Catalytic and regulatory properties of pyruvate kinase isozymes from octopus mantle muscle and liver H. E. GUDERLEY,' K. B. STOREY , 2 J. H. A. FIELDS,' AND P. W. HOCHACHKA]
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Pac$c Biomedical Research Center, University of Hawaii, Honolulu, HZ, U . S . A . Received December 10. 1975
H. E., K. B. STOREY, J. H. A. FIELDS,and P. W. HOCHACHKA. 1976. Catalytic and GUDERLEY, regulatory properties of pyruvate kinase isozymes from octopus mantle muscle and liver. Can. J. Zool. 54: 863-870. The octopus is a relatively slow-moving animal that relies upon burst swimming to power its predatory activities. Pyruvate kinase (EC, 2.7.1.40), as one of the major glycolytic control sites, must be regulated in such a fashion to allow the increased glycolytic rate characteristic of burst metabolism. The mantle enzyme is regulated by the concerted action of ATP, arginine phosphate, and citrate. The K, for ADP was 0.28 mM and that for phosphoenolpyruvate (PEP), 0.25 mM. In contrast to many other invertebrate muscle pyruvate kinases, the enzyme is insensitive to fructose- l,6-diphosphate (FDP) activation. The pyruvate kinase from the liver is kinetically and electrophoretically distinct from the mantle enzyme. The liver isozyme has a considerably lower affinity for PEP (K, = 0.85 mM), is inhibited by ATP, citrate, and arginine phosphate, and is subject to a strong activation by FDP ( K , = 1 x lo-"). These differences between the pyruvate kinases from catabolic and synthetic tissues are reminiscent of the distinctions between mammalian muscle and liver pyruvate kinases. GUDERLEY, H. E., K. B. STOREY, J . H. A. FIELDSet P. W. HOCHACHKA. 1976. Catalytic and regulatory properties of pyruvate kinase isozymes from octopus mantle muscle and liver. Can. J. Zool. 54: 863-870. La pieuvre est un animal qui se meut relativement lentement et qui depend de ses 'poussees' de nage pour exercer ses activitks predatrices. La pyruvate-kinase (EC 2.7.1.40), I'un des principaux facteurs de contr8le de la glycolyse, doit Ctre reglee de f a ~ o na permettre le taux de glycolyse plus eleve caracteristique du metabolisme accompli lors de la pousste. La regulation de I'enzyme du manteau se fait par I'action combinee de I'ATP, ainsi que de la phosphate d'arginine et de la citrate. La valeur de K,, dans le cas de I'ADP, est de 0.28 mM; elle est de 0.25 mM pour ce qui est du PEP (phosphoenolpyruvate). Contrairement aux pyruvate-kinases musculaires de plusieurs autres invertebres, cet enzyme est insensible a l'activation par le FDP (fructose-1,6-diphosphate). La pyruvate-kinase venant du foie a des proprietts kinesiques differentes de celles de I'enzyme du manteau et rtagit diffkremment a I'electrophorese. L'isoenzyme du foie a une affinite beaucoup plus faible pour le PEP (K, = 0.85 mM), est inhibe par I'ATP, par le citrate et le Ces phosphate d'arginine, et peut subir une forte activation par le FDP ( K , = 1 x differences entre les pyruvate-kinases de tissus catabolique et synthetique s'apparentent i celles qu'on observe chez les mammiferes, entre les pyruvate-kinases musculaire et hepatique. [Traduit par le journal]
Introduction A number of highly differentiated life-styles that rely upon distinctive metabolic control mechanisms have evolved within cephalopods. For example, the pelagic squid, Symplectoteuthis oualaniensis, sustains continual high-speed swimming by a highly aerobic metabolism. As one novel feature of their metabolism, squids use an 'Permanent address: Department of Zoology, University of British Columbia, Vancouver, B.C., Canada V6T 1W5. 'Current address: Department of Biochemistry, Oxford University, Oxford, England.
active a-glycerol phosphate shunt to provide efficient regulation of cytoplasmic redox balance in the face of continual carbohydrate breakdown (Hochachka et al. 1975). The squid also uses reduced nicotinamide adenine dinucleotide (NADH) as a prime regulatory metabolite. When NADH levels are high, flux through glycolysis and the Krebs cycle is limited (Storey and Hochachka 1975a). However, the squid mantle retains the capacity for redox balance via octopine formation (Fields et al., in preparation), and thus must have some residual anaerobic capacity. The life history of the octopus is distinct from
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that of the squid. Octopus cyanea is a bottom dweller in shallow waters, and its predatory behaviour usually involves anaerobic burst activity, a strategy which metabolically entails high power but low efficiency. In light of these major ecological and physiological differences we sought to compare metabolic organization in the octopus mantle with that in the previously studied squid mantle. Since carbohydrate is the prime metabolic fuel in these animals (Moon and Hulbert 1975), the glycolytic control mechanisms that usually reside at the phosphofructokinase (EC 2.7.1.1 1) and pyruvate kinase loci must be so adapted to facilitate the rapid fluctuations in carbon flux that characterize 'burst' metabolism. This study of octopus mantle and liver pyruvate kinases had two major goals: (1) the elucidation of the control mechanisms of mantle pyruvate kinase that would allow the increased glycolytic rate characteristic of the octopus's burst swimming; and (2) a comparison of the regulation of pyruvate kinase from a gluconeogenic versus a catabolic tissue. In gluconeogenic tissues, the pyruvate kinase reac~ionis bypassed by pyruvate carboxylase (EC 6.4.1.1 ) and phosphoenoipyruvate carboxykinase (EC 4.1.1.32) (Scrutton and Utter 1968). Pyruvate kinase in these tissues must be inhibited to allow net gluconeogenic flux. In mammals, liver pyruvate kinase is controlled by potent adenosine triphosphate (ATP), alanine, and citrate inhibition, by its low phosphoenolpyruvate (PEP) affinity, and by a high sensitivity to fructose-I,6-diphosphate (FDP) "feed forward" activation (Van Berkel er ol. 1974). I n contrast to the mammalian muscle enzyme, many invertebrate and lower vertebrate muscle pyruvate kinases show strong FDP activation, and potent inhibition by ATP, amino acids, and citrate (Flanders et al. 1971; Johnston 1975; Mustafa et al. 1971; Somero and Hochachka 1968; Mustafa and Hochachka 1971 ; Storey and Hochachka 1974). These regulatory properties are much like those of the mammalian liver enzyme. Whether these animals use the same enzyme in both their muscle and their 'liver,' and if not, what regulatory strategies are used by their 'liver' enzymes, are two generally unanswered questions. We found that the mantle enzyme was insensitive to FDP activation, and was inhibited by ATP, arginine phosphate, and citrate. The concerted action of ATP and arginine phosphate
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probably provides the major means by which pyruvate kinase is controlled in the resting mantle. The action of citrate may be important in integrating maximal rates of Krebs cycle and pyruvate kinase function. In contrast with the squid enzyme, which is strongly inhibited by NADH, the octopus enzyme showed little response to this compound. The octopus liver enzyme showed a considerably lower affinity for PEP than the mantle enzyme, and was subject to activation by FDP and inhibition by ATP, arginine phosphate, and citrate. FDP strongly affected the activity of the liver enzyme, and may be the major regulator of its activity in the cell.
