme and subunits are 234,000 and 58,000 respectively. The enzyme is a tetramer of apparently identical subunits. 4. The enzyme shows slight sigmoidal kinetics with phosphoenolpyruvate as substrate and is strongly inhibited by alanine and phenylalanine. Fructose 1,6-biphosphate is an activator. 5. The regulatory properties of the enzyme are very similar to M2-type pyruvate kinase in mammalian muscle and other mollusc isozymes.
INTRODUCTION
Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) catalyzes an essentially irreversible step in the glycolytic pathway. Some reports have shown that mammalian enzymes can be separated into, at least, three noninterconvertible forms (Tanaka et al., 1967; Inamura et al., 1972a; Carbonell et al., 1973) which have been designated types L, Mt and M 2 by lnamura et al. (1972a). Multiple enzyme forms are also found in other tissues. Thus, the hybrid set L-M 2 exists in rat kidney and intestine (Inamura et al., 1972b) whereas the hybrid set M1-M2 exists in rat fetal muscle (Susor et al., 1971) and some adult tissues (Strandholm et al., 1976). The L, M I and M 2 isozymes differ in their kinetic and regulatory properties and in their localization in the different tissues (Strandholm et al., 1976). The L-type enzyme shows sigmoidal kinetic with PEP as substrate and is inhibited by alanine and activated by fructose 1,6-biphosphate. The M2-type isozyme shows slightly sigmoidal kinetics with regard to phosphoenolpyruvate and is activated by fructose-l,6-biphosphate and inhibited by alanine and phenylalanine. The M I type isozyme shows hyperbolic kinetics, is inhibited by phenylalanine and is not affected by fructose 1-6-biphosphate. Other pyruvate kinases have been described in sea molluscs. Holwerda & de Zwaan (1973) characterized the enzyme from Mytilus edulis as L-type. Mustafa et al. (1971), reported the presence of two isozymes in the oyster Cassosheagigas that differed in their kinetic constants. Zammit et al. (1978a, b) studied the regulatory properties of kinases and phosphoenoipyruvate carboxykinase from muscle of several invertebrates and postulated a probable regulatory mechanism of carbohydrate metabolism which would explain the ability of oysters to withstand anaerobic conditions. Giles et al. (1977) described the allosteric properties .~.l' 72 IB I
65
and the kinetic mechanism (Giles et al., 1980) of pyruvate kinase obtained from the hepatopancreas of Carcinus maenas. In this paper we report on the purification and some properties of muscle pyruvate kinase obtained from the abalone-like sea mollusc Concholepasconcholepas. We show that this enzyme is allosteric with respect to phosphoenolpyruvate and that it is inhibited by alanine and phenylalanine. Fructose 1,6-bisphosphate counteracts this inhibition and the kinetics become hyperbolic.
MATERIAL AND METHODS
Chemicals Phosphoenolpyruvate (PEP), tetrasodium fructose 1,6-bisphosphate (F-I, 6-P2), disodium ADP, NADH, alanine, lactic dehydrogenase, glutamic deshydrogenase, serum albumin, ovalbumin, DEAE-cellulose and CNBractivated Sepharose-4B were purchased from Sigma Chemical Co. Sephadex G-25 medium, G-200 and Blue Dextran 2000 were obtained from Pharmacia Fine Chemicals. Acrylamide and N,N'-methylembis-acrylamide were from Fluka. All other chemical were of analytical grade and were obtained from either Sigma or Merk & Company. Protein determination Protein concentration was measured by the method of Lowry et al. (1971). At lower protein concentrations the method of Bradford et al. (1976) as modified by Bearden (1978) was also used. Enzyme assay Pyruvate kinase activity was measured according to Bucber & Pfieiderer (1955) at 20°C in a Gilford 240 spectrophotometer. The assay mixture contained final concentrations of 100mM Tris-HCl buffer, pH 7.4; 100mM KCI 5mM MgSO4; lmM PEP; lmM ADP; 0.15mM NADH and 7 units/ml of lactic dehydrogenase.
OSCAR LEONet al.
66
A unit of pyruvate kinase activity is defined as the amount of enzyme required to oxidize l ,umole of NADH per minute under conditions for the coupled-assay.
