Properties of octopine dehydrogenase from the foot muscle of concholepas concholepas

Comp. Biochem. Physiol. Vol. 90B, No. 1, pp. 77-79, 1988

0305-0491/88$3.00+ 0.00 © 1988PergamonPress pie

Printedin Great Britain

PROPERTIES OF OCTOPINE DEHYDROGENASE FROM THE FOOT MUSCLE OF C O N C H O L E P A S C O N C H O L E P A S NELSON CARVAJALand EDUARDOKESSI Departamento de Biologia Molecular, Facultad de Ciencias Biol6gicas y de Recursos Naturales, Universidad de Concepci6n, Casilla 2407, Concepci6n, Chile

(Received 21 May 1987) Abstract--1. The activities of lactate dehydrogenase and octopine dehydrogenases were assayed in homogenates of the foot muscle tissue of Concholepas concholepas. The results indicated that in Concholepas concholepas, octopine dehydrogenase replaces lactate dehydrogenase as the terminal enzyme of anaerobic glycolysis in the muscle. 2. Two molecular forms of octopine dehydrogenase were detected in ion exchangechromatographic and electrophoretic studies of preparations of foot muscle tissue. These forms had the same mol. wt, pH optimum and kinetic properties. 3. The Km values for arginine and pyruvate were decreased by increasing concentrations of the co-substrate. Synergistic substrate inhibition was observed at high concentrations of both arginine and pyruvate. Substrate inhibition by octopine was not observed.

INTRODUCTION

were obtained from commercial sources (most from Sigma Chemical Co.) and were the purest available.

Octopine dehydrogenase (EC 1.5.5.11) catalyzes the reaction

Enzyme activity

Pyruvate + L-arginine + NADH + H + ~octopine + NAD + + H20 This enzyme replaces lactate dehydrogenase in maintaining the redox balance in the muscle of many molluscs (G/ide, 1980). Octopine and not lactate is, therefore, the end product of anaerobic glycolysis in these species. Octopine dehydrogenase has been shown to occur in molluscs, sipunculids and coelenterates (Regnouf and van Thoai, 1970; Grieshaber and Giide, 1976, 1977; Giide et al., 1978; Grieshaber, 1978; G/ide, 1980; Koormann and Grieshaber, 1980; Baldwin et al., 1981; G/ide and Carlsson, 1984). Other opine dehydrogenases utilizing glycine (strombine dehydrogenase) or alanine (alanopine dehydrogenase) have also been shown in some invertebrates (Collicutt and Hochachka, 1977; Dando et al., 1981; Dando, 1981; de Zwaan and Zurburg, 1981; Gfide, 1986). We are studying the metabolic aspects of phosphoenolpyruvate and pyruvate in tissues of the marine gastropod Concholepas concholepas (Carvajal et al., 1986). As part of these studies, we have examined the dehydrogenases acting at the pyruvate branch point in the muscle of the mollusc. This report demonstrates the absence of lactate dehydrogenase and the presence of two molecular forms of octopine dehydrogenase in this tissue. The properties of Concholepas concholepas octopine dehydrogenases are compared with those of the enzymes from other sources. MATERIALS AND METHODS

Animals and chemicals Specimens of Concholepas concholepas were collected in the bay of Concepci6n by local fishermen. All chemicals 77

Standard assay conditions for octopine formation were 100mM potassium phosphate buffer (pH 7.0), 2 mM arginine, 1 mM pyruvate and 0.1 mM NADH and for octopine oxidation, 100mM Tris-HC1 (pH 8.7), 2mM octopine and 0.2 mM NAD, All assays were performed at 20°C. The reaction was followed by measuring the disappearance or appearance of NADH at 340 rim. Initial velocities were determined from the slopes of the recorded tracings and kinetic data were analyzed by double reciprocal plots.

