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Glutamate dehydrogenase of the shrimp Crangon crangon L. effects of shrimp weight and season upon its activity in the oxidative and reductive function
Comp. Biochem. Physiol. Vol. 86B, No. 3, pp. 525-530, 1987 Printed in Great Britain
0305-0491/87 $3.00+0.00 © 1987 Pergamon Journals Ltd
GLUTAMATE DEHYDROGENASE OF THE SHRIMP CRANGON CRANGON L. EFFECTS OF SHRIMP W E I G H T AND SEASON UPON ITS ACTIVITY IN THE OXIDATIVE AND REDUCTIVE F U N C T I O N MICHELE REGNAULT and YVES BATREL Station Biologique, CNRS and Univ. P. & M. Curie (Paris 6), F-29211 Roscoff, France and Lab. Biologie Marine, Coll6ge de France, F-29110 Concarneau, France (Received 21 April 1986)
Abstraet--l. Enzymatic activity of GDH of C. crangon was measured in the reductive and oxidative function in crude homogenates of winter shrimp and summer shrimp. 2. The specific activity of the NAD-dependent GDH was /n vitro significantly (P = 0.01) higher (30-40%) than the specific activity of the NADH-dependent GDH. 3. Changes in GDH specific activity and in the enzyme activity of the whole shrimp were studied as a function of shrimp weight and of season. GDH activity in the reductive function (glutamate synthesis) appeared to be relatively independent on these factors but GDH activity in the reverse function (glutamate deamination) varied. 4. Seasonal variations of the NAD-dependent GDH are discussed in terms of environmental factors and the metabolic level of shrimp. It is suggested that they could be explained in terms of the control of the energy charge.
INTRODUCTION In crustaceans, glutamate dehydrogenase (GDH) has received considerable attention with respect to its role in osmoregulation. Through glutamate synthesis, the enzyme contributes to the regulation of the pool of intracellular amino-acids during salinity changes of the external medium (Schoffeniels, 1964, 1966; Schoffeniels and Gilles, 1970). It was also suggested that G D H would favour the amino-acid storage into protein--especially haemocyanin--when the animal was transferred into a hypoosmotic medium (Schoffeniels, 1976). The reaction catalyzed by the G D H is indicated by the equation (1) below: glutamate + N A D ÷ ~-~~-ketoglutarate + N A D H + NH4 +. Thus, only one role (the reductive function) was considered. The reverse function (oxidative deamination of glutamate and ammonia formation) was scarcely studied as the previous authors reported that its activity, in vitro, was either insignificant or non-existent (Schoffeniels, 1965) and that in vivo the oxido-reduction potential values would strongly oppose this function (Schoffeniels, 1976; 1984). However, Chaplin et al. (1965) succeeded in recording the activity of G D H of Carcinus maenas in both funcitons, even though the KM values, obtained by these authors for the substrates, demonstrated a higher affinity of G D H for ~-ketoglutarate than for glutamate. Later, a relationship between the activity level of the NAD-dependent G D H and the ammonia excretion rate was observed by Bidigare and King (1981) in a mysid, Praunus flexuosus, and by Batrel and Regnault (1985) in a shrimp, Crangon
crangon, thus suggesting a possible role of the G D H in ammoniogenesis. In marine invertebrates, few studies have dealt with the G D H activity in its twin functions. However, the activity ratio (glutamate synthesis:glutamate deamination) showed that the enzymatic activity when in the reductive function was usually higher than when in the oxidative function (Reiss et aL, 1977; Male and Storey, 1983) except for the squid, Loligo pealeii, in which an activity ratio = 1:1 was found (Storey et al., 1978). From our previous study (Batrel and Regnault, 1985), it appeared that the specific activity of the NAD-dependent G D H was much higher in C. crangon than quoted for other marine invertebrates in the literature. The aim of the present study was to .compare the specific activities of the NADH-dependent and NAD-dependent G D H of the shrimp Crangon crangon. Furthermore, as the activity level of the NAD-dependent G D H of this species was observed to vary during the year, we have looked for any seasonal changes in the G D H activity level.
MATERIALAND METHODS Animals Shrimp were collected in the Penze estuary (N. Brittany) then held in the laboratory in running sea-water for a week prior to enzymatic measurements. During this period they were fed daily. The shrimp size was selected according to the prevailing size-group at each sampling period. Experiments were run in February 1985 (sea-water T = 8-10°) and in August 1985 (17-18°C). Particular moult stages were not controlled but freshly moulted shrimp were discarded.
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MICH~LE REGNAULT a n d YVES BATREL
Extraction procedure The enzyme activity was determined on shrimp groups (4-5 shrimps/group); only 4 groups or samples were analysed daily as the enzyme activity of the extract was constant for only 1 or 2 hr. Shrimp were gently dried on filter paper and weighed (Mettler: 0.01 mg), then they were sacrificed, cut into several pieces and homogenized in an ice-cold solution containing 50 mM imidazole-HC1 buffer, pH: 7.6, 0.5mM phenylmethyl sulfonyl fluoride (PMSF), 0.1% n-cetyl trimethylammonium bromide, 0.5 mM dithiothreitol (Clealand's reagent: DTT). Homogenization and centrifugation were carried out as previously described (Batrel and Regnault, 1985). Enzyme assays GDH activity was determined from the reduction of NAD + into NADH (oxidative function of GDH) or the oxidation of NADH into NAD + (reductive function of GDH). For the NAD-dependent GDH the standard assay mixture was 42mM imidazole-HCl buffer, pH: 8.55, 0.5mM NAD +, 33 mM glutamate; volume of shrimp extract=250/zl (final volume: 3ml). The reaction was initiated by the addition of glutamate. For the NADHdependent GDH the standard assay mixture was 42mM imidazole-HC1 buffer, pH: 7.65, 0.08 mM ~-ketoglutarate, 280mM NH4 acetate; volume of shrimp extract = 250 #1 (final volume: 3 ml). These optimal conditions were defined after the study of the characteristics of the enzyme (see Results). The reaction was initiated by the addition of -ketoglutarate. Both assays were subsequently recorded for each individual sample. Increase or decrease of NADH concentration was recorded at 340 nm and 20°C using a double-beamed spectrophotometer (SP 1700 PYE UNICAM). Supernatant protein was estimated by Bradford's (1976) method using BSA as a reference standard. Chemicals were obtained from Sigma (NAD, glutamate) and Boehringer (NADH, ADP, ~-ketoglutarate). Results are expressed as international unit [U.I. =/tmol NADH (formed or oxidized), rain -I] (Kaufman, 1955). Sample activity is expressed as U.I. g i fresh weight and U . I . mg -] protein.