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Molecular weight determination The molecular weight of pyruvate kinase was estimated by the method of Fish et al. (1964) (without guanidine hydrocloride), on a Sephadex G-200 (60 x 2cm) column using bovine serum albumin (tool. wt 68,0001, ovalbumin (tool. wt 45,000), Lactic dehydrogenase (tool. wt 140,000) and glutamic dehydrogenase (tool. wt 250,000) as marker proteins. The molecular weights of the subunits were determined by SDS polyacrylamide gel electrophoresis according to Weber & Osborn (1969) with some minor modifications. Bovine serum albumin, ovalbumin, lactic dehydrogenase and glutamic dehydrogenase were used as standards. Protein samples (5 20pg) were disolved in 10mM sodium phosphate buffer, pH 7.0, containing 1'~,;glycerol and 0.1 M 2-mercaptoethanol and were allowed to stand at room temperature for 20 rain. After electrophoresis, the gels were stained with Coomasie Blue R-250 (0.3g in 500ml of 50'!i, v/v methanol and 7'!o v v acetic acid} for two hours at room temperature and destained with 75% acetic acid and 5"~; butanol overnight.
Blue Dextran Sepharo,w column Blue Dextran-2000 was attached to CNBr-activated Sepharose-4B according to the method of Ryan & Vestling (1974). RESULTS
AND
DISCUSSION
PuHlication of pyrurate kinase All purification steps were carried out at room temperature except the centrifugation and extraction steps which were done at 4~'C. Step 1.. Extraction. Muscle tissue ( l l 0 g ) was homogenized in a Waring blendor for 2 min. with three volumes of 10mM T r i s H C 1 buffer; pH 7.4 containing 1 m M E D T A and 1 m M 2-mercaptoethanol. The extract was centrifuged at 15,000 0 for 15 min. The resulting pellet was discarded and the supernatant was centrifuged at 105,0000 for 1 hour. Step 2: DEAE-cellulose chromato,qraphy. The supernatant fraction obtained in step 1 was applied to a DEAE-cellulose column (60 × 3.5 cm) equilibrated in the above buffer. The column was resolved with a linear gradient of 0 to 1 M KCI in the same buffer (Fig. 1). Fractions with activity were pooled and concentrated with 65°o (NH4)zSO 4, and centrifuged. Step 3: Desaltin9. The pellet was resuspended in 10ml of the same buffer and desalted on a Sephadex G-25 column (65 x 2.4 cm).
Step 4: Alfinity chromatography on Blue Dextran Sepharose 4B. The desalted eluate, containing pyruvate kinase activity was absorbed onto a Blue Dextran Sepharose-4B column (45.5 x 2.2cm) equilibrated in the same buffer and was eluted with 1 mM fructose 1,6-bisphosphate and 1 0 m M KCI in the same buffer (Fig. 2). The active fractions were pooled and concentrated with sucrose in a dialysis sack. Step 5: Gel filtration on Sephadex G-200. The concentrated enzyme was applied to a sephadex G-200 column (75 x 2cm) equilibrated with 1 0 m M potasium phosphate buffer, p H 7 . 4 containing 1 mM E D T A and 1 mM 2-mercaptoethanol. The active fractions were pooled, concentrated with solid sucrose and stored at 4~C.
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Fig. 1. DEAE-cellulose chromatography of Concholepas concholepas muscle pyruvate kinase. The supernatant fraction (see Materials and Methods) was applied to a DEAE cellulose column equilibrated in 10mM Tris HC1 buffer, pH 7.4 containing 1 mM EDTA and 1 mM 2-mercaptoethanol. The enzyme was eluted with a linear concentration gradient of (~1 M KC1 m the same buffer. Typical results of the purification scheme are given in Table I. The DEAE cellulose step not only removed a large amount of protein (Fig. 1) but also removed most of the glycogen found in the muscle extract. In addition a large amount of material absorbing at 260nm was removed by the Sephadex G-25 filtration step. The main step in the purification scheme was the affinity column in spite of the low capacity of the absorbent (Fig. 2). The retention of the enzyme was very sensitive to the concentration of salt, When high salt concentration were used the enzyme was not retained and if the temperature was lowered a colacomitant loss of retention was also observed. Harada et al. (1978) reported the purification of the M1, M2 and L-type pyruvate kinases from rat tissues by affinity elution on phosphocelluloses, however, our enzyme was inactivated by this procedure since it is unstable at pH values below 6.5. Contaminating protein was removed by gel filtration on Sephadex G-200. The final specific activity was 177U/mg of protein and the preparation showed essentially a single band after SDS-gel electrophoresis (Fig. 3). The purification procedure can be performed in two weeks and the enzyme obtained is stable for 3 months when stored at 4°C in the presence of 20°4; sucrose.
