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Pyruvate kinase isozymes in oocytes and embryos from the frog Xenopus laevis
Comp. Btochem. Physiol. Vol. 88B, No. 3, pp. 743-749, 1987 Printed in Great Britain
0305-0491/87 $3.00+ 0.00 © 1987PergamonJournals Ltd
PYRUVATE KINASE ISOZYMES IN OOCYTES A N D EMBRYOS F R O M THE F R O G XENOPUS LAEVIS MARK B. DWORKIN, NEIL SEGIL and EVA DWORKIN-RASTL Department of Biological Sciences, Sherman Fairchild Center, Columbia University, New York, NY 10027, USA (Tel.: 212 280-8281) (Received 7 January 1987)
Ala~traet--1. The kinetic characteristics of pyruvate kinase isozymes from oocytes, embryos, liver and skeletal muscle from the clawed frog Xenopus laevis were measured in cell extracts. 2. The muscle and liver isozymes display Michaelis-Menten kinetics with Kms for phosphoenolpyruvate (PEP) of 0.02 and 0.05 raM, respectively. 3. Pyruvate kinase from oocytes and embryos displays cooperative kinetics for PEP with a K~ of about 0.15 raM; the kinetics become hyperbolic and the Km for PEP is reduced to 0.05 mM in the presence of /zM concentrations of fruetose-l,6-bisphosphate. 4. These data serve to characterize pyruvate kinase activity in oocytes and embryos and the kinetics are compared to mammalian pyruvate kinase isozymes.
INTRODUCTION Oocyte maturation in vertebrates is a progesterone dependent process in which the full-grown oocyte (stage VI oocyte in the clawed frog Xenopus laevis: Dumont, 1972), arrested in prophase of the first meiotic division, is induced to continue through its meiotic cell cycle and then arrest at metaphase of the second meiotic division, when the now mature oocyte can be fertilized (Mailer, 1985). Oocyte maturation involves the breakdown of the large oocyte nucleus, the germinal vesicle, as well as a number of ionic and other biochemical changes including several cAMP dependent and independent protein phosphorylations. Oocyte maturation is accompanied by a decrease in cAMP and inhibitors of adenylate cyclase, when injected into oocytes, result in maturation in the absence of hormone (Mailer, 1985). Since a decrease in cAMP concentration seems necessary and sufficient to induce oocyte maturation and a decrease in cAMP would be predicted to increase glycolytic flux, there might be an important relationship between increased glycolysis and oocyte maturation. Consistent with the possible importance of glycolysis in oogenesis and/or early development, we recently identified a cDNA clone for the glycolytic enzyme enolase in an embryonic cDNA library and showed that enolase m R N A is very abundant in early oocytes and decreases in concentration during oocyte growth and early embryogenesis (Dworkin and DworkinRastl, 1985; Dworkin et aL, 1985). Glucose is oxidized in cells by two alternative pathways. Glucose-6-phosphate can be oxidized to pyruvate via glycolysis and this process is linked to the production of a small amount of ATP. Three carbon compounds generated in this pathway are precursors to cellular macromolecules. Alternatively,
the oxidation of glucose-6-phosphate by glucose-6phosphate dehydrogenase and the subsequent reactions of the phosphogluconate pathway provide the cell with N A D P H and a variety of sugars, which are key substances required for biosynthetic processes such as fatty acid and nucleic acid synthesis. A recent study of Xenopus laevis full-grown oocytes shows a predominance of metabolism of glucose via the phosphogluconate pathway rather than via glycolysis (Cicirelli and Smith, 1985). It has been suggested that the phosphogluconate pathway is necessary to support the synthesis of ribose required for the large amount of deoxyribonucleotides needed during cleavage. Consistent with this idea, ribonucleotide reductase activity is greatly increased at fertilization in some species (Standart et al., 1985). However, classical studies by Warburg more than 50 fifty years ago showed that lactate formation is also increased upon fertilization of many types of eggs (see Warburg, 1956). The significance of increased rates of fermentation in newly fertilized eggs, as well as in serum or growth factor stimulated cells (Diamond et al., 1978) and neoplastically transformed cells (Warburg, 1956; Burk et al., 1967) is still not clear. In many cells glycolytic activity is partly regulated by hormones whose receptors act through cAMP, with high cAMP levels acting to inhibit glycolysis (discussed in Claus et al., 1984). Two key sites in the regulation of glycolytic flux by cAMP-dependent protein kinase are 6-phosphofructo-2-kinase/phosphofructo-2,6-bisphosphatase, which regulates the intracellular concentration of F2,6P2 (Claus et al., 1984), and pyruvate kinase (Engstrrm, 1980). Phosphorylation by cAMP-dependent protein kinase increases phosphofructo-2,6-bisphosphatase activity, reducing the level of F2,6P2. F2,6P2 is a key intracellular activator of 6-phosphofructo-l-kinase and in Abbreviations used: PEP, phosphoenolpyruvate; F1,6P2, the absence of F2,6P2, 6-phosphofructo-l-kinase is fructose- 1,6-bisphosphate; F2,6P 2, fructose-2,6-bisphos- virtually inactive in many cells under normal intracellular concentrations of ATP (Claus et al., 1984). phate. 743
744
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MARK B. DWORKINet al.
The product of the 6-phosphofructo-l-kinase reaction, F1,6P2, is in turn a potent activator of pyruvate kinase (Ramaiah, 1974). Three of the four pyruvate kinase isozymes which have been described in m a m malian tissues (liver, erythrocyte, and fetal) display sigrnoidal kinetics with respect to PEP, but are activated to hyperbolic kinetics by micromolar quantities o f F1,6P2 (Kahn and Marie, 1982; I m a m u r a and Tanaka, 1982; Cardenas, 1982). M a m m a l i a n muscle pyruvate kinase displays hyperbolic kinetics for PEP. All forms of pyruvate kinase are inhibited by A T P in the absence of F1,6P2. Further, several pyruvate kinase isozymes from rat, pig and h u m a n can be directly phosphorylated by c A M P - d e p e n d e n t protein kinase, which increases the K~ for PEP by a factor of two to three in the absence of F1,6P2. In this paper we describe the kinetics o f Xenopus laevis oocyte and embryonic pyruvate kinase, as well as adult liver and leg muscle pyruvate kinase. MATERIALS AND METHODS
