Control of phosphofructokinase from the phasic adductor muscle of the bay scallop, Argopecten irradians concentricus

Comp. Biochem. PhysioL Vol. 87B, No. 4, pp. 767-772, 1987 Printed in Great Britain

0305-0491/87 $3.00+ 0.00 © 1987PergamonJournals Ltd

CONTROL OF PHOSPHOFRUCTOKINASE FROM THE PHASIC A D D U C T O R MUSCLE OF THE BAY SCALLOP, A R G O P E C T E N I R R A D I A N S C O N C E N T R I C U S C. P. CHIH and W. R. ELLINGTON* Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA (Received 15 July 1986) Abstract--l. Phosphofructokinase (PFK) purified from the adductor muscle of Argopecten irradians concentricus showed sigmoidal saturation kinetics with respect to F-6-P in the physiological range of pH from 6.8 to 7.2. The saturation curve with respect to ATP showed typical substrate inhibition. 2. Decreases in pH resulted in lower binding capacity of PFK for F-6-P as well as stronger inhibition by ATP. 3. Both AMP and F-2,6-P activated adductor muscle PFK by shifting the F-6-P saturation curve from sigmoidal to hyperbolic. The presence of AMP also relieved the inhibitory effect of ATP. 4. Phosphoenolpyruvate, fructose-l,6-P and inorganic phosphate were inhibitory to adductor muscle PFK. Arginine phosphate had no effect on the activity of PFK.

INTRODUCTION Phosphofructokinase (PFK) is known to be an important site of regulation of glycolysis in vertebrate muscle (Newsholme and Start, 1976). Regulation of PFK in vivo is very complicated since the enzyme is modulated by a variety of effectors (Uyeda, 1979; Hers and Hue, 1983). PFKs purified from a number of systems have been found to be extremely sensitive to pH. Pettigrew and Frieden (1979a,b) have proposed a model for the inactivation of rabbit muscle PFK by H +. According to this model, changes in pH shift the equilibrium of protonated and unprotonated forms of the enzyme. Negative modulators such as ATP and citrate bind preferentially to the protonated form of PFK, while positive modulators such as AMP bind primarily to the unprotonated form. Decreases in pH not only decrease the activity of PFK, but also increase the degree of regulatory behavior of the enzyme. At low pH, PICK purified from the rabbit muscle showed sigmoidal kinetics with respect to fructose-6-phosphate. Activity of PFK is also strongly affected by adenylates (Pettigrew and Frieden, 1979b). High levels of ATP inhibit the enzyme, while increases of AMP and ADP relieve the inhibition by ATP. In this way, PFK responds rapidly to the changes in energy status of the cell and the rate of glycolysis is regulated. In a number of molluscan systems, PFK also appears to be an important regulatory enzyme (Ebberink, 1982; Chih and Ellington, 1986). The kinetic properties of PFK purified from the adductor muscle of a sessile mussel Mytilus edulis were quite similar to that of vertebrate muscle. The enzyme showed sigmoidal kinetics with respect to F-6-P at low pH values and was strongly activated by AMP. It was suggested that during long term anaerobiosis, the changes in negative effectors (such as H +) and *To whom all editorial correspondence and reprint requests should be sent. 767

positive effectors (such as AMP) counteracted each other and resulted in a lack of activation of PFK, which in turn caused a lack of Pasteur effect in the adductor muscle of M. edulis. In contrast, PFK purified from the phasic adductor muscle of a motile scallop, Placopecten magellanicus was found to exhibit hyperbolic kinetics with respect to F-6-P even at low pH values (Ebberink et al., 1983). This property of PFK did not seem to favor a rapid activation of the enzyme during muscle work. In the phasic adductor muscle of the bay scallop, A. irradians concentricus, PFK was found to be the main rate limiting enzyme in the later stages of contractile activity (Chih and Ellington, 1986). Increases in PFK activity during muscle contraction were evident by a movement of mass action ratio towards equilibrium. To understand the regulation of PFK and hence the control of glycolysis, kinetic properties of PFK purified from the phasic adductor muscle of A. irradians concentricus were studied. The results communicated in this paper indicate that changes in pH and concentrations of positive modulators such as AMP may play an important role in activating PFK in the adductor muscle during contractile activity. MATERIALS AND METHODS

Animals Animals were collected at St Joseph's Bay, Florida, and were maintained in running seawater at the Florida State University Marine Laboratory, Turkey Point. A few days before the experiments,animals were transferred to the main campus of Florida State University and maintained for short time periods in recirculating aquaria. Materials All biochemicals were purchased from Sigma (St. Louis, MO) and Boehringer Mannheim (Indianapolis, IN). Cibacron blue Sepharose was purchased from Pharmacia Fine Chemicals (Piscataway, New Jersey). All other chemicals were of reagent grade quality.

