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Properties and possible role of malate dehydrogenase in the foot muscle of the sea mollusc Concholepas concholepas (Gastropoda: Muricidae)
Comp. Biochem. Physiol. Vol. 9911,No. 1, pp. 83-86, 1991 Printed in Great Britain
0305-0491/91 $3.00+ 0.00 © 1991 PergamonPress plc
PROPERTIES ANI~ POSSIBLE ROLE OF MALATE DEHYDROGENASE I IN THE FOOT MUSCLE OF THE SEA MOLLUSC CONCHOLEPAS CONCHOLEPAS (GASTROPODA: MURICIDAE) NELSON CARVAJAL,EDUARDOKESSI,* CLAUD10TORRES,OSCARMAR~N,VIVIANATORRES,'~ ALEJANDROCAMPOSand CLAUDIAPOSADA Departamento de Biologia Molecular, Facultad de Ciencias Biol6gicas y de Recursos Naturales, Universidad de Concepci6n, Casilla 2407, Concepci6n, Chile (Received 3 October 1990) Abstract--1. Very few mitochondria, undetectable levels of succinate dehydrogenase and glutamate dehydrogenase and significant activities of glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and malate dehydrogenase were observed in the deeper foot muscle tissue of Concholepas concholepas. 2. Malate dehydrogenase activity was resolved into two molecular forms, which differ in K~ values for oxaloacetate and malate, and sensitivity to inhibition by ~-ketoglutarate. 3. In addition to reoxidation of NADH generated from glycolysis, the function of malate dehydrogenase is discussed in connection with octopine synthesis and oxidation in the foot muscle of C. concholepas. 4. The results of initial rate and product inhibition studies of both forms of malate dehydrogenase are consistent with an Ordered Bi Bi kinetic mechanism, with NADH adding first and NAD ÷ leaving last. Oxaloacetate substrate inhibition is suggested to result from formation of an enzyme-NAD+-oxaloacetate dead end complex.
through the phosphoenolpyruvate carboxykinase reaction to the succinate pathway of glycogen degradation. Anoxia induced changes have been shown to occur in pyruvate kinase from several marine molluscs and the effect is believed to play an important role in the adaptation of these animals to anoxic conditions (Plaxton and Storey, 1985; Michaelidis et al., 1988; Holwerda et al., 1989). Recently we have shown that pyruvate kinase from the deeper foot muscle of Concholepas concholepas is not altered when the animals are exposed to prolonged environmental anoxia. Dramatic changes were, however, observed in the enzyme from the heart tissue of the mollusc. Moreover, that a glycogen-succinate pathway does not operate in the deeper foot muscle, was indicated by the absence of phosphoenolpyruvate carboxykinase activity in this tissue (Carvajal, 1988, 1990). Continuing with our studies, we have now turned our attention to the enzymatic aspects of aspartate metabolism in the deeper foot muscle tissue of C. concholepas. Data in this report show a significant enzymatic capacity for conversion of aspartate to malate but not to succinate in this tissue. The properties of two molecular forms of malate dehydrogenase are described and their possible functions are discussed.
INTRODUCTION
While malate dehydrogenase (EC 1.1.1.37) has been isolated and extensively studied in many species (Storer et al., 1981; Mullinax et al., 1982; Waters et aL, 1985; Ohshima and Sakuraba, 1986; Forn6s et al., 1987; Basaglia, 1989), knowledge of the enzyme from molluscan tissues is very limited (Swift and Lakshmanan, 1982; Paynter et al., 1985; Bolognani Fantin et al., 1987). One specially documented aspect is the presence of cytosolic and mitochondrial forms of malate dehydrogenase in tissues of many vertebrates and invertebrates (Basaglia, 1989). In addition to the well known roles of malate dehydrogenase in the tricarboxylic acid cycle and the exchange of reducing equivalents between the cytosol and the mitochondria (Crow et aL, 1982, 1983; Forn6s et aL, 1987), the enzyme is involved in the glycogen-succinate and aspartate-succinate pathways, which operate in several anoxia-tolerant molluscs (Gade, 1983). This is important because glycogen and aspartate are known to be the fuels and succinate and alanine the major end products of anaerobic metabolism in these species (Gade, 1983; Plaxton and Storey, 1985; Eberlee and Storey, 1988). Briefly, at the early stages of anoxia, there is a coupled conversion of glycogen to alanine and aspartate to succinate; when the aspartate reserves are exhausted, phosphoenolpyruvate is channelled
MATERIALS AND METHODS
Animals and chemicals Specimens of C. concholepas were collected by local fishermen from the Bay of Concepci6n. All chemicals were obtained from commercial sources (most from Sigma Chem.