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RESULTS i
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L Kinetic characteristics o f the GDH of C. crangon The kinetic characteristics of the N A D - d e p e n d e n t G D H of C. crangon have been described previously (Batrel and Regnault, 1985) and are briefly summarized here. The optimal p H for the oxidative function ranged between 8.18-8.20 using an imidazole-HCl buffer (pH: 8.55). The cofactor ( N A D ÷) dependence of G D H presented no linear relationship as two plateaux of enzyme saturation were recorded for N A D concentrations ranging from 0 to 1.3 mM. The enzyme saturation by substrate was obtained with 24 m M glutamate and an inhibition of G D H activity by an excess of substrate was observed for glutamate concentrations higher than 50 mM. The kinetic characteristics of the N A D H dependent G D H of C. crangon, undescribed until now, are shown in Fig. 1. The optimal conditions of the standard assay were obtained using an imidazole-HC1 buffer (pH = 7.6-7.7). The cofactor dependence of G D H in the reductive function differed from that in the oxidative function. The enzyme saturation was obtained with 0.05raM N A D H when the first plateau of enzyme saturation
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527
GDH activity in Crangon crangon L. was obtained with 0.5 mM N A D +. Furthermore, an inhibition of the enzyme activity was observed for N A D H concentrations higher than 0.15 mM when no inhibition appeared for N A D concentrations ranging from 0 to 1.4mM. The enzyme saturation by ~-ketoglutarate was obtained with 0.35 mM concentrations of substrate thus much more quickly than with glutamate. The higher affinity of the G D H of C. crangon to ~t-ketoglutarate (KM = 1.43 mM) than to glutamate (KM = 5.16 mM) was in agreement with the data of Chaplin et al. (1965) in Carcinus maenas. An inhibition of G D H activity by an excess of substrate was observed for ~-ketoglutarate concentrations higher than 12mM. For the other substrate of the NADH-dependent G D H (NH4+), ammonium acetate was used rather than ammonium chloride, following observations of Male and Storey (1983). High concentrations of ammonium salt (250mM minimum) were necessary to obtain the enzyme saturation and no inhibition by an excess of substrate was observed in the experimental range of concentrations (from 0 to 300 mM). This corroborates the weak G D H affinity for ammonium ions (Chapman and Atkinson, 1973). IL Activity o f the GDH o f C. crangon The G D H activity in the reductive function (A1) and in the oxidative function (A2) were measured simultaneously in crude homogenates of shrimp. Activity measurements were performed on winter animals (February) and summer animals (August). In February (T = 8-10°C), the mean value of the G D H specific activity (X +- S.E.M., N = 24 shrimp groups) was 0.565 +_ 0.030 U.I. g ~ fresh wt and 0.0140 + 0.0009 U.I. mg -~ protein for A1 (glutamate synthesis), 0.736 +- 0.024 U.I. g - l fresh wt and 0.0182 +- 0.0006 U.I. mg ] protein for A2 (glutamate deamination). In August (T = 17-18°C), the mean value (n = 27) of the G D H specific activity was respectively 0.606 +__0.033 and 0.0137 +_ 0.0008 for A1, 0.863 __+0.026 and 0.0196 +- 0.0007 for A2. The A1 activity was clearly distinct from the A2 activity (Fig. 2). From the Student's t-test both activities differed from each other with a 99% prob-
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Fig. 2. Specific activity of the GDH of C. crangon. A1 = activity of the NADH-dependent GDH; A2 = activity of the NAD-dependent GDH; mean values +_SEM (plain vertical bars); the confidence interval with a 99% probability is figured by the dotted vertical bars. Winter: n = 24; summer: n = 27.
ability whatever was the season and the expression of results (fresh weight or protein). Under the present experimental conditions, the G D H of C. erangon was 1.3 or 1.4 fold more active in the oxidative function than in the reductive one, in winter and summer, respectively. Ill. Relationship between GDH activity and shrimp weight The mean fresh weight of shrimp studied in summer (364.07+_17.30mg) was distinct with a 95% probability from that of shrimp studied in winter (439.75 +_ 11.32 rag). This change in weight is related to the biological cycle of this species: recruitment in April-May, high growth during summer then reproduction and low growth in winter (Regnault, 1977). A weak and negative correlation was observed between the G D H specific activity and shrimp weight. The G D H activity of whole shrimp was calculated as the specific activity was measured on crude homogenates of whole shrimp. The mean value (A' = U.I. shrimp -]) was 0.246 +_ 0.014 for A I ' and 0.323+_0.013 for A2' in winter ( n = 2 4 ) , 0.216 +_ 0.014 and 0.309 -t- 0.014 respectively in summer (n = 27). The G D H activity of whole shrimp was also significantly higher (with a 99% probability) in the oxidative function than in the reductive function. However, the correlation between the A I ' and A2' activities as shown by the following regression equations: A2' = 0.493 A I ' + 0.202
r = 0.516 (in summer)
A2' = 0.306 A I ' + 0.248
r = 0.321 (in winter)
was significant (P = 0.01) only in summer. In contrast to the G D H specific activity, the enzymatic activity of the whole shrimp was positively and strongly correlated with the shrimp weight, except for A I ' in winter (Fig. 3). IV. Seasonal changes in GDH activity The specific activity of G D H in the reductive function (AI) did not vary significantly with season as shown in Fig. 2, but activity in the reverse function (A2) was significantly (P = 0.05) higher in summer than in winter. Here, a 17% increase of the mean specific activity A2 was observed in summer. The distinct responses of the two functions of the G D H to seasonal differences explains the change in the activity ratio (A1 :A2) noted above. As the G D H activity of the whole shrimp was strongly correlated with shrimp weight, as far as A2' was concerned, it was of interest to know to what extent the weight effect interacted with the seasonal effect. Figure 4 shows that for any selected shrimp weight, the summer activity of G D H in the oxidative function was 13% higher than the winter activity. Thus, the weight effect poorly contributed to the seasonal change in activity as it was reducing rather than increasing it. On the other hand, we noticed that the activation of the NAD-dependent G D H by the adenosine 5'-diphosphate (ADP) was more pronounced in winter than in summer (Fig. 5). In both cases, the optimal activation was obtained by the addition of 1.5 #mol
MICH~LE REONAULT and YVES BATREL
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DISCUSSION
Most studies on glutamate dehydrogenase have been focused on its role in glutamate synthesis as the enzyme activity in this function was strongly prevailing over its activity in the reverse function (glutamate deamination). In vertebrates and marine invertebrates, the activity ratio (glutamate synthesis:glutamate deamination) was usually much higher than 1, as shown by the following examples-A1 :A2 = 10:1 in the rat (McGivan and Chappell, 1976), 4:1 in the eel (Hayashi et al., 1982), 10:1 in the sea anemone Anthopleura xanthogrammica (Male and Storey, 1983), 6.5 : 1 in the mussel Modiolus demissus (Reiss et al., 1977). Furthermore, synthesis is favoured by the thermodynamic laws, as mentioned earlier (see Introduction). The kinetic characteristics of the GDH of Crangon crangon as defined in this study support this opinion. In vitro and under optimal conditions the NADHdependent GDH required cofactor concentrations 10 fold lower than those required by the NAD-dependent GDH; the GDH affinity to
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Fig. 3. Relationship between the GDH activity of the whole shrimp and shrimp weight, in summer (A) and winter (B). (AI') GDH activity in the reductive function (glutamate synthesis), (A2') GDH activity in the oxidative function (glutamate deamination).