Methods All reagents used in this study were purchased from Sigma Chemical Company, St. Louis, Missouri. Octopus were obtained by diving, and were courtesy of Mr. Howell Mahoe, Kailua, Oahu. Preparation of Mantle Pyruvate Kinase The mantle was cut into small pieces and homogenized in 50 mM imidazole chloride, pH 7.0, in a Virtis homogenizer, for 2 min. The homogenate was centrifuged at 20000g for 15 min, the supernatant brought to 45% ammonium sulphate, stirred at 4 'C for 30 min, centrifuged as above, and the pellet discarded. The supernatant was then brought to 60% ammonium sulphate, stirred for 30 min, centrifuged as above, and the supernatant discarded. The pellet was resuspended in 50 m M imidazole chloride, pH 7.0, and dialyzed against two changes of the same buffer. The pH was reduced to 6.5, and damp, precycled phosphocellulose was added, keeping the pH at 6.5, just until loss of pyruvate kinase activity could be detected. The sluny was centrifuged as above, the supernatant was removed, and the pH was adjusted to 7.0. This preparation was used for kinetic studies. Although only severalfold purified, it was free of any enzymes that would interfere with the basic assay, or that would interconvert any of the added metabolites. Preparation of the Octopus Liver Pyruvate Kinase Liver was homogenized in 50 mM imidazole chloride, 100 m M KCI, 3 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0, in a Virtis homogenizer for 2 min. The homogenate was centrifuged for 15 min at 20000g. Ammonium sulphate was added to 5 0 z saturation, the solution was stirred for 30 min at 4 'C, and centrifuged as above. The pellet was discarded and the supernatant was brought to 65% ammonium sulphate, stirred for 30 min at 4 "C, and centrifuged as above. The pellet from this step was resuspended in the extraction buffer. Diethylaminoethyl (DEAE) Sephadex, equilibrated in 100 mM imidazole chloride at pH 7.1. was graduallv added until a slight loss of pyruvate kinas; actLity couid be detected. The slurry was centrifuged at 10 000g for 20 min, and the supernatant was carefully decanted. To this supernatant, hydroxyl apatite was added until a slight loss of pyruvate kinase activity was detected. This slurry was
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abductor enzyme (Mustafa and Hochachka 1971). Pyruvate kinases require a divalent cation for catalysis, and this requirement can be satisfied with Mg2+ or Mn2+ (Kayne 1973). The octopus mantle enzyme adhered to this pattern, with Mg2+ saturation of the enzyme following hyperbolic kinetics, with an apparent Km of 0.55 mM. Pyruvate kinases generally either require or are activated by monovalent cations (Kayne 1973), with K + and NH,+ being physiologically most important. In keeping with this behaviour, octopus mantle pyruvate kinase showed a marked Michaelis-Menten activation by K f with a K,,, of 14.3 mM. In contrast to the squid mantle enzyme, this activation was not absolute (Storey and Hochachka 19753).
Starch-gel Electrophoresis Electrophoresis was carried out on 13% starch gels, using tris (hydroxymethyl)aminomethane (Tris) citrate, pH 7.8, with KC1 as both the gel and the tank buffer. Freshly prepared high-speed supernatants were used, and the gels were run at 40 mA, 200 V for 12 h. The gels were stained for pyruvate kinase activity by a negative method, which follows the appearance of dark bands under ultraviolet light caused by the disappearance of NADH and its fluorescence. The staining solution, which consisted of 5 m M ADP, 5 m M PEP, 10 m M MgC12, 72 mM KCI, 3 m M NADH, and excess lactate dehydrogenase, was applied to a filter paper overlying the gel and monitored with an ultraviolet light for the appearance of the bands of pyruvate kinase activity.
Substrate Afinities and Metabolic Effectors Substrate saturation curves followed regular Michaelis-Menten hyperbolas, with the Km for ADP being 0.28 m M and that for PEP being 0.25 mM. The affinity of each substrate was unaffected by varying the concentration of the other, indicating the random-order binding mechanism, which is characteristic of mammalian muscle pyruvate kinases (Kayne 1973). The molluscan pyruvate kinases that have previously been examined provide distinct contrasts in terms of regulatory strategies. The bivalve mantle and muscle enzymes show kinetics where FDP, alanine, and pH, as the chief regulatory signals, provide a sensitive 'throttle' on the reaction (Mustafa and Hochachka 1971). In contrast to the facultative anaerobe, the highly aerobic squid showed a pyruvate kinase with completely different regulatory characteristics. Neither alanine nor FDP affects the activity of the squid pyruvate kinase; instead, control was vested in NADH, citrate, and ATP (Storey and Hochachka 1975b). Similarly, neither alanine nor FDP was found to exert a significant effect upon the activity of the octopus mantle enzyme. In checking for an effect of FDP on enzyme-substrate and enzyme-inhibitor interactions, we varied the concentration of FDP from 0.005 m M to 0.05 mM. Only in the case of the NADH inhibition was FDP able to reverse partially the inhibitory effects. Alanine was tested at concentrations ranging up to 20 mM, where a 7% inhibition was evident. Other compounds which were found to be without effect on the
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centrifuged, and the supernatant used for kinetic studies. This preparation, only severalfold purified, was free of enzymes that would interfere with the assay; however, it needed to be used quickly, for it was not stable for prolonged periods. Pyruvate kinase was assayed by the method of Biicher and Pfleiderer (1955). Pyruvate formation was coupled to lactate dehydrogenase, and the rate of pyruvate kinase activity was measured as the decrease in OD,,, due to NADH disappearance. Imidazole chloride buffers were used in all reactions. Standard assay mixtures contained Mg2+,K + , adenosine diphosphate (ADP), PEP, NADH, and excess of lactate dehydrogenase (Sigma) in the concentrations specified in each figure legend. For the mantle enzyme, saturating concentrations of each of the reactants were 100 m M K + , 10 mM Mg2+, 3 m M PEP, and 2.5 m M ADP. For the liver enzyme the corresponding concentrations were 100 m M K + , 10 mM Mg2+, 6 m M PEP, and 3 m M ADP. Reactions were started by the addition of pyruvate kinase. All experiments were performed at 25 "C.
Calculation of the Michaelis Constant The Michaelis constant (K,) was determined in the presence and absence of metabolite effectors by Lineweaver-Burk plots l/velocity versus l/substrate concentration. The K, values obtained are reproducible to within f 10%.
Results and Discussion Octopus Mantle Pyruvate Kinase The specific activity of pyruvate kinase in the mantle was 50.4 units per gram wet weight of tissue. The enzyme activity in the mantle was confined to a single anodally migrating band on starch-gel electrophoresis.
