Inhibition of tubifex pyruvate kinase by storage phosphagens and adenosine triphosphate

Comp. Biochem. Physiol. Vol. 70B, pp. 77 to 83, 1981

0305-0491/81/090077-07102.00/0 Copyright © 1981 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

INHIBITION OF TUBIFEX PYRUVATE KINASE BY STORAGE PHOSPHAGENS AND ADENOSINE TRIPHOSPHATE KLAUS H. HOFFMANN Allgemeine Zoologic (Biologic I), Universit~it Ulm, Oberer Eselsberg, D-7900 Ulm/Donau, West Germany

(Received 29 December 1980) Abstract--1. Within the first 6-12 hr of experimental anaerobiosis the concentration of Tubifex phos-

phagens, phospholombricine and phosphoarginine, decrease to 10--30To of the values under aerobic conditions (7.5 and 3.5 ~uM/g dry wt). 2. High concentrations of both phosphagens (5-25 mM) inhibit Tubifex pyruvate kinase (PK) activity, the inhibitory mechanisms seem to be different. The inhibition is not mediated by the corresponding phosphagen kinases. Inhibition by phosphoarginine obviously requires an additional factor, which chemical nature is still unknown. 3. Certain phosphocreatine preparations also inhibit Tubifex PK. This inhibition, however, is likely due to contaminants than to phosphocreatine. 4. Mg-ATP demonstrates the strongest inhibitory effect on PK activity with a mixed competitive inhibition vs PEP and a non competitive vs ADP.

INTRODUCTION The glycolytic enzyme pyruvate kinase (PK) from tissues of several facultative anaerobic invertebrates shows a high degree of regulatory refinement (see de Zwaan & Holwerda, 1972; Hoffmann, 1976; Hoffmann, 1977). In the oligochaete Tubifex during the first hours of an experimental anaerobiosis the rate of glycolysis increases (Sch6ttler, 1978) and lactate accumulates as a major anaerobic endproduct. As anoxic conditions persist, decreasing concentrations of hexosephosphates indicate a diminished flux in glycolysis. Long-term anoxia leads to further anaerobic reactions with a higher total ATP yield, as a consequence of the production of succinate, propionate and acetate. The rate and amount of the endproduct accumulation largely depend on the regulating properties of the pyruvate kinase. Regulation of pyruvate kinase activity is achieved, at least partly, by mechanisms that differ from those present in other invertebrates capable of anaerobic excursions. Changes in the concentrations of Fe 2÷, fructose-l.6-diphosphate, adenylate energy charge (AEC) and also in the HCO3- + CO2 system during anaerobic incubation can be considered to regulate Tubifex pyruvate kinase activity (Hoffmann et al., 1979). Furthermore, within the first 6-12 hr of experimental anaerobiosis, the concentrations of phosphagens change considerably (Hoffmann, 1981). Concentrations of phospholombricine and phosphoarginine

Abbreviations used: ADP--adenosine diphosphate; ATP-adenosine triphosphate; DTT~dithiothreitol; EDTA-ethylene diamine tetraacetic acid; FDP--fructose-l.6diphosphate; K~--half saturation of inhibitor; PEP-phosphoenolpyruvate; PK--pyruvate kinase; LK--lombricine kinase; AK--arginine kinase; CK--creatine kinase; P-arg--phosphoarginine; P-crea--phosphocreatine; Plom--phospholombricine. 77

decrease to 10--30To of the values under aerobic conditions. Wu et al. (1978) demonstrated that pyruvate kinase from several vertebrate and invertebrate species was inhibited by physiological concentrations of muscle phosphagens. Arginine phosphate could act either as a competitive inhibitor (PK from Oplophorus 9racilirostris muscle: Guderley et al., 1976) or by phosphorylating or inducing a protein-protein interaction resulting in a decreased affinity for phosphoenolpyruvate (PK from Helix pomatia foot muscle: Wieser & Lackner, 1977). De Zwaan & Ebberink (1978), however, assert that the inhibitory effect of phosphoarginine on pyruvate kinase from the adductor muscle of the sea mussel, Mytilus edulis, is probably only due to a side reaction with arginine kinase contaminating the pyruvate kinase preparation. Wu et al. (1979) also demonstrated that the muscle PK from a polychaete worm, Marphysa sanyuinea, lost most of its sensitivity to phosphagen inhibition, when it was partially purified. The PK inhibition in crude enzyme extract was mediated through creatine kinase by depleting adenosine diphosphate. Fitch et al. (1979) further established that certain preparations of phosphocreatine are potent inhibitors in the PK assay but that the inhibition was due to a contaminant rather than to phosphocreatine. Owing to these contradictory results we determined the effect of different phosphagens (arginine phosphate, creatine phosphate, lombricine phosphate) and ATP on crude, partially and highly purified pyruvate kinases extracted from the tissues of Tubifex. MATERIALS AND METHODS