of ADP in the standard assay (same experimental conditions). The ADP activation represented a 26% increase of enzyme activity in summer and a 70% increase in winter. The activity increase appeared especially marked as the initial value (without ADP addition) of the enzyme activity was low. However, the optimal enzyme activity resulting from ADP activation was rcmarkedly independent of the activity initial level and of season.
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GDH activity in Crangon crangon L. ct-ketoglutarate was much higher than to glutamate as shown by the respective KM values. However, in these optimal conditions, the specific activity of the GDH of C. crangon was higher for glutamate deamination than for glutamate synthesis. The statistical treatment of our data allows us to state that in C. crangon the mean activity of G D H in the oxidative function is significantly higher (30--40%) than in the reverse function. However, if the value of the activity ratio was actually around 0.7 on account of the mean activity values, this ratio value was not systematically obtained for each sample. Thus, it would be better to say that in vitro the GDH of C. crangon is equally efficient to catalyze oxidation and synthesis of glutamate. It is possible that in vivo the G D H did not find requisite conditions to express its potential activity in both functions. However, it is worth noting that, except for the squid Loligo pealeii in which a 1 : 1 activity ratio was found (Storey et al., 1978), all species studied to date have shown a G D H activity ratio equal or inferior to 1, and all such activities have been measured in vitro. The variations of G D H activity of C. crangon as a function of shrimp weight and season are especially interesting. As for all metabolic functions, the specific activity of GDH was negatively correlated with shrimp weight and the GDH activity of the whole shrimp was positively weight-dependent. The last correlation was very significant in summer and winter for A2 but only in summer for AI (P = 0.05 in all cases) (Fig. 3). Curiously, the G D H activity in the reductive function was not influenced by shrimp weight in winter. Furthermore, the GDH reductive activity was constant in both seasons whilst the G D H deaminative activity was significantly higher in summer. Our previous values obtained in June (18°C) for the specific A2 activity were markedly higher (l.313+_0.053U.I.g -1 fresh wt, 0.032_+ 0.020 U.I. mg -1 protein) (Batrel and Regnault, 1985) which suggests that the seasonal variation of the A2 activity could be more pronounced than that exhibited in this study. In fact, it seems that the optimal activity of the G D H in the oxidative function would occur early in the summer and that the A2 activity would already be declining at the end of summer. Seasonal variations of A2 activity call for some comments. The different seasonal activity levels of the NADdependent G D H of Crangon crangon were not the result of changes in shrimp weight as we found that this factor would reduce rather than increase such variability. From examination of the summer values of A2 activity recorded at 18°C in June and August, respectively, it is also unlikely that the temperature of the environmental medium was responsible for the seasonal level of this activity. Furthermore, Batrel and LeGal (1984) observed that the Vmax for the NAD-dependent G D H of Arenicola marina acclimated at 9°C was constant from 10 to 30°C. On the other hand, as the specific activity of the NADdependent G D H is strongly influenced by the nutritional level of shrimp, one could suggest that the A2 seasonal variation was produced indirectly by the trophic conditions which characterized the 2 envisaged seasons. However, this possible effect could have been lessened by the acclimatization week im-
529
posed on the shrimp prior to enzymatic measurements. Finally, it seems much more likely that seasonal variations of A2 are directly related to the metabolic level of shrimp. In this species, high moult frequency and growth are observed in spring and summer periods but not in the winter period. This is corroborated by the corresponding metabolic rates. The mean ammonia excretion rate of shrimp of weight similar to those of this study, ranged from 1.10 to 1.40 #mol NH3 • g-~ fresh wthr -~ in summer to 0.520-0.750~mol NH3 in winter (Regnault, 1983a; 1984). The mean 02 consumption rate indicated parallel variations, 470-520#1 02 "g-~ fresh wthr -~ and 150-200/~1 02 in summer and winter respectively (Regnault, 1983b; Regnault and Lagardere, 1983). Thus, the energy requirements of shrimp are correspondingly higher in summer than in winter. As GDH via the glutamate deamination was proposed as an ATP regenerating system (Matsushima and Kado, 1983; Campbell et al., 1983), an activity increase of the NAD-dependent GDH when energy expenditures are high would agree with Atkinson's (1968) theory of the energy charge of the adenylate pool. From the activation curves of NADdependent GDH by ADP (Fig. 5) it is suggested that the high summer activity of A2 in C. crangon would result from high in vivo ADP concentrations and conversely that its low winter activity would be the result of low in vivo ADP concentrations. Thus, the seasonal variation of GDH oxidative activity could be explained in terms of the regulation of the energy charge. In Crangon crangon, the GDH responses to weight and season factors were different depending on the enzyme function. The relative independence of the enzyme reductive activity to shrimp weight and season is in accord with its key-role in osmoregulation and amino-acid storage. In the same way, the constant reductive activity level of GDH explains why, as yet, most studies have been devoted to it in this role. In contrast, the variability of the enzyme activity in the reverse deaminative function according to shrimp weight, season and possibly energy charge, leads to a consideration of the NAD-dependent GDH as a regulatory system. Such a role was previously suggested by Storey et al. (1978). According to these authors, the primary role of GDH would be in the supply of energy via the glutamate oxidation in order to restore the adenylate pool when depleted by intense metabolic activity. On the other hand, another possible role of GDH, in ammoniogenesis of C. crangon, was proposed (Batrel and Regnault, 1985). The variability of the A2 activity here supports our previous reserve that the GDH could not constantly take over the ammonia excretion of this species. Other metabolic pathways for ammoniogenesis might possibly be needed at some periods of the year or the biological cycle of this species.