The p H Optimum and Cation Requirements The pH optimum of mantle pyruvate kinase under saturating substrate and cofactor conditions was pH 7.0. This is similar to the pH optimum for the squid mantle pyruvate kinase (Storey and Hochachka 1975b), but considerably lower than the pH optimum for the oyster
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enzyme activity were malate (5 mM), glutamate (10 mM), octopine (5 mM), a-glycerol phosphate (10 mM), proline (60 mM), aspartate (10 mM), 3-phosphoglyceric acid (5 mM), arginine (5 mM), and acetyl coenzyme A (0.01 mM). However, three other compounds were found to exert a marked inhibitory action on enzyme activity. These were MgATP, arginine phosphate, and citrate. In contrast to its effect on the squid enzyme, NADH produced only a relatively weak inhibition of the octopus pyruvate kinase (Fig. 1). MgATP and arginine phosphate showed mixed competitive inhibition with regard to the two substrates, and both showed a greater effect on the binding of the adenylate substrate. The Ki of MgATP versus ADP was 8.5 mM, while its Ki versus PEP was 12.5 mM. ATP inhibition was manifest primarily as a Vma, effect in the latter case. Arginine phosphate, which provides the high-energy phosphate store for the resting mantle, exhibited a Ki of 9.5 m M versus ADP and a 28 m M Ki versus PEP (Figs. 2 and 3). Citrate, tested as the Mg,citrate complex to avoid chelation effects, showed linear competitive inhibition versus ADP, with a Ki of 17 mM. Citrate inhibition with PEP is complex. Its two major facets are a change in the PEP saturation curves from hyperbolic to sigmoidal, and a decrease in.,,,/I The clearest demonstration of the effectiveness of these inhibitors came from an examination of individual and additive effects on the enzyme at physiological concentrations of substrates (Table
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1). At K, concentrations of PEP and ADP, each of the inhibitors, added singly, caused a marked inhibition of enzyme activity. The inhibitory effects of ATP and arginine phosphate were additive. At 10 m M ATP and 10 m M arginine phosphate, an approximation of the levels found in the resting mantle, only 30% of the enzyme activity was evident. While citrate demonstrated additive inhibition with either ATP or arginine phosphate, adding all three inhibitors together caused no further reduction of enzyme activity. NADH also inhibited the reaction; 0.45 m M inhibited enzyme activity by 32%, with FDP partially reversing this inhibition. When 0.45 m M NADH was added at 10 m M ATP and 10 mM arginine phosphate, the inhibition increased to 75%. Thus, between the inhibited and deinhibited state the pyruvate kinase can undergo at least a fourfold change in activity. In the resting mantle, the pyruvate kinase would probably be inhibited by the high arginine phosphate and ATP levels. With the onset of muscular action, dropping arginine phosphate and eventually dropping ATP levels would lead to an increase in pyruvate kinase activity. If flux through the reaction were limiting overall glycolytic flux, the buildup of PEP and ADP would further deinhibit the reaction. Pyruvate formed by the pyruvate kinase reaction has two possible major metabolic fates; one is its conversion via pyruvate dehydrogenase into acetyl CoA, which then, by condensation with oxaloacetate, forms citrate. Pyruvate's other fate as substrate for octopine dehydrogenase in the following reaction,
pyruvate
+ arginine + NADH o
FDP
octopine
+ NAD
TABLE 1 . The effect o f various inhibitors on the activity o f octopus mantle pyruvate kinase Activity as
% o f control
Treatment
0.1
0.2
0.3
0.4
[NADH] rnM
FIG. 1 . NADH inhibition o f octopus and squid mantle pyruvate kinase. Assay conditions for octopus mantle enzyme are 0.3 mM ADP, 0.25 mM PEP, 10 mM MgC12, 100 mM KCl, and excess lactate dehydrogenase (Sigma). Conditions for the squid mantle enzyme are given in Storey and Hochachka (19756).
10 mM citrate 10 mM arginine phosphate 1 0 m M ATP All three inhibitors at 10 mM 10 mM ATP 10 mM citrate 10 mM ATP 10 mM arginine phosphate 10 mM arginine phosphate 10 mM citrate 10 mM citrate 10 mM arginine phosphate + 10 mM ATP + 0.45 mM NADH
+ + +
+
53 74 51 30 44 30 44 26
NOTE:These tests were run at the following substrate and cofactor concentrations: 0.3 mMADP, 0.25 m M PEP, I0 m M MgCI2, 100 mM KCI. 0.15 mM NADH. and excess lactate dehydrogenase (Sigma). The values are the mean of three determinations.
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GUDERLEY ET AL.
FIG.2. Arginine phosphate (ARG-PHOS) inhibition of octopus mantle pyruvate kinase, interaction with ADP. Assay conditions are 3 m M PEP, 10 m M MgC12, 100 m M KCI, 0.15 m M NADH, and excess lactate dehydrogenase (Sigma). ADP and arginine phosphate concentrations are varied as shown. Vindicates AOD,,,.
FIG.3. Arginine phosphate (ARG-PHOS) inhibition of octopus mantle pyruvate kinase, interaction with PEP. Assay conditions are 2.5 m M ADP, 10 mM MgC12, 100 mM KC], 0.15 m M NADH, and excess lactate dehydrogenase (Sigma). PEP and arginine phosphate concentrations are varied as shown. Vindicates AOD340.
is important in cytoplasmic redox balance. Since octopus utilizes carbohydrate as its major fuel, Krebs-cycle function must be integrated with glycolysis under aerobic conditions. Therefore it is reasonable that-the buildup of citrate should serve as a signal for limiting its own production, both by inhibiting the pyruvate kinase reaction as well as by inhibiting citrate synthase (Fields et al., in preparation).
Octopus Liver Pyruvate Kinase The specific activity of the liver pyruvate kinase was considerably lower than that of the mantle enzyme, 1.02 units per gram fresh weight tissue and 50.4 units per gram fresh weight being the respective quantities. The enzyme in the liver was present as a single band on starch-gel electrophoresis. This band showed slower anodal migration than that from the mantle, indicating
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CAN. J. ZOOL. VOL. 54, 1976
FIG.4. MgATP and FDP effects on PEP affinity of octopus liver pyruvate kinase. Assay conditions are 3 m M ADP, 10 mM MgCI2, 100 rnM KCI, 0.15 rnM NADH, and excess lactate dehydrogenase (Sigma). PEP, MgATP, and FDP concentrations are varied as shown. Vindicates AOD340.
the presence of two pyruvate kinase isozymes in the octopus.