Preparation of cell extracts and enzyme assays Oocytes, embryos, and adult tissues (leg muscle, liver, and brain) from female frogs were homogenized at 0°C in a Dounce homogenizer (oocytes and embryos) or in a Sorvall Omnimix (adult tissues) in 2-3 volumes of 20 mM Tris-HC1, pH 7.5 (25°C), 100 mM KCI, 5 mM MgSO4, 1 mM EDTA, 10raM mercaptoethanol, 0.5mM phenylmethylsulfonylfluoride. Homogenates were centrifuged 5 rain at 2 K in the Sorvall SS34 rotor and the supernatants recentdfuged 5 min at 10K in the same rotor. These 12,000g supernatants (referred to as cell extracts) were either immediately frozen in aliquots in a dry ice/ethanol bath and stored at -80°C, or first dialyzed by gel filtration. Approximately 0.5 ml aliquots containing 5 #Ci ct-32p-dCTP as tracer for small molecules were filtered through 3 ml Sephadex G-50 in homogenization buffer and the colored extract devoid of radioactivity was frozen in aliquots as described above. Cell extracts were analyzed for enolase, pyruvate kinase and lactate dehydrogenase activity in 50 mM imidazoleHC1, pH 7.0 (25°C), 70mM KC1, 10mM MgSO4, 3 mM EDTA according to Lowry and Passonneau (1964). All assays were linked to the oxidation of NADH in the lactate dehydrogenase reaction. Reactions were carried out in 1 ml at room temperature and the change in absorbance at 340 nm was recorded. Commercial enzymes and substrates were all from Sigma; all enzymes were found to be free of contaminating activities. Substrates (2-phosphoglycerate, PEP, pyruvate, F1,6P2, ADP and ATP) were dissolved, neutralized with NaOH if necessary, and frozen in aliquots; NADH was dissolved fresh for each experiment. ADP, ATP and NADH concentrations were determined spectrophotometrically; 2-phosphoglycerate, PEP and pyruvate concentrations were determined with the lactate dehydrogenase assay. For pyruvate kinase assays a typical reaction consisted of 0.12mM NADH, 0.4mM ADP, 9u lactate dehydrogenase, and PEP and FI,6P2 as necessary. The reaction was initiated by addition of 1-3#1 of cell extract so that the reaction at maximum velocity would support the oxidation of 50 nmol NADH/min. Rates (v) are given in nmot NADH oxidized per min in a 1 ml reaction at 20°C. K a (activation constant) for F1,6P2 is the concentration of F1,6P2 that produces half maximal rate at 0.02 mM PEP and 150 (inhibition constant) for ATP is the concentration of ATP that inhibits the reaction by 50% at 0.4 mM PEP. Cellulose acetate electrophoresis Cellulose acetate electrophoresis was used to separate the isozymic forms of pyruvate kinase. The following proc,edure was modified from published protocols (Susor et al., 1975;
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Fig. 1. Velocity profiles of oocyte, liver and muscle pyruvate kinase. (A). Activity of pyruvate kinase as a function of PEP concentration for cell extracts prepared from full-grown oocytes ( O - - O ) , adult leg muscle ( © - - © ) and adult liver (x--x). Assay conditions are described in Materials and Methods, except that ADP was 0.4 mM for the oocyte curve and 0.2 mM for the liver and muscle curves in this experiment. All measurements were done in the absence of FI,6P 2. (B), (C) and (D) are Lineweaver-Burk plots for the muscle, liver and oocyte curves, respectively. Oskam et al., 1985). Cell lysates (0.25#1) were applied to cellulose acetate strips (Sepraphore III, Gelman Sciences, Ann Arbor, Michigan). Strips were electrophorescd at room temperature in 20 mM sodium-phosphate buffer, pH 7.0, for 1.5 hr at 120V (2 mA). Pyruvate kinase activity was localized by incubating strips against a substrate transfer membrane (Gelman Sciences) which had been soaked in the following solution: 100mM Tris-HC1, pH7.0, 10raM MgC12, 100 mM KCI, 0.5 mM EDTA, 1.5 mM ADP, 20 mM AMP, 10mM NADP +, 1 mM glucose, 0.5mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 0.17 mM phenazine methosulphate, 10mM PEP, l mM FI,6P 2, 6.5 u/ml hexokinase and 2.5 u/ml glucose-6-phosphate dehydrogenase. Incubation was at 37°C for approx. 20 min under conditions of high humidity and low fight. In control experiments, PEP and FI,6P2 were omitted from the incubation solution. Incubation was stopped by washing in 5% acetic acid for 2 min followed by a wash with elcctrophoresis buffer. RESULTS AND DISCUSSION Rates of pyruvate formation from PEP were measured as described in Materials and Methods in cell extracts of adult leg muscle, adult liver, small oocytes (stages I and II, D u m o n t , 1972), full-grown oocytes (stage VI), unfertilized eggs, and 6-8 hr blastulae. The velocity profiles of X e n o p ~ laevis muscle, liver and fu11-grown oocyte pyruvate kinase as functions o f P E P concentration are shown in Fig. IA. The Lineweaver-Burk plots (Figs I B - D ) show that the muscle and liver pyruvate kinases display MichaelisMenten kinetics, but the oocyte enzyme does not. Thus, on the basis of these plots the oocyte pyruvate kinase can be distinguished from both the muscle and liver forms. In contrast to mammalian liver pyruvate kinase, the X e n o p ~ liver enzyme does not display
745
Pyruvate kinase in Xenopus Table 1. Kinetic characteristics of pyruvate kinase isozyrnes from Xenopus laevis. The data were obtained as described in Materials and Methods .and the figures. The concentration of F I , 6 P 2 that increases the rate of the reaction to 1/2 V ~ , at a PEP concentration of 0.02 mM is shown as I ~ . The concentration of A T P that inhibits the reaction to 1/2 V ~ at a PEP concentration of 0.4 mM is shown as I50. The K~ shown for the sigmoidal enzyme is the concentration of PEP that supports the reaction to I/2 Vmx
(PEP) kinetics Hill coefficient Kra (PEP), mM Km (ADP), mM (F1,6P2) kinetics (FI,6P2), K,, ,aM (ATP), Iso, mM
Muscle hyperbolic 1.1 0.02 0.1 no effect
Liver hyperbolic 1.3 0.05 0.1 small effect
3.3
2.6
sigmoidal kinetics. Liver extracts in which small molecules have been removed by Sephadex G-50 gel filtration show similar kinetics. T h e / ~ of the oocyte enzyme for P E P is about 0.15 m M , while the /(mS of the muscle and liver enzymes are, respectively, approx. 0.02 and 0 . 0 5 m M (Table 1). Additional curves describing the oocyte isozyme are shown in Figs 2 and 3. The velocity profile in Fig. 2A shows
Oocyte/embryo sigmoidal 2.3 0.15 0.1 activated 0.1 2.2
that in the presence o f 5# M F 1,6P 2 the kinetics o f the oocyte enzyme assume a hyperbolic shape. The a m o u n t of F I , 6 P 2 necessary to completely activate A
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Fig. 2. Kinetic analysis of ooc~e py~vatc kinase. (A). Activity of pyruvate kinase in full-grown oocyte extracts as shown in Fig. 1 ( 0 - - 0 ) , with the addition of 5pM F1,6P 2 ( O - - O ) , or with the addition of 0.25 rnM ATP (x--x). (B). Activity of oocyte pyrnvate kinase as a function of FI,6P 2 concentration in the presence of 0.02raM PEP, 0.4raM ADP. (C). Lineweaver-Burk plot of the oocyte curve in the presence of 5#M F1,6P 2. (D), (E). Hill plots of the oocyte enzyme in the absence of FI,6P2 (D) and in the presence of 5pM FI,6P2 (E). The Hill coefficient, n, is shown in the top left corner of each graph.
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Fig. 3. Effect of ATP on oocyte velocity profiles. (A). Activity of pyruvate kinase in full-grown oocytes in the presence of 0.25 mM ATP. The first three points are also plotted on the profile shown in Fig. 2A. (B). Activity of the oocyte pyruvate kinase as a function of ATP concentration at 0.4 mM PEP and 0.4 mM ADP. (C). Activity of oocyte pyruvate kinase as a function of FI,6P2 concentration in the presence of 0.4raM PEP, 0.8raM ADP, 3raM ATP. 1/2 V~, occurs at 0.1pM F1,6P2.