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C.P. CmH and W. R. ELLINGTON Table 1. Purification of phosphofructokinase from the phasic adductor muscle of Argopecten irradians concentricus Total Specific activity activity Fold of Recovery (unit) (unit/mg) purification (%) Crude extract 160.5 0.08 -100 60% ammonium sulfate precipitation 70.9 0.14 1.8 44 Eluate from Cibacron blue column 67.5 4.83 60 42 Eluate from Sephadex G-200 45.2 22.63 283 28

Enzyme assays Activity of PFK was determined using either the F-1, 6-P coupled assay or the ADP coupled assay. The F-I, 6-P coupled assay solution contained 50mM imidazole/HC1 (pH indicated in Results section), 8 mM MgC12, 0.2 mM NADH and an excess amount of aldolase, triosephosphate isomerase and glycerol-3-P dehydrogenase. Concentrations ofATP, F-6-P and other modulators are listed in the Results section. The ADP-coupled assay solution contained 50 mM imidazole/HC1, 8 m M MgCI2, 0.2mM NADH, 0.2mM PEP, 50mM KC1, 8 mM potassium phosphate and an excess amount of lactate dehydrogenase and pyruvate kinase. Again, the concentrations of F-6-P, ATP and other modulators are given in the Results section. All auxiliary enzymes were dialyzed before use to remove ammonium sulfate. Assays were determined at 25°C using a Gilford 252-1 spectrophotometer or a Beckman Acta CIII spectrophotometer. Purification of phosphofructokinase Freshly thawed phasic aductor muscle of A. irradians concentricus was extracted in five volumes of 50 mM potassium phosphate (pH = 7.8) containing I mM dithiothreitol (DTT) and 2 mM EDTA. The extract was centrifuged at 10,000 g for 20 min. The resulting supernatant was subjected to a 60% ammonium sulfate cut. Ammonium sulfate was added slowly to the sample with gental stirring at 4°C. After about 1 hr of stirring, the sample was centrifuged at 10,000 g for 20 min. The resulting pellet was then resuspended in 50 mM potassium phosphate (pH = 7.8) containing 1 mM DTT and 1 mM F-6-P (buffer I) and dialysed overnight against the same buffer. After dialysis, the sample was centrifuged at 10,000 g for I0 min. The resulting supernatant was applied to Cibacron blue column (2.5 × 23 cm) which had been equilibrated with buffer I. The column was then washed thoroughly to remove unbound protein. Phosphofructokinase was eluted with 20 mM ATP. The eluate was concentrated by pressure filtration using an Amicon stirred cell with a PM-10 membrane. The concentrated sample was then run through a Sephadex G-200 (1.5 × 45cm) column, which had been equilibrated with buffer I. The collected fractions of PFK were used for the kinetics studies. The whole purification procedure was carried out at 4°C. Concentration of protein of crude extracts was monitored by absorbance at 280 nm. Concentration of protein of eluates from columns was determined according to the method described by Bradford (1976).

lators, scallop muscle P F K exhibited a sigmoidal s a t u r a t i o n curve with respect to F-6-P with a Hill coefficient of 2.81. B o t h the m a x i m u m activity a n d binding capacity o f F-6-P were significantly altered by changes in p H from 6.8 to 7.2 (Fig. 2). The difference in P F K activity due to p H was more p r o n o u n c e d at low F-6-P concentration. S a t u r a t i o n curves with respect to A T P showed typical substrate inhibition at a high c o n c e n t r a t i o n s o f ATP. Decreases in p H also increased the substrate inhibition o f A T P (Fig. 3). The presence of 0.1 m M A M P converted the F-6-P s a t u r a t i o n curve from sigmoidal to hyperbolic (Fig. 4). The a p p a r e n t K m value determined by a L i n e w e a v e r - B u r k plot was 0.07 m M . The inhibition by A T P was relieved by A M P (Fig. 5). Fructose-2,6-P also h a d a p r o n o u n c e d activating effect o n P F K (Figs 4,5). In the presence of I / ~ M F-2,6-P, the F-6-P s a t u r a t i o n curve shifted from sigmoidal to hyperbolic yielding a n a p p a r e n t K m o f 0.7 m M (Fig. 4). The activation curve o f A M P was hyperbolic with a K, of 0 . 0 2 2 m M (Fig. 6). The activation curve of F-2,6-P was also hyperbolic with a Ka value of 0.65 # M (Fig. 7). F r u c t o s e - l , 6 - P was f o u n d to be inhibitory at concentrations above 0.1 m M (Figs 8, 9). Inorganic p h o s p h a t e (Pi) was inhibitory at physiological concentrations ( 1 0 m M ) . The inhibition by Pi was relieved by the presence of 0.l m M A M P (Fig. 10). Arginine p h o s p h a t e was f o u n d to have no effect o n scallop a d d u c t o r muscle P F K (Fig. 10). Phosp h o e n o l p y r u v a t e exerted an inhibitory effect o n P F K