Acknowledgements--The authors wish to thank Dr Roger
Uglow (University of Hull) and Dr Lars Hagerman (University of Copenhagen) for their critical comments on the manuscript.
530
MICHI~LE REGNAULT and YVES BATREL REFERENCES
Atkinson D. E. (1968) The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7, 4030-4034. Barrel Y. and LeGal Y. (1984) Nitrogen metabolism in Arenicola marina. Characterization of a NAD-dependent glutamate dehydrogenase. Comp. Biochem. Physiol. 78B, 119-124. Barrel Y. and Regnault M. (1985) Metabolic pathways of ammoniogenesis in the shrimp Crangon crangon L.: Possible role of glutamate dehydrogenase. Comp. Biochem. Physiol. 82B, 217-222. Bidigare R. R. and King F. D. (1981) The measurement of glutamate dehydrogenase activity in Praunus flexuosus and its role in the regulation of ammonium excretion. Comp. Biochem. Physiol. 70B, 409-413. Bradford M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analyst. Biochem. 72, 248-254. Campbell J. W., Aster P. L. and Vorhaben J. E. (1983) Mitochondrial ammoniogenesis in liver of channel catfish Ictalurus punctatus. Am. J. Physiol. 244, R709-R717. Chaplin A. E., Huggins A. K. and Munday K. A. (1965) Ionic effects on glutamate dehydrogenase activity from beef liver, lobster muscle and crab muscle. Comp. Biochem. Physiol. 16, 49-62. Chapman A. G. and Atkinson D. E. (1973) Stabilization of adenylate deaminase reaction. J. biol. Chem. 248, 8309-8312. Hayashi S., Ise K., Itakura T. and Ooshiro Z. (1982) Biochemicals properties of glutamate dehydrogenase purified from eel liver. Bull. Jap. Soc. scient. Fish. 48, 697-701. Kaufman S. (1955) ~-Ketoglutaric dehydrogenase system and phosphorylating enzyme from heart muscle. In Methods in Enzymology (Edited by Colowick S. P. and Kaplan N. O.), Vol. 1, pp. 714-722. Academic Press, New York. McGivan J. D. and Chappell J. B. (1975) On the metabolic function of glutamate dehydrogenase in rat liver. FEBS Lett 52, 1-7. Male K. B. and Storey K. B. (1983) Kinetic characterization of NADP-specific glutamate dehydrogenase from the sea anemone Anthopleura xanthogrammica: control of amino acid biosynthesis during osmotic stress. Comp. Biochem. Physiol. 76B, 823-829.
Matsushima O. and Kado Y. (1983) Effect of adenine nucleotides on glutamate dehydrogenase activities of the brackish and freshwater clams, Corbicula japonica and C. leana. Annot. Zool. Jap. 56, 3-9. Regnault M. (1977) Etude de la croissance chez la crevette Crangon crangon L. d'apr~s les variations quantitatives de ses acides nucl~iques. Influence de l'alimentation. Th~se Doct. Etat, Sci. nat. Univ. P. & M. Curie (Paris 6), 180 pp. Regnault M. (1983a) Influence des variations de salinit~ cons6cutives au cycle de marie sur l'excr&ion ammoniacale de Crangon crangon L. Oceanologica Acta 6, 297-302. Regnault M. (1983b) Influence ~ long terme du taux prot~ique du r~gime sur l'excr&ion d'azote et le m6tabolisme de la crevette Crangon crangon L. Oc~anis 9, 241-255. Regnault M. (1984) Salinity-induced changes in ammonia excretion rate of the shrimp Crangon crangon L. over a winter tidal cycle. Mar. Ecol. Progr. Ser 20, 119-125. Regnault M. and Lagard~re J. P. (1983) Effects of ambient noise on the metabolic level of Crangon crangon (Decapoda, Natantia). Mar. Ecol. Progr. Ser. l l , 71-78. Reiss P. M., Pierce S. K. and Bishop S. H (1977) Glutamate dehydrogenase from tissues of the ribbed mussel Modiolus demissus: ADP activation and possible physiological significance. J. exp. Zool. 202, 253-258. Schoffeniels E. (1964) Effect of inorganic ions on the activity of L-glutamic acid dehydrogenase. Life Sci. 3, 845-850. Schoffeniels E. (1965) L-Glutamic acid dehydrogenase activity in the gills of Palinurus vulgaris Latr. Archs Int. Physiol. Biochim. 73, 73 80. Schoffeniels E. (1966) Activit6 de la deshydrogenase de l'acide L-glutamique et osmoregulation. Archs Int. Physiol. Biochim. 74, 333-335. Schoffeniels E. (1976) Adaptation with respect to salinity. Biochem. Soc. Syrup. 41, 179-204. Schoffeniels E. (1984) Biochimie compar6e. In Collection de Biologie Evolutive, Vol. 9, Ch. 7, pp. 123 131. Masson, Paris. Schoffeniels E. and Gilles R. (1970) Osmoregulation in aquatic Arthropods. In Chemical Zoology (Edited by Florkin M. and Scheer B. T.), Vol. 5A, pp. 255-286. Academic Press, New York. Storey K. B., Fields J. H. A. and Hochachka P. W. (1978) Purification and properties of glutamate dehydrogenase from the mantle muscle of the squid Loligo pealeii. Role of the enzyme in energy production from amino acids. J. exp. Zool. 205, 111-118.