AnimaLs Tubifex were obtained from Fa. Honka, Munich and kept at 8-10°C as in Hoffmann (1981) until they were used for enzyme studies.

KLAUSH. HOFFMANN

78

cine kinase), however, no detectable amount of creatine kinase in crude extract and in the 100,000g supernatant. In the 100,000g supernatant activity of lombricine kinase was about 2 times that of pyruvate kinase. After Biogel treatment the pyruvate kinase was essentially free of phosphagen kinase activities. The effects of various phosphagens (sodium salts) and of ATP on the pyruvate kinase assay with enzyme of different purity grade are shown in Fig. 1. In the presence of 25 mM phosphoarginine or 5 mM of phospholombricine pyruvate kinase activity (from crude extract and from 100,00 g pellet) was depressed to ca. 50-60% of the activity in the control. Six or 12mM of A D P in the assay instead of 2 m M had little effect on the activity of the pyruvate kinase preparations. The phosphagen inhibition (by phosphoarginine and phospholombricine) in both the crude extract and in the 100,000g pellet persisted (Fig. 2). All progress curves are linear in the presence of the phosphagens as well as in the absence of the inhibitors. When the pH value of the assay was lowered from pH 7.2 to pH 6.5, the inhibitory effect of both phosphagens increased slightly (Ki for phosphoarginine: 25 mM at pH 7.2 as against 14 mM at pH 6.5) (Fig. 3). Phosphoarginine inhibition is non competitive vs ADP, and phospholombricine inhibition vs ADP is of a mixed competitive type. Both phosphagens are mixed competitive inhibitors vs PEP (Table 2). High concentrations of both phosphagens slightly increased the allosteric behaviour of the pyrurate kinase. Addition of phosphoarginine (25 mM) exhibited no inhibitory effect when using PK after the 100,000 ff centrifugation step (PK 100,000g supernatant), or after the Biogel treatment (Fig. 1). Phospholombricine, on the other hand, also inhibited activity of highly purified PK preparations. In the presence of 25 mM of phospholombricine, enzyme activity of pyruvate kinase after Biogel treatment decreased to 20% of the value in the control. The commercial preparation of creatine phosphate (Sigma P-6502, Lot 98 C-5017) was found to be a potent inhibitor in all pyruvate kinase assays. 25 mM of phosphocreatine inhibited PK activity between 30 and 55% (Fig. 1 ; Fig. 3). A partial purification of the Sigma phosphocreatine preparation by anion exchange chromatography after Fitch et al. (1979), however, eliminated all inhibitory effects (Fig. 1). Of all phosphorylated compounds that have been tested, ATP demonstrated the strongest inhibitory RESULTS effect on PK activity. The inhibition rate is indepenTable 1 shows the presence of both pyruvate kinase and phosphagen kinases (arginine kinase and lombri- dent of the purity grade of the enzyme (Fig. 1).