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RESULTS P h o s p h o f r u c t o k i n a s e from the phasic a d d u c t o r muscle of A. irradians concentricus was purified a b o u t 283-fold to a final specific activity of 22.63 u n i t / m g protein with a 2 8 % recovery (Table 1). T h e enzyme was stable for a b o u t 2 weeks at 4°C. The activity of P F K increased as the p H of assay solution increased (Fig. 1). In the absence of m o d u -

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P h o s p h o f r u c t o k i n a s e purified from the a d d u c t o r muscle o f A. irradians concentricus is very similar to vertebrate skeletal muscle P F K in m a n y respects. The enzyme shows sigmoidal s a t u r a t i o n kinetics with respect to F-6-P in the physiological range o f p H from 6.8 to 7.2 (Fig. 10). The activity o f P F K decreases a b o u t 3-fold as the p H is lowered from 7.2 to 6.8. Lowering of p H decreases the affinity of P F K for F-6-P. The s a t u r a t i o n curve with respect to A T P shows typical substrate inhibition at high concen-

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Fig. 9. Effect of F-I,6-P on the ATP saturation curve of scallop adductor muscle PFK. Initial rates were measured using ADP coupled assay containing 50 mM imidazole/HC1 (pH 7.0) and 1.0mM F-6-P. (3: Control; I : with 0.I mM F-I,6-P.

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Fructose- 2.6- P [ aM ] Fig. 7. Initial velocities of scallop adductor muscle PFK vs the concentrations of F-2,6-P. Assay solution contained 50 mM imidazole (pH 7.0), 0.2 mM F-6-P and 0.1 mM ATP. Initial rates were measured using F-I,6-P coupled assay. trations of ATP. The inhibitory effect is also stronger in lower p H range. The intracellular pH (pHi) of the phasic adductor muscle was found to increase from 7.06 to 7.15 in the initial period of contraction due to the dephosphorylation of arginine phosphate (Chih

and Ellington, 1985). The increase of pH may result in a higher binding capacity for F-6-P and less inhibition by ATP. This combined effect could cause a significant increase in P F K activity. The activating effect of increasing pHi on P F K during the initial period of contractive activity is very important, since changes of most other activators such as A M P are very small in this stage (Chih and Ellington, 1986). In the later period of contraction in scallop adductor muscle the intracellular pH fell from 7.16 to 6.94. The drop of pH can lead to a significant decrease of P F K activity. However, the negative effects may be compensated for by the increase of activators such as A M P in the adductor muscle. A M P is a potent activator of scallop adductor muscle P F K . In the presence of 0.1 m M A M P , the F-6-P saturation curve becomes hyperbolic with an apparent K m for F-6-P of 0.07 mM, which is close to the physiological concentration range of F-6-P (Chih and Ellington, 1986). At lower concentrations of

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scallop adductor muscle PFK. Initial rates were measured using ADP coupled assay containing 50 mM imidazole-HCl (pH 7.0) and 0.1 rnM ATP. (3: Control; O: with 0.1 mM F-1,6-P.

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Fructose-B- P [mM ] Fig. 10. Effect of negative modulators on F-6-P saturation curve of scallop adductor muscle PFK. Assay solution contained 50raM imidazole/HCl (pH 7.0) and 0.1 mM ATP. O: Control; A: with 10mM Pi; ×: with 10mM arginine phosphate; +: with 0.5 mM phosphoenolpyruvate; O: with 0.1 mM AMP and 20mM Pi. Initial rates were measured using F-I,6-P coupled assay.