Determination qlthe apparent molecular weight A molecular weight of 234,000 was obtained alter gel filtration on Sephadex as described in Materials and Methods (Fig. 4). This value is similar to the molecular weight of 237,000 obtained by Warner et al. (1958) for Mr-type pyruvate kinase from rabbit muscle. When the purified enzyme was subjected to SDS-polyacrylamide gel electrophoresis, a single band corresponding to a molecular weight of 57,000 was obtained suggesting that the enzyme is a tetramer composed of apparently identical subunits (Fig. 5).
Effect of pH The pH activity profiles of the purified enzyme in the presence of 0.2 and 1 m M PEP are shown in Fig. 6. At 1 m M PEP the enzyme shows a broad pH opli-
Purification and characterization of pyruvate kinase
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Fig. 2. Affinity chromatography of pyruvate kinase on a Blue Dextran-Sepharose-4B column. The column was equilibrated as described in Fig. 1 and was resolved by including 1 mM F-I, 6-P2 and 10mM KCI in the buffer. mum between 7.0 and 8.0. However, with 0.2 mM PEP a much narrower pH optimum is observed (7.3-7.4). Figure 6 also shows that 0.I mM F-I, 6-P2 in the presence of 0.2 mM PEP lowers the pH optimum to 7.1. These effects were also reported in the adductor muscle pyruvate kinase from oyster by Mustafa et al. (1971).
Kinetic properties of pyruvate kinase Apparent K= for phosphoenolpyruvate.
Figure 7 shows the effect of PEP concentration on pyruvate kinase activity in the presence and absence of F-I, 6-P2. Without F-I, 6-P2 the curve is slightly sigmoidal. A So.5 value of 0.15 mM for PEP and a nn of 1.64 were calculated from the Hill plot. Nevertheless, the kinetics become hyperbolic in the presence of 0.1 mM F-I, 6-P2 and the So.s and nn values decrease to 0.07 mM and 1.0 respectively. Effect of alanine and phenylalanine. Fig. 7 also shows the inhibitory effect of 0.5 mM phenylalanine on pyruvate kinase activity. Both the cooperative effect and the So.5 are increased while V=~ is not significantly affected. Alanine also inhibited the enzyme; albeit not as effectively (results not shown). The K~ values for alanine and phenylalanine are 2 and 1.4 mM respectively at 0.5 mM PEP, These properties are similar to M2-type pyruvate kinase from bovine (C~irdenas et al., 1975) and oyster
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Fig. 4. Determination of the molecular weight of muscle pyruvate kinase by filtration on Sephadex G-200. The arrow indicates the K d value for the enzyme. LDH: lactic dehydrogenase, GDH: glutamic dehydrogenase. and which explains the ability of this mollusc to tolerate falls in pH when it is subjected to anaerobic conditions (Zammit et al., 1978). Effect of K +. As is the case for enzymes from other species (Jimenez de Asfia et al., 1970; Somero & Hochachka 1968) the Coneholepas-coneholepas pyruvate kinase also requires K + and Mg 2÷ ions for its activity. In the presence of 0.2 mM PEP the activation by K + is slightly sigmoidal (nn = 1.3) and become hyperbolic (nn = 0.93) when 0.1mM F-l, 6-P2 is added (Fig. 8). On the other hand, with 1 mM PEP hyperbolic kinetics of activation were obtained whether F-l, 6-P2 was present or not (results not shown). In all cases, maximum activation was obtained at 60mM K ÷. The extent of the activation was lower at K + concentrations higher than 100 mM. The effect of K + could not be replaced by NH,~ as in oyster pyruvate kinase (Mustafa et al., 1971). The inhibitory effects of alanine and phenylalanine were reversed by 0.1 mM F-l, 6-P2 (results not shown). Apparent Kufor ADP. Figure 9 shows that a hyperbolic curve is obtained when ADP concentration are increased. The apparent K= for ADP is 0.38 mM. At ADP concentrations higher than 5 mM the enzyme is inhibited. The inhibitory effect of ADP has also been
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Fig. 3. Scan of pyruvate kinase after SDS-gel electrophoresis 15/~g of protein from the Sephadex G-200 step were applied to the top of the gel.