*Present address: Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile. ?Present address: Department of Physiology and Endocrinology, Medical College, Georgia, USA.
Co.) and were the purest available. 83
84
NELSON CARVAJALet al.
Tissue content of the enzymes Freshly excised sections of the deeper foot muscle tissue were minced and then homogenized using five 20 sec bursts at high speed in a Virtis tissue grinder. Homogenates (25%, w/v) were prepared in 10 mM Tris-HC1 (pH 7.4) containing 1 mM EDTA, 1 mM 2-mercaptoethanol and 0.1 mM phenylmethylsulfonyl fluoride. All the enzyme activities were assayed at pH 7.4 and 25°C, by following the changes of absorbance at 340 nm. Glutamate pyruvate transaminase and glutamate oxaloacetate transaminase were determined in coupled assays with lactate dehydrogenase and malate dehydrogenase, respectively. Glutamate dehydrogenase activity was assayed both in the direction of ~-ketoglutarate and glutamate formation. Succinate dehydrogenase activity was assayed by the spectrophotometric method with ferricyanide (Veeger et al., 1969). The malate dehydrogenase reaction in the direction of oxaloacetate reduction was followed in a medium containing 50 mM Tris-HC1 (pH 7.5), 1.0 mM oxaloacetate and 0.2 mM NADH; for the analysis of the reverse reaction, substrate concentrations were 10 mM malate and 0.4 mM NAD +. Kinetic mechanism of malate dehydrogenase For the analysis of the kinetic mechanism of the malate dehydrogenase reaction, initial velocities in the direction of oxaloacetate reduction were determined at 25°C in 50 mM glycine-NaOH buffer (pH 9.0). Duplicate rate measurements differed by less than 2%. Kinetic data were analyzed by double reciprocal plots and computer-fitted to the appropriate rate equation by means of the least-squares method, assuming equal variances for the velocities. The nomenclature used in this paper is that of Cleland (1970) and initial velocity data yielding intersecting patterns, competitive inhibition and non-competitive inhibition were fitted to equations (1), (2) and (3), respectively. v = VAB/[K,aKb + KaB + KbA + AB]
(1)
v = VA/[Ka (1 + I/Kgs) + A]
(2)
v = VA/[Ka(1 + I / K ~ s ) + A (1 +I/K,i)].
(3)
Electron microscopy Small pieces of muscle tissue from the external edge and the internal zones of the foot of C. concholepas were dissected and examined by electron microscopy using standard techniques following glutaraldehyde fixation.
RESULTS Enzyme activities and electron microscopy o f the f o o t
As determined in the direction of oxaloacetate reduction, and at pH 7.4, the malate dehydrogenase activity was 18.4 _ 1.2 units/g (wet wt) of deeper foot muscle tissue. Activities of glutamate pyruvate transaminase and glutamate oxaloacetate transaminase were 1.3 + 0.2 and 1.1 _ 0.1 units/g (wet wt) of tissue, respectively. Values are mean + SD of determinations in three animals. Succinate dehydrogenase and glutamate dehydrogenase were absent or too low to be detected in the deeper foot muscle tissue. Moreover, electron microscopy studies revealed that mitochondria in this tissue are very scarce and contain very little cristae (Fig. 1). In contrast, numerous enlarged mitochondria with fine and compacted cristae were observed in the smooth muscle tissue which is immediately below the surface epithelium. Separation and properties o f two molecular f o r m s o f malate dehydrogenase
Malate dehydrogenase activity was partially purified by a procedure involving the following steps: (a) fractionation of the homogenate with a m m o n i u m sulfate (30~55% saturation); (b) chromatography on a phenyl-Sepharose column equilibrated with homogenizing buffer containing 1.5 M a m m o n i u m sulfate and eluted with a linear gradient of a m m o n i u m
Fig. 1. Electron micrograph of the deeper foot muscle tissue of Coneholepas concholepas. Magnification 5720 x. Typical mitochondria (m) in this tissue are indicated.
85
M a l a t e d e h y d r o g e n a s e in m o l l u s c f o o t m u s c l e Table 1. Apparent K,~ values (mM) of malate dehydrogenases Substrate
pH
Enzyme I
Enzyme II
Oxalaeetate NADH Malate NAD ÷
7.4 7.4 9.0 9.0
1.00 0.03 1.40 0.14
0.15 0.04 6.45 0.21
When used as fixed substrates, concentrations were I mM oxaloacetate, 0.2 mM N A D H , 10 mM malate and 0.4 mM N A D ÷.