Chemicals Enzymes, coenzymes,and substrates were obtained from Sigma GmbM, Munich except lombricine kinase and phospholombricine which had been isolated from Tubifex worms after Hoffmann (1981). Pyrucate kinase preparation The worms were blotted dry and homogenized in 5 vol of cold 0.02 M Tris/HC1 buffer (pH 7.2), containing 1 mM EDTA and 0.5 mM DTT. After centrifugation for 30 rain at 10,000 g and 0°C the supernatant was filtered through glass wool (= PK crude extract). Then the hemoglobine was separated from the crude extract following centrifugation at 100,000g (= PK lO0,O00 g supernatant extract). The pellet from the 100,000g centrifugation step was dissolved in a small volume of 0.01 M phosphate buffer, pH 7.0 (= PK lO0,O00g pellet extract). Another part of the 100,000g supernatant was brought to 65% saturation with solid ammonium sulfate, and after stirring for 1 hr the suspension was centrifuged at 37,000g for 15 min. The pellet was dissolved in a small volume of 0.01 M phosphate buffer, pH 7.0, dialysed and passed through a column (80 x 2.6 cm) of Sepharose CL- 6 B equilibrated with 0.04 M phosphate buffer, pH 7.0 containing I mM EDTA and 0.5 mM DTT. Fractions containing PK activity were pooled and applied to a column (30 x 0.9 cm) of hydroxylapatite (Biogel), which was equilibrated with the same buffer as the Sepharose column. The column was developed with a phosphate buffer (pH 7.0) gradient of 40--400mM within 24 hr. Fractions with PK activity were combined and frozen until use (= PK Bioyel extract). Enzyme assays Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was assayed according to Hoffmann et al. (1979), accept a phosphoenolpyruvate (PEP) concentration of 1 mM and an ADP concentration of 2 raM. When PEP was added to complete the assay the mixture without PEP was preincubated for 5 min at 25°C. The pH of the final reaction medium was regular monitored to ensure that the enzyme activity was assayed at pH 7.2 Arginine kinase (ATP: L-arginine phosphotransferase, EC 2.7.3.3) and lombricine kinase (ATP: lombricine phosphotransferase, EC 2.7.3.5) activities were measured as in Hoffmann (1981). All measurements were started by addition of the corresponding substrate. All enzyme activities are the average of at least two separate determinations with less than 10% variability between values. Enzyme activities are expressed as AOD/ rain or as/~mol substrate converted per rain per mg protein. Protein concentrations were determined after Lowry et a l. (1951) and using the "280/260" method of Layne (1957).

Table 1. Activities of pyruvate kinase and phosphagen kinases in various Tubifex preparations (mU/mg protein) Fraction Crude extract 100,000 ff supernatant 100,000 ff pellet Biogel treatment

PK activity 35 85 7 3914

LK activity P-lom--~lom (79)* 160 ND ND

AK activity P-arg--,arg (19)* 93 ND ND

CK activity P-crea---*crea ND ND ND ND

* Adenylate kinase (mmkinase) activity interfered the assay in the "crude extract" even in the presence of 2 mmol of P~, p5 diadenosine pentaphosphate. (P)-lom = (phospho)lombricine; (P)-arg = (phospho)arginine; (P)-crea = (phospho)creatine; LK--lombricine kinase; AK--arginine kinase; CK--creatine kinase. ND--Not detectable.

PK inhibition by phosphagens

79

crude extract

IOO 8o 6c

H

4C 2c

I00,000 X g I00 supernatant 80

I]

60

R

40 2C

I00,000 X 9 pellet I00 8060-

I H

40 20I

+2U Biogel u°° treatment

H

AK

i"

80 60 40 20

control

R[I

5mM

25mM

P-lomb

M IOmM

25mM

P - arg

25mM P - crea up

5mM p

IOmM ATP

Fig. 1. Effect of various phosphagens and ATP on Tubifex pyruvate kinase assay with enzyme of different purity grade. P-Iom--phospholombricine, P-arg--phosphoarginine, P-crea--phosphocreatine, up---unpurified phosphocreatine, l>-purified phosphocreatine, ATP--adenosine triphosphate, AK-Sigma arginine kinase.

80

KLAUS H. HOFFMANN

2 mM

6 mM

12 mM

A DP

A OD/min 0.200

P-Iombricine

1

0.100

0.02C

0.20C

1

lq

P - arginine

:I1

O.IOC

0.02C

P- creatine

0200

0.10C

I I 0.02C I

0.200 ATP

0 i00

002C r

~

L

[] 0

5

10

[] 0

5

10

0

5

I0

mM

Fig. 2. Effect of different ADP concentrations on phosphagen and ATP inhibition rate of pyruvate kinase from the crude extract.

