Some properties of malic enzyme and glutamate dehydrogenase activities in the shrimp Pandalus borealis from the Saguenay Fjord (Québec, Canada)

Comp. Biochem.Physiol.Vol. 99B, No. 4, pp. 903-909, 1991 Printed in Great Britain

0305-0491/91 $3.00+ 0.00 © 1991PergamonPress pie

SOME PROPERTIES OF MALIC ENZYME AND GLUTAMATE DEHYDROGENASE ACTIVITIES IN THE SHRIMP PANDALUS BOREALIS FROM THE SAGUENAY FJORD (QUI~BEC, CANADA) CLAUDE ROULEAU,*JOCELYNE PELLERIN-MASSICOTTE*and ]~MILIENPELLETIER~" *Universit6du Qu6bec fi Rimouki, D6partement d'Oc~anographie, 310 des Ursulines, Rimouski, Qu6bec, G5L 3A1 Canada and i'INRS-Oc~anologie, 310 des Ursulines, Rimouski, Qu6bec, G5L 3A1 Canada (Tel: 418 724 1650); (Fax: 418 723 7234) (Received 19 February 1991) A~traet--1. The activitiesof malic enzymein the direction of oxidative decarboxylation, and of glutamate dehydrogenase in the direction of reductive amination, have been characterized in the abdominal muscle of Pandalus borealis. 2. These two enzymes were used as stress indicators during experiments on the trophic transfer of sublethal doses of methylmercury. The Km and Vmaxof both enzymes were not modified for the two concentrations of mercury (0.5 and 2.7/Jg Hg/g wet wt) tested in the shrimps' food.

INTRODUCTION Malic enzyme (ME) catalyzes the oxidative decarboxylation of L-malate to pyruvate, as well as the reductive carboxylation of pyruvate to L-malate. The major role of this enzyme is generally believed to be the supplier of NADPH for the reductive steps of lipogenesis (Lehninger, 1982). ME also gives an alternative path for the oxidation of L-malate (Outlaw and Springer, 1983). Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate to 2-ketoglutarate and the reductive amination of 2-ketoglutarate to L-glutamate. G D H is an important mitochondrial enzyme producing 2-ketoglutarate, which is used for ATP production in the Krebs cycle and plays an important role in ammoniac transport and the urea cycle; GDH is also involved in amino acids metabolism (Lehninger, 1982). Their kinetic parameters and cofactors specificity toward NAD and NADP depend upon the source of the enzymes. Few studies have been devoted to these two enzymes in marine crustaceans and, to our knowledge, none to the shrimp Pandalus borealis. Pandalus borealis is an epibenthic shrimp species largely distributed in boreal and subarctic zones (Butler and Boutillier, 1983), and is the most common shrimp species found in the St Lawrence gulf and the Saguenay t]ord (Qu6bec, Canada) (Couture and Trudel, 1968). The fjord of the Saguenay river was heavily contaminated by mercurial outfall from a chior-alkali plant from 1947 to 1976 (Loring and Bewers, 1978), and the shrimp population of the fjord has been exposed to mercury for many generations. In the course of our study on the trophic transfer of sublethal doses of methylmercury, in the presence or absence of selenium, we intend to use ME and GDH as indicators of stress induced by a sublethal contamination (Bayne, 1985). In the present work, some properties of the activity of these two enzymes, in crude homogenates obtained from the abdominal

muscle of P. boreal& are investigated. The effects of these two enzymes on behaviour when mercury was introduced into the food of P. borealis are discussed. MATERIALS AND