sulfate (1.5 M to 0); (c) chromatography on D E A E cellulose equilibrated with homogenizing buffer and eluted with a linear gradient of KC1 (0-1 M). Two active fractions were eluted from the DEAE-cellulose column. About 65% of the enzyme activity eluted with the washing buffer (enzyme I) and the remaining 35% (enzyme II) eluted in the salt gradient at a concentration of about 0.1 M KC1. Malate dehydrogenase activity of both enzyme forms was maximal at pH 8.9-9.2. At all pH values examined (7.5-10), both molecular forms exhibited Michaelis-Menten kinetics. To gain some understanding of the functioning of the enzyme in vivo, kinetic studies were performed at pH 7.4. At this pH value, the reaction in the direction of oxaloacetate reduction was clearly detected, but the reverse reaction was very slow. The reaction in the direction of malate oxidation was, therefore, examined at pH 9.0. Calculated apparent Kmvalues for the substrates are those given in Table 1. Substrate inhibition of both enzymic forms was observed at concentrations of oxaloacetate higher than 5 mM. Of several compounds tested, ct-ketoglutarate was found to be inhibitory, but only to the enzyme I. A 50% inhibition of enzyme I was caused by about 3.5 mM ct-ketoglutarate. Citrate was slightly inhibitory to both enzymic forms, with concentrations higher than 40 mM being required for 50% inhibition of the enzymes.
Kinetic mechanism of malate dehydrogenase For the analysis of the kinetic mechanism of the enzyme, initial velocity and product inhibition studies were performed at pH 9.0. Results described here were those specifically obtained with the enzyme I; in any case, kinetic patterns were the same for both enzyme forms. All the lines of double reciprocal plots intersected to the left of the 1Iv axis and substrate inhibition was clearly observed at concentrations of oxaloacetate higher than 5 mM, the inhibition being uncompetitive with respect to NADH. Secondary plots of the data at non-inhibitory levels of oxaloacetate were linear. Kinetic parameters calculated from these plots were: Ka = 0.027 raM; Kia = 0.016 mM; Kb = 0.43 mM.
Varied substrate
NAD + NAD + Malate Malate
NADH oxaloacetate NADH oxaloacetate
DISCUSSION
In the light of our present findings of undetectable levels of succinate dehydrogenase and a very low amount of mitochondria in the deeper foot muscle of C. concholepas, metabolism in this tissue is expected to be essentially anaerobic under all circumstances. The observation that exposure of the animals to anoxic conditions has no effect on foot muscle pyruvate kinase (Carvajal et al., 1988, 1990) is, therefore, not unexpected. The low mitochondrial density also has interesting consequences for the functioning of malate dehydrogenase in the foot muscle of C. concholepas. Even though the significance of the existence of molecular forms of malate dehydrogenase in this tissue is not yet clear, they are expected to be cytosolic enzymes. Moreover, certain trends become clear from a comparison of their kinetic properties. Differences in K~ values for oxaloacetate and malate are probably not too large, but they would be useful for the tissue to handle a wider range of substrate concentrations. Further work is evidently required to examine whether the effect of ~t-ketoglutarate on the enzyme I has any physiological relevance. However, it is clear that even in the presence of considerably high concentrations of ~t-ketoglutarate, a significant malate dehydrogenase activity is expected in the muscle tissue, because only one of the enzymic forms is inhibited by this compound. In this connection, ~t-ketoglutarate inhibition of malate dehydrogenase is not a common finding; one exception is the enzyme from Methanospirillum hungatti (Storer et al., 1981). Finally, the effect of citrate is most probably of no physiological importance because of the considerably high concentrations which are required for significant inhibition of the enzymes. In contrast, citrate has been suggested to be a controlling factor of mitochondrial malate dehydrogenase (Mullinax et al., 1982). Previously, we have shown the absence of lactate dehydrogenase and the presence of octopine dehydrogenase in the deeper foot muscle tissue of C. concholepas (Carvajal and Kessi, 1988). It is now clear that, in addition to octopine dehydrogenase, malate dehydrogenase would also be involved in reoxidation of N A D H generated from glycolysis in this tissue. In comparison with lactate dehydrogenase, malate and octopine dehydrogenases would maintain a lower N A D H / N A D ÷ ratio during anoxia (Fields, 1983), which is evidently advantageous for the essentially anaerobic deeper foot muscle tissue of
C. concholepas.
Table 2. Product inhibition patterns Inhibitor
The results of the product inhibition studies are shown in Table 2. All patterns were found to give linear inhibition.
Pattern
K~s (mM)
Kfi (mM)
C NC NC NC
3.35 2.50 2.30 4.57
5.20 2.45 2.47
C, competitive inhibition; NC, non-competitive inhibition. K~, apparent slope inhibition constant; Kfi, apparent intercept inhibition constant. When used as fixed substrates, concentrations were 1 mM oxaloacetate and 0.1 mM N A D H .