0305-0491/87 $3.00+0.00 © 1987 Pergamon Journals Ltd
GLUTAMATE DEHYDROGENASE OF THE SHRIMP CRANGON CRANGON L. EFFECTS OF SHRIMP W E I G H T AND SEASON UPON ITS ACTIVITY IN THE OXIDATIVE AND REDUCTIVE F U N C T I O N MICHELE REGNAULT and YVES BATREL Station Biologique, CNRS and Univ. P. & M. Curie (Paris 6), F-29211 Roscoff, France and Lab. Biologie Marine, Coll6ge de France, F-29110 Concarneau, France (Received 21 April 1986)
Abstraet--l. Enzymatic activity of GDH of C. crangon was measured in the reductive and oxidative function in crude homogenates of winter shrimp and summer shrimp. 2. The specific activity of the NAD-dependent GDH was /n vitro significantly (P = 0.01) higher (30-40%) than the specific activity of the NADH-dependent GDH. 3. Changes in GDH specific activity and in the enzyme activity of the whole shrimp were studied as a function of shrimp weight and of season. GDH activity in the reductive function (glutamate synthesis) appeared to be relatively independent on these factors but GDH activity in the reverse function (glutamate deamination) varied. 4. Seasonal variations of the NAD-dependent GDH are discussed in terms of environmental factors and the metabolic level of shrimp. It is suggested that they could be explained in terms of the control of the energy charge.
INTRODUCTION In crustaceans, glutamate dehydrogenase (GDH) has received considerable attention with respect to its role in osmoregulation. Through glutamate synthesis, the enzyme contributes to the regulation of the pool of intracellular amino-acids during salinity changes of the external medium (Schoffeniels, 1964, 1966; Schoffeniels and Gilles, 1970). It was also suggested that G D H would favour the amino-acid storage into protein--especially haemocyanin--when the animal was transferred into a hypoosmotic medium (Schoffeniels, 1976). The reaction catalyzed by the G D H is indicated by the equation (1) below: glutamate + N A D ÷ ~-~~-ketoglutarate + N A D H + NH4 +. Thus, only one role (the reductive function) was considered. The reverse function (oxidative deamination of glutamate and ammonia formation) was scarcely studied as the previous authors reported that its activity, in vitro, was either insignificant or non-existent (Schoffeniels, 1965) and that in vivo the oxido-reduction potential values would strongly oppose this function (Schoffeniels, 1976; 1984). However, Chaplin et al. (1965) succeeded in recording the activity of G D H of Carcinus maenas in both funcitons, even though the KM values, obtained by these authors for the substrates, demonstrated a higher affinity of G D H for ~-ketoglutarate than for glutamate. Later, a relationship between the activity level of the NAD-dependent G D H and the ammonia excretion rate was observed by Bidigare and King (1981) in a mysid, Praunus flexuosus, and by Batrel and Regnault (1985) in a shrimp, Crangon
crangon, thus suggesting a possible role of the G D H in ammoniogenesis. In marine invertebrates, few studies have dealt with the G D H activity in its twin functions. However, the activity ratio (glutamate synthesis:glutamate deamination) showed that the enzymatic activity when in the reductive function was usually higher than when in the oxidative function (Reiss et aL, 1977; Male and Storey, 1983) except for the squid, Loligo pealeii, in which an activity ratio = 1:1 was found (Storey et al., 1978). From our previous study (Batrel and Regnault, 1985), it appeared that the specific activity of the NAD-dependent G D H was much higher in C. crangon than quoted for other marine invertebrates in the literature. The aim of the present study was to .compare the specific activities of the NADH-dependent and NAD-dependent G D H of the shrimp Crangon crangon. Furthermore, as the activity level of the NAD-dependent G D H of this species was observed to vary during the year, we have looked for any seasonal changes in the G D H activity level.
MATERIALAND METHODS Animals Shrimp were collected in the Penze estuary (N. Brittany) then held in the laboratory in running sea-water for a week prior to enzymatic measurements. During this period they were fed daily. The shrimp size was selected according to the prevailing size-group at each sampling period. Experiments were run in February 1985 (sea-water T = 8-10°) and in August 1985 (17-18°C). Particular moult stages were not controlled but freshly moulted shrimp were discarded.
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MICH~LE REGNAULT a n d YVES BATREL
Extraction procedure The enzyme activity was determined on shrimp groups (4-5 shrimps/group); only 4 groups or samples were analysed daily as the enzyme activity of the extract was constant for only 1 or 2 hr. Shrimp were gently dried on filter paper and weighed (Mettler: 0.01 mg), then they were sacrificed, cut into several pieces and homogenized in an ice-cold solution containing 50 mM imidazole-HC1 buffer, pH: 7.6, 0.5mM phenylmethyl sulfonyl fluoride (PMSF), 0.1% n-cetyl trimethylammonium bromide, 0.5 mM dithiothreitol (Clealand's reagent: DTT). Homogenization and centrifugation were carried out as previously described (Batrel and Regnault, 1985). Enzyme assays GDH activity was determined from the reduction of NAD + into NADH (oxidative function of GDH) or the oxidation of NADH into NAD + (reductive function of GDH). For the NAD-dependent GDH the standard assay mixture was 42mM imidazole-HCl buffer, pH: 8.55, 0.5mM NAD +, 33 mM glutamate; volume of shrimp extract=250/zl (final volume: 3ml). The reaction was initiated by the addition of glutamate. For the NADHdependent GDH the standard assay mixture was 42mM imidazole-HC1 buffer, pH: 7.65, 0.08 mM ~-ketoglutarate, 280mM NH4 acetate; volume of shrimp extract = 250 #1 (final volume: 3 ml). These optimal conditions were defined after the study of the characteristics of the enzyme (see Results). The reaction was initiated by the addition of -ketoglutarate. Both assays were subsequently recorded for each individual sample. Increase or decrease of NADH concentration was recorded at 340 nm and 20°C using a double-beamed spectrophotometer (SP 1700 PYE UNICAM). Supernatant protein was estimated by Bradford's (1976) method using BSA as a reference standard. Chemicals were obtained from Sigma (NAD, glutamate) and Boehringer (NADH, ADP, ~-ketoglutarate). Results are expressed as international unit [U.I. =/tmol NADH (formed or oxidized), rain -I] (Kaufman, 1955). Sample activity is expressed as U.I. g i fresh weight and U . I . mg -] protein.
