Metabolic pathways of ammoniogenesis in the shrimp Crangon crangon L.: Possible role of glutamate dehydrogenase

Comp. Biochem. Physiol. Vol. 82B, No. 2, pp. 217-222, 1985 Printed in Great Britain

0305-0491/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd

METABOLIC P A T H W A Y S OF A M M O N I O G E N E S I S IN THE SHRIMP CRANGON CRANGON L." POSSIBLE ROLE OF GLUTAMATE DEHYDROGENASE YVES BATREL* a n d M1CH~LE REGNAULT'~ *Lab. Biologie Marine, Coll6ge de France, F-29110 Concarneau, France; and tStation Biologique, CNRS and Univ. P. and M. Curie, F-29211 Roscoff, France (Received 7 February 1985) Abstraet--l. The oxidative deamination of glutamate by glutamate dehydrogenase (GDH) was determined in crude homogenates of the shrimp Crangon crangon. 2. The GDH activity of whole shrimps (1.192 _+0.164 U1/g wet wt and 0.032 + 0.004 UI mg protein) (_+ SD) is probably sufficient to account for all the ammonia excretion of this species. 3. Starvation markedly influenced GDH activity. A 50~o decrease of GDH activity was observed following 7 days of fasting but subsequently no further decrease in GDH activity was noticed during starvation up to a maximum of 17 days.

INTRODUCTION A m m o n o t e l i s m is a m a j o r characteristic of marine invertebrates a n d teleostean fishes. The metabolic pathways involved in a m m o n i a excretion are still poorly understood. According to C a m p b e l l (1973) a m i n o acids would represent one of the main sources of a m m o n i a , resulting either directly from a m i n o acid oxidation or dehydration, or indirectly from transa m i n a t i o n reactions. G i b b s a n d Bishop (1977) demonstrated that the purine nucleotide cycle, originally described by Lowenstein (1972) in Vertebrates, was mainly s u p p o r t i n g the a m m o n i a excretion o f the lugw o r m Arenieola cristata. Recently, V a n W a a r d e (1981, 1983) a n d Van W a a r d e a n d De Wilde-Van Berge H e n e g o u w e n (1982) recognized two m a j o r metabolic pathways as responsible for a m m o n i a excretion in Carassius auratus: the oxidative dea m i n a t i o n of glutamate via G D H a n d the purine nucleotide cycle via A M P - d e a m i n a s e . Moreover, some specific amides a n d a m i n o acids might play a key role in ammoniogenesis (Van W a a r d e and Kesbeke, 1981, 1982). In Crustaceans, g l u t a m a t e dehydrogenase received considerable a t t e n t i o n with respect to its role in osmoregulation, especially in a d a p t a t i o n to hyperosmotic stress (Campbell, 1973). Consequently, only the reductive function of G D H (glutamate forming reaction) was recognized (Schoffeniels, 1976). However, the enzyme which catalyses the reaction: glutamate + N A D + ~ 7-ketoglutarate + NADH + NH 4 is a completely reversible enzyme as observed by Chaplin et al. (1965) in the muscle o f Carcinus maenas a n d Homarus vulgaris. F u r t h e r m o r e , Bidigare a n d King (1981) f o u n d a strong correlation between G D H activity a n d a m m o n i a excretion rate in a mysid Praunus flexuosus. The aim of the present study has been to elucidate the potential role of the glutamate dehydrogenase (EC 1.4.1.3) in the a m m o n i a excretion of the shrimp 217

Crangon crangon since more t h a n 94~o o f total nitrogen excreted by this species was released as a m m o n i a (Regnault, 1983a). Thus, only the oxidative function (glutamate d e a m i n a t i n g reaction) of G D H has been considered. The effect of the shrimp's nutritional level u p o n the G D H activity has also been investigated. MATERIAL AND M E T H O D S