Tubifex

PK inhibition by phosphagens

81

A0D/rnln 0.15o f

pH

6.5

o.IOO

0.050

t P- arginine

°

° I 2 3

2 5

7.5

I0

°

~ 20

°" P-creatine . P- Iombricine , 30

mM 40

[phosphagens

]

AOD/min o.15o~

pH

7.2

0.100

\ \. P-arginine

0.050

*- P-

ATP I I I I 2 3

I 5

I I "/'.5 I0

I 20

creafine

•

P- Iombricine, I 30

mM

l 40

[phosphagens

]

Fig. 3. Effect of concentrations of phosphagens and ATP on Tubifex pyruvate kinase reaction rate at pH 6.5 and 7.2 (crude extract). Mg-ATP is a mixed competitive inhibitor vs PEP and a non competitive inhibitor vs ADP (Ki = 3.75 raM) (Table 2). The degree of inhibition is not significantly different between pH 6.5 and 7.2 (Fig. 3) and fructose-l.6-diphosphate did not reverse the inhibition caused by Mg-ATP. Concentrations of 25 mM of phosphagens as well as of ATP did not demonstrate any effects on activity of the auxiliary enzyme in the coupled PK assay, the lactate dehydrogenase. Also no significant changes in c.a.P. 7 ~ l a - - r

the PK reaction rates have been detected in the presence of 100 mM of sodium (chloride) ions in exchange to the phosphagens. DISCUSSION

Phosphoarginine and phospholombricine are the two naturally occurring storage phosphagens in Tubifex (Hoffmann, 1981). Several authors have recently demonstrated that physiological concentrations of muscle phosphagens inhibit pyruvate

82

KLAUSH. HOFFMANN Table 2. Apparaent K,~ values, maximum velocity values (V) and Hill coefficients (nil) of Tubifex PK reaction (partially purified enzyme) in the presence of various concentrations of phosphagens and ATP Inhibitor (mM)

apparent Km (/zM)

V (AOD/min)

n,

170 312 284 157 192 267 170 317

0.229 0.136 0.080 0.212 0.131 0.106 0.229 0.080

1.18 1.02 1.44 1.21 1.19 1.59 1.19 1.27

416 393 367 288 225 366 390 310

0.332 0.175 0.287 0.121 0.059 0.222 0.075 0.017

0.96 1.19 0.95 1.08 1.00 1.05 1.03 1.09

PEP as variable substrate: mM Phosphoarginine 0 7.5 15 Phospholombricine 0 5 10 ATP 0 5 ADP as variable substrate: mM Phosphoarginine 0 7.5 Phospholombricine 0 5 10 ATP 0 5 10

kinase from a number of vertebrate and invertebrate species (Guderley et al., 1976; Wieser & Lackner, 1977; Wu et al., 1978). Wieser & Lackner (1977) reported that in the snail Helix pomatia the inhibition of pyruvate kinase by arginine phosphate is mediated by a protein, which either decreases the affinity to PEP (Eigenbrodt & Schoner, 1979) or may transfer a phosphate group from the phosphagen onto the pyruvate kinase, thus inactivating the enzyme. Phosphorylation and dephosphorylation serves as a possible regulatory mechanism also in some mammalian L-type isoenzymes (Eigenbrodt & Schoner, 1975; Titanj et al., 1976; Berg et al., 1978). In contrast, de Zwaan & Ebberink (1978) showed that phosphoarginine by itself does not effect the pyruvate kinase activity of the sea mussel, Mytilus edulis. An inhibitory effect of phosphoarginine which can be found only in crude PK preparations, is probably due to a side reaction in the presence of arginine kinase; ADP is converted into ATP which leads to a concomitant reduction of the PK reaction rate. An excess of 12 mM of ADP in the PK assay instead of 2 mM abolishes any inhibitory effect of phosphoarginine on the P K reaction rate. Wu et al. (1979) reported strong evidence that the inhibition of rat muscle pyruvate kinase by phosphocreatine is also mediated via the phosphagen (creatine) kinase activity. Similar results have been published recently for an apparent inhibition of Carcinus maenas type-M PK by a less pure form of arginine phosphate (Poat et al., 1980). In contrast to these data our results let suggest that phosphoarginine and phospholombricine inhibition on Tubifex PK activity is not mediated by the corresponding phosphagen kinases. Inhibition of enzyme from the crude extract and from the 100,000 # pellet by phosphoarginine persists in the presence of 6 or 12 mM of ADP. PK activity in the 100,000 g supexnatant, on the other hand, is not affected by phosphoarginine concentrations up to 25 mmol, although the