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L Kinetic characteristics o f the GDH of C. crangon The kinetic characteristics of the N A D - d e p e n d e n t G D H of C. crangon have been described previously (Batrel and Regnault, 1985) and are briefly summarized here. The optimal p H for the oxidative function ranged between 8.18-8.20 using an imidazole-HCl buffer (pH: 8.55). The cofactor ( N A D ÷) dependence of G D H presented no linear relationship as two plateaux of enzyme saturation were recorded for N A D concentrations ranging from 0 to 1.3 mM. The enzyme saturation by substrate was obtained with 24 m M glutamate and an inhibition of G D H activity by an excess of substrate was observed for glutamate concentrations higher than 50 mM. The kinetic characteristics of the N A D H dependent G D H of C. crangon, undescribed until now, are shown in Fig. 1. The optimal conditions of the standard assay were obtained using an imidazole-HC1 buffer (pH = 7.6-7.7). The cofactor dependence of G D H in the reductive function differed from that in the oxidative function. The enzyme saturation was obtained with 0.05raM N A D H when the first plateau of enzyme saturation
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Fig. 1. Kinetic characteristics of the NADH-dependent GDH of C. crangon. (A) Optimal pH of the imidazole-HCl buffer used in the standard assay. (B) Effect of various amounts of the NADH cofactor in the assay (final concentrations ranging from 0 to 0.23 raM). (C and D) Substrate dependence of the enzyme. GDH activity expressed as U.I. m1-1 shrimp extract; see Methods for conditions of the assay standard.
527
GDH activity in Crangon crangon L. was obtained with 0.5 mM N A D +. Furthermore, an inhibition of the enzyme activity was observed for N A D H concentrations higher than 0.15 mM when no inhibition appeared for N A D concentrations ranging from 0 to 1.4mM. The enzyme saturation by ~-ketoglutarate was obtained with 0.35 mM concentrations of substrate thus much more quickly than with glutamate. The higher affinity of the G D H of C. crangon to ~t-ketoglutarate (KM = 1.43 mM) than to glutamate (KM = 5.16 mM) was in agreement with the data of Chaplin et al. (1965) in Carcinus maenas. An inhibition of G D H activity by an excess of substrate was observed for ~-ketoglutarate concentrations higher than 12mM. For the other substrate of the NADH-dependent G D H (NH4+), ammonium acetate was used rather than ammonium chloride, following observations of Male and Storey (1983). High concentrations of ammonium salt (250mM minimum) were necessary to obtain the enzyme saturation and no inhibition by an excess of substrate was observed in the experimental range of concentrations (from 0 to 300 mM). This corroborates the weak G D H affinity for ammonium ions (Chapman and Atkinson, 1973). IL Activity o f the GDH o f C. crangon The G D H activity in the reductive function (A1) and in the oxidative function (A2) were measured simultaneously in crude homogenates of shrimp. Activity measurements were performed on winter animals (February) and summer animals (August). In February (T = 8-10°C), the mean value of the G D H specific activity (X +- S.E.M., N = 24 shrimp groups) was 0.565 +_ 0.030 U.I. g ~ fresh wt and 0.0140 + 0.0009 U.I. mg -~ protein for A1 (glutamate synthesis), 0.736 +- 0.024 U.I. g - l fresh wt and 0.0182 +- 0.0006 U.I. mg ] protein for A2 (glutamate deamination). In August (T = 17-18°C), the mean value (n = 27) of the G D H specific activity was respectively 0.606 +__0.033 and 0.0137 +_ 0.0008 for A1, 0.863 __+0.026 and 0.0196 +- 0.0007 for A2. The A1 activity was clearly distinct from the A2 activity (Fig. 2). From the Student's t-test both activities differed from each other with a 99% prob-
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Fig. 2. Specific activity of the GDH of C. crangon. A1 = activity of the NADH-dependent GDH; A2 = activity of the NAD-dependent GDH; mean values +_SEM (plain vertical bars); the confidence interval with a 99% probability is figured by the dotted vertical bars. Winter: n = 24; summer: n = 27.
ability whatever was the season and the expression of results (fresh weight or protein). Under the present experimental conditions, the G D H of C. erangon was 1.3 or 1.4 fold more active in the oxidative function than in the reductive one, in winter and summer, respectively. Ill. Relationship between GDH activity and shrimp weight The mean fresh weight of shrimp studied in summer (364.07+_17.30mg) was distinct with a 95% probability from that of shrimp studied in winter (439.75 +_ 11.32 rag). This change in weight is related to the biological cycle of this species: recruitment in April-May, high growth during summer then reproduction and low growth in winter (Regnault, 1977). A weak and negative correlation was observed between the G D H specific activity and shrimp weight. The G D H activity of whole shrimp was calculated as the specific activity was measured on crude homogenates of whole shrimp. The mean value (A' = U.I. shrimp -]) was 0.246 +_ 0.014 for A I ' and 0.323+_0.013 for A2' in winter ( n = 2 4 ) , 0.216 +_ 0.014 and 0.309 -t- 0.014 respectively in summer (n = 27). The G D H activity of whole shrimp was also significantly higher (with a 99% probability) in the oxidative function than in the reductive function. However, the correlation between the A I ' and A2' activities as shown by the following regression equations: A2' = 0.493 A I ' + 0.202
r = 0.516 (in summer)
A2' = 0.306 A I ' + 0.248
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was significant (P = 0.01) only in summer. In contrast to the G D H specific activity, the enzymatic activity of the whole shrimp was positively and strongly correlated with the shrimp weight, except for A I ' in winter (Fig. 3). IV. Seasonal changes in GDH activity The specific activity of G D H in the reductive function (AI) did not vary significantly with season as shown in Fig. 2, but activity in the reverse function (A2) was significantly (P = 0.05) higher in summer than in winter. Here, a 17% increase of the mean specific activity A2 was observed in summer. The distinct responses of the two functions of the G D H to seasonal differences explains the change in the activity ratio (A1 :A2) noted above. As the G D H activity of the whole shrimp was strongly correlated with shrimp weight, as far as A2' was concerned, it was of interest to know to what extent the weight effect interacted with the seasonal effect. Figure 4 shows that for any selected shrimp weight, the summer activity of G D H in the oxidative function was 13% higher than the winter activity. Thus, the weight effect poorly contributed to the seasonal change in activity as it was reducing rather than increasing it. On the other hand, we noticed that the activation of the NAD-dependent G D H by the adenosine 5'-diphosphate (ADP) was more pronounced in winter than in summer (Fig. 5). In both cases, the optimal activation was obtained by the addition of 1.5 #mol
MICH~LE REONAULT and YVES BATREL
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DISCUSSION