746
MARK B. DWORKINet al. B
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Fig. 4. Kinetic analysis of liver pyruvate kinase. (A). Velocity profiles of liver pyruvate kinase in the absence (O--O) and presence (O--O) of 5#M F1,6P2. (B, C). Hill plots of the liver enzyme in the absence of F1,6P2 (B) and in the presence of 5#M FI,6P 2 (C). the oocyte enzyme is less than I # M with a /Ca of about 0.1/~M (Fig. 2B; Table 1). The LineweaverBurk plot of the oocyte enzyme in the presence of 5gM F1,6P2 is a straight line (Fig. 2C) and the Km of the enzyme for PEP is reduced to 0.05 mM. In the absence of F1,6P2 the Hill coefficient of the oocyte isozyme is 2.3 (Fig. 2D). The Hill coefficient is reduced to approx. 1.0 by the addition of the activator F1,6P2 (Fig. 2E). These results for the Xenopus oocyte pyruvate kinase are in complete agreement with the kinetic descriptions of the fetal/tumor and liver forms of pyruvate kinase in mammalian tissues (Engstr6m, 1980; Kahn and Marie, 1982; Imamura and Tanaka, 1982; Cardenas, 1982). In the presence of ATP the velocity of the pyruvate kinase reaction is reduced at concentrations of PEP less that 1 mM. Some of these data at 0.25 mM ATP are shown for the oocyte enzyme in Figs 2A and 3A. An inhibition curve for ATP is shown in Fig. 3B. The ATP concentration which inhibits the reaction by 50% at 0.4mM PEP (I50) is 2.2 mM (Table 1). Slightly higher Is0s were obtained by this analysis for the liver and muscle enzymes (Table 1). In the presence of 5#M F1,6P2 and 2 mM ATP, the oocyte pyruvate kinase, as well as the liver and muscle isozymes, show hyperbolic kinetics with KmS for PEP of 0.02-0.05mM (data not shown). Less than I # M of FI,6P2 fully activates the oocyte enzyme at 0.4mM PEP and 3 mM ATP (Fig. 3C). Thus, micromolar levels of FI,6P: relieve the ATP inhibition of the oocyte pyruvate kinase as well as lower the Km of the enzyme for PEP. The effect of F1,6P2 on the kinetics of the liver enzyme is shown in Fig. 4. A small effect is observed on both the velocity profile and the Hill plots. However, the Xenopus liver enzyme clearly does not show the cooperative kinetics described for mammalian liver pyruvate kinases. The unusual behavior of this isozyme has been previously cited (Ibsen, 1977; also see Ibsen et al., 1980) and is presented here to show a clear distinction between the liver and oocyte forms of pyruvate kinase in Xenopus.
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Fig. 5. Kinetic analysis of blastula pyruvate kinase. (A). Activity of pyruvate kinase from blastulae in the absence (O--O) and presence (O--©) of 5#M F1,6P2. (B, C). Lineweaver-Burk plots of the blastula enzyme in the absence (B) and presence (C) of5#M F1,tP2. (D, E). Hill plots of the blastula enzyme in the absence (D) and presence (E) of 5#M F1,6P 2. Kinetic data for the early oocyte, egg and blastula enzymes were virtually indistinguishable from the full-grown oocyte form. Velocity measurements for the blastula enzyme are shown in Fig. 5. The enzyme shows sigmoidal kinetics with respect to PEP with a Km for PEP of about 0.15 mM; these kinetics are converted to standard Michaelis-Menten kinetics by the addition of F1,6P2 and the Km for PEP is reduced to 0.05 mM (Fig. 5A, C). This activator decreases the Hill coefficient from 2.3 to 1.2. We have found no way to kinetically distinguish oocyte and embryonic pyruvate kinase isozymes and conclude that the pyruvate kinase form does not change during oocyte growth, maturation, or during very early embryogenesis. This conclusion is substantiated by cellulose acetate electrophoresis of pyruvate kinase isozymes from oocytes, eggs and adult tissues (Fig. 6). The
747
Pyruvate kinase in Xenopus
I
2
3
4
-I-
Fig. 6. Cellulose acetate electrophoresis o f pyruvate kinase isoforms. Pyruvate kinase isozymes in cell extracts from adult leg muscle (1), small ooeytes (stages I, II; Dumont, 1972) (2), full-grown oocytes (3) and eggs (4) were separated by electrophoresis on cellulose acetate strips and pyruvate kinase activity localized as described in Materials and Methods.
pattern of bands seen with the oocyte and egg preparations is identical; the pattern of bands seen with muscle is different. The liver pattern (not shown) is unexpectedly similar to the oocyte/embryo pattern. Such a complex pattern of bands in amphibians has been previously reported in eggs and adult tissues of Rana pipiens (Schloen et al., 1974). Since pyruvate kinase is a tetramer, two electrophoretically distinct subunits can form five different combinations which can be separated by cellulose acetate electrophoresis (Cardenas, 1982). The kinetics that we measure in these cell extracts are a composite of the kinetics of these pyruvate kinase isoforms. Ibsen et al. (1980) have separated several isoforms of pyruvate kinase from Xenopus embryos and tissues and their data also reveal three isoforms in muscle and four major and two minor isoforms in liver. However, their data show a single major isoform in blastulae, whereas
1.0
>E 0.5
•
•
I
l
I
0
•
•
I
I
I
I
0.I
0.2
0.3
0.4
[ADP] , mM Fig. 7. Velocity profiles o f occyte and liver pyruvate kinases as functions o f A D P concentration. Measurements shown were made at 0.2 m M PEP for both the full-grown oocyte enzyme ( O - - O ) and the adult liver enzyme ( O - - O ) .
our study indicates four or more isoforms for oocytes and eggs, which persist to blastula (not shown). The isoform they describe in blastula also shows sigmoidal kinetics, though not as extreme as what we observe in oocytes, eggs and blastulae, and is activated by F1,6P2. The velocity profiles of the oocyte and liver pyruvate kinases for ADP are shown in Fig. 7. They are superimposable, as is the muscle profile (not shown) and are unaffected by 5/zM F1,6P2 at I mM PEP (not shown). T h e / ~ of these isozymes for ADP is about 0.1 mM. All of these kinetic parameters are listed in Table 1. The characteristics of the Xenopus muscle and liver isozymes are very similar to mammalian muscle pyruvate kinase. The Xenopus oocyte enzyme best resembles the type M 2 rat isozyme characteristic of fetal and tumor tissue. In particular its high I50 for ATP seems to distinguish it from the mammalian liver form of pyruvate kinase, which is inhibited at lower concentrations of ATP (Imamura and Tanaka, 1982). The activities of cell extracts from leg muscle, liver, brain, oocytes and eggs for lactate dehydrogenase, pyruvate kinase and enolase are listed in Table 2. Some of these data for representative individual Table 2. Activities of lactate dehydrogenase (LDH), pyruvate kinase (PK) and enolase in nmoles NADH oxidized/rain per g protein. Protein concentrations were determined according to Lowry et al. (1951). The data for leg muscle, brain, and small oocytes (stages I, II; Dumont, 1972) are from single lysates, the liver data from two independent lysates and the data for full-grown (stage VI) oocytes/eggs from four different lysates prepared from either oocytes or eggs from different females. One of these preparations showed a particularly high level of LDH activity, thereby making the average LDH activity higher than in most individual preparations (Fig. 8) LDH PK Enolase Leg muscle Liver Brain Oocytes (I, lI) Oocytes (VI), eggs
1.9 1.3 0.38 1.7 1.9
2.6 0.30 1.0 5.2 3.5
2.2 0.055 0.29 0.42 1.4
MARK B. DWORKIN et al.