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Fig. 5. Molecular weight determination of pyruvate kinase subunits by SDS-polyacrylamide gel elcctrophoresis according to the method of Weber & Osborn (1969). The arrow indicates the mobility of the enzymes.
OSCAR LEON et al.
68
Table 1. Purification of pyruvate kinase from Concholepas concholepas muscle
Crude extract Supernatant (105,000 0) DEAE-cellulose 65% (NH,~)2SO4 fraction Sephadex G-25 Blue Dextran-Sepharose Sephadex G-200
Total activity (units)
Total protein (mg)
Sp. act. (U/mg)
Purification
Yield ('!~,)
4000 3200 2600 2400 1760 1538 800
22,000 l 8,000 691 592 219 16.7 4.5
0.18 0. l 7 3.76 4.05 8.0 92 177
1 1 10 22 44 511 983
100 80 65 60 44 38 20
reported for pyruvate kinases from oyster adductor and sea anemone basilar muscles (Zammit et al., 1978a).
Effect of A T P on pyruvate kinase Like pyruvate kinase from other sources (Tanaka et al., 1967; Rosengurt et al., 1969; Mustafa et al., 1971)
A T P inhibits the enzyme from Concholepas concholepas muscle and the extent of this inhibition depends on the pH. As in oyster pyruvate kinase (Mustafa et al., 1971) the inhibition is greater at acid pH values. The inhibition could be reversed by F D P (results not shown). An allosteric pyruvate kinase in muscle tissue of the
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Fig. 7. Effect of F-l, 6-P 2 and phenylalanine on the pyluvate kinase activity with regard to PEP concentration at pH 7.4. O O Control; • • Plus 0.1 mM F-I. 6-P2; • • Plus 0.5 mM phenylalanine.
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Fig. 8. Effect of K + on the activation of pyruvate kinase by K + ion. Hill plots of the data are inserted. O -- O0.2mM P E P n u = 1 . 3 : • - - • 0 . 2 m M PEP plus 0.1mM F-I, 6-P:.
Purification and characterization of pyruvate kinase
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Fig. 9. Effect of ADP concentration on pyruvate kinase activity (PEP = 0.4mM). Lineweaver-Burk plot is included. Concholepas concholepas m a y be of physiological significance in the regulation of glycolysis. The main factors on the activity of pyruvate kinase seem to be the p H a n d the concentration of the substrate (phosphoenol pyruvate) a n d of the effectors fructose 1,6 biphosphate, phenylalanine, alanine and ATP.
Acknowledgements--We thank Dr Peter Ward for his help in the elaboration of the manuscript. This work was supported by grant 1.09.64 of the office of the Dean of Research of the University of Concepi6n (Chile).