phate (AMP). To see which compounds affected the octopus liver pyruvate kinase, we tested a number of compounds, of which the following were found to be without effect: arginine (6 mM), The pH Optimum and Cation Requirements The liver enzyme showed a pH profile with an malate (10 mM), octopine (5 mM), a-ketogluoptimum at pH 7.0, both in the absence and in tarate (5 mM), 3-phosphoglyceric acid (2 mM), the presence of FDP. It showed an absolute inorganic phosphate (20 mM), a-glycerol phoscation requirement that could be fulfilled either phate (10 mM), nicotinamide adenine dinucleoby Mg2+or by Mn2+,with Km values of 0.5 mM tide phosphate (NADP) (1 mM), fructose-6and 1.1 m M respectively. The liver enzyme phosphate (5 mM), AMP (I0 mM), proline showed a hyperbolic activation by K + with a Km (20 mM), NADH (0.45 mM), and acetyl CoA (0.01 mM). Alanine at 20 m M led to an 8% of 13 mM. inhibition. These tests were run at 0.5 m M ADP and 1.5 m M PEP, i.e. just slightly above the Km Substrate AfJinities and Metabolic Effectors The substrate saturation curves for both PEP levels for the two substrates. Four compounds and ADP followed regular hyperbolas, with the were found to be good effectors: FDP, citrate, Km for PEP being 0.85 m M and that for ADP arginine phosphate, and ATP. As an activator, FDP functioned in two ways : being 0.125 mM. The Km for PEP was profoundly affected by FDP, with the addition of first, it facilitated the binding of the substrate 0.05 m M FDP dropping the PEP Kmto 0.17 mM. PEP (Fig. 4), and second, it reversed the inThe ADP affinity was unaffected by FDP. The hibition caused by arginine phosphate and ATP, FDP effects are reminiscent of the regulation and to some extent that caused by citrate shown by mammalian liver pyruvate kinases, (Table 2). In the absence of FDP, 20 m M with the distinction that the unactivated mam- arginine phosphate reduced the activity to 15% malian enzymes show sigmoidal kinetics. FDP of the control, but in the presence of 0.05 m M FDP, 20 m M arginine phosphate could not drop saturation indicates a K, of about 1 pM. Regulation of liver pyruvate kinases in mam- the activity below the control level. While FDP malian systems involves a complex series of exerted a protective action in the face of MgATP different metabolic effectors, including various inhibition, its effect was not as marked as with amino acids, ATP, pH, FDP, glucose-6-phos- the arginine phosphate inhibition. The inhibitory phate, glucose-l,6-phosphate, inorganic phos- action of Mg2citrate was only slightly affected by phate, citrate, malate, and adenosine monophos- the presence of FDP.
GUDERLEY ET AL. TABLE2. The inhibition of octopus liver pyruvate kinase by MgATP, arginine phosphate, and citrate Activity as % of control Treatment
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Arginine phosphate
MgATP
Mg,citrate
Control
Concn., mM 5 10 15 20 5 10 15 20 5 10 15 20
t 0.05 mM -FDP
FDP
63 42 22 15
150 132 127 108
-
NOTE: These tests were conducted at the following substrate con-
centrations: 0.5 mMADP, 1.5 mMPEP, 10 mM MgCI2, 10 mM KCI, 0.15 mM NADH, and excess lactate dehydrogenase (Sigma).
The metabolic significance of these facts is that within the liver cell, control of pyruvate kinase activity is probably vested in changing FDP, ATP, and presumably arginine phosphate levels. Since the specific activities of the enzyme are low, the inhibition of the reaction, allowing carbon flow through an enzymatic bypass such as that catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase in vertebrates, is feasible. When gluconeogenesis is occurring, the low intracellular FDP levels would have the effect of keeping pyruvate kinase in its low-affinity, easily inhibited state. As ATP (and presumably arginine phosphate) levels are depleted as a result of synthetic activity, gluconeogenesis would slow, and FDP levels would rise. The drop in ATP and the rise in FDP would simultaneously activate pyruvate kinase activity, thus producing more ATP both through the action of pyruvate kinase and through the augmentation of Krebs-cycle activity. Control by citrate would come into play here and prevent an overshoot of pyruvate (and thus acetyl CoA) production. In this way, control of liver pyruvate kinase would involve an integration of feedforward signals from FDP and feedback signals from ATP, arginine phosphate, and citrate. The control properties of both the mantle and liver pyruvate kinases are similar to those of the mammalian enzymes. The mantle enzyme lacked the complex FDP activation and alanine in-
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hibition found in many invertebrate and lowervertebrate muscle enzymes, but instead showed control mechanisms much like those found in the mammalian muscle enzyme: control by ATP and the storage phosphagen, in this case arginine phosphate. Like mammals, octopus had a separate liver isozyme that is FDP-sensitive, allowing modulation of the enzyme from a lowaffinity to a high-affinity state. Unlike the mammalian liver enzyme, this enzyme did not show sigmoidal kinetics, nor was it characterized by a high sensitivity to amino acid inhibition. Instead, control of the liver enzyme seemed to revolve primarily around an interaction between t* FDP activation and ATP, citrate, and possible arginine phosphate inhibition. There are sdme similarities between the regulation of the sq(uid mantle and the octopus mantle enzymes, pdimarily their mutual responsiveness to NADH add citrate inhibition. However, while these rebponses form the crux of the enzyme's regulatiOn in squid mantle, they are peripheral for the oqtopus mantle pyruvate kinase. This difference in1 regulatory design correlates well with the differing degrees of aerobic metabolism found in these two animals. BUCHER,T., and G. PFLEIDERER. 1955. Pyruvate kinase from muscle. Methods Enzymol. 1: 435-441. L. E., J. R. BAMBURG. and H. Y. SALLACH. FLANDERS, 1971. Pyruvate kinase isozymes in adult tissue and eggs of Rana pipiens. 11. Physical and kinetic studies of purified skeletal and heart muscle pyruvate kinases. Biochim. Biophys. Acta, 242: 566-579. HOCHACHKA, P. W., T. MOON,T. MUSTAFA,and K. B. STOREY.1975. Metabolic sources of power for mantle muscle of a fast swimming squid. Comp. Biochem. Physiol. 52B: 151-158. JOHNSTON, I. A. 1975. Pyruvate kinase from the red skeletal musculature of the carp (Carassius carassius L.). Biochem. Biophys. Res. Commun. 63: 115-120. KAYNE, F. J. 1973. Pyruvate kinase. In The enzymes. Vol. 8. 3rd ed. Edited by P. D . Boyer. Academic Press, New York, San Francisco, London. pp. 353-382. 1975. The ultrastrucMOON,T. W., and W. C. HULBERT. ture of the mantle musculature of the squid Symplectoteuthis oualaniensis (Lesson). Comp. Biochem. Physiol. 52B: 145-151. T., and P. W. HOCHACHKA. 1971. Catalytic and MUSTAFA, regulatory properties of pyruvate kinases in tissues of a marine bivalve. J. Biol. Chem. 246: 3196-3203. MUSTAFA, T., T. W. MOON,and P. W. HOCHACHKA. 1971. Effects of pressure and temperature on the catalytic and regulatory properties of muscle pyruvate kinase from an off-shore fish. Am. Zool. 11: 451-466. SCRUTTON, M. C., and M. F. UTTER.1968. The regulation of glycolysis and gluconeogenesis in animal tissues. Annu. Rev. Biochem. 37: 249-302. SOMERO, G. N., and P. W. HOCHACHKA. 1968. Effect of
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temperature on the catalytic and regulatory properties of pyruvate kinases of the rainbow trout and the antarctic fish, Trematomus bernachii. Biochem. J . 110: 395400. STOREY, K. B., and P. W. HOCHACHKA. 1974. Enzymes of energy metabolism in a vertebrate facultative anaerobe, Pseudemys scripta. Turtle heart and pyruvate kinase. J. Biol. Chem. 249: 1423-1427. 1975a. Redox regulation of muscle phosphofructokinase in a fast swimming squid. Comp. Biochem. Physiol. 52B: 159-163.
1975b. Squid muscle pyruvate kiiase: control properties in a tissue with an active aGP cycle. Comp. Biochem. Physiol. S2B: 187-193. VAN BERKEL, T. H. J. C., J. F. KOSTER, J. K. KRUYT,and W.C. H U L S ~ N1974. . On the regulation and allosteric model of L-typepyruvate kinase from rat liver. Biochem. Biophys. Acta, 370: 450-458.