METHODS

Tested organisms All the tested organisms were caught in the Ste-Rose-duNord on the Saguenay fjord. Shrimps used for characterization of ME and GDH were immediately frozen and kept at -70°C until homogenization. Shrimps used for uptake experiments with methylmercury were rapidly transported to the laboratory and acclimated to conditions similar to those prevailing in the deepest zone of the Saguenay fjord: 30-31%o salinity, 4°C and very good aeration in the absence of light. Shrimps sampled during the course of the experiments were also frozen and kept at -70°C until homogenization. Tissue homogenization The abdominal muscle was removed, finelychopped with scissors and portions of tissue (0.5-1.5 g) were homogenized with a glass homogenizer in buffer at pH 7.60, containing 20mM triethanolamine (TEA), 0.5mM phenylmethylsulphonyl fluoride (PMSF) and 0.5mM dithiothreitol (DTT), in the proportion of 7 ml per g tissue. The thawing of abdominal muscle samples caused disruption of mitochondria and liberated mitochondrial enzymes into the homogenization medium. Homogenates were centrifuged at 700g for 10min. The supernatant was collected and centrifuged at 29,000g for 30 min. All these operations were done at 4°C. The resulting supematant was then distributed in 1.5 ml polyethylene micro-test-tubes and kept at -70°C until the determination of enzymatic activity and protein concentration by the method of Bradford (1976) using BSA as standard. Enzymatic assays Malic enzyme activity has been characterized in the direction of oxidative decarboxylation of L-malate, and GDH activity in the direction of reductive amination of 2-ketoglutarate. The activities of these two enzymes in the crude homogenates were characterized by measuring their

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Fig. 1. The effect of TEA concentration on oxidative decarboxylation of L-malate catalyzed by ME, from the abdominal muscle of P. borealis at 25°C. The assay mixture consisted of 0.5 mM NADP, 10 mM L-malate, 1 mM Mn 2÷, I00 #1 of abdominal muscle homogenate and various concentrations of TEA at pH 8.0.

Fig. 3. The effect of L-malate concentration on oxidative decarboxylation of L-malate catalyzed by ME from the abdominal muscle of P. borealis at 25°C. The assay mixture consisted of 50 mM TEA, 0.5 mM NADP, I mM Mn 2+, 100 #1 of abdominal muscle homogenate and various concentrations of L-malate at pH 8.0.

spec. act. as a function of the concentration of each component of the assay medium, and keeping constant the concentration of others. Assays were carried out at 25°C with a Perkin-Elmer spectrophotometer (model Coleman 3) at wavelength 340 n m (eNADH_NADP H = 6.22 cm2/gmol). Quartz or polystyrene spcctrophotometer cells (d = 1 cm) were used. The final volume of the assay was 3 ml and the volume of the homogenate was 0.1 ml. Results have been standardized by expressing them as activity units per mg protein (spec. act. following Bergmeyer and Grabl, 1983). The activity unit (U) was then equivalent to 1 #tool of NADP reduced per min for ME, or 1 #mol of NADH oxidized per min for GDH. For ME activity measurement, the homogenate, TEA, NADH, L-malate and NADP were pre-incubated for 5 rain. The reaction was then started by the addition of Mn 2+ (as MnCI2'4H20), and absorbance was noted every 30 see for 3 rain. GDH activity was measured in the homogenate, in a medium containing TEA, ADP, NADH and ammonium acetate, and pre-incubated for 5 min. The reaction was then started by the addition of 2-ketoglutarate and the absorbance was monitored every 30 sec for 3 min.

Chemicals used for these assays were purchased from Sigma ® (TEA, 2-ketoglutarate, ammonium acetate, Lmalate, MnCI2.4H20, DTT) and Boehringer-Mannheim¢ (NADH, NADP, ADP, PMSF). Calculation of kinetic constants and statistics The Michaelis-Menten constant (Kin) and maximum activity (Vm~x) for ME and GDH were calculated from spec. act. measured at increasing concentrations of substrate (L-malate for ME and 2-ketoglutarate for GDH), with the programme Enzyme-PC kindly provided by Dr Rudolf A. Lutz (Laboratory of Theoretical and Physical Biology, NIH, Bethesda, MD). Comparisons between groups for the kinetic parameters calculated were made with the Student t-test for non-paired data (Lutz et al., 1986). Uptake experiments Shrimps were acclimated for 1 week before each experiment and were fed non-contaminated blue mussels (Mytilus edulis) daily. During the experiments, shrimps were fed blue mussels contaminated with methylmercury and/or selenium (Rouleau, 1989) every day. During experiment A, shrimps o.010

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Fig. 4. Rate of NADP reduction at 25°C, expressed as/~ mol NADPH produced per rain, as a function of NADH concentration in the assay medium in the absence of Mn 2+ . The assay mixture consisted of 50mM TEA, 0.5 mM NADP, 5 mM L-malate, 100/J1 of abdominal muscle homogenate and various concentrations of NADH at pH 8.0.