In the absence of phosphoenolpyruvate carboxykinase, aspartate would be the immediate source of the oxaloacetate involved in N A D H reoxidation in the deeper foot muscle tissue of C. concholepas. The required glutamate oxaloacetate transaminase activity was detected in this study. With respect to the metabolic fate of malate, the compound would be transported for metabolization in other tissues, including the adjacent tissues in the foot, in which
86
NELSON CARVAJALet al.
mitochondria were found to be very abundant. Another interesting possibility is malate oxidation in the same deeper foot muscle tissue. Even though malate oxidation was found to be very slow in vitro, significant oxidation would be allowed in vivo by removal of oxaloacetate and NADH via the glutamate oxaloacetate transaminase and the octopine dehydrogenase reactions, respectively. Another interesting conclusion emerges if one considers that octopine oxidation in the deeper foot muscle tissue would be coupled to reoxidation of NADH in the malate dehydrogenase reaction. As discussed previously (Carvajal and Kessi, 1988), octopine synthesis and oxidation in the same tissue are advantageous for retaining the muscle pool of arginine for arginine phosphate synthesis. There is considerable evidence for an ordered Bi Bi kinetic mechanism for malate dehydrogenase from several sources (Crow et al., 1983; Waters et al., 1985). There are, however, some discrepancies in the explanations for the oxaloacetate substrate inhibition of the enzyme. These include formation of binary and ternary complexes (Bernstein et al., 1978) and the lower Vmaxof an alternative pathway which becomes dominant at high concentrations of oxaloacetate (Muller, 1985). Our present results also indicate an Ordered Bi Bi kinetic mechanism for malate dehydrogenase from the foot muscle of C. concholepas. Dead end combination of oxaloacetate to the enzymeNAD ÷ complex, resulting in uncompetitive substrate inhibition, is also indicated by our results. The absence of a slope effect in the substrate inhibition by oxaloacetate excludes a dead end enzyme-oxaloacetate complex. In fact, a slope effect is predicted by dead end combination prior to the binding of the variable substrate NADH (Cleland, 1970). Acknowledgements--This research was supported by Grant 82/87 from FONDECYT and Grant 20.31.31 from the Direccirn de Investigaci6n, Universidad de Concepci6n. REFERENCES
Basaglia F. (1989) Some aspects of isozymes of lactate dehydrogenase, malate dehydrogenase and glucosephosphate isomerase in fish. Comp. Biochem. Physiol. 92B, 213-226. Bemstein L. H., Grisham M. B., Cole K. D. and Everse J. (1978) Substrate inhibition of the mitochondrial and cytoplasmic malate dehydrogenases. J. biol. Chem. 253, 8697-8701. Bolognani Fantin A. M., Bolognani L., Ottaviani E. and Franchini A. (1987) Histochemical research on metabolic pathways of glucose in some species of Mollusca Gastropoda. Acta Histochem. 81, 155-162. Carvajal N., Gonzailez R. and Kessi E. (1988) Enzymatic aspects of phosphoenolpyruvate metabolism in Concholepas concholepas: effect of environmental anoxia. Fifth PAABS Meeting. Villa Giardino-C6rdoba, Argentina. Carvajal N., Gonz~tlezR. and Kessi E. (1990) Tissue-specific regulation of pyruvate kinase during environmental anoxia in Concholepas concholepas (Gastropoda: Muricidae). J. exp. Zool. 225, 280-285. Carvajal N. and Kessi E. (1988) Properties of octopine dehydrogenase from the foot muscle of Concholepas concholepas. Comp. Biochem. Physiol. 90B, 77-79.