Animals Shrimp were maintained in the laboratory for 1 or 2 weeks prior to experiments. During this acclimatization period they were held in running sea water (34%0 S) at 18°C and were fed daily. The enzyme activity was measured on groups of 3-4 shrimp. Shrimp between 40 and 45 mm total length (mean wet weight 500 mg) were used. Moult stage was not controlled but freshly moulted shrimp were discarded. Enzyme activity was determined in whole shrimp. However, for one experiment living shrimp were dissected on a ice-bath to remove the hepatopancreas; hepatopancreas-ectomized shrimp and isolated hepatopancreas received the same treatment as whole shrimp. Extraction procedure Shrimp were gently dried on filter paper, weighed (_+0.1 mg) and then homogenized in an ice-cold solution containing 50mM imidazole-HC1 buffer, pH 7.6, 0.5mM phenylmethyl sulfonyl fluoride (PMSF), 0.1% n-cetyl trimethylammonium bromide, 0.5mM dithiothreitol (Clealand's reagent: DTT). The ratio of shrimp wet weight to extractive solution was 1:2.5; homogenization was carried out (2 x 30 sec) using a Polytron (type PTA 20 SM). Homogenates were centrifuged at 24,000 g for 30 min at 4°C. Enzyme activities were determined in the supernatant fluid 2hr after centrifugation. Preliminary assays demonstrated that enzyme activity was constant for 1 or 2 hr from this time. Enzyme assays GDH activity was determined from the reduction of NAD + into NADH (E =6.22cm2/~mol at 340nm). The standard assay contained 42 mM imidazole-HCl buffer, pH 8.55, 0.5mM NAD, 33raM glutamate and 0.250ml of shrimp extract (final volume: 3.1 ml). These optimal conditions were defined after the study of the characteristics of the NAD-dependent GDH of Crangon (see Results). The reaction was initiated by the addition of glutamate; increase of

218

YVES BATREL and

absorption was recorded at 340 nm and 20°C using a SP 1700 Pye Unicam spectrophotometer. Supernatant protein was estimated by Bradford's (1976) method using BSA as a reference standard. Chemicals were obtained from Merck (imidazole; PMSF) BDH Chemicals (DTT) Sigma (NAD; glutamate) and Boehringer (ADP). Results are expressed as international unit (UI = #mol NADH/min). Sample activity is expressed as UI/g wet wt and Ul/mg protein.

MICHI~LE R E G N A U L T

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Optimal pH. The effect of the buffer pH on G D H activity is shown in Fig. 1. The optimal activity was observed in the 8.55-8.60 pH range. These values, obtained with an imidazole-HC1 buffer, were consistent with those found in literature when the buffer was either imidazole-HCl or Tris: optimal pH 8.6 (Chaplin et al., 1965; Bidigare and King, 1981; Batrel and Le Gal, 1984) or 8.5 (Reiss et al., 1977; Hayashi et al., 1982; Male and Storey, 1983). However, the optimal pH was 8.0 with a K-phosphate buffer (Van Waarde, 1981). On the other hand, the pH of the standard assay mixture was measured. Its value (8.18-8.20) indicated the pH that was actually required for the oxidative deamination of glutamate. Cofactor dependence. In lower invertebrates, N A D P is a much less effective cofactor for G D H than N A D (Van Waarde, 1981); therefore, only N A D was tested as cofactor. No linear relationship was observed between G D H activity and N A D concentration (Fig. 2). The final concentration of N A D in our standard assay corresponded to the first plateau of enzyme saturation exhibited in Fig. 2. Substrate dependence. The saturation of G D H by substrate was obtained with 75/tmol of glutamate (final concentration: 24 mM) (Fig. 3). An inhibition of G D H activity by an excess of substrate was observed for glutamate concentrations higher than 50 mM.

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Effect o f ADP. Some authors have reported an activation of G D H by adenine dinucleotide phosphate (ADP); in some cases, enzyme activity was unmeasurable without A D P addition (Bidigare and King, 1981; McBean et al., 1966; Walton and Cowey, 1977). Thus, various amounts of A D P (from 0.5 to 5/~M) were added to the enzyme assay (Fig. 4). Optimal activation (26~o increase of G D H activity measured without A D P ) was obtained by the addition of 1.5/aM of the nucleotide. On account of this low activation, A D P was omitted for the G D H activity determinations in C. crangon.