748 muscle
liver
brain
1.0~ 0.5
L PE
L PE
LPE
Fig. 8. Relative activities of lactate dehydrogenase, pyruvate kinase and enolase in cell extracts. The relative activities of lactate dehydrogenase (L), pyruvate kinase (P) and enolase (E) on a per g of protein basis for six individual and representative lysates are shown. The activity of the most active enzyme for each lysate was set at 1.0. Activity measurements were made in the imidazole buffer as described in Materials and Methods and were all linked to the oxidation of NADH by lactate dehydrogenase. Commercial lactate dehydrogenase (9u/mi) and pyruvate kinase (4 u/ml) were used as required. NADH, ADP, 2-phosphoglycerate, PEP and pyruvate were added as needed at, respectively, 0.12, 0.8, 0.5, 0.4 and 0.4mM. lysates are depicted graphically in Fig. 8. The relative levels of these three enzymes in full-grown oocytes, eggs and blastulae are more similar to adult brain than to muscle or liver. On a per g of protein basis, oocytes and embryos contain high amounts of all three enzymes at levels close to those of leg muscle. Oocytes and eggs have considerably more enolase activity than adult liver. In this report we have described the cooperative kinetics of the oocyte and embryonic form of pyruvate kinase which distinguish it from adult liver and muscle isozymes. Pyruvate kinase isozymes showing these kinetic characteristics in mammals are key regulatory enzymes in the glycolytic pathway and the oocyte pyruvate kinase is by analogy likely to be a regulatory enzyme in Xenopus. It has been suggested that pyruvate kinase activity is hormonally regulated via cAMP and cAMP-dependent protein kinase both directly by phosphorylation, as well as indirectly through F2,6P2 levels (see Introduction). In these cases cAMP serves to lower pyruvate kinase activity and reduce glycolytic flux. Since we have observed no difference in the kinetics of pyruvate kinase from ooctyes, eggs and embryonic cells and, in particular, no difference in the Kms of pyruvate kinase for PEP among those cells, we do not think there is a functionally significant change in the phosphorylation state of the enzyme up to blastula stage. Also, we do not observe any changes in the isozyme profiles by cellulose acetate electrophoresis. Since decreased adenylate cyclase activity is a necessary and sufficient trigger for oocyte matu-
ration, changes in glycolytic flux that are expected to result from decreases in cAMP levels could be intimately involved in maturation or subsequent activation of the egg at fertilization. Schneider et al. (1978) have suggested that in quiescent 3T3 cells stimulated to divide by serum or growth factors the significance of the activation of 6-phosphofructo-1-kinase activity and, consequently, of glycolytic flux may be regulation of cell proliferation rather than the energetic needs of the cell. Recently, it has been shown that some growth factors, whose receptors are linked to the breakdown of phosphatidylinositol-4,5-bisphosphate, may also be linked to the activation of 6-phosphofructo-2-kinase (Bosca et al., 1985). Oocyte and embryonic pyruvate kinase is ailosterically regulated by F1,6P2 and would be expected to respond to an increase in F2,6P2 caused by such factors. Further studies of glycolytic flux in this developmental system are underway. Acknowledgements--We thank Roberta Lieber for per-
forming several kinetic measurements and Drs Anthony Infante and Efralm Racker for critical reviews of the manuscript. This work was supported by a grant from the National Institutes of Health (HD 17234-03) and a gift from Boehringer Ingelheim, GmbH. REFERENCES
Bosca L., Rousseau G. G. and Hue L. (1985) Phorbol 12-myristate 13-acetate and insulin increase the concentration of fructose 2,6-bisphosphate and stimulate giycolysis in chicken embryo fibroblasts. Proc. natn. Acad. Sci. USA 82, 6440--6444. Burk D., Woods M. and Hunter J. (1967) On the significance of giucolysis for cancer growth, with special reference to Morris rat hepatomas. J. natn. Cancer Inst. 38, 839-863. Cardenas J. (1982) Pyruvate kinase from bovine muscle and liver. Meth. EnzymoL 90, 140-149. CicireUi M. F. and Smith L. D. (1985) Energy metabolism and pyridine nucleotide levels during Xenopus oocyte maturation. Develop, Growth and Differentiation 27, 283-294. Claus T. H., EI-Maghrabi M. R., Regen D. M., Stewart H. B., McGrane M., Kountz P. D., Nyfeler F., Pilkis J. and Pilkis S. J. (1984) The role of fructose 2,6-bisphospilate in the regulation of carbohydrate metabolism. Curr. Top. Cell. Reg. 23, 57-86. Diamond I., Legg A., Schneider J. A. and Rozengurt E. (1978) Glycolysis in quiescent cultures of 3T3 cells. J. biol. Chem. 253, 866-871. Dumont J. N. (1972) Oogenesis in Xenopus laevis (Daudin)--1. Stages of oocyte development in laboratory maintained animals. J. Morph. 136, 153-164. Dworkin M. B. and Dworkin-Rastl E. (1985) Changes in RNA titers and polyadenylation during oogenesis and oocyte maturation in Xenopus laevis. Dev. Biol. 112, 451-457. Dworkin M. B., Shrutkowski A. and Dworkin-Rastl E. (1985) Mobilization of specific maternal RNA species into polysomes after fertilization in Xenopus laevis. Proc. natn. Acad. Sci. USA 82, 7636-7640. Engstr6m L. (1980) Regulation of liver pyruvate kinase by phosphorylation-dephosphorylation. In Recently Discovered Systems of Enzyme Regulation by Reversible Phosphorylation (Edited by Cohen), pp. 11-31. Elsevier/
North-Holland. Ibsen K. H. (1977) Interrelationships and functions of the pyruvate kinase isozymes and their variant forms: a review. Cancer Res. 37, 341-353.
Pyruvate kinase in Xenopus Ibsen K. H., Cardin Jr. J. P., Chiu R. H-C., Garratt K. N., Morles S. W. and Doty J. R. (1980) Distribution of pyruvate kinase isozymes in adult and developing Xenopus laevis. Comp. Bioehem. Physiol. 65B, 473-480. Imamura K. and Tanaka T. (1982) Pyruvate kinase isozymes from rat. Meth. Enzymol. 90, 150-165. Kahn A. and Marie J. (1982) Pyruvate kinases from human erythrocytes and liver. Meth. Enzymol. 90, 131-139. Lowry O. H. and Passonneau J. V. (1964) The relationships between substrates and enzymes of glycolysis in brain. J. biol. chem. 239, 31-42. Lowry O. H. Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Maller J. L. (1985) Regulation of amphibian oocyte maturation. Cell Differentiation 16, 211-221. Oskam R., Rijksen G., Lips C. J. M. and Staal G. E. J. 0985) Enolase isozymes in differentiated and undifferentiated medullary thyroid carcinomas. Cancer 55, 394-399.
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Ramaiah A. (1974) Pasteur effect and phosphofructokinase. Curr. Top. Cell. Reg. 8, 297-345. Schlocn L. H., Kmiotek E. H. and SaUach H. J. (1974) Pyruvate kinase isozymes in adult tissues and eggs of Rana pipiens. Archs Biochem. Biophys. 164, 254-265. Schneider, J. A., Diamond I. and Rozengurt E. (1978) Glycolysis in quiescent cultures of 3T3 cells. J. biol. Chem. 253, 872-877. Standart N. M., Bray S. J., George E. L., Hunt T. and Ruderman J. V. (1985) The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs. J. Cell Biol. 100, 1968-1976. Susor W. A., Penhoet E. and Rutter W. J. (1975) Fructosediphosphate aldolase, pyruvate kinase, and pyridine nucleotide-linked activities after electrophoresis. Meth. Enzymol. 41, 66-73. Warburg O. (1956) On the origin of cancer cells. Science 123, 309-314.