Electrophoresis Homogenates and partially purified enzyme preparations were examined by starch (12.5%) and polyacrylamide(7.5% acrylamide) gel electrophoresis. Octopine dehydrogenase was detected by immersing the gel for 30 min in the dark at 37°C in a solution containing 2raM octopine, 0.2mM NAD, 0.02 mg/ml phenazine methosulfate, 1 mg/ml nitroblue tetrazolium and 100 mM Tris-HC1 (pH 8.0). A duplicate set of gels was stained for protein with a 0.1% solution of Coomassie Brilliant Blue in 50% trichloroacetic acid.

Molecular weights Molecular weights were determined by gel filtration on Sephadex G-100. The column (100 x 2 cm) was equilibrated and eluted with 10mM Tris-HC1 (pH7.5) containing 20mM KCI. The column was standardized with Blue Dextran 2000 and several mol. wt markers. RESULTS AND DISCUSSION

Separation of two molecular forms of octopine dehydrogenase Opine dehydrogenases and lactate dehydrogenase activities were measured in homogenates (20% w/v) of foot muscle tissue of Concholepas concholepas in 10raM Tris-HC1 (pH 7.5) containing 150 mM KC1 and 0.2 mM phenylmethylsulfonyl fluoride (PMSF). Lactate, alanopine and strombine dehydrogenases were absent and the level of octopine dehydrogenase

78

NELSON CARVAJAL a n d EDUARDO KESSI

activity was 53.8 units/g (wet wt) of tissue. It is clear, therefore, that in Concholepas concholepas octopine dehydrogenase replaces lactate dehydrogenase as the terminal enzyme of the anaerobic glycolysis in the muscle. Octopine production might be advantageous for this mollusc for energy supply during activity in a hypoxic micro habitat. Lactate dehydrogenase activity is usually low or absent in mollusc muscles with high octopine dehydrogenase activity (Regnouf and van Thoai, 1970). Exceptions are the marine molluscs Cardium tuberculatum, Nassarius coronatus and Nassa mutabilis. In these species, octopine dehydrogenase and lactate dehydrogenase can function to catalyze the terminal step of glycolysis during muscle anoxia associated with different physiological states (Baldwin et aL, 1981). In the foot of the marine gastropod Nassa mutabilis, octopine is produced only during exercise, while lactate accumulates during recovery from exercise and following exposure to air (G/ide et al., 1984). The homogenate of muscle tissue of Concholepas concholepas was fractionated by ammonium sulfate precipitation (35-70% saturation), gel filtration on Sephadex G-100 equilibrated and eluted with 10 mM Tris-HCI (pH7.5) containing 20mM KC1 and ion exchange chromatography on DEAE-cellulose. The column was equilibrated with 5 mM Tris-HC1 (pH 7.5) and eluted with a linear gradient of KC1. As shown in Fig. 1, two active fractions eluting at about 70 and 110 mM KC1, respectively, were resolved by the DEAE-cellulose chromatography. These were designated enzymes A and B. Evidence for two active fractions was also obtained by starch and polyacrylamide gel electrophoresis followed by staining for octopine dehydrogenase activity. Similar results have been reported for other gastropod and bivalve molluscs (G/ide, 1980).

Molecular weight As determined by gel filtration on Sephadex G-100, the mol. wt of both enzymes was found to be 42,000. All opine dehydrogenases so far studied are monomeric proteins with mol. wt of about 40,000 (Olomucki et al., 1972; Gfide and Carlsson, 1984; G/ide, 1986). p H optimum With both enzymes A and B, octopine formation 0.20

2 A

KCI,mM

so

0

10

~ 20 30 40 FRACTION NUMBER

50

60

0

0

Fig. 1. Separation of two enzyme forms (A and B) by DEAE-cellulose chromatography of a partially purified preparation of octoNne dehydrogenase. (3: enzyme activity; O: A280.