Phosphofructokinase from scallop adductor muscle F-6-P, the presence of 0.1 mM AMP increases the activity of PFK up to 100-fold. AMP also relieves the inhibitory effect of ATP. The activation curve of AMP is hyperbolic with an apparent Ka of 0.022 mM. This value is lower than the resting level of AMP (0.1 mM, assuming intracellular water makes up 50% of muscle weight) according to the measurement of perchloric acid extracts (Chih and Ellington, 1986). However, the AMP concentrations calculated from myokinase equilibrium are in the range between 0.6 pM (under resting conditions) and 14.4 pM (during contractile activity) in the scallop adductor muscle (Chih and Ellington, 1986). This calculation indicates a 24-fold increase of AMP levels, reflecting an extremely low activity of PFK under resting conditions and a much larger activation effect during contractile activity. Whether this is the case in vivo is still unclear. Fructose-2,6-phosphate is another important activator of scallop adductor muscle PFK. Both the maximum activity and binding capacity for F-6-P are affected by F-2,6-P. The fructose-6-P saturation curve shifts from sigrnoidal to a hyperbolic curve with an apparent Kr~ of 0.7 mM in the presence of 1 pM F-2,6-P. The inhibitory effect of ATP is less in the presence of F-2,6-P. The general activation effect resulting from F-2,6-P appears to be more pronounced than that of AMP. PFKs purified from a number of systems have been found to be activated by F-2,6-P at very low concentrations ( < I / ~ M ) (Hers and Van Schaftingen, 1982). However, the physiological role of F-2,6-P during contractile activity is not very clear. In some muscle systems, F-2,6-P levels increase as the glycolytic flux increases (Storey, 1983), while in other cases there is no relationship between the levels of F-2,6-P and the rates of glycogenolysis (Hue et al., 1982; Minatogawa and Hue, 1984). Increases in F-2,6-P levels have been found in the foot muscles of a marine gastropod, Littorina littorea during long term anoxia (Storey, 1985). In contrast, the levels of F-2,6-P remain low in a sessile mussle M. edulis after 6 hr of anoxia. The physiological role of F-2,6-P in the adductor muscle of .4. irradians concentricus is not very clear. In contrast to the vertebrate muscle PFK, F-I,6-P and Pi have both been found to be inhibitory to scallop adductor muscle PFK. It has been suggested that the activation of PFK by its product (fructose1,6-P) leads to the oscillation behavior of glycolysis in the vertebrate muscle system (Tornheim, 1979). Tornheim (1985) has suggested that F-I,6-P is more likely to play a role in the physiological activation of PFK than F-2,6-P in rat skeletal muscle. However, the role of F-1,6-P as a physiological activator of PFK in other tissue, such as liver, has been questioned (Hers and Hue, 1983). In yeast and erythrocytes, F-I,6-P has been found to counteract the effect of F-2,6-P (Hers and Hue, 1983). In M. edulis, PFK purified from the adductor muscle has also been found to be inhibited by F-1,6-P at high concentrations (Ebberink, 1982). In the adductor muscle of A. irradians concentricus, F-1,6-P began to exert significant product inhibition on PFK at concentrations above 0.1 mM. The physiological concentration of F-1,6-P rises from 0.02 to 0.72 mM after 80 contractions (Chih and Ellington, 1986). In contrast