0305-0491/87 $3.00+ 0.00 © 1987PergamonJournals Ltd
PYRUVATE KINASE ISOZYMES IN OOCYTES A N D EMBRYOS F R O M THE F R O G XENOPUS LAEVIS MARK B. DWORKIN, NEIL SEGIL and EVA DWORKIN-RASTL Department of Biological Sciences, Sherman Fairchild Center, Columbia University, New York, NY 10027, USA (Tel.: 212 280-8281) (Received 7 January 1987)
Ala~traet--1. The kinetic characteristics of pyruvate kinase isozymes from oocytes, embryos, liver and skeletal muscle from the clawed frog Xenopus laevis were measured in cell extracts. 2. The muscle and liver isozymes display Michaelis-Menten kinetics with Kms for phosphoenolpyruvate (PEP) of 0.02 and 0.05 raM, respectively. 3. Pyruvate kinase from oocytes and embryos displays cooperative kinetics for PEP with a K~ of about 0.15 raM; the kinetics become hyperbolic and the Km for PEP is reduced to 0.05 mM in the presence of /zM concentrations of fruetose-l,6-bisphosphate. 4. These data serve to characterize pyruvate kinase activity in oocytes and embryos and the kinetics are compared to mammalian pyruvate kinase isozymes.
INTRODUCTION Oocyte maturation in vertebrates is a progesterone dependent process in which the full-grown oocyte (stage VI oocyte in the clawed frog Xenopus laevis: Dumont, 1972), arrested in prophase of the first meiotic division, is induced to continue through its meiotic cell cycle and then arrest at metaphase of the second meiotic division, when the now mature oocyte can be fertilized (Mailer, 1985). Oocyte maturation involves the breakdown of the large oocyte nucleus, the germinal vesicle, as well as a number of ionic and other biochemical changes including several cAMP dependent and independent protein phosphorylations. Oocyte maturation is accompanied by a decrease in cAMP and inhibitors of adenylate cyclase, when injected into oocytes, result in maturation in the absence of hormone (Mailer, 1985). Since a decrease in cAMP concentration seems necessary and sufficient to induce oocyte maturation and a decrease in cAMP would be predicted to increase glycolytic flux, there might be an important relationship between increased glycolysis and oocyte maturation. Consistent with the possible importance of glycolysis in oogenesis and/or early development, we recently identified a cDNA clone for the glycolytic enzyme enolase in an embryonic cDNA library and showed that enolase m R N A is very abundant in early oocytes and decreases in concentration during oocyte growth and early embryogenesis (Dworkin and DworkinRastl, 1985; Dworkin et aL, 1985). Glucose is oxidized in cells by two alternative pathways. Glucose-6-phosphate can be oxidized to pyruvate via glycolysis and this process is linked to the production of a small amount of ATP. Three carbon compounds generated in this pathway are precursors to cellular macromolecules. Alternatively,
the oxidation of glucose-6-phosphate by glucose-6phosphate dehydrogenase and the subsequent reactions of the phosphogluconate pathway provide the cell with N A D P H and a variety of sugars, which are key substances required for biosynthetic processes such as fatty acid and nucleic acid synthesis. A recent study of Xenopus laevis full-grown oocytes shows a predominance of metabolism of glucose via the phosphogluconate pathway rather than via glycolysis (Cicirelli and Smith, 1985). It has been suggested that the phosphogluconate pathway is necessary to support the synthesis of ribose required for the large amount of deoxyribonucleotides needed during cleavage. Consistent with this idea, ribonucleotide reductase activity is greatly increased at fertilization in some species (Standart et al., 1985). However, classical studies by Warburg more than 50 fifty years ago showed that lactate formation is also increased upon fertilization of many types of eggs (see Warburg, 1956). The significance of increased rates of fermentation in newly fertilized eggs, as well as in serum or growth factor stimulated cells (Diamond et al., 1978) and neoplastically transformed cells (Warburg, 1956; Burk et al., 1967) is still not clear. In many cells glycolytic activity is partly regulated by hormones whose receptors act through cAMP, with high cAMP levels acting to inhibit glycolysis (discussed in Claus et al., 1984). Two key sites in the regulation of glycolytic flux by cAMP-dependent protein kinase are 6-phosphofructo-2-kinase/phosphofructo-2,6-bisphosphatase, which regulates the intracellular concentration of F2,6P2 (Claus et al., 1984), and pyruvate kinase (Engstrrm, 1980). Phosphorylation by cAMP-dependent protein kinase increases phosphofructo-2,6-bisphosphatase activity, reducing the level of F2,6P2. F2,6P2 is a key intracellular activator of 6-phosphofructo-l-kinase and in Abbreviations used: PEP, phosphoenolpyruvate; F1,6P2, the absence of F2,6P2, 6-phosphofructo-l-kinase is fructose- 1,6-bisphosphate; F2,6P 2, fructose-2,6-bisphos- virtually inactive in many cells under normal intracellular concentrations of ATP (Claus et al., 1984). phate. 743
744
10 ,~ ~
MARK B. DWORKINet al.
The product of the 6-phosphofructo-l-kinase reaction, F1,6P2, is in turn a potent activator of pyruvate kinase (Ramaiah, 1974). Three of the four pyruvate kinase isozymes which have been described in m a m malian tissues (liver, erythrocyte, and fetal) display sigrnoidal kinetics with respect to PEP, but are activated to hyperbolic kinetics by micromolar quantities o f F1,6P2 (Kahn and Marie, 1982; I m a m u r a and Tanaka, 1982; Cardenas, 1982). M a m m a l i a n muscle pyruvate kinase displays hyperbolic kinetics for PEP. All forms of pyruvate kinase are inhibited by A T P in the absence of F1,6P2. Further, several pyruvate kinase isozymes from rat, pig and h u m a n can be directly phosphorylated by c A M P - d e p e n d e n t protein kinase, which increases the K~ for PEP by a factor of two to three in the absence of F1,6P2. In this paper we describe the kinetics o f Xenopus laevis oocyte and embryonic pyruvate kinase, as well as adult liver and leg muscle pyruvate kinase. MATERIALS AND METHODS