Cleland W. W. (1970) Steady state kinetics. In The Enzymes (Edited by Boyer P. D,), 3rd edn, Vol. 2, pp. 1-65. Academic Press, New York. Crow K. E., Braggins T. J., Batt R. D. and Hardman M. J. (1982) Rat liver cytosolic malate dehydrogenase: purification, kinetic properties, role in control of free cytosolic NADH concentrations. Analysis of control of ethanol metabolism using computer simulation. J. bioL Chem. 257, 14,217-14,225. Crow K. E., Braggins T. J. and Hardman M. J. (1983) Human liver cytosolic malate dehydrogenase: purification, kinetic properties and role in ethanol metabolism. Archs Biochem. Biophys. 225, 621-629. Eberlee J. C. and Storey K. B. (1988) Tissue-specific biochemical responses during anoxia and recovery in the channeled whelk. J. exp. mar. Biol. Ecol. 121, 165-176. Fields J. H. A. (1983) Alternatives to lactic acid: possible advantages. J. exp. Zool. 228, 445-457. Forn6s M., Bozal X., Abante J., Cortrs A. and Bozal J. (1987) Comparative analysis of the malate dehydrogenases isolated from the cytosolic fraction of several tissues of guinea-pig Cavia porcellus. Comp. Biochem. Physiol. 86B, 89-94. Gade G. (1983) Energy metabolism of arthropods and mollusks during environmental and functional anaerobiosis. J. exp. Zool. 228, 415-429. Holwerda D. A., Veldhuizen-Tsoerkan M., Veenhof P. R. and Evers E. (1989) In vivo and in vitro studies on the pathway of modification of mussel pyruvate kinase. Comp. Biochem. Physiol. 92B, 375-380. Michaelidis B., Gaitanaki C. and Beis Is. (1988) Modification of pyruvate kinase from the foot muscle of Patella caerulea (L) during anaerobiosis. J. exp. Zool. 248, 264-271. Muller J. (1985) Binary and ternary complexes of malate dehydrogenases with substrates and substrate analogs. Biochim. biophys. Acta 830, 95-100. Mullinax T. R., Mock J. N,, McEvily A. J. and Harrison J. H. (1982) Regulation of mitochondrial malate dehydrogenase. Evidence for an allosteric citrate binding site. J. biol. Chem. 257, 13,233-13,239. Ohshima T. and Sakuraba H. (1986) Purification and characterization of malate dehydrogenase from the phototrophic bacterium Rhodopseudomonas capsulata. Biochim. biophys. Acta 869, 171-177. Paynter K. T., Karam of G. A., Ellis L. L. and Bishop S. H. (1985) Subcellular distribution of aminotransferases, and pyruvate branch point enzymes in gill tissue from four bivalves. Comp. Biochem. Physiol. 82B, 129-132. Plaxton W. C. and Storey K. B. (1985) Purification and properties of aerobic and anoxic forms of pyruvate kinase from the hepatopancreas of the channelled whelk, Busycotypus canaliculatum. Archs. Biochem. Biophys. 2,43, 195-205. Storer A. C., Sprot G. D. and Martin W. G. (1981) Kinetic and physical properties of the L-malate-NAD+ oxidoreductase from Methanospirillum hungatii and comparison with the enzyme from other sources. Biochem. J. 193, 235-244. Swift M. L. and Lakshmanan S. (1982) Isolation and partial characterization of a malate dehydrogenase from Crassostrea virginica. J. Shellfish Res. 3, 102. Veeger C., Der Vartanian D. V. and Zeylemaker W. P. (1969) Succinic dehydrogenase. In Methods Enzymol (Edited by Lowenstein J. M.), Vol XIII, pp. 81-90. Academic Press. Waters J. K., Karr D. B. and Emerich D. W. (1985) Malate dehydrogenase from Rhizobium japonicus 3I lb-143 bacteroids and Glycine max root-nodule mitochondria. Biochemistry 2,4, 6479-6486.
0305-0491/91 $3.00+ 0.00 © 1991 PergamonPress plc
PROPERTIES ANI~ POSSIBLE ROLE OF MALATE DEHYDROGENASE I IN THE FOOT MUSCLE OF THE SEA MOLLUSC CONCHOLEPAS CONCHOLEPAS (GASTROPODA: MURICIDAE) NELSON CARVAJAL,EDUARDOKESSI,* CLAUD10TORRES,OSCARMAR~N,VIVIANATORRES,'~ ALEJANDROCAMPOSand CLAUDIAPOSADA Departamento de Biologia Molecular, Facultad de Ciencias Biol6gicas y de Recursos Naturales, Universidad de Concepci6n, Casilla 2407, Concepci6n, Chile (Received 3 October 1990) Abstract--1. Very few mitochondria, undetectable levels of succinate dehydrogenase and glutamate dehydrogenase and significant activities of glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and malate dehydrogenase were observed in the deeper foot muscle tissue of Concholepas concholepas. 2. Malate dehydrogenase activity was resolved into two molecular forms, which differ in K~ values for oxaloacetate and malate, and sensitivity to inhibition by ~-ketoglutarate. 3. In addition to reoxidation of NADH generated from glycolysis, the function of malate dehydrogenase is discussed in connection with octopine synthesis and oxidation in the foot muscle of C. concholepas. 4. The results of initial rate and product inhibition studies of both forms of malate dehydrogenase are consistent with an Ordered Bi Bi kinetic mechanism, with NADH adding first and NAD ÷ leaving last. Oxaloacetate substrate inhibition is suggested to result from formation of an enzyme-NAD+-oxaloacetate dead end complex.