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Pac$c Biomedical Research Center, University of Hawaii, Honolulu, HZ, U . S . A . Received December 10. 1975
H. E., K. B. STOREY, J. H. A. FIELDS,and P. W. HOCHACHKA. 1976. Catalytic and GUDERLEY, regulatory properties of pyruvate kinase isozymes from octopus mantle muscle and liver. Can. J. Zool. 54: 863-870. The octopus is a relatively slow-moving animal that relies upon burst swimming to power its predatory activities. Pyruvate kinase (EC, 2.7.1.40), as one of the major glycolytic control sites, must be regulated in such a fashion to allow the increased glycolytic rate characteristic of burst metabolism. The mantle enzyme is regulated by the concerted action of ATP, arginine phosphate, and citrate. The K, for ADP was 0.28 mM and that for phosphoenolpyruvate (PEP), 0.25 mM. In contrast to many other invertebrate muscle pyruvate kinases, the enzyme is insensitive to fructose- l,6-diphosphate (FDP) activation. The pyruvate kinase from the liver is kinetically and electrophoretically distinct from the mantle enzyme. The liver isozyme has a considerably lower affinity for PEP (K, = 0.85 mM), is inhibited by ATP, citrate, and arginine phosphate, and is subject to a strong activation by FDP ( K , = 1 x lo-"). These differences between the pyruvate kinases from catabolic and synthetic tissues are reminiscent of the distinctions between mammalian muscle and liver pyruvate kinases. GUDERLEY, H. E., K. B. STOREY, J . H. A. FIELDSet P. W. HOCHACHKA. 1976. Catalytic and regulatory properties of pyruvate kinase isozymes from octopus mantle muscle and liver. Can. J. Zool. 54: 863-870. La pieuvre est un animal qui se meut relativement lentement et qui depend de ses 'poussees' de nage pour exercer ses activitks predatrices. La pyruvate-kinase (EC 2.7.1.40), I'un des principaux facteurs de contr8le de la glycolyse, doit Ctre reglee de f a ~ o na permettre le taux de glycolyse plus eleve caracteristique du metabolisme accompli lors de la pousste. La regulation de I'enzyme du manteau se fait par I'action combinee de I'ATP, ainsi que de la phosphate d'arginine et de la citrate. La valeur de K,, dans le cas de I'ADP, est de 0.28 mM; elle est de 0.25 mM pour ce qui est du PEP (phosphoenolpyruvate). Contrairement aux pyruvate-kinases musculaires de plusieurs autres invertebres, cet enzyme est insensible a l'activation par le FDP (fructose-1,6-diphosphate). La pyruvate-kinase venant du foie a des proprietts kinesiques differentes de celles de I'enzyme du manteau et rtagit diffkremment a I'electrophorese. L'isoenzyme du foie a une affinite beaucoup plus faible pour le PEP (K, = 0.85 mM), est inhibe par I'ATP, par le citrate et le Ces phosphate d'arginine, et peut subir une forte activation par le FDP ( K , = 1 x differences entre les pyruvate-kinases de tissus catabolique et synthetique s'apparentent i celles qu'on observe chez les mammiferes, entre les pyruvate-kinases musculaire et hepatique. [Traduit par le journal]
Introduction A number of highly differentiated life-styles that rely upon distinctive metabolic control mechanisms have evolved within cephalopods. For example, the pelagic squid, Symplectoteuthis oualaniensis, sustains continual high-speed swimming by a highly aerobic metabolism. As one novel feature of their metabolism, squids use an 'Permanent address: Department of Zoology, University of British Columbia, Vancouver, B.C., Canada V6T 1W5. 'Current address: Department of Biochemistry, Oxford University, Oxford, England.
active a-glycerol phosphate shunt to provide efficient regulation of cytoplasmic redox balance in the face of continual carbohydrate breakdown (Hochachka et al. 1975). The squid also uses reduced nicotinamide adenine dinucleotide (NADH) as a prime regulatory metabolite. When NADH levels are high, flux through glycolysis and the Krebs cycle is limited (Storey and Hochachka 1975a). However, the squid mantle retains the capacity for redox balance via octopine formation (Fields et al., in preparation), and thus must have some residual anaerobic capacity. The life history of the octopus is distinct from
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that of the squid. Octopus cyanea is a bottom dweller in shallow waters, and its predatory behaviour usually involves anaerobic burst activity, a strategy which metabolically entails high power but low efficiency. In light of these major ecological and physiological differences we sought to compare metabolic organization in the octopus mantle with that in the previously studied squid mantle. Since carbohydrate is the prime metabolic fuel in these animals (Moon and Hulbert 1975), the glycolytic control mechanisms that usually reside at the phosphofructokinase (EC 2.7.1.1 1) and pyruvate kinase loci must be so adapted to facilitate the rapid fluctuations in carbon flux that characterize 'burst' metabolism. This study of octopus mantle and liver pyruvate kinases had two major goals: (1) the elucidation of the control mechanisms of mantle pyruvate kinase that would allow the increased glycolytic rate characteristic of the octopus's burst swimming; and (2) a comparison of the regulation of pyruvate kinase from a gluconeogenic versus a catabolic tissue. In gluconeogenic tissues, the pyruvate kinase reac~ionis bypassed by pyruvate carboxylase (EC 6.4.1.1 ) and phosphoenoipyruvate carboxykinase (EC 4.1.1.32) (Scrutton and Utter 1968). Pyruvate kinase in these tissues must be inhibited to allow net gluconeogenic flux. In mammals, liver pyruvate kinase is controlled by potent adenosine triphosphate (ATP), alanine, and citrate inhibition, by its low phosphoenolpyruvate (PEP) affinity, and by a high sensitivity to fructose-I,6-diphosphate (FDP) "feed forward" activation (Van Berkel er ol. 1974). I n contrast to the mammalian muscle enzyme, many invertebrate and lower vertebrate muscle pyruvate kinases show strong FDP activation, and potent inhibition by ATP, amino acids, and citrate (Flanders et al. 1971; Johnston 1975; Mustafa et al. 1971; Somero and Hochachka 1968; Mustafa and Hochachka 1971 ; Storey and Hochachka 1974). These regulatory properties are much like those of the mammalian liver enzyme. Whether these animals use the same enzyme in both their muscle and their 'liver,' and if not, what regulatory strategies are used by their 'liver' enzymes, are two generally unanswered questions. We found that the mantle enzyme was insensitive to FDP activation, and was inhibited by ATP, arginine phosphate, and citrate. The concerted action of ATP and arginine phosphate
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probably provides the major means by which pyruvate kinase is controlled in the resting mantle. The action of citrate may be important in integrating maximal rates of Krebs cycle and pyruvate kinase function. In contrast with the squid enzyme, which is strongly inhibited by NADH, the octopus enzyme showed little response to this compound. The octopus liver enzyme showed a considerably lower affinity for PEP than the mantle enzyme, and was subject to activation by FDP and inhibition by ATP, arginine phosphate, and citrate. FDP strongly affected the activity of the liver enzyme, and may be the major regulator of its activity in the cell.