Preparation of cell extracts and enzyme assays Oocytes, embryos, and adult tissues (leg muscle, liver, and brain) from female frogs were homogenized at 0°C in a Dounce homogenizer (oocytes and embryos) or in a Sorvall Omnimix (adult tissues) in 2-3 volumes of 20 mM Tris-HC1, pH 7.5 (25°C), 100 mM KCI, 5 mM MgSO4, 1 mM EDTA, 10raM mercaptoethanol, 0.5mM phenylmethylsulfonylfluoride. Homogenates were centrifuged 5 rain at 2 K in the Sorvall SS34 rotor and the supernatants recentdfuged 5 min at 10K in the same rotor. These 12,000g supernatants (referred to as cell extracts) were either immediately frozen in aliquots in a dry ice/ethanol bath and stored at -80°C, or first dialyzed by gel filtration. Approximately 0.5 ml aliquots containing 5 #Ci ct-32p-dCTP as tracer for small molecules were filtered through 3 ml Sephadex G-50 in homogenization buffer and the colored extract devoid of radioactivity was frozen in aliquots as described above. Cell extracts were analyzed for enolase, pyruvate kinase and lactate dehydrogenase activity in 50 mM imidazoleHC1, pH 7.0 (25°C), 70mM KC1, 10mM MgSO4, 3 mM EDTA according to Lowry and Passonneau (1964). All assays were linked to the oxidation of NADH in the lactate dehydrogenase reaction. Reactions were carried out in 1 ml at room temperature and the change in absorbance at 340 nm was recorded. Commercial enzymes and substrates were all from Sigma; all enzymes were found to be free of contaminating activities. Substrates (2-phosphoglycerate, PEP, pyruvate, F1,6P2, ADP and ATP) were dissolved, neutralized with NaOH if necessary, and frozen in aliquots; NADH was dissolved fresh for each experiment. ADP, ATP and NADH concentrations were determined spectrophotometrically; 2-phosphoglycerate, PEP and pyruvate concentrations were determined with the lactate dehydrogenase assay. For pyruvate kinase assays a typical reaction consisted of 0.12mM NADH, 0.4mM ADP, 9u lactate dehydrogenase, and PEP and FI,6P2 as necessary. The reaction was initiated by addition of 1-3#1 of cell extract so that the reaction at maximum velocity would support the oxidation of 50 nmol NADH/min. Rates (v) are given in nmot NADH oxidized per min in a 1 ml reaction at 20°C. K a (activation constant) for F1,6P2 is the concentration of F1,6P2 that produces half maximal rate at 0.02 mM PEP and 150 (inhibition constant) for ATP is the concentration of ATP that inhibits the reaction by 50% at 0.4 mM PEP. Cellulose acetate electrophoresis Cellulose acetate electrophoresis was used to separate the isozymic forms of pyruvate kinase. The following proc,edure was modified from published protocols (Susor et al., 1975;
/I , "/ /
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Fig. 1. Velocity profiles of oocyte, liver and muscle pyruvate kinase. (A). Activity of pyruvate kinase as a function of PEP concentration for cell extracts prepared from full-grown oocytes ( O - - O ) , adult leg muscle ( © - - © ) and adult liver (x--x). Assay conditions are described in Materials and Methods, except that ADP was 0.4 mM for the oocyte curve and 0.2 mM for the liver and muscle curves in this experiment. All measurements were done in the absence of FI,6P 2. (B), (C) and (D) are Lineweaver-Burk plots for the muscle, liver and oocyte curves, respectively. Oskam et al., 1985). Cell lysates (0.25#1) were applied to cellulose acetate strips (Sepraphore III, Gelman Sciences, Ann Arbor, Michigan). Strips were electrophorescd at room temperature in 20 mM sodium-phosphate buffer, pH 7.0, for 1.5 hr at 120V (2 mA). Pyruvate kinase activity was localized by incubating strips against a substrate transfer membrane (Gelman Sciences) which had been soaked in the following solution: 100mM Tris-HC1, pH7.0, 10raM MgC12, 100 mM KCI, 0.5 mM EDTA, 1.5 mM ADP, 20 mM AMP, 10mM NADP +, 1 mM glucose, 0.5mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 0.17 mM phenazine methosulphate, 10mM PEP, l mM FI,6P 2, 6.5 u/ml hexokinase and 2.5 u/ml glucose-6-phosphate dehydrogenase. Incubation was at 37°C for approx. 20 min under conditions of high humidity and low fight. In control experiments, PEP and FI,6P2 were omitted from the incubation solution. Incubation was stopped by washing in 5% acetic acid for 2 min followed by a wash with elcctrophoresis buffer. RESULTS AND DISCUSSION Rates of pyruvate formation from PEP were measured as described in Materials and Methods in cell extracts of adult leg muscle, adult liver, small oocytes (stages I and II, D u m o n t , 1972), full-grown oocytes (stage VI), unfertilized eggs, and 6-8 hr blastulae. The velocity profiles of X e n o p ~ laevis muscle, liver and fu11-grown oocyte pyruvate kinase as functions o f P E P concentration are shown in Fig. IA. The Lineweaver-Burk plots (Figs I B - D ) show that the muscle and liver pyruvate kinases display MichaelisMenten kinetics, but the oocyte enzyme does not. Thus, on the basis of these plots the oocyte pyruvate kinase can be distinguished from both the muscle and liver forms. In contrast to mammalian liver pyruvate kinase, the X e n o p ~ liver enzyme does not display
745
Pyruvate kinase in Xenopus Table 1. Kinetic characteristics of pyruvate kinase isozyrnes from Xenopus laevis. The data were obtained as described in Materials and Methods .and the figures. The concentration of F I , 6 P 2 that increases the rate of the reaction to 1/2 V ~ , at a PEP concentration of 0.02 mM is shown as I ~ . The concentration of A T P that inhibits the reaction to 1/2 V ~ at a PEP concentration of 0.4 mM is shown as I50. The K~ shown for the sigmoidal enzyme is the concentration of PEP that supports the reaction to I/2 Vmx
(PEP) kinetics Hill coefficient Kra (PEP), mM Km (ADP), mM (F1,6P2) kinetics (FI,6P2), K,, ,aM (ATP), Iso, mM
Muscle hyperbolic 1.1 0.02 0.1 no effect
Liver hyperbolic 1.3 0.05 0.1 small effect
3.3
2.6
sigmoidal kinetics. Liver extracts in which small molecules have been removed by Sephadex G-50 gel filtration show similar kinetics. T h e / ~ of the oocyte enzyme for P E P is about 0.15 m M , while the /(mS of the muscle and liver enzymes are, respectively, approx. 0.02 and 0 . 0 5 m M (Table 1). Additional curves describing the oocyte isozyme are shown in Figs 2 and 3. The velocity profile in Fig. 2A shows
Oocyte/embryo sigmoidal 2.3 0.15 0.1 activated 0.1 2.2
that in the presence o f 5# M F 1,6P 2 the kinetics o f the oocyte enzyme assume a hyperbolic shape. The a m o u n t of F I , 6 P 2 necessary to completely activate A
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Fig. 2. Kinetic analysis of ooc~e py~vatc kinase. (A). Activity of pyruvate kinase in full-grown oocyte extracts as shown in Fig. 1 ( 0 - - 0 ) , with the addition of 5pM F1,6P 2 ( O - - O ) , or with the addition of 0.25 rnM ATP (x--x). (B). Activity of oocyte pyrnvate kinase as a function of FI,6P 2 concentration in the presence of 0.02raM PEP, 0.4raM ADP. (C). Lineweaver-Burk plot of the oocyte curve in the presence of 5#M F1,6P 2. (D), (E). Hill plots of the oocyte enzyme in the absence of FI,6P2 (D) and in the presence of 5pM FI,6P2 (E). The Hill coefficient, n, is shown in the top left corner of each graph.
J 0.2
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Fig. 3. Effect of ATP on oocyte velocity profiles. (A). Activity of pyruvate kinase in full-grown oocytes in the presence of 0.25 mM ATP. The first three points are also plotted on the profile shown in Fig. 2A. (B). Activity of the oocyte pyruvate kinase as a function of ATP concentration at 0.4 mM PEP and 0.4 mM ADP. (C). Activity of oocyte pyruvate kinase as a function of FI,6P2 concentration in the presence of 0.4raM PEP, 0.8raM ADP, 3raM ATP. 1/2 V~, occurs at 0.1pM F1,6P2.