771

to the vertebrate skeletal muscle, increases of F-I,6-P during contractile activity seems to exert a negative effect on PFK in the scallop adductor muscle. The inhibitory effect of Pi on scallop adductor muscle PFK is contradictory to the general belief that Pi is a requisite for the activation of glycolysis. The maximum activity and binding capacity for F-6-P of PFK are both significantly affected by Pi. However, the inhibitory effect of Pi is relieved in the presence of 0.1 mM AMP. Since both AMP and Pi rise during contractile activity (Chih and Ellington, 1986), the inhibitory affect of increasing Pi on PFK can be eliminated by the simultaneous increase of AMP levels. Arginine phosphate was suggested to be the main regulator of PFK in the adductor muscle of oyster, Crassostrea virginica (Storey, 1976) and the mantle muscle of Sepia officinalis (Storey, 1981). The decline of arginine phosphate during muscle contraction was thought to relieve its inhibitory effect of PFK and hence accelerate the glycolytic rate. Creatine phosphate was also found to be inhibitory to PFK in the vertebrate muscle system. However, Fitch et al. (1979) have indicated that the inhibitory effect of creatine phosphate was due to contamination of creatine phosphate preparations. The inhibitory effect was eliminated when the contaminants were separated from creatine phosphate by anion exchange chromatography. PFK purified from adductor muscle of M. edulis was not affected by arginine-P at concentrations from 1 to 20 mM (Ebberink, 1982). In adductor muscle of A. irradians concentricus, 10 mM arginine phosphate has no effect on the activity of PFK. There is no evidence that arginine phosphate serves as a regulator in the scallop adductor muscle. Phosphoenolpyruvate (PEP) was found to be an inhibitor of PFK of both rabbit muscle (Uyeda and Racker, 1965) and adductor muscle of M. edulis (Ebberink, 1980). It was suggested that PEP is an important regulator in the later stages of anaerobiosis of M. edulis. PFK from scallop adductor muscle is also significantly inhibited by high levels of PEP (0.5 raM). However, the physiological concentrations of PEP are quite low throughout the period of contraction (Chih and Ellington, 1986). Whether PEP serves as a feed back regulator of PFK in the scallop adductor muscle during muscle contraction is still questionable. In conclusion, the increase of PFK activity in the scallop adductor muscle during contractile activity, as evident by the movement of the mass action ratio towards equilibrium, is probably brought about by the increases of positive modulators and changes in intracellular pH. The positive modulators exert their effects by influencing the binding of substrates, enhancing the maximum activity and releasing the inhibitory effect of negative effectors. The most important effect of these positive modulators is the shifting of F-6-P saturation curve from sigmoidal to hyperbolic. The sigmoidal kinetics of PFK in the absence of activators results in an extremely low activity at physiological concentrations of F-6-P ( < 0.05 mM). The initial increase of intracellular pH during contractile activity may be important in activating PFK since the increase in levels of positive

772

C . P . CHIH and W. R. ELLINGTON

m o d u l a t o r s was n o t as p r o n o u n c e d in the initial stage of contractions. The later fall o f p H m a y be compensated for by the further increase in levels of positive modulators. Acknowledgements--This study was supported by funds from the U.S. National Science Foundation (DCB8401258) to W. Ross Ellington.

REFERENCES

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Hers H. G. and Hue L. (1983) Gluconeogenesis and related aspects of glycolysis. A. Rev. Biochem. 52, 617~53. Hers H. G. and Van Schaftingen E. (1982) Fructose 2,6-bisphosphate two years after its discovery. Biochem. J. 206, 1-12. Hue L., Blackmore P. F., Shikama H., Robinson-Steiner A. and Exton J. H. (1982) Regulation of fructose-2,6bisphosphate content in rat hepatocytes, perfused hearts and perfused hindlimbs. J. biol. Chem. 257, 4308-4313. Minatogawa Y. and Hue L. (1984) Fructose 2,6-bisphosphate in rat skeletal muscle during contraction. Biochem. J. 223, 73-79. Newsholme E. A. and Start C. (1976) Regulation in Metabolism. John Wiley, London. Pettigrew D. W. and Frieden C. (1979a) Rabbit Muscle Phosphofructokinase. A model for regulatory kinetic behavior. J. biol. Chem. 254, 1896-1901. Pettigrew D. W. and Frieden C. (1979b) Binding of regulatory ligands to rabbit muscle phosphofructokinase. A model for nucleotide binding as a function of temperature and pH. J. biol. Chem. 254, 1887 1895. Storey K. B. (1976) Purification and properties of adductor muscle phosphofructokinase from the oyster Crassostrea virsinica. Eur. J. Biochem. 70, 331 337. Storey K. B. (1981) Effects of arginine phosphate and octopine on glycolytic enzyme activities from Sepia officinalis mantle muscle. J. comp. Physiol. 207, 7 13. Storey K. B. (1983) Regulation of cockroach flight muscle phosphofructokinase by fructose-2,6-bisphosphate. FEBS Lett. 161,265 268. Storey K. B. (1985) Fructose-2,6-bisphosphate and anaerobic metabolism in marine molluscs. FEBS Lett. 179, 1-4. Tornheim K. (1979) Oscillation of the glycolytic pathway and the purine nucleotide cycle. J. theor. Biol. 79, 491-541. Tornheim K. (1985) Activation of muscle phosphofructokinase by fructose 2,6-bisphosphate and fructose 1,6-bisphosphate is differently affected by other regulatory metabolites. J. biol. Chem. 260, 7985 7989. Uyeda K. (1979) Phosphofructokinase. Adv. Enzym. 48, 193-244. Uyeda K. and Racker E. (1965) Regulatory mechanism in carbohydrate metabolism. J. biol. Chem. 240, 4682-4688.