activity of arginine kinase in this preparation is high. The same is true for PK activity in a Biogel treated enzyme preparation in the presence of 25 mM of phosphoarginine and after addition of 2 U of arginine kinase (see Fig. 1). Inhibition of Tubifex PK by phosphoarginine obviously requires an additional factor which has either a high mol wt or is bound to a corresponding protein that may be separated from the enzyme by an uitracentrifugation step. Activities of all the four Tubifex PK preparations were significantly lowered in the presence of phospholombricine and inhibition was also not removed by addition of excess amounts of ADP. No evidence could be presented that PK-inhibition by phospholombricine is due to a contaminant rather than to the phosphagen, or that the inhibitory effects require the presence of an additional factor which can be separated from the pyruvate kinase. In agreement with earlier reports (see in Fitch et al., 1979) we found a Sigma preparation of creatine phosphate to be also a potent inhibitor in the PK assay, although phosphocreatine is not a naturally occurring phosphagen in Tubifex. However, the inhibition is due to a contaminant rather than to phosphocreatine; the nature of the contaminant remains to be identified. A variability in the presence of the contaminant would explain why P K inhibition by commercial phosphocreatine has been observed by some investigators but not by others (Wu et al., 1979). ATP exhibits the most effective inhibition of all phosphorylated compounds that have been tested. In the presence of Mg 2+, Kt values are on a level consistent with a possible regulatory control and agree well with values reported for other invertebrates (Hoffmann, 1975; Guderley et al., 1976; Hoffmann, 1977). As Mg 2+ is replaced by Mn 2+, Ki values increase drastically (Hoffmann, 1977), suggesting that under these conditions ATP has noregulatory role. In contrast with many other invertebrate pyruvate

PK inhibition by phosphagens kinases, the Tubifex enzyme shows no significant reversal of ATP inhibition by fructose-l.6-diphosphate. The varying levels of phosphagens as well as of ATP in Tubifex during an experimental short-term anaerobiosis (Hoffmann, 1981) probably provide a major means of which Tubifex pyruvate kinase is regulated, also in vivo. A significant drop in concentrations of phospholombricine and phosphoarginine at the onset of anoxia would deinhibit the pyruvate kinase, as would also a drop in the ATP level. The increasing rate of glycolysis (Scht~ttler, 1978) leads to an accumulation of lactate as a major anaerobic endproduct during this period. An increased amount of energy will be needed for the corkscrew motions, manifested by Tubifex observed during oxygen lack within the first hours of anaerobiosis. If anaerobiosis persists (long-term anaerobiosis) PK activity again will be reduced with succinate and propionate accumulated as endproducts via the PEPCK reaction (Hoffmann, 1977). This inhibition, however, is realized by other properties of the enzyme, such as its concentration and allosteric behaviour, or its sensitivity towards ferrous-ions and chinolinic acid (Hoffmann and Seul3, 1979), fructose-l.6-diphosphate and total CO2 (Hoffmann et al., 1979). SUMMARY

The facultative anaerobic Tubifex is capable of degrading carbohydrates via different channels, depending on the duration of anoxic exposure. Shortterm anoxia leads to lactate production via pyruvate kinase (Hoffmann et al., 1979). Within the first 6-12 hr of an experimental anaerobiosis the concentrations of the two naturally occurring phosphagens, lombricine phosphate and arginine phosphate, change considerably (Hoffmann, 1981). High concentrations of both phosphagens inhibit Tubifex pyruvate kinase activity, the inhibitory mechanisms, however, seem to be different. The inhibition is not mediated by the corresponding phosphagen kinases. Inhibition by phosphoarginine may be mediated by another "high molecular weight protein". Certain phosphocreatine preparations also inhibit Tubifex pyruvate kinase. This inhibitory effect, however, is likely due to a contaminant rather than to phosphocreatine. ATP is the most effective inhibitor even on highly purified Tubifex pyruvate kinase. Mg-ATP is a mixed competitive inhibitor vs PEP, and a non competitive inhibitor vs ADP.

Acknowled#ement--This work was supported by the Deutsche Forschungsgemeinschaft (Ho 631/7-8). REFERENCES BERG VAN DEN G. B., BERKELVAN T.'J. C. 86 KOSTER J. F.