Most studies on glutamate dehydrogenase have been focused on its role in glutamate synthesis as the enzyme activity in this function was strongly prevailing over its activity in the reverse function (glutamate deamination). In vertebrates and marine invertebrates, the activity ratio (glutamate synthesis:glutamate deamination) was usually much higher than 1, as shown by the following examples-A1 :A2 = 10:1 in the rat (McGivan and Chappell, 1976), 4:1 in the eel (Hayashi et al., 1982), 10:1 in the sea anemone Anthopleura xanthogrammica (Male and Storey, 1983), 6.5 : 1 in the mussel Modiolus demissus (Reiss et al., 1977). Furthermore, synthesis is favoured by the thermodynamic laws, as mentioned earlier (see Introduction). The kinetic characteristics of the GDH of Crangon crangon as defined in this study support this opinion. In vitro and under optimal conditions the NADHdependent GDH required cofactor concentrations 10 fold lower than those required by the NAD-dependent GDH; the GDH affinity to
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of ADP in the standard assay (same experimental conditions). The ADP activation represented a 26% increase of enzyme activity in summer and a 70% increase in winter. The activity increase appeared especially marked as the initial value (without ADP addition) of the enzyme activity was low. However, the optimal enzyme activity resulting from ADP activation was rcmarkedly independent of the activity initial level and of season.
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GDH activity in Crangon crangon L. ct-ketoglutarate was much higher than to glutamate as shown by the respective KM values. However, in these optimal conditions, the specific activity of the GDH of C. crangon was higher for glutamate deamination than for glutamate synthesis. The statistical treatment of our data allows us to state that in C. crangon the mean activity of G D H in the oxidative function is significantly higher (30--40%) than in the reverse function. However, if the value of the activity ratio was actually around 0.7 on account of the mean activity values, this ratio value was not systematically obtained for each sample. Thus, it would be better to say that in vitro the GDH of C. crangon is equally efficient to catalyze oxidation and synthesis of glutamate. It is possible that in vivo the G D H did not find requisite conditions to express its potential activity in both functions. However, it is worth noting that, except for the squid Loligo pealeii in which a 1 : 1 activity ratio was found (Storey et al., 1978), all species studied to date have shown a G D H activity ratio equal or inferior to 1, and all such activities have been measured in vitro. The variations of G D H activity of C. crangon as a function of shrimp weight and season are especially interesting. As for all metabolic functions, the specific activity of GDH was negatively correlated with shrimp weight and the GDH activity of the whole shrimp was positively weight-dependent. The last correlation was very significant in summer and winter for A2 but only in summer for AI (P = 0.05 in all cases) (Fig. 3). Curiously, the G D H activity in the reductive function was not influenced by shrimp weight in winter. Furthermore, the GDH reductive activity was constant in both seasons whilst the G D H deaminative activity was significantly higher in summer. Our previous values obtained in June (18°C) for the specific A2 activity were markedly higher (l.313+_0.053U.I.g -1 fresh wt, 0.032_+ 0.020 U.I. mg -1 protein) (Batrel and Regnault, 1985) which suggests that the seasonal variation of the A2 activity could be more pronounced than that exhibited in this study. In fact, it seems that the optimal activity of the G D H in the oxidative function would occur early in the summer and that the A2 activity would already be declining at the end of summer. Seasonal variations of A2 activity call for some comments. The different seasonal activity levels of the NADdependent G D H of Crangon crangon were not the result of changes in shrimp weight as we found that this factor would reduce rather than increase such variability. From examination of the summer values of A2 activity recorded at 18°C in June and August, respectively, it is also unlikely that the temperature of the environmental medium was responsible for the seasonal level of this activity. Furthermore, Batrel and LeGal (1984) observed that the Vmax for the NAD-dependent G D H of Arenicola marina acclimated at 9°C was constant from 10 to 30°C. On the other hand, as the specific activity of the NADdependent G D H is strongly influenced by the nutritional level of shrimp, one could suggest that the A2 seasonal variation was produced indirectly by the trophic conditions which characterized the 2 envisaged seasons. However, this possible effect could have been lessened by the acclimatization week im-
529
posed on the shrimp prior to enzymatic measurements. Finally, it seems much more likely that seasonal variations of A2 are directly related to the metabolic level of shrimp. In this species, high moult frequency and growth are observed in spring and summer periods but not in the winter period. This is corroborated by the corresponding metabolic rates. The mean ammonia excretion rate of shrimp of weight similar to those of this study, ranged from 1.10 to 1.40 #mol NH3 • g-~ fresh wthr -~ in summer to 0.520-0.750~mol NH3 in winter (Regnault, 1983a; 1984). The mean 02 consumption rate indicated parallel variations, 470-520#1 02 "g-~ fresh wthr -~ and 150-200/~1 02 in summer and winter respectively (Regnault, 1983b; Regnault and Lagardere, 1983). Thus, the energy requirements of shrimp are correspondingly higher in summer than in winter. As GDH via the glutamate deamination was proposed as an ATP regenerating system (Matsushima and Kado, 1983; Campbell et al., 1983), an activity increase of the NAD-dependent GDH when energy expenditures are high would agree with Atkinson's (1968) theory of the energy charge of the adenylate pool. From the activation curves of NADdependent GDH by ADP (Fig. 5) it is suggested that the high summer activity of A2 in C. crangon would result from high in vivo ADP concentrations and conversely that its low winter activity would be the result of low in vivo ADP concentrations. Thus, the seasonal variation of GDH oxidative activity could be explained in terms of the regulation of the energy charge. In Crangon crangon, the GDH responses to weight and season factors were different depending on the enzyme function. The relative independence of the enzyme reductive activity to shrimp weight and season is in accord with its key-role in osmoregulation and amino-acid storage. In the same way, the constant reductive activity level of GDH explains why, as yet, most studies have been devoted to it in this role. In contrast, the variability of the enzyme activity in the reverse deaminative function according to shrimp weight, season and possibly energy charge, leads to a consideration of the NAD-dependent GDH as a regulatory system. Such a role was previously suggested by Storey et al. (1978). According to these authors, the primary role of GDH would be in the supply of energy via the glutamate oxidation in order to restore the adenylate pool when depleted by intense metabolic activity. On the other hand, another possible role of GDH, in ammoniogenesis of C. crangon, was proposed (Batrel and Regnault, 1985). The variability of the A2 activity here supports our previous reserve that the GDH could not constantly take over the ammonia excretion of this species. Other metabolic pathways for ammoniogenesis might possibly be needed at some periods of the year or the biological cycle of this species.