Table 1. Apparent Km values for Concholepas concholepas octopine dehydrogenases Substrate

Co-substrates

Arginine

0.1 mM NADH 1.0 mM pyruvate 0.1 mM NADH 2.0 mM pyruvate 0.1 mM NADH 10.0 mM pyruvate 0.1 mM NADH 1.0 mM arginine 0.1 mM NADH 2.0 mM arginine 0.1 mM NADH 25.0 mM arginine 1.0 mM arginine 2.0 mM pyruvate 0.05 mM NAD 0.20 rnM NAD 0.50 mM octopine 1.00 mM octopine

Pyruvate

NADH Octopine NAD

K~. (mM) 3.0 2.0 1.0 2.0 1.0 0.5 0.025 1.25 1.00 0.33 0.29

was maximal at pH 7.0 whereas a pH optimum of 8.7 was observed for the octopine dehydrogenase activity. Similar values have been reported for the enzyme from several other sources (Gfide and Carlsson, 1984).

Kinetic studies The effect of varying concentrations of the substrate for the forward and reverse reactions were examined. Both molecular forms exhibited Michaelis-Menten kinetics and had essentially the same kinetic properties. Two molecular forms of octopine dehydrogenase differing in electrical charge but not in kinetic properties has been also reported for the enzyme from Pecten maximus L. (MonneuseDoublet et al., 1980). The apparent Km values for the substrates are given in Table 1. These values are in the range of those commonly observed from other octopine dehydrogenases (Storey and Dando, 1982). Table 1 also shows that the Km values for arginine and pyruvate decreased with increasing concentrations of the cosubstrate. As discussed for other opine dehydrogenases (Gfide and Carlsson, 1984), the effect would be of importance in promoting octopine synthesis as the terminal product of glycolysis during burst muscular work. As shown in Fig. 2, the enzymes exhibited decreased activity in the presence of high concentrations of both substrates. Double synergistic substrate inhibition by pyruvate and arginine has been also observed with enzymes from other sources (Storey and Storey, 1979; G/ide, 1980; Schrimsher and Taylor, 1984). The effect would be explained by highly synergistic separate binding of the substrates or the formation of an inhibitory complex between pyruvate and arginine (e.g. the imine), as suggested for the enzyme from Pecten maximus (Schrimsher and Taylor, 1984). In any case, substrate inhibitions of the Concholepas concholepas enzymes were not very strong. In fact, at 50 mM arginine and 10 mM pyruvate, only about 30% inhibition was observed. Of special physiological importance would be, however, the effects of octopine. Substrate inhibition by octopine was not observed and product inhibition by this compound was non competitive with respect to arginine. A 50% inhibition was caused by 1.5 mM octopine. It seems, therefore, that only limited