ME and GDH activities in P. borealis Table 1. Optimal conditions for the assay of ME and G D H from the abdominal muscle of P. borealis. After 5-min incubation, assay was started by the addition of Mn :+ for ME and 2-ketoglutarat¢ for G D H

pH TEA L-Malate 2-Ketoglutarate CH 3COONH4 NADH NADP ADP Proteins

ME

GDH

8.00 50 mM 0.125-2.0 mM --0.03 mM 0.5 mM -150 #g in assay

7.80 30 mM -0.125-3.0 mM 150 mM 0.15 mM -0.75 mM 80 #g in assay

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0.02 were fed mussels containing 0.5 + 0.1/~g Hg/g (wet wt) for I0 days. Eight to ten shrimps were randomly sampled at days 0, 5 and 10. The mercury concentration and the activities of ME and GDH in the abdominal muscle were determined. Experiment B lasted 32 days: shrimps were divided into three groups and received mussels containing 2.5 + 1.1 gg Hg/g, 2.9 + 1.2 #g Hg/g and 1.6 + 0.5 #g Se/g (wet wt), respectively,and 2.6 _+0.6 #g Se/g during 21 days. Then only uncontaminated mussels were provided during an I 1-day depuration period. Two to six shrimps were sampled at days 0, 4, 9, 15, 21, 26 and 32. All samples were analyzed for total mercury and selenium and the activity of GDH in the abdominal muscle was measured at 0, 21 and 32 days.

Mercury and selenium analyses About 1 g abdominal muscle was used for the duplicate determination of mercury concentration by cold vapour AAS, according to Hatch and Ott (1968), and the determination of selenium concentration by GC-ECD, following the method described by Cappon and Smith (I 978). Concentrations are expressed as #g mercury or selenium per g wet tissue (pgHg/g and #g Se/g). The detection limit for mercury was 0.05 #g Hg/g and the coefficientof variation of the method was + 10%. The detection limit for selenium was 0.03 #g Se/g and the coefficient of variation of the method was + 15%. Comparison of the data was made using a Student t-test. RESULTS AND DISCUSSION

Characterization o f M E and GDH Malic enzyme activity was maximum with TEA concentration between 50 and 100 mM (Fig. 1). The 0.03 _,-.,

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Fig. 5. The effect of TEA concentration on reductive amination of 2-ketoglutarate catalyzed by GDH, for the abdominal muscle of P. borealis at 25°C. The assay mixture consisted of 0.25mM NADH, 3mM 2-ketoglutarate, 100 mM CH3COONH4, 100 ~1 of abdominal muscle homogenate and various concentrations of TEA at pH 7.8.

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Fig. 6. The effect of pH on reductive amination of 2-ketoglutarate catalyzed by GDH, for the abdominal muscle of P. borealis at 25°C. The assay mixture consisted of 30 mM TEA, 0.25mM NADH, 3 mM 2-ketoglutarate, 225mM CH3COONH4and 100 #l of abdominal muscle homogenate at various pH. effect of pH variation on the oxidative decarboxylation of L-malate by ME was determined from pH 7.0 to 9.0 (Fig. 2) and the maximum activity was observed at pH 8.0; a value similar to previously reported data by Biegniewska and Skorkowski (1983), where the authors found maximum ME activity at pH 7.5-8.0 in the abdominal muscle of the shrimp Crangon crangon, The same value was also found for mitochondrial ME from the heart of the cod Gadus morhua (Biegniewska and Skorkowski, 1987). Mitochondrial ME from the abdominal muscle of the crayfish, Orconectes limosus, showed maximum activity at pH 7.5 (Skorkowski et al., 1977), while pH was optimum at 7.9 for ME from Drosophila melanogaster (Geer et al., 1980). Maximal ME activity was obtained with 2 mM L-malate; no inhibition of the enzyme by the substrate was observed up to l0 mM (Fig. 3). Increased concentrations of NADP (0.25-5.0 mM) as well as Mn 2+ (0.1-5.0mM) did not modify the activity of ME. Previous Kmvalues reported for NADP and Mn 2+ are generally in the # M order; Biegniewska and Skorkowski 0983) found Km values of 16 and 3 #M for NADP and Mn 2+, respectively, in the abdominal muscle of C. crangon. In the abdominal muscle of the crayfish O. limosus, ME had Kmvalues of 12.5 # M for NADP and 2.5/~M for Mn 2+ (Skorkowski et al., 1977). The lack of variation of ME activity in P. borealis may be due to the high concentrations of NADP and Mn 2+ used. To avoid interference from malate dehydrogenase, which has the same substrate as ME (Smith, 1983), a small amount of NADH was added to the assay medium to block the transformation of L-malate by malate dehydrogenase (Outlaw and Springer, 1983). After the addition of 30 # M of NADH (Fig. 4), the activity was partially lowered in the absence of Mn 2+, but not completely, probably due to the presence of endogenous Mn 2+ in the homogenates. To avoid ambiguous results, ME activity was operationally defined as activity in the presence of Mn 2+ minus activity without Mn 2+. A fixed amount of proteins determined to have a maximal change in absorbance of 0.03-0.04/min was used