through the phosphoenolpyruvate carboxykinase reaction to the succinate pathway of glycogen degradation. Anoxia induced changes have been shown to occur in pyruvate kinase from several marine molluscs and the effect is believed to play an important role in the adaptation of these animals to anoxic conditions (Plaxton and Storey, 1985; Michaelidis et al., 1988; Holwerda et al., 1989). Recently we have shown that pyruvate kinase from the deeper foot muscle of Concholepas concholepas is not altered when the animals are exposed to prolonged environmental anoxia. Dramatic changes were, however, observed in the enzyme from the heart tissue of the mollusc. Moreover, that a glycogen-succinate pathway does not operate in the deeper foot muscle, was indicated by the absence of phosphoenolpyruvate carboxykinase activity in this tissue (Carvajal, 1988, 1990). Continuing with our studies, we have now turned our attention to the enzymatic aspects of aspartate metabolism in the deeper foot muscle tissue of C. concholepas. Data in this report show a significant enzymatic capacity for conversion of aspartate to malate but not to succinate in this tissue. The properties of two molecular forms of malate dehydrogenase are described and their possible functions are discussed.
INTRODUCTION
While malate dehydrogenase (EC 1.1.1.37) has been isolated and extensively studied in many species (Storer et al., 1981; Mullinax et al., 1982; Waters et aL, 1985; Ohshima and Sakuraba, 1986; Forn6s et al., 1987; Basaglia, 1989), knowledge of the enzyme from molluscan tissues is very limited (Swift and Lakshmanan, 1982; Paynter et al., 1985; Bolognani Fantin et al., 1987). One specially documented aspect is the presence of cytosolic and mitochondrial forms of malate dehydrogenase in tissues of many vertebrates and invertebrates (Basaglia, 1989). In addition to the well known roles of malate dehydrogenase in the tricarboxylic acid cycle and the exchange of reducing equivalents between the cytosol and the mitochondria (Crow et aL, 1982, 1983; Forn6s et aL, 1987), the enzyme is involved in the glycogen-succinate and aspartate-succinate pathways, which operate in several anoxia-tolerant molluscs (Gade, 1983). This is important because glycogen and aspartate are known to be the fuels and succinate and alanine the major end products of anaerobic metabolism in these species (Gade, 1983; Plaxton and Storey, 1985; Eberlee and Storey, 1988). Briefly, at the early stages of anoxia, there is a coupled conversion of glycogen to alanine and aspartate to succinate; when the aspartate reserves are exhausted, phosphoenolpyruvate is channelled
MATERIALS AND METHODS
Animals and chemicals Specimens of C. concholepas were collected by local fishermen from the Bay of Concepci6n. All chemicals were obtained from commercial sources (most from Sigma Chem.
*Present address: Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile. ?Present address: Department of Physiology and Endocrinology, Medical College, Georgia, USA.
Co.) and were the purest available. 83
84
NELSON CARVAJALet al.
Tissue content of the enzymes Freshly excised sections of the deeper foot muscle tissue were minced and then homogenized using five 20 sec bursts at high speed in a Virtis tissue grinder. Homogenates (25%, w/v) were prepared in 10 mM Tris-HC1 (pH 7.4) containing 1 mM EDTA, 1 mM 2-mercaptoethanol and 0.1 mM phenylmethylsulfonyl fluoride. All the enzyme activities were assayed at pH 7.4 and 25°C, by following the changes of absorbance at 340 nm. Glutamate pyruvate transaminase and glutamate oxaloacetate transaminase were determined in coupled assays with lactate dehydrogenase and malate dehydrogenase, respectively. Glutamate dehydrogenase activity was assayed both in the direction of ~-ketoglutarate and glutamate formation. Succinate dehydrogenase activity was assayed by the spectrophotometric method with ferricyanide (Veeger et al., 1969). The malate dehydrogenase reaction in the direction of oxaloacetate reduction was followed in a medium containing 50 mM Tris-HC1 (pH 7.5), 1.0 mM oxaloacetate and 0.2 mM NADH; for the analysis of the reverse reaction, substrate concentrations were 10 mM malate and 0.4 mM NAD +. Kinetic mechanism of malate dehydrogenase For the analysis of the kinetic mechanism of the malate dehydrogenase reaction, initial velocities in the direction of oxaloacetate reduction were determined at 25°C in 50 mM glycine-NaOH buffer (pH 9.0). Duplicate rate measurements differed by less than 2%. Kinetic data were analyzed by double reciprocal plots and computer-fitted to the appropriate rate equation by means of the least-squares method, assuming equal variances for the velocities. The nomenclature used in this paper is that of Cleland (1970) and initial velocity data yielding intersecting patterns, competitive inhibition and non-competitive inhibition were fitted to equations (1), (2) and (3), respectively. v = VAB/[K,aKb + KaB + KbA + AB]
(1)
v = VA/[Ka (1 + I/Kgs) + A]
(2)
v = VA/[Ka(1 + I / K ~ s ) + A (1 +I/K,i)].