Properties of octopine dehydrogenase

79

Dando P. R., Storey K. B., Hochachka P. W. and Storey J, M. (1981) Multiple dehydrogenases in marine molluscs: Electrophoretic analysis of alanopine dehydrogenase, strombine dehydrogenase, octopine dehydrogenase and lmM ARGININE lactate dehydrogenase. Mar. Biol. Lett. 2, 249-257. 1 Dando P. R. (1981) Strombine N-(carboxymethyl)o-alanine, dehydrogenase and alanopine meso-N(1-carboxyethyl)-alaninedehydrogenase from the mussel x 0 i I 3O 10 20 Mytilus edulis L. Biochem, Soc. Trans. 9~ 297-298, Z PYRUVATE~mM G/ide G. (1980) A comparative study of octopine dehydrogenase isoenzymesin gastropod, bivalve and cephalopod 3 molluscs. Comp. Biochem. Physiol. 67B, 575-582. G/ide G. (1986) Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the 2 ormer, Haliotis lamellosa. Eur. J. Biochem. 1611,311-318. 1~ 2mM PYRUVATE G~.de G., Carlsson K. H. and Meinardus G. (1984) Energy metabolism in the foot of the marine gastropod Nassa mutabilis during environmental and functional anaerI f 0| I I i L J obiosis. Mar. Biol. 80, 49-56. 0 ~0 20 30 t,O 5O G~ide G. and Carlsson K. H. (1984) Purification and characterization of octopine dehydrogenase from the ARGININE/mM marine nemertean Cerebratulus lacteus (Anopla: HeteroFig. 2. Substrate inhibition of octopine dehydrogenase by nemerta): comparison with scallop octopine dehydropyruvate and arginine. genase. Mar. Biol. 79, 39-45. G~de G., Weeda E. and Gabbott G. (•978) Changes in the level of octopine during the escape responses of the scallop, Pecten maximus (L.). J. Comp. PhysioL 124, amounts of octopine would be accumulated in the 121-127. muscle in vivo. Octopine formed in the muscle would be re-oxidized in this or other tissues. Reversal of Grieshaber M. and G/ide G. (1976) The biological role of octopine in the squid, Loligo vulgaris (Lamarck). J. Comp. octopine dehydrogenase reaction in muscle would be Physiol. 108, 225-232. advantageous for retaining the muscle pool of ar- Grieshaber M. and G/ide G. (1977) Energy supply and the ginine for arginine phosphate synthesis. Future work formation of octopine in the adductor muscle of the is required to clarify this aspect. Nevertheless, it is of scallop, Pecten jacobaeus (Lamarck). Comp. Biochem. interest the suggestion made by G/ide (1980) that Physiol. 588, 249-252. both bivalve and gastropod molluscs have the ability Grieshaber M. (1978) Breakdown and formation of highenergy phosphates and octopine in the adductor muscle to form and re-oxidize octopine in their muscles. of the scallop, Chlamys opercularis (L.) during escape The partially purified enzymes were inactive with swimmingand recovery. J. Comp. Physiol. 126, 269-276, glycine, alanine, aspartic acid, branched chain amino acids, ornithine and citrulline, but used lysine as a Koormann R. and Grieshaber M. (1980) Investigations on the energy metabolism and on octopine formation on the substrate. The Vm~/K m value for lysine was about 10 common whelk, Buccinum undatum L., during escape and times lower than the corresponding value for arrecovery. Comp. Biochem. Physiol. 658, 543-547. ginine. Because of this and their low levels in tissues Monneuse-Doublet M.-O., Lefebure F. and Olomucki A. of marine molluscs (Storey and Dando, 1982), lysine (1980) Isolation and characterization of two molecular is unlikely to be a physiological substrate for the forms of octopine dehydrogenase from Pecten maximus L. Eur. J. Biochem, 1118,261-269. enzymes from the muscle of Concholepas concholepas. Olomucki A., Huc C., Lefebure F. and van Thoai N. (1972) Octopine dehydrogenase. Evidence for a single-chain Acknowledgements--This work was supported by Grant structure. Eur. J. Biochem. 28, 261-268. No. 20.31.19 from the Direccibn de Investigaci6n, UniverRegnouf F. and van Thoai N. (1970) Octopine and lactate sidad de Concepcirn, Chile. dehydrogenase in mollusc muscles. Comp. Biochem. Physiol. 32, 411-416. Schrimsher J. L. and Taylor K. B. (1984) Octopine dehyREFERENCES drogenase from Pecten maximus: Steady-state mechanism. Biochemistry 23, 1348-1353. Baldwin J., Lee A. K. and England W. R. (1981) The functions of octopine dehydrogenase and o-lactate de- Storey K. B. and Dando P. R. (1982) Substrate specificities of octopine dehydrogenases from marine invertebrates. hydrogenase in the pedal retractor muscle of the dog Comp. Biochem. Physiol. 73B, 521-528. whelk Nassarius coronatus (Gastropoda: Nassariidae). Storey K. B. and Storey J. M. (1979) Kinetic characterizMar. Biol. 62, 235-238. ation of tissue-specific isozymes of octopine dehydroCarvajal N., Gonzfilez R. and Morfin A. (1986) Properties genase from mantle muscle and brain of Sepia officinalis. of pyruvate kinase from the heart of Concholepas conEur. J. Biochem. 93, 545-552. cholepas. Comp. Biochem. Physiol. 85B, 577-580. Collicutt J. M. and Hochachka P. W. (1977) The anaerobic Zwaan A. de and Zurburg W. (1981) The formation of strombine in the adductor muscle of the sea mussel, oyster heart: Coupling of glucose and aspartate ferMytilus edulis L. Mar. Biol. Lett. 2, 179-192. mentation. J. Comp. PhysioL 115, 147-157. f"~"~25

mM ARGININE