746
MARK B. DWORKINet al. B
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Fig. 4. Kinetic analysis of liver pyruvate kinase. (A). Velocity profiles of liver pyruvate kinase in the absence (O--O) and presence (O--O) of 5#M F1,6P2. (B, C). Hill plots of the liver enzyme in the absence of F1,6P2 (B) and in the presence of 5#M FI,6P 2 (C). the oocyte enzyme is less than I # M with a /Ca of about 0.1/~M (Fig. 2B; Table 1). The LineweaverBurk plot of the oocyte enzyme in the presence of 5gM F1,6P2 is a straight line (Fig. 2C) and the Km of the enzyme for PEP is reduced to 0.05 mM. In the absence of F1,6P2 the Hill coefficient of the oocyte isozyme is 2.3 (Fig. 2D). The Hill coefficient is reduced to approx. 1.0 by the addition of the activator F1,6P2 (Fig. 2E). These results for the Xenopus oocyte pyruvate kinase are in complete agreement with the kinetic descriptions of the fetal/tumor and liver forms of pyruvate kinase in mammalian tissues (Engstr6m, 1980; Kahn and Marie, 1982; Imamura and Tanaka, 1982; Cardenas, 1982). In the presence of ATP the velocity of the pyruvate kinase reaction is reduced at concentrations of PEP less that 1 mM. Some of these data at 0.25 mM ATP are shown for the oocyte enzyme in Figs 2A and 3A. An inhibition curve for ATP is shown in Fig. 3B. The ATP concentration which inhibits the reaction by 50% at 0.4mM PEP (I50) is 2.2 mM (Table 1). Slightly higher Is0s were obtained by this analysis for the liver and muscle enzymes (Table 1). In the presence of 5#M F1,6P2 and 2 mM ATP, the oocyte pyruvate kinase, as well as the liver and muscle isozymes, show hyperbolic kinetics with KmS for PEP of 0.02-0.05mM (data not shown). Less than I # M of FI,6P2 fully activates the oocyte enzyme at 0.4mM PEP and 3 mM ATP (Fig. 3C). Thus, micromolar levels of FI,6P: relieve the ATP inhibition of the oocyte pyruvate kinase as well as lower the Km of the enzyme for PEP. The effect of F1,6P2 on the kinetics of the liver enzyme is shown in Fig. 4. A small effect is observed on both the velocity profile and the Hill plots. However, the Xenopus liver enzyme clearly does not show the cooperative kinetics described for mammalian liver pyruvate kinases. The unusual behavior of this isozyme has been previously cited (Ibsen, 1977; also see Ibsen et al., 1980) and is presented here to show a clear distinction between the liver and oocyte forms of pyruvate kinase in Xenopus.
I
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o
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Fig. 5. Kinetic analysis of blastula pyruvate kinase. (A). Activity of pyruvate kinase from blastulae in the absence (O--O) and presence (O--©) of 5#M F1,6P2. (B, C). Lineweaver-Burk plots of the blastula enzyme in the absence (B) and presence (C) of5#M F1,tP2. (D, E). Hill plots of the blastula enzyme in the absence (D) and presence (E) of 5#M F1,6P 2. Kinetic data for the early oocyte, egg and blastula enzymes were virtually indistinguishable from the full-grown oocyte form. Velocity measurements for the blastula enzyme are shown in Fig. 5. The enzyme shows sigmoidal kinetics with respect to PEP with a Km for PEP of about 0.15 mM; these kinetics are converted to standard Michaelis-Menten kinetics by the addition of F1,6P2 and the Km for PEP is reduced to 0.05 mM (Fig. 5A, C). This activator decreases the Hill coefficient from 2.3 to 1.2. We have found no way to kinetically distinguish oocyte and embryonic pyruvate kinase isozymes and conclude that the pyruvate kinase form does not change during oocyte growth, maturation, or during very early embryogenesis. This conclusion is substantiated by cellulose acetate electrophoresis of pyruvate kinase isozymes from oocytes, eggs and adult tissues (Fig. 6). The
747
Pyruvate kinase in Xenopus
I
2
3
4
-I-
Fig. 6. Cellulose acetate electrophoresis o f pyruvate kinase isoforms. Pyruvate kinase isozymes in cell extracts from adult leg muscle (1), small ooeytes (stages I, II; Dumont, 1972) (2), full-grown oocytes (3) and eggs (4) were separated by electrophoresis on cellulose acetate strips and pyruvate kinase activity localized as described in Materials and Methods.
pattern of bands seen with the oocyte and egg preparations is identical; the pattern of bands seen with muscle is different. The liver pattern (not shown) is unexpectedly similar to the oocyte/embryo pattern. Such a complex pattern of bands in amphibians has been previously reported in eggs and adult tissues of Rana pipiens (Schloen et al., 1974). Since pyruvate kinase is a tetramer, two electrophoretically distinct subunits can form five different combinations which can be separated by cellulose acetate electrophoresis (Cardenas, 1982). The kinetics that we measure in these cell extracts are a composite of the kinetics of these pyruvate kinase isoforms. Ibsen et al. (1980) have separated several isoforms of pyruvate kinase from Xenopus embryos and tissues and their data also reveal three isoforms in muscle and four major and two minor isoforms in liver. However, their data show a single major isoform in blastulae, whereas
1.0
>E 0.5
•
•
I
l
I
0
•
•
I
I
I
I
0.I
0.2
0.3
0.4
[ADP] , mM Fig. 7. Velocity profiles o f occyte and liver pyruvate kinases as functions o f A D P concentration. Measurements shown were made at 0.2 m M PEP for both the full-grown oocyte enzyme ( O - - O ) and the adult liver enzyme ( O - - O ) .
our study indicates four or more isoforms for oocytes and eggs, which persist to blastula (not shown). The isoform they describe in blastula also shows sigmoidal kinetics, though not as extreme as what we observe in oocytes, eggs and blastulae, and is activated by F1,6P2. The velocity profiles of the oocyte and liver pyruvate kinases for ADP are shown in Fig. 7. They are superimposable, as is the muscle profile (not shown) and are unaffected by 5/zM F1,6P2 at I mM PEP (not shown). T h e / ~ of these isozymes for ADP is about 0.1 mM. All of these kinetic parameters are listed in Table 1. The characteristics of the Xenopus muscle and liver isozymes are very similar to mammalian muscle pyruvate kinase. The Xenopus oocyte enzyme best resembles the type M 2 rat isozyme characteristic of fetal and tumor tissue. In particular its high I50 for ATP seems to distinguish it from the mammalian liver form of pyruvate kinase, which is inhibited at lower concentrations of ATP (Imamura and Tanaka, 1982). The activities of cell extracts from leg muscle, liver, brain, oocytes and eggs for lactate dehydrogenase, pyruvate kinase and enolase are listed in Table 2. Some of these data for representative individual Table 2. Activities of lactate dehydrogenase (LDH), pyruvate kinase (PK) and enolase in nmoles NADH oxidized/rain per g protein. Protein concentrations were determined according to Lowry et al. (1951). The data for leg muscle, brain, and small oocytes (stages I, II; Dumont, 1972) are from single lysates, the liver data from two independent lysates and the data for full-grown (stage VI) oocytes/eggs from four different lysates prepared from either oocytes or eggs from different females. One of these preparations showed a particularly high level of LDH activity, thereby making the average LDH activity higher than in most individual preparations (Fig. 8) LDH PK Enolase Leg muscle Liver Brain Oocytes (I, lI) Oocytes (VI), eggs
1.9 1.3 0.38 1.7 1.9
2.6 0.30 1.0 5.2 3.5
2.2 0.055 0.29 0.42 1.4
MARK B. DWORKIN et al.