REFERENCES
BEARDEN J. C. (1978) Quantitation of submicrogram quantities of protein by an improved protein dye binding assay. Biochem. biophys. Acta 533, 525-529. BRADFORD M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. BUCHER T. & PFLEIDERER G. (1955) in Methods in Enzymology, Vol. 1 (Edited by COLOWlCK S. P. & KAPLAN N. D.), pp. 435 440. Academic Press, New York. CARBONELL J., FELIU J. E., MARCO R. & SOLS A. (1973) Pyruvate kinase classes of regulatory isozymes in mammalian tissues. Eur. J. Biochem. 37, 148-156. CARDENAS J. M., STRANDHOLMJ. J. t~¢ MILLER J. M. (1975) Effects of phenylalanine and alanine on the kinetics of bovine pyruvate kinases isozymes. Biochemistry 14, 4041-4045. FISH W. W., MANN K. G. ,~¢ TANEORD C. T. (1969) The estimation of polypeptide chains molecular weight by gel filtration in 6M guanidine HCI. J. biol. Chem. 244, 4989-4994. GILES I. G., POAT P. C. t~¢ MUNDAY K. A. (1977) An investigation of the interactions of the allosteric modifiers of
69
pyruvate kinase from Carcinus maenas hepatopancreas. Biochem. J. 165, 97-105. GILES L. G. & POAT P. C. (1980) A steady state kinetic analysis of the fructose 1,6-biphosphate activated pyruvate kinase from Carcinus maenas hepatopancreas. BiDchem. J. 185, 289-299. HARADA K., SAHEKI S., WADA K. & TANAKA T. (1978) Purification of four pyruvate kinase isozymes of rats by affinity elution chromatography. Biochim. biophys. Acta 524, 327 339. HOLWERDA D. A. • DE ZWAAN A. (1973) Kinetic and molecular characteristic of allosteric pyruvate kinase from muscle tissue of the sea mussel Mytilus edulis L. Biochim. biophys. Acta 276, 430-433. [NAMURA K., TANIUCHI K. ,~¢ TANAKA T. (1972a) Purification of Mz-type pyruvate kinase from Yoshida ascites hepatoma 130 cells and comparative studies on the enzymology and inmunological properties of the three types of pyruvate kinases L, M 1 and M 2. J. Biochem. 72, 1001-1015. INAMURA K. t~¢ TANAKA T. (1972b) Multimolecular forms of pyruvate kinase from rat and other mammalian tissues. J. Biochem. 71, 1043-1051. JIMENEZ DE ASUA L., ROZENGURT E. & CARMINATTI H. (1970) Some kinetic properties of liver pyruvate kinase. III. Effect of monovalent cation on its allosteric behaviour. J. biol. Chem. 245, 3901-3905. LOWRY O. H., ROSENBROUGHN. J., FARR A. L. ~¢ RANDALL R. J. (1951) Protein measurement with the Folin reagent. J. biol. Chem. 193, 265 275. MUSTAFA T. & HOCHACHKA P. W. (1971) Catalysis and regulatory properties of pyruvate kinase of a marine bivalve. J. biol. Chem. 246, 3196-3203. RYAN L. D. ~¢ VESTLING C. S. (1974) Rapid purification of lactate dehydrogenase from rat liver and hepatoma: A new approach. Archs Biochem. Biophys. 160, 279 284. STRANDHOLM J. J., DYSON R. D. & CARDENAS J. M. (1976) Bovine pyruvate kinase isozymcs and hybrid isozymes electrophoretic studies and tissue distribution. Archs Biochem. Biophys. 173, 125-131. SOMERO G. N. & HOCHACHKA P. W. (1968) The effect of temperature on catalytic and regulatory functions of pyruvate kinases of the Rainbow Trout and the antartic fish Trematomus bernacchii. Biochem. J. ll0, 395-399. SUSOR W. A. & RUTTER W. J. (1971) Method for the detection of pyruvate kinase, aldolase and other pyridine nucleotide linked enzymes activities after electrophoresis. Analyt. Biochem. 43, 147. TANAKA T., HARANO Y., SUE F. & MORIMURA H. (1967) Crystallization, characterization and metabolic regulation of two types of pyruvate kinases isolated from rat tissues. J. Biochem. 62, 71-91. WARNER R. C. (1958) Physical properties of crystalline "Fluorokinase". Archs Biochem. Biophys. 78, 494. WEBER K. & OSBORN M. (1969) The reliability of molecular weight determinations by dodecy] sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412. ZAMMIT V. A. t~¢ NEWSHOLME E. A. (1978a) Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates. Biochem. J. 174, 979-987. ZAMMIT V. A., BEIS I. & NEWSHOLME E. A. (1978b) Maximum activities and effects of fructose bisphosphate on pyruvate kinase from muscle of vertebrates and invertebrates in relation to the control of glycolysis. Biochem. J. 174, 989-998.