Methods All reagents used in this study were purchased from Sigma Chemical Company, St. Louis, Missouri. Octopus were obtained by diving, and were courtesy of Mr. Howell Mahoe, Kailua, Oahu. Preparation of Mantle Pyruvate Kinase The mantle was cut into small pieces and homogenized in 50 mM imidazole chloride, pH 7.0, in a Virtis homogenizer, for 2 min. The homogenate was centrifuged at 20000g for 15 min, the supernatant brought to 45% ammonium sulphate, stirred at 4 'C for 30 min, centrifuged as above, and the pellet discarded. The supernatant was then brought to 60% ammonium sulphate, stirred for 30 min, centrifuged as above, and the supernatant discarded. The pellet was resuspended in 50 m M imidazole chloride, pH 7.0, and dialyzed against two changes of the same buffer. The pH was reduced to 6.5, and damp, precycled phosphocellulose was added, keeping the pH at 6.5, just until loss of pyruvate kinase activity could be detected. The sluny was centrifuged as above, the supernatant was removed, and the pH was adjusted to 7.0. This preparation was used for kinetic studies. Although only severalfold purified, it was free of any enzymes that would interfere with the basic assay, or that would interconvert any of the added metabolites. Preparation of the Octopus Liver Pyruvate Kinase Liver was homogenized in 50 mM imidazole chloride, 100 m M KCI, 3 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0, in a Virtis homogenizer for 2 min. The homogenate was centrifuged for 15 min at 20000g. Ammonium sulphate was added to 5 0 z saturation, the solution was stirred for 30 min at 4 'C, and centrifuged as above. The pellet was discarded and the supernatant was brought to 65% ammonium sulphate, stirred for 30 min at 4 "C, and centrifuged as above. The pellet from this step was resuspended in the extraction buffer. Diethylaminoethyl (DEAE) Sephadex, equilibrated in 100 mM imidazole chloride at pH 7.1. was graduallv added until a slight loss of pyruvate kinas; actLity couid be detected. The slurry was centrifuged at 10 000g for 20 min, and the supernatant was carefully decanted. To this supernatant, hydroxyl apatite was added until a slight loss of pyruvate kinase activity was detected. This slurry was
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abductor enzyme (Mustafa and Hochachka 1971). Pyruvate kinases require a divalent cation for catalysis, and this requirement can be satisfied with Mg2+ or Mn2+ (Kayne 1973). The octopus mantle enzyme adhered to this pattern, with Mg2+ saturation of the enzyme following hyperbolic kinetics, with an apparent Km of 0.55 mM. Pyruvate kinases generally either require or are activated by monovalent cations (Kayne 1973), with K + and NH,+ being physiologically most important. In keeping with this behaviour, octopus mantle pyruvate kinase showed a marked Michaelis-Menten activation by K f with a K,,, of 14.3 mM. In contrast to the squid mantle enzyme, this activation was not absolute (Storey and Hochachka 19753).
Starch-gel Electrophoresis Electrophoresis was carried out on 13% starch gels, using tris (hydroxymethyl)aminomethane (Tris) citrate, pH 7.8, with KC1 as both the gel and the tank buffer. Freshly prepared high-speed supernatants were used, and the gels were run at 40 mA, 200 V for 12 h. The gels were stained for pyruvate kinase activity by a negative method, which follows the appearance of dark bands under ultraviolet light caused by the disappearance of NADH and its fluorescence. The staining solution, which consisted of 5 m M ADP, 5 m M PEP, 10 m M MgC12, 72 mM KCI, 3 m M NADH, and excess lactate dehydrogenase, was applied to a filter paper overlying the gel and monitored with an ultraviolet light for the appearance of the bands of pyruvate kinase activity.
Substrate Afinities and Metabolic Effectors Substrate saturation curves followed regular Michaelis-Menten hyperbolas, with the Km for ADP being 0.28 m M and that for PEP being 0.25 mM. The affinity of each substrate was unaffected by varying the concentration of the other, indicating the random-order binding mechanism, which is characteristic of mammalian muscle pyruvate kinases (Kayne 1973). The molluscan pyruvate kinases that have previously been examined provide distinct contrasts in terms of regulatory strategies. The bivalve mantle and muscle enzymes show kinetics where FDP, alanine, and pH, as the chief regulatory signals, provide a sensitive 'throttle' on the reaction (Mustafa and Hochachka 1971). In contrast to the facultative anaerobe, the highly aerobic squid showed a pyruvate kinase with completely different regulatory characteristics. Neither alanine nor FDP affects the activity of the squid pyruvate kinase; instead, control was vested in NADH, citrate, and ATP (Storey and Hochachka 1975b). Similarly, neither alanine nor FDP was found to exert a significant effect upon the activity of the octopus mantle enzyme. In checking for an effect of FDP on enzyme-substrate and enzyme-inhibitor interactions, we varied the concentration of FDP from 0.005 m M to 0.05 mM. Only in the case of the NADH inhibition was FDP able to reverse partially the inhibitory effects. Alanine was tested at concentrations ranging up to 20 mM, where a 7% inhibition was evident. Other compounds which were found to be without effect on the
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centrifuged, and the supernatant used for kinetic studies. This preparation, only severalfold purified, was free of enzymes that would interfere with the assay; however, it needed to be used quickly, for it was not stable for prolonged periods. Pyruvate kinase was assayed by the method of Biicher and Pfleiderer (1955). Pyruvate formation was coupled to lactate dehydrogenase, and the rate of pyruvate kinase activity was measured as the decrease in OD,,, due to NADH disappearance. Imidazole chloride buffers were used in all reactions. Standard assay mixtures contained Mg2+,K + , adenosine diphosphate (ADP), PEP, NADH, and excess of lactate dehydrogenase (Sigma) in the concentrations specified in each figure legend. For the mantle enzyme, saturating concentrations of each of the reactants were 100 m M K + , 10 mM Mg2+, 3 m M PEP, and 2.5 m M ADP. For the liver enzyme the corresponding concentrations were 100 m M K + , 10 mM Mg2+, 6 m M PEP, and 3 m M ADP. Reactions were started by the addition of pyruvate kinase. All experiments were performed at 25 "C.
Calculation of the Michaelis Constant The Michaelis constant (K,) was determined in the presence and absence of metabolite effectors by Lineweaver-Burk plots l/velocity versus l/substrate concentration. The K, values obtained are reproducible to within f 10%.
Results and Discussion Octopus Mantle Pyruvate Kinase The specific activity of pyruvate kinase in the mantle was 50.4 units per gram wet weight of tissue. The enzyme activity in the mantle was confined to a single anodally migrating band on starch-gel electrophoresis.