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feeding state of shrimp. Two experiments were run in July to verify this assumption. For the first experiment, shrimp were used about 2 months after collecting; they were irregularly fed except for the week prior to experiment when they were fed daily. They were deprived of food as experiment 1 started and then maintained unfed for 14 days. Samples were taken every 3 or 4 days for enzyme activity determination. In the second experiment, shrimp were held as described in Materials and Methods for a week after collecting. Then, they were deprived of food for 17 days and sampled for enzyme activity determination every 2 days. The shrimp mean wet weight at the beginning of both experiments was 515 and 440 mg, respectively. At day 0, before food removal, G D H activity was 1.194_+ 0.089 UI/g wet wt (,( _+ SD) (experiment 1) and 1.192 + 0.22 UI/g wet wt (,~ _+ SD) (experiment 2). Thus, the nutritional past of experimental shrimp had no effect on G D H activity. A similar observation was made about the specific activity of the G D H (0.034 _+ 0.002 and 0.032 + 0.005 UI/mg protein, respectively). Furthermore, similar changes either in enzyme activity or in protein content of the supernatant were noticed during both starvation periods. Thus, only the results of the more detailed experiment 2 are discussed below and shown in Fig. 6. After a first decrease of G D H activity in the 24 hr following food deprivation, enzyme activity was reduced by 30~o for 5 or 6 days. Then, from day 7 to day 17 enzyme activity was only half as great as its initial value. Similar changes were observed in the specific activity of G D H (Fig. 6B). In contrast, no significant changes were observed in protein content of the crude enzyme extract (Fig. 6C); its mean value was maintained around 11 mg protein/ml. DISCUSSION

Most studies on glutamate dehydrogenase in Crustaceans have been concerned with its key role in glutamate synthesis. Although the reverse function (glutamate deamination) of G D H was first observed in 1965 by Chaplin et al. in crab and lobster muscle, Schoffeniels and his coworkers claimed that in Crustaceans the properties of G D H strongly favour reductive amination of keto-glutarate and prevent this enzyme from being involved in ammonia production (Schoffeniels, 1965, 1976, 1984; Schoffeniels and Gilles, 1970). According to this author, glutamate deamination is an endergonic reaction and the oxidoreduction potential values for the systems N A D H / N A D + ( - 0 . 3 2 V ) and gluta~nate/2-ketoglutarate ( - 0 . 1 0 8 V ) would strongly oppose this reaction. This point of view was supported by Lowenstein (1972) on account of the free energy exchanges observed in Vertebrates. By contrast, Reiss et al. (1977), Storey et al. (1978), Bidigare and King (1981) and Batrel and Le Gal (1982, 1984) noticed that in various marine invertebrates G D H was efficient in the glutamate deaminating reaction as well as in the glutamate forming reaction. McBean et al. (1966), Walton and Cowey (1977) and Van Waarde (1981) measured G D H activity in the oxidative deamination reaction in teleostean fishes. From all the above studies and

220

YVES BATREL and MICHI~LE REGNAULT

according to Campbell (1973) it appeared that glutamate d e a m i n a t i o n via G D H could contribute significantly to the a m m o n i a excretion of a m m o notelic species. Moreover, according to the A t k i n s o n ' s (1968) theory, the oxidative function of G D H was recognized as an A T P regenerating system by M a t s u s h i m a and K a d o (1983) in the mollusc C o r b i c r l a sp. and by Campbell et al. (1983) in the fish