(3)
Electron microscopy Small pieces of muscle tissue from the external edge and the internal zones of the foot of C. concholepas were dissected and examined by electron microscopy using standard techniques following glutaraldehyde fixation.
RESULTS Enzyme activities and electron microscopy o f the f o o t
As determined in the direction of oxaloacetate reduction, and at pH 7.4, the malate dehydrogenase activity was 18.4 _ 1.2 units/g (wet wt) of deeper foot muscle tissue. Activities of glutamate pyruvate transaminase and glutamate oxaloacetate transaminase were 1.3 + 0.2 and 1.1 _ 0.1 units/g (wet wt) of tissue, respectively. Values are mean + SD of determinations in three animals. Succinate dehydrogenase and glutamate dehydrogenase were absent or too low to be detected in the deeper foot muscle tissue. Moreover, electron microscopy studies revealed that mitochondria in this tissue are very scarce and contain very little cristae (Fig. 1). In contrast, numerous enlarged mitochondria with fine and compacted cristae were observed in the smooth muscle tissue which is immediately below the surface epithelium. Separation and properties o f two molecular f o r m s o f malate dehydrogenase
Malate dehydrogenase activity was partially purified by a procedure involving the following steps: (a) fractionation of the homogenate with a m m o n i u m sulfate (30~55% saturation); (b) chromatography on a phenyl-Sepharose column equilibrated with homogenizing buffer containing 1.5 M a m m o n i u m sulfate and eluted with a linear gradient of a m m o n i u m
Fig. 1. Electron micrograph of the deeper foot muscle tissue of Coneholepas concholepas. Magnification 5720 x. Typical mitochondria (m) in this tissue are indicated.
85
M a l a t e d e h y d r o g e n a s e in m o l l u s c f o o t m u s c l e Table 1. Apparent K,~ values (mM) of malate dehydrogenases Substrate
pH
Enzyme I
Enzyme II
Oxalaeetate NADH Malate NAD ÷
7.4 7.4 9.0 9.0
1.00 0.03 1.40 0.14
0.15 0.04 6.45 0.21
When used as fixed substrates, concentrations were I mM oxaloacetate, 0.2 mM N A D H , 10 mM malate and 0.4 mM N A D ÷.
sulfate (1.5 M to 0); (c) chromatography on D E A E cellulose equilibrated with homogenizing buffer and eluted with a linear gradient of KC1 (0-1 M). Two active fractions were eluted from the DEAE-cellulose column. About 65% of the enzyme activity eluted with the washing buffer (enzyme I) and the remaining 35% (enzyme II) eluted in the salt gradient at a concentration of about 0.1 M KC1. Malate dehydrogenase activity of both enzyme forms was maximal at pH 8.9-9.2. At all pH values examined (7.5-10), both molecular forms exhibited Michaelis-Menten kinetics. To gain some understanding of the functioning of the enzyme in vivo, kinetic studies were performed at pH 7.4. At this pH value, the reaction in the direction of oxaloacetate reduction was clearly detected, but the reverse reaction was very slow. The reaction in the direction of malate oxidation was, therefore, examined at pH 9.0. Calculated apparent Kmvalues for the substrates are those given in Table 1. Substrate inhibition of both enzymic forms was observed at concentrations of oxaloacetate higher than 5 mM. Of several compounds tested, ct-ketoglutarate was found to be inhibitory, but only to the enzyme I. A 50% inhibition of enzyme I was caused by about 3.5 mM ct-ketoglutarate. Citrate was slightly inhibitory to both enzymic forms, with concentrations higher than 40 mM being required for 50% inhibition of the enzymes.
Kinetic mechanism of malate dehydrogenase For the analysis of the kinetic mechanism of the enzyme, initial velocity and product inhibition studies were performed at pH 9.0. Results described here were those specifically obtained with the enzyme I; in any case, kinetic patterns were the same for both enzyme forms. All the lines of double reciprocal plots intersected to the left of the 1Iv axis and substrate inhibition was clearly observed at concentrations of oxaloacetate higher than 5 mM, the inhibition being uncompetitive with respect to NADH. Secondary plots of the data at non-inhibitory levels of oxaloacetate were linear. Kinetic parameters calculated from these plots were: Ka = 0.027 raM; Kia = 0.016 mM; Kb = 0.43 mM.