The p H Optimum and Cation Requirements The pH optimum of mantle pyruvate kinase under saturating substrate and cofactor conditions was pH 7.0. This is similar to the pH optimum for the squid mantle pyruvate kinase (Storey and Hochachka 1975b), but considerably lower than the pH optimum for the oyster
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enzyme activity were malate (5 mM), glutamate (10 mM), octopine (5 mM), a-glycerol phosphate (10 mM), proline (60 mM), aspartate (10 mM), 3-phosphoglyceric acid (5 mM), arginine (5 mM), and acetyl coenzyme A (0.01 mM). However, three other compounds were found to exert a marked inhibitory action on enzyme activity. These were MgATP, arginine phosphate, and citrate. In contrast to its effect on the squid enzyme, NADH produced only a relatively weak inhibition of the octopus pyruvate kinase (Fig. 1). MgATP and arginine phosphate showed mixed competitive inhibition with regard to the two substrates, and both showed a greater effect on the binding of the adenylate substrate. The Ki of MgATP versus ADP was 8.5 mM, while its Ki versus PEP was 12.5 mM. ATP inhibition was manifest primarily as a Vma, effect in the latter case. Arginine phosphate, which provides the high-energy phosphate store for the resting mantle, exhibited a Ki of 9.5 m M versus ADP and a 28 m M Ki versus PEP (Figs. 2 and 3). Citrate, tested as the Mg,citrate complex to avoid chelation effects, showed linear competitive inhibition versus ADP, with a Ki of 17 mM. Citrate inhibition with PEP is complex. Its two major facets are a change in the PEP saturation curves from hyperbolic to sigmoidal, and a decrease in.,,,/I The clearest demonstration of the effectiveness of these inhibitors came from an examination of individual and additive effects on the enzyme at physiological concentrations of substrates (Table
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1). At K, concentrations of PEP and ADP, each of the inhibitors, added singly, caused a marked inhibition of enzyme activity. The inhibitory effects of ATP and arginine phosphate were additive. At 10 m M ATP and 10 m M arginine phosphate, an approximation of the levels found in the resting mantle, only 30% of the enzyme activity was evident. While citrate demonstrated additive inhibition with either ATP or arginine phosphate, adding all three inhibitors together caused no further reduction of enzyme activity. NADH also inhibited the reaction; 0.45 m M inhibited enzyme activity by 32%, with FDP partially reversing this inhibition. When 0.45 m M NADH was added at 10 m M ATP and 10 mM arginine phosphate, the inhibition increased to 75%. Thus, between the inhibited and deinhibited state the pyruvate kinase can undergo at least a fourfold change in activity. In the resting mantle, the pyruvate kinase would probably be inhibited by the high arginine phosphate and ATP levels. With the onset of muscular action, dropping arginine phosphate and eventually dropping ATP levels would lead to an increase in pyruvate kinase activity. If flux through the reaction were limiting overall glycolytic flux, the buildup of PEP and ADP would further deinhibit the reaction. Pyruvate formed by the pyruvate kinase reaction has two possible major metabolic fates; one is its conversion via pyruvate dehydrogenase into acetyl CoA, which then, by condensation with oxaloacetate, forms citrate. Pyruvate's other fate as substrate for octopine dehydrogenase in the following reaction,
pyruvate
+ arginine + NADH o
FDP
octopine
+ NAD
TABLE 1 . The effect o f various inhibitors on the activity o f octopus mantle pyruvate kinase Activity as
% o f control
Treatment
0.1
0.2
0.3
0.4
[NADH] rnM
FIG. 1 . NADH inhibition o f octopus and squid mantle pyruvate kinase. Assay conditions for octopus mantle enzyme are 0.3 mM ADP, 0.25 mM PEP, 10 mM MgC12, 100 mM KCl, and excess lactate dehydrogenase (Sigma). Conditions for the squid mantle enzyme are given in Storey and Hochachka (19756).
10 mM citrate 10 mM arginine phosphate 1 0 m M ATP All three inhibitors at 10 mM 10 mM ATP 10 mM citrate 10 mM ATP 10 mM arginine phosphate 10 mM arginine phosphate 10 mM citrate 10 mM citrate 10 mM arginine phosphate + 10 mM ATP + 0.45 mM NADH
+ + +
+
53 74 51 30 44 30 44 26
NOTE:These tests were run at the following substrate and cofactor concentrations: 0.3 mMADP, 0.25 m M PEP, I0 m M MgCI2, 100 mM KCI. 0.15 mM NADH. and excess lactate dehydrogenase (Sigma). The values are the mean of three determinations.
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GUDERLEY ET AL.
FIG.2. Arginine phosphate (ARG-PHOS) inhibition of octopus mantle pyruvate kinase, interaction with ADP. Assay conditions are 3 m M PEP, 10 m M MgC12, 100 m M KCI, 0.15 m M NADH, and excess lactate dehydrogenase (Sigma). ADP and arginine phosphate concentrations are varied as shown. Vindicates AOD,,,.
FIG.3. Arginine phosphate (ARG-PHOS) inhibition of octopus mantle pyruvate kinase, interaction with PEP. Assay conditions are 2.5 m M ADP, 10 mM MgC12, 100 mM KC], 0.15 m M NADH, and excess lactate dehydrogenase (Sigma). PEP and arginine phosphate concentrations are varied as shown. Vindicates AOD340.
is important in cytoplasmic redox balance. Since octopus utilizes carbohydrate as its major fuel, Krebs-cycle function must be integrated with glycolysis under aerobic conditions. Therefore it is reasonable that-the buildup of citrate should serve as a signal for limiting its own production, both by inhibiting the pyruvate kinase reaction as well as by inhibiting citrate synthase (Fields et al., in preparation).
Octopus Liver Pyruvate Kinase The specific activity of the liver pyruvate kinase was considerably lower than that of the mantle enzyme, 1.02 units per gram fresh weight tissue and 50.4 units per gram fresh weight being the respective quantities. The enzyme in the liver was present as a single band on starch-gel electrophoresis. This band showed slower anodal migration than that from the mantle, indicating
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CAN. J. ZOOL. VOL. 54, 1976
FIG.4. MgATP and FDP effects on PEP affinity of octopus liver pyruvate kinase. Assay conditions are 3 m M ADP, 10 mM MgCI2, 100 rnM KCI, 0.15 rnM NADH, and excess lactate dehydrogenase (Sigma). PEP, MgATP, and FDP concentrations are varied as shown. Vindicates AOD340.
the presence of two pyruvate kinase isozymes in the octopus.