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The present study shows that the glutamate dehydrogenase , f the shrimp C. crangon can, in vitro, oxidize glutamate and consequently liberate a m m o nia. The in L'itro pH of this reaction was 8.20 at the highest, thus close to the physiological values reported for the blood pH of P a l a e m o n e t e s varians ( H a g e r m a n and Uglow, 1984). The G D H response to cofactor c o n c e n t r a t i o n (Fig. 2) displayed a transitory plateau of saturation as already observed in Arenicola m a r i n a by Batrel and Le Gal (1984). This saturation plateau might be related to some negative cooperativity processes involved in intracellular regulations. O n the other hand, activation of the N A D - d e p e n d e n t G D H of C. crangon by A D P was weak in c o m p a r i s o n with activation by this nucleotide in M o d i o l u s d e m i s sus (Reiss et al., 1977) and in Loligo pealeii (Storey et al., 1978). However, its amplitude (Fig. 3) was similar to that reported by Hayashi et al. (1982) in the eel. The m e a n value of G D H activity of crude homogehates of daily fed C. crangon was a r o u n d 1.2 UI/g wet wt and 0.032 U I / m g protein. The values for G D H activity in the oxidative reaction found in literature for marine Invertebrates a n d for some Teleosts are shown in Table 1. As the reported activities were determined either for whole animals or for specific tissues and as methods used were not quite similar, these values are given more for i n f o r m a t i o n t h a n for comparison. It is worth noting that the G D H activities recorded in C. crangon are in the range of those reported in Table 1. O u r results are quite similar to those of Bidigare and King (1981) for a mysid except that A D P was added to the enzyme assay by these authors. It appears from Table 1 that G D H activity in Teleosts as well as in the cephalopod L. pealeii is

Table 1. GDH activity in the oxidative deamination reaction. All values are expressed as UI (#tool NADH/min); values from the literature expressed as O.D. (optical density) are not reported• ADP addition to the enzyme assay is mentioned UI/g wet wt UI/mg protein ADP References Species Invertebrates Modiolus demissus Loligo pealeii Anthopleura xanthogrammica Arenicoht marina Prauntts [~eXltostts

Crangon crangon

Teleosts Anguilkz rosgr¢ll~l

Salmo gairdnerii Carassius oltrtlllIS

0.02-0.06 0.320 (muscle) 1.350 (liver) --

-1.970 1.313 _+0.053 (SEM) 1.l 93 + 0.054 (SEM) 3.50 (liver) traces (muscle) 0.95 _ 0.17 (liver) traces (muscle) 2.30 + 1.23 (liver) 0.31 + 0.17 (muscle)

0.004-0.023

+

Male and Storey (1983) Barrel and Le Gal (1984)

0.014 0.01 0.03 0.032

Reiss et al. (1977) Storey et al. (1978)

+

Bidigareand King (1981) This study

0.032 _+0.002 +

McBeanet al. (1966)

+

Walton and Cowey (1977)

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Van Waarde (L981)

221

NAD+-dependent GDH of Crangon crangon always higher in liver than in muscle. However, a reverse situation was observed in the freshwater crab Oziotelphusa senex as the activity of the NADdependent G D H was 4 times higher in muscle than in hepatopancreas (Ramamurthi et al., 1982). In C. crangon the G D H activity of whole shrimp was significantly higher than that of hepatoectomized shrimp. This suggests that G D H was partly located in the hepatopancreas. Yet, no enzyme activity was recorded in isolated hepatopancreas under the present experimental conditions. Some G D H inactivation or lysis by proteolytic enzymes has probably occurred and a partial purification of the G D H would be needed to appreciate its activity level in hepatopancreas. Few authors have studied the relationship between G D H activity and ammonia excretion rate in Crustaceans. Fellows and Hird (1979) did not find any correlation between these processes in Cherax destructor in contrast to Bidigare and King (1981) who observed a strong correlation in Praunus flexuosus (r = 0.916). According to McBean et al. (1966) and Campbell et al. (1983) the in vitro G D H activity of fish liver can more than account for the ammonia excreted by the animal. Van Waarde (1981) recognized two ammonia-producing systems in goldfish tissues: glutamate deamination and AMPdeamination; from his data, the capacity of the glutamate deamination reaction was from 4 to 5 times greater than the capacity of the purine nucleotide cycle. In C. crangon, G D H activity in our experimental conditions would be able to supply an ammonia production equal to 71.52/~mol NH3/g wet wt/hr. From previous studies the mean ammonia excretion rate of C. crangon at 18°C ranged between 1.34pmol NH3/gwt wt/hr(Regnault, 1983b) and 1.40/~M (Regnault and Lagard6re, 1983). Thus, G D H activity as it was measured in vitro would be fully sufficient to justify the excretion rate of this species. However, it is difficult to prejudge the actual activity of G D H in vivo. This study has demonstrated the effect of the nutritional level of animals upon G D H activity. It is worth noting that the nutritional past of shrimp had no effect when they were fed daily for a few days prior to experiments. After a week of starvation, the G D H activity was only half as great. However, the decrease in activity did not continue with prolonged starvation (until the 17th day at least). Curiously, the nutritional level of animals did not receive any attention in the previous studies on G D H activity. The very low activities observed in Modiolus (Table 1) could partly result from this factor. This could also explain the nonsignificant activities observed for the oxidative reaction in Astacusfluviatilis (Schoffeniels and Gilles, 1963) and in Palinurus vulgaris (Schoffeniels, 1965) when the G D H activities in the reverse reaction were easily measurable. It is a matter of fact that G D H is more active in the glutamate synthesis than in the glutamate oxidation reaction. In marine Invertebrates, the activity ratio of the oxidative:reductive reaction was 1:6.5 in mussel (Reiss et al., 1977) and 1:10 in sea anemone (Male and Storey, 1983); in eel a 1:4 ratio was found (Hayashi et al., 1982). The enzyme decrease observed in the few days