Varied substrate
NAD + NAD + Malate Malate
NADH oxaloacetate NADH oxaloacetate
DISCUSSION
In the light of our present findings of undetectable levels of succinate dehydrogenase and a very low amount of mitochondria in the deeper foot muscle of C. concholepas, metabolism in this tissue is expected to be essentially anaerobic under all circumstances. The observation that exposure of the animals to anoxic conditions has no effect on foot muscle pyruvate kinase (Carvajal et al., 1988, 1990) is, therefore, not unexpected. The low mitochondrial density also has interesting consequences for the functioning of malate dehydrogenase in the foot muscle of C. concholepas. Even though the significance of the existence of molecular forms of malate dehydrogenase in this tissue is not yet clear, they are expected to be cytosolic enzymes. Moreover, certain trends become clear from a comparison of their kinetic properties. Differences in K~ values for oxaloacetate and malate are probably not too large, but they would be useful for the tissue to handle a wider range of substrate concentrations. Further work is evidently required to examine whether the effect of ~t-ketoglutarate on the enzyme I has any physiological relevance. However, it is clear that even in the presence of considerably high concentrations of ~t-ketoglutarate, a significant malate dehydrogenase activity is expected in the muscle tissue, because only one of the enzymic forms is inhibited by this compound. In this connection, ~t-ketoglutarate inhibition of malate dehydrogenase is not a common finding; one exception is the enzyme from Methanospirillum hungatti (Storer et al., 1981). Finally, the effect of citrate is most probably of no physiological importance because of the considerably high concentrations which are required for significant inhibition of the enzymes. In contrast, citrate has been suggested to be a controlling factor of mitochondrial malate dehydrogenase (Mullinax et al., 1982). Previously, we have shown the absence of lactate dehydrogenase and the presence of octopine dehydrogenase in the deeper foot muscle tissue of C. concholepas (Carvajal and Kessi, 1988). It is now clear that, in addition to octopine dehydrogenase, malate dehydrogenase would also be involved in reoxidation of N A D H generated from glycolysis in this tissue. In comparison with lactate dehydrogenase, malate and octopine dehydrogenases would maintain a lower N A D H / N A D ÷ ratio during anoxia (Fields, 1983), which is evidently advantageous for the essentially anaerobic deeper foot muscle tissue of
C. concholepas.
Table 2. Product inhibition patterns Inhibitor
The results of the product inhibition studies are shown in Table 2. All patterns were found to give linear inhibition.
Pattern
K~s (mM)
Kfi (mM)
C NC NC NC
3.35 2.50 2.30 4.57
5.20 2.45 2.47
C, competitive inhibition; NC, non-competitive inhibition. K~, apparent slope inhibition constant; Kfi, apparent intercept inhibition constant. When used as fixed substrates, concentrations were 1 mM oxaloacetate and 0.1 mM N A D H .
In the absence of phosphoenolpyruvate carboxykinase, aspartate would be the immediate source of the oxaloacetate involved in N A D H reoxidation in the deeper foot muscle tissue of C. concholepas. The required glutamate oxaloacetate transaminase activity was detected in this study. With respect to the metabolic fate of malate, the compound would be transported for metabolization in other tissues, including the adjacent tissues in the foot, in which
86
NELSON CARVAJALet al.
mitochondria were found to be very abundant. Another interesting possibility is malate oxidation in the same deeper foot muscle tissue. Even though malate oxidation was found to be very slow in vitro, significant oxidation would be allowed in vivo by removal of oxaloacetate and NADH via the glutamate oxaloacetate transaminase and the octopine dehydrogenase reactions, respectively. Another interesting conclusion emerges if one considers that octopine oxidation in the deeper foot muscle tissue would be coupled to reoxidation of NADH in the malate dehydrogenase reaction. As discussed previously (Carvajal and Kessi, 1988), octopine synthesis and oxidation in the same tissue are advantageous for retaining the muscle pool of arginine for arginine phosphate synthesis. There is considerable evidence for an ordered Bi Bi kinetic mechanism for malate dehydrogenase from several sources (Crow et al., 1983; Waters et al., 1985). There are, however, some discrepancies in the explanations for the oxaloacetate substrate inhibition of the enzyme. These include formation of binary and ternary complexes (Bernstein et al., 1978) and the lower Vmaxof an alternative pathway which becomes dominant at high concentrations of oxaloacetate (Muller, 1985). Our present results also indicate an Ordered Bi Bi kinetic mechanism for malate dehydrogenase from the foot muscle of C. concholepas. Dead end combination of oxaloacetate to the enzymeNAD ÷ complex, resulting in uncompetitive substrate inhibition, is also indicated by our results. The absence of a slope effect in the substrate inhibition by oxaloacetate excludes a dead end enzyme-oxaloacetate complex. In fact, a slope effect is predicted by dead end combination prior to the binding of the variable substrate NADH (Cleland, 1970). Acknowledgements--This research was supported by Grant 82/87 from FONDECYT and Grant 20.31.31 from the Direccirn de Investigaci6n, Universidad de Concepci6n. REFERENCES
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