phate (AMP). To see which compounds affected the octopus liver pyruvate kinase, we tested a number of compounds, of which the following were found to be without effect: arginine (6 mM), The pH Optimum and Cation Requirements The liver enzyme showed a pH profile with an malate (10 mM), octopine (5 mM), a-ketogluoptimum at pH 7.0, both in the absence and in tarate (5 mM), 3-phosphoglyceric acid (2 mM), the presence of FDP. It showed an absolute inorganic phosphate (20 mM), a-glycerol phoscation requirement that could be fulfilled either phate (10 mM), nicotinamide adenine dinucleoby Mg2+or by Mn2+,with Km values of 0.5 mM tide phosphate (NADP) (1 mM), fructose-6and 1.1 m M respectively. The liver enzyme phosphate (5 mM), AMP (I0 mM), proline showed a hyperbolic activation by K + with a Km (20 mM), NADH (0.45 mM), and acetyl CoA (0.01 mM). Alanine at 20 m M led to an 8% of 13 mM. inhibition. These tests were run at 0.5 m M ADP and 1.5 m M PEP, i.e. just slightly above the Km Substrate AfJinities and Metabolic Effectors The substrate saturation curves for both PEP levels for the two substrates. Four compounds and ADP followed regular hyperbolas, with the were found to be good effectors: FDP, citrate, Km for PEP being 0.85 m M and that for ADP arginine phosphate, and ATP. As an activator, FDP functioned in two ways : being 0.125 mM. The Km for PEP was profoundly affected by FDP, with the addition of first, it facilitated the binding of the substrate 0.05 m M FDP dropping the PEP Kmto 0.17 mM. PEP (Fig. 4), and second, it reversed the inThe ADP affinity was unaffected by FDP. The hibition caused by arginine phosphate and ATP, FDP effects are reminiscent of the regulation and to some extent that caused by citrate shown by mammalian liver pyruvate kinases, (Table 2). In the absence of FDP, 20 m M with the distinction that the unactivated mam- arginine phosphate reduced the activity to 15% malian enzymes show sigmoidal kinetics. FDP of the control, but in the presence of 0.05 m M FDP, 20 m M arginine phosphate could not drop saturation indicates a K, of about 1 pM. Regulation of liver pyruvate kinases in mam- the activity below the control level. While FDP malian systems involves a complex series of exerted a protective action in the face of MgATP different metabolic effectors, including various inhibition, its effect was not as marked as with amino acids, ATP, pH, FDP, glucose-6-phos- the arginine phosphate inhibition. The inhibitory phate, glucose-l,6-phosphate, inorganic phos- action of Mg2citrate was only slightly affected by phate, citrate, malate, and adenosine monophos- the presence of FDP.
GUDERLEY ET AL. TABLE2. The inhibition of octopus liver pyruvate kinase by MgATP, arginine phosphate, and citrate Activity as % of control Treatment
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Arginine phosphate
MgATP
Mg,citrate
Control
Concn., mM 5 10 15 20 5 10 15 20 5 10 15 20
t 0.05 mM -FDP
FDP
63 42 22 15
150 132 127 108
-
NOTE: These tests were conducted at the following substrate con-
centrations: 0.5 mMADP, 1.5 mMPEP, 10 mM MgCI2, 10 mM KCI, 0.15 mM NADH, and excess lactate dehydrogenase (Sigma).
The metabolic significance of these facts is that within the liver cell, control of pyruvate kinase activity is probably vested in changing FDP, ATP, and presumably arginine phosphate levels. Since the specific activities of the enzyme are low, the inhibition of the reaction, allowing carbon flow through an enzymatic bypass such as that catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase in vertebrates, is feasible. When gluconeogenesis is occurring, the low intracellular FDP levels would have the effect of keeping pyruvate kinase in its low-affinity, easily inhibited state. As ATP (and presumably arginine phosphate) levels are depleted as a result of synthetic activity, gluconeogenesis would slow, and FDP levels would rise. The drop in ATP and the rise in FDP would simultaneously activate pyruvate kinase activity, thus producing more ATP both through the action of pyruvate kinase and through the augmentation of Krebs-cycle activity. Control by citrate would come into play here and prevent an overshoot of pyruvate (and thus acetyl CoA) production. In this way, control of liver pyruvate kinase would involve an integration of feedforward signals from FDP and feedback signals from ATP, arginine phosphate, and citrate. The control properties of both the mantle and liver pyruvate kinases are similar to those of the mammalian enzymes. The mantle enzyme lacked the complex FDP activation and alanine in-
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hibition found in many invertebrate and lowervertebrate muscle enzymes, but instead showed control mechanisms much like those found in the mammalian muscle enzyme: control by ATP and the storage phosphagen, in this case arginine phosphate. Like mammals, octopus had a separate liver isozyme that is FDP-sensitive, allowing modulation of the enzyme from a lowaffinity to a high-affinity state. Unlike the mammalian liver enzyme, this enzyme did not show sigmoidal kinetics, nor was it characterized by a high sensitivity to amino acid inhibition. Instead, control of the liver enzyme seemed to revolve primarily around an interaction between t* FDP activation and ATP, citrate, and possible arginine phosphate inhibition. There are sdme similarities between the regulation of the sq(uid mantle and the octopus mantle enzymes, pdimarily their mutual responsiveness to NADH add citrate inhibition. However, while these rebponses form the crux of the enzyme's regulatiOn in squid mantle, they are peripheral for the oqtopus mantle pyruvate kinase. This difference in1 regulatory design correlates well with the differing degrees of aerobic metabolism found in these two animals. BUCHER,T., and G. PFLEIDERER. 1955. Pyruvate kinase from muscle. Methods Enzymol. 1: 435-441. L. E., J. R. BAMBURG. and H. Y. SALLACH. FLANDERS, 1971. Pyruvate kinase isozymes in adult tissue and eggs of Rana pipiens. 11. Physical and kinetic studies of purified skeletal and heart muscle pyruvate kinases. Biochim. Biophys. Acta, 242: 566-579. HOCHACHKA, P. W., T. MOON,T. MUSTAFA,and K. B. STOREY.1975. Metabolic sources of power for mantle muscle of a fast swimming squid. Comp. Biochem. Physiol. 52B: 151-158. JOHNSTON, I. A. 1975. Pyruvate kinase from the red skeletal musculature of the carp (Carassius carassius L.). Biochem. Biophys. Res. Commun. 63: 115-120. KAYNE, F. J. 1973. Pyruvate kinase. In The enzymes. Vol. 8. 3rd ed. Edited by P. D . Boyer. Academic Press, New York, San Francisco, London. pp. 353-382. 1975. The ultrastrucMOON,T. W., and W. C. HULBERT. ture of the mantle musculature of the squid Symplectoteuthis oualaniensis (Lesson). Comp. Biochem. Physiol. 52B: 145-151. T., and P. W. HOCHACHKA. 1971. Catalytic and MUSTAFA, regulatory properties of pyruvate kinases in tissues of a marine bivalve. J. Biol. Chem. 246: 3196-3203. MUSTAFA, T., T. W. MOON,and P. W. HOCHACHKA. 1971. Effects of pressure and temperature on the catalytic and regulatory properties of muscle pyruvate kinase from an off-shore fish. Am. Zool. 11: 451-466. SCRUTTON, M. C., and M. F. UTTER.1968. The regulation of glycolysis and gluconeogenesis in animal tissues. Annu. Rev. Biochem. 37: 249-302. SOMERO, G. N., and P. W. HOCHACHKA. 1968. Effect of
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temperature on the catalytic and regulatory properties of pyruvate kinases of the rainbow trout and the antarctic fish, Trematomus bernachii. Biochem. J . 110: 395400. STOREY, K. B., and P. W. HOCHACHKA. 1974. Enzymes of energy metabolism in a vertebrate facultative anaerobe, Pseudemys scripta. Turtle heart and pyruvate kinase. J. Biol. Chem. 249: 1423-1427. 1975a. Redox regulation of muscle phosphofructokinase in a fast swimming squid. Comp. Biochem. Physiol. 52B: 159-163.
1975b. Squid muscle pyruvate kiiase: control properties in a tissue with an active aGP cycle. Comp. Biochem. Physiol. S2B: 187-193. VAN BERKEL, T. H. J. C., J. F. KOSTER, J. K. KRUYT,and W.C. H U L S ~ N1974. . On the regulation and allosteric model of L-typepyruvate kinase from rat liver. Biochem. Biophys. Acta, 370: 450-458.