following food removal in C. crangon was especially interesting when compared to the excretory response of this species under similar conditions. A previous study demonstrated a decrease in ammonia excretion rate of C. crangon in the 24 hr following food deprivation, the new rate (about 25% of the initial rate) being maintained for 5-6 days (Regnault, 1981). This similarity in the change of G D H activity and excretion rate at the beginning of a starvation period supported the assumption that G D H in C. crangon might be fully responsible for the ammonia excretion. However, as starvation was prolonged, opposite changes were observed: the enzyme activity was at its lowest when the excretion rate was higher than before starvation. In these conditions, glutamate deamination by GDH cannot justify the excreted amounts of ammonia and a shift to other metabolic pathways has to be envisaged. It was observed in C. crangon that proteins were the main substrate oxidized to meet the energetic requirements during prolonged starvation (Regnault, 1981). Ammonia formation from amino acid or amide catabolism was emphasized by Campbell (1973). Some enzymes such as serine dehydratase (Fellows and Hird, 1979; Schoffeniels, 1976), leucine transaminase, glutaminase and asparginase (Van Waarde and Kesbeke, 1981; Campbell et al., 1983) were shown to be playing a key role in ammonia formation. On the other hand, according to Chapman and Atkinson (1973) a decrease of the adenylate energy charge, subsequent to a metabolic stress such as starvation, involved a sharp increase of AM P-deaminase activity to prevent a depletion of the energy charge. Activation of AMPdeaminase over a 7-day starvation period was also noticed in the trout (Raffin, 1983). Thus, several metabolic pathways including the purine nucleotide cycle could possibly relieve or replace glutamate deamination by G D H when the G D H activity is depleted by a low nutritional level. In summary, the in vitro activity of glutamate dehydrogenase of C. crangon could assume the ammonia excretion of daily fed shrimp. However, G D H activity in the oxidative reaction was dependent on the shrimp's nutritional level; a week of starvation was sufficient to decrease it by 50%. A possible shift to other metabolic pathways could be envisaged with prolonged starvation.

REFERENCES

Atkinson D. E. (1968) The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7, 4030-4034. Batrel Y. and Le Gal Y. (1982) La glutamate deshydrog~nase d'Arenicole: un indicateur du niveau d'eutrophisation du milieu et de sa pollution. C. r. S~anc. Soc. Biol. 176, 619-623. Batrel Y. and Le Gal Y. (1984) Nitrogen metabolism in Arenicola marina. Characterization of a NAD-dependent glutamate dehydrogenase. Comp. Biochem. Physiol. 78B, 119-124. 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~,13. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing

222

YVES BATREL and MICHI~LE REGNAULT

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