748 muscle
liver
brain
1.0~ 0.5
L PE
L PE
LPE
Fig. 8. Relative activities of lactate dehydrogenase, pyruvate kinase and enolase in cell extracts. The relative activities of lactate dehydrogenase (L), pyruvate kinase (P) and enolase (E) on a per g of protein basis for six individual and representative lysates are shown. The activity of the most active enzyme for each lysate was set at 1.0. Activity measurements were made in the imidazole buffer as described in Materials and Methods and were all linked to the oxidation of NADH by lactate dehydrogenase. Commercial lactate dehydrogenase (9u/mi) and pyruvate kinase (4 u/ml) were used as required. NADH, ADP, 2-phosphoglycerate, PEP and pyruvate were added as needed at, respectively, 0.12, 0.8, 0.5, 0.4 and 0.4mM. lysates are depicted graphically in Fig. 8. The relative levels of these three enzymes in full-grown oocytes, eggs and blastulae are more similar to adult brain than to muscle or liver. On a per g of protein basis, oocytes and embryos contain high amounts of all three enzymes at levels close to those of leg muscle. Oocytes and eggs have considerably more enolase activity than adult liver. In this report we have described the cooperative kinetics of the oocyte and embryonic form of pyruvate kinase which distinguish it from adult liver and muscle isozymes. Pyruvate kinase isozymes showing these kinetic characteristics in mammals are key regulatory enzymes in the glycolytic pathway and the oocyte pyruvate kinase is by analogy likely to be a regulatory enzyme in Xenopus. It has been suggested that pyruvate kinase activity is hormonally regulated via cAMP and cAMP-dependent protein kinase both directly by phosphorylation, as well as indirectly through F2,6P2 levels (see Introduction). In these cases cAMP serves to lower pyruvate kinase activity and reduce glycolytic flux. Since we have observed no difference in the kinetics of pyruvate kinase from ooctyes, eggs and embryonic cells and, in particular, no difference in the Kms of pyruvate kinase for PEP among those cells, we do not think there is a functionally significant change in the phosphorylation state of the enzyme up to blastula stage. Also, we do not observe any changes in the isozyme profiles by cellulose acetate electrophoresis. Since decreased adenylate cyclase activity is a necessary and sufficient trigger for oocyte matu-
ration, changes in glycolytic flux that are expected to result from decreases in cAMP levels could be intimately involved in maturation or subsequent activation of the egg at fertilization. Schneider et al. (1978) have suggested that in quiescent 3T3 cells stimulated to divide by serum or growth factors the significance of the activation of 6-phosphofructo-1-kinase activity and, consequently, of glycolytic flux may be regulation of cell proliferation rather than the energetic needs of the cell. Recently, it has been shown that some growth factors, whose receptors are linked to the breakdown of phosphatidylinositol-4,5-bisphosphate, may also be linked to the activation of 6-phosphofructo-2-kinase (Bosca et al., 1985). Oocyte and embryonic pyruvate kinase is ailosterically regulated by F1,6P2 and would be expected to respond to an increase in F2,6P2 caused by such factors. Further studies of glycolytic flux in this developmental system are underway. Acknowledgements--We thank Roberta Lieber for per-
forming several kinetic measurements and Drs Anthony Infante and Efralm Racker for critical reviews of the manuscript. This work was supported by a grant from the National Institutes of Health (HD 17234-03) and a gift from Boehringer Ingelheim, GmbH. REFERENCES
Bosca L., Rousseau G. G. and Hue L. (1985) Phorbol 12-myristate 13-acetate and insulin increase the concentration of fructose 2,6-bisphosphate and stimulate giycolysis in chicken embryo fibroblasts. Proc. natn. Acad. Sci. USA 82, 6440--6444. Burk D., Woods M. and Hunter J. (1967) On the significance of giucolysis for cancer growth, with special reference to Morris rat hepatomas. J. natn. Cancer Inst. 38, 839-863. Cardenas J. (1982) Pyruvate kinase from bovine muscle and liver. Meth. EnzymoL 90, 140-149. CicireUi M. F. and Smith L. D. (1985) Energy metabolism and pyridine nucleotide levels during Xenopus oocyte maturation. Develop, Growth and Differentiation 27, 283-294. Claus T. H., EI-Maghrabi M. R., Regen D. M., Stewart H. B., McGrane M., Kountz P. D., Nyfeler F., Pilkis J. and Pilkis S. J. (1984) The role of fructose 2,6-bisphospilate in the regulation of carbohydrate metabolism. Curr. Top. Cell. Reg. 23, 57-86. Diamond I., Legg A., Schneider J. A. and Rozengurt E. (1978) Glycolysis in quiescent cultures of 3T3 cells. J. biol. Chem. 253, 866-871. Dumont J. N. (1972) Oogenesis in Xenopus laevis (Daudin)--1. Stages of oocyte development in laboratory maintained animals. J. Morph. 136, 153-164. Dworkin M. B. and Dworkin-Rastl E. (1985) Changes in RNA titers and polyadenylation during oogenesis and oocyte maturation in Xenopus laevis. Dev. Biol. 112, 451-457. Dworkin M. B., Shrutkowski A. and Dworkin-Rastl E. (1985) Mobilization of specific maternal RNA species into polysomes after fertilization in Xenopus laevis. Proc. natn. Acad. Sci. USA 82, 7636-7640. Engstr6m L. (1980) Regulation of liver pyruvate kinase by phosphorylation-dephosphorylation. In Recently Discovered Systems of Enzyme Regulation by Reversible Phosphorylation (Edited by Cohen), pp. 11-31. Elsevier/
North-Holland. Ibsen K. H. (1977) Interrelationships and functions of the pyruvate kinase isozymes and their variant forms: a review. Cancer Res. 37, 341-353.
Pyruvate kinase in Xenopus Ibsen K. H., Cardin Jr. J. P., Chiu R. H-C., Garratt K. N., Morles S. W. and Doty J. R. (1980) Distribution of pyruvate kinase isozymes in adult and developing Xenopus laevis. Comp. Bioehem. Physiol. 65B, 473-480. Imamura K. and Tanaka T. (1982) Pyruvate kinase isozymes from rat. Meth. Enzymol. 90, 150-165. Kahn A. and Marie J. (1982) Pyruvate kinases from human erythrocytes and liver. Meth. Enzymol. 90, 131-139. Lowry O. H. and Passonneau J. V. (1964) The relationships between substrates and enzymes of glycolysis in brain. J. biol. chem. 239, 31-42. Lowry O. H. Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Maller J. L. (1985) Regulation of amphibian oocyte maturation. Cell Differentiation 16, 211-221. Oskam R., Rijksen G., Lips C. J. M. and Staal G. E. J. 0985) Enolase isozymes in differentiated and undifferentiated medullary thyroid carcinomas. Cancer 55, 394-399.
C.B,P. 88/3B---D
749
Ramaiah A. (1974) Pasteur effect and phosphofructokinase. Curr. Top. Cell. Reg. 8, 297-345. Schlocn L. H., Kmiotek E. H. and SaUach H. J. (1974) Pyruvate kinase isozymes in adult tissues and eggs of Rana pipiens. Archs Biochem. Biophys. 164, 254-265. Schneider, J. A., Diamond I. and Rozengurt E. (1978) Glycolysis in quiescent cultures of 3T3 cells. J. biol. Chem. 253, 872-877. Standart N. M., Bray S. J., George E. L., Hunt T. and Ruderman J. V. (1985) The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs. J. Cell Biol. 100, 1968-1976. Susor W. A., Penhoet E. and Rutter W. J. (1975) Fructosediphosphate aldolase, pyruvate kinase, and pyridine nucleotide-linked activities after electrophoresis. Meth. Enzymol. 41, 66-73. Warburg O. (1956) On the origin of cancer cells. Science 123, 309-314.