Acknowledgements--The authors wish to thank Dr Roger
Uglow (University of Hull) and Dr Lars Hagerman (University of Copenhagen) for their critical comments on the manuscript.
530
MICHI~LE REGNAULT and YVES BATREL REFERENCES
Atkinson D. E. (1968) The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7, 4030-4034. Barrel Y. and LeGal Y. (1984) Nitrogen metabolism in Arenicola marina. Characterization of a NAD-dependent glutamate dehydrogenase. Comp. Biochem. Physiol. 78B, 119-124. Barrel Y. and Regnault M. (1985) Metabolic pathways of ammoniogenesis in the shrimp Crangon crangon L.: Possible role of glutamate dehydrogenase. Comp. Biochem. Physiol. 82B, 217-222. Bidigare R. R. and King F. D. (1981) The measurement of glutamate dehydrogenase activity in Praunus flexuosus and its role in the regulation of ammonium excretion. Comp. Biochem. Physiol. 70B, 409-413. Bradford M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analyst. Biochem. 72, 248-254. Campbell J. W., Aster P. L. and Vorhaben J. E. (1983) Mitochondrial ammoniogenesis in liver of channel catfish Ictalurus punctatus. Am. J. Physiol. 244, R709-R717. Chaplin A. E., Huggins A. K. and Munday K. A. (1965) Ionic effects on glutamate dehydrogenase activity from beef liver, lobster muscle and crab muscle. Comp. Biochem. Physiol. 16, 49-62. Chapman A. G. and Atkinson D. E. (1973) Stabilization of adenylate deaminase reaction. J. biol. Chem. 248, 8309-8312. Hayashi S., Ise K., Itakura T. and Ooshiro Z. (1982) Biochemicals properties of glutamate dehydrogenase purified from eel liver. Bull. Jap. Soc. scient. Fish. 48, 697-701. Kaufman S. (1955) ~-Ketoglutaric dehydrogenase system and phosphorylating enzyme from heart muscle. In Methods in Enzymology (Edited by Colowick S. P. and Kaplan N. O.), Vol. 1, pp. 714-722. Academic Press, New York. McGivan J. D. and Chappell J. B. (1975) On the metabolic function of glutamate dehydrogenase in rat liver. FEBS Lett 52, 1-7. Male K. B. and Storey K. B. (1983) Kinetic characterization of NADP-specific glutamate dehydrogenase from the sea anemone Anthopleura xanthogrammica: control of amino acid biosynthesis during osmotic stress. Comp. Biochem. Physiol. 76B, 823-829.
Matsushima O. and Kado Y. (1983) Effect of adenine nucleotides on glutamate dehydrogenase activities of the brackish and freshwater clams, Corbicula japonica and C. leana. Annot. Zool. Jap. 56, 3-9. Regnault M. (1977) Etude de la croissance chez la crevette Crangon crangon L. d'apr~s les variations quantitatives de ses acides nucl~iques. Influence de l'alimentation. Th~se Doct. Etat, Sci. nat. Univ. P. & M. Curie (Paris 6), 180 pp. Regnault M. (1983a) Influence des variations de salinit~ cons6cutives au cycle de marie sur l'excr&ion ammoniacale de Crangon crangon L. Oceanologica Acta 6, 297-302. Regnault M. (1983b) Influence ~ long terme du taux prot~ique du r~gime sur l'excr&ion d'azote et le m6tabolisme de la crevette Crangon crangon L. Oc~anis 9, 241-255. Regnault M. (1984) Salinity-induced changes in ammonia excretion rate of the shrimp Crangon crangon L. over a winter tidal cycle. Mar. Ecol. Progr. Ser 20, 119-125. Regnault M. and Lagard~re J. P. (1983) Effects of ambient noise on the metabolic level of Crangon crangon (Decapoda, Natantia). Mar. Ecol. Progr. Ser. l l , 71-78. Reiss P. M., Pierce S. K. and Bishop S. H (1977) Glutamate dehydrogenase from tissues of the ribbed mussel Modiolus demissus: ADP activation and possible physiological significance. J. exp. Zool. 202, 253-258. Schoffeniels E. (1964) Effect of inorganic ions on the activity of L-glutamic acid dehydrogenase. Life Sci. 3, 845-850. Schoffeniels E. (1965) L-Glutamic acid dehydrogenase activity in the gills of Palinurus vulgaris Latr. Archs Int. Physiol. Biochim. 73, 73 80. Schoffeniels E. (1966) Activit6 de la deshydrogenase de l'acide L-glutamique et osmoregulation. Archs Int. Physiol. Biochim. 74, 333-335. Schoffeniels E. (1976) Adaptation with respect to salinity. Biochem. Soc. Syrup. 41, 179-204. Schoffeniels E. (1984) Biochimie compar6e. In Collection de Biologie Evolutive, Vol. 9, Ch. 7, pp. 123 131. Masson, Paris. Schoffeniels E. and Gilles R. (1970) Osmoregulation in aquatic Arthropods. In Chemical Zoology (Edited by Florkin M. and Scheer B. T.), Vol. 5A, pp. 255-286. Academic Press, New York. Storey K. B., Fields J. H. A. and Hochachka P. W. (1978) Purification and properties of glutamate dehydrogenase from the mantle muscle of the squid Loligo pealeii. Role of the enzyme in energy production from amino acids. J. exp. Zool. 205, 111-118.