(1978) Cyclic AMP-dependent inactivation of human liver pyruvate kinase. Biochem. biophys. Res. Commun. 82~ 859-864. EIGENBRODT, E. 86 SCHONERW. (1975) Demonstration of a phosphorylation and dephosphorylation of chicken liver pyruvate kinase isoenzymes type L and M2. Z. Physiol. Chem. 356, 227-228. EIGENBRODT E. 86 SCRONERW. (1979) Modification of pyr-

83

uvate kinase activity by proteins from chicken liver. Hoppe-Seyler's Z. Physiol. Chem. 360, 1243-1252. FITCH C. D., CHEVLIR. & JELLINEK M. (1979) Phosphocreatine does not inhibit rabbit muscle phosphofructokinase or pyruvate kinase. J. biol. Chem. 254 (22), 11357-11359. GUDERLEY H., STOREY K. B., FIELDS J. H. A. 8£

HOCHACHKAP. W. (1976) Pyruvate kinase from Oplophorus oracilirostris muscle. Comp. Biochem. Physiol. 55B, 475-478. HOFFMANNK. H. (1975) Pyruvate kinase from muscle and fat body of the house cricket Acheta domesticus L.: Purification and catalytic studies. J. comp. Physiol. 104, 59-69. HOFFMANNK. H. (1976) Catalytic efficiency and structural properties of invertebrate muscle pyruvate kinases: correlation with body temperature and oxygen consumption rates. J. eomp. Physiol. 110, 185-195. HOFFMANNK. H. (1977) The regulatory role of muscle pyruvate kinase in carbohydrate metabolism of invertebrates: A comparative study in catalytic properties of enzymes isolated from Tubifex tubifex (Oligochaeta) and Tenebrio molitor (Coleoptera). Physiol. Zool. 50, 142-155. HOFFMANNK. H. (1981) Phasphagens and phosphokinases in Tubifex sp. J. comp. Physiol. in press. HOFFMANN K. H., MUSTAFAT. 86 JORGENSENJ. B. (1979) Role of pyruvate kinase, phosphoenolypyruvate carboxykinase, malic enzyme and lactate dehydrogenase in anaerobic energy metabolism of Tubifex sp. J. comp. Physiol. 130, 337-345. HOFFMANNK. H. 86 SEUI3J. (1979) Zum Energiestoffwechsel yon Tubifex sp. (Annelida: Oligochaeta): Umsatz von Phosphoenolpyruvat unter aeroben und anaeroben Bedingungen. Verh. Dtsch. Zool. Ges. p. 281. LAYNE E. (1957) Spectrophotometric and turbidimetric methods for measuring proteins. In Methods in Enzymoloyy (Edited by COLOWICKS. P. & KAPLANN. O.) VOL. III, pp. 451-455. Academic Press, New York. LOWRY O., ROSENBROUGHN., FARR A. & RANDALL R. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. POATP. C., GILESJ. G. 86 MUNDAYK. A. (1980) An investigation into the apparent inhibition by arginine phosphate to the activity of Carcinus maenas type-M pyruvate kinase. Biochem. biophys. Acta 613, 410-419. SCHOTTLERU. (1978) The influence of anaerobiosis on the levels of adenosine nucleotides and some glycolytic metabolites in Tubifex sp. (Annelida, Oligochaeta). Comp. Biochem. Physiol. 61B, 29-32. TITANJ V. P., ZELTERGVISTO., ENGSTROML. (1976) Regulation in vitro of rat liver pyruvate kinase by phosphorylation-dephosphorylation reactions, catalyzed by cyclicAMP dependent protein kinases and a histone phosphatase. Biochim biophys. Acta 422, 98-108. WIESER W. 86 LACKNERR. (1977) Inhibition of the pyruvate kinase of Helix pomatia L. by phospho-L-arginine. Phosphorylation or a novel mechanism? FEBS Lett. 80, 299-302. Wu S. W. N., WONG S. C. 86 YEUNGD. (1978) Comparative studies of vertebrate and invertebrate pyruvate kinases. Comp. Biochem. Physiol. 61B, 93-98. Wu S. W. N., WONGS. C. 86 YEUNGD. (1979) Apparent inhibition of pyruvate kinase by phosphocreatine and phosphoarginine. Comp. Biochem. Physiol. 6311,29-34. ZWAAN A. DE 86 EBBERINK H. M. (1978) Apparent inhibition of pyruvate kinase by arginine phosphate. A problem of kinetic studies on partially purified extracts. FEBS Left. 89, 301-303. ZWAAN A. DE 86 HOLWERDAD. A. (1972) The effect of phosphoenolpyruvate, fructose-l.6-diphosphate and pH on allosteric pyruvate kinase in muscle tissue of the bivalve Mytilus edulis L. Biochim. biophys. Acta 276, 430-433.