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Fig. 9. The effect of ADP concentration on reductive amination of 2-ketoglutarate catalyzed by GDH for the abdominal muscle of P. borealis at 25°C. The assay mixture consisted of 30 mM TEA, 0.25 mM NADH, 3 mM 2-ketoglutarate, 225mM CH3COONH4, 100#l of abdominal muscle homogenate and various concentrations of ADP at pH 7.8. 50% at a concentration of 2-ketoglutarate as high as 60 mM (Ruiz Ruano et al., 1985). The inhibition of the activity of G D H in C. crangon was effective for 2-ketoglutarate concentration above 12mM (Regnault and Barrel, 1987). The amount of NH~ ions in the assay required to obtain a maximal G D H activity reached 150 mM (Fig. 8). The decrease in activity observed for higher concentrations of NH + was probably due to the increase in the ionic strength of the assay medium; /~ = 0.20 (molar unit) for 150 mM of CHaCOONH 4 and p =0.45 (molar unit) for 400mM of CH 3COOHN 4. The concentration of N A D H had no effect on G D H activity for the range of concentrations tested (0.05-0.50 mM), probably due to the high concentrations used relative to the K mmeasured. K m values for the shrimp C. crangon were approximately 2 5 p M (Regnault and Batrel, 1987), and ranged from 18 to 34/~M for many other species (Fields et al., 1978; Walton and Cowey, 1977; Ruiz Ruano et al., 1985). G D H from P. borealis was not inhibited by N A D H , while G D H from C. crangon was inhibited at concentrations higher than 0.15 mM (Regnault and Batrel, 1987). The addition of 0.75 mM of ADP to the assay improved G D H activity by a factor of four (Fig. 9). G D H from the hepatopancreas of M. edulis, measured in the direction of reductive amination, showed an absolute requirement for A D P as a cofactor (Ruiz Ruano et al., 1985), while the activity of G D H from C. crangon weakly improved (26-70%) with 0.5 mM ADP in the assay (Regnault and Batrel, 1987). The G D H activity of P. borealis lay between these two extremes as the increase in activity with ADP was important, but the activity of G D H without A D P was high enough to demonstrate that the G D H activity of P. borealis did not have an absolute requirement for this cofactor. The quantity of proteins in the assay was fixed to 80/~g. The optimal conditions of the assay for the determination of G D H activity are summarized in Table I.

907

ME and GDH activities in P. borealis Table 2. Total mercury concentration (/z g Hg/g wet wt) in abdominal muscle of P. borealis feeding on contaminated mussels. Values are means ± SD and the number of shrimps is given in parentheses

et

ExperimentA

Sampling~ [Hgl= 0.5/~g/g day "~ 0 4

0.46 _ 0.I 9 (9)

5 9 10 10(control) 15 21 21 (control) 26 32 32 (control)

0.34 ± 0.15(9) 0.45 + 0.15 (10) 0.38 + 0.15(8)

Experiment B Group 1 [Hg] = 2 . 5 / ~ g / g

Group 2 [Hgl = 2.9 #g/g

[Se] = 1.6#g/g 0.46 _+0.04(4) 0.39+ 0.03(4) 0.54+0.10(4) 0.37+0.09(4)

0.42+0.12(4) 0.42+0.05(3)

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0.38+0.05(4) 0.49+0.10(4) 0.62 _ 0.16(5) 1.00__.0.43(2) 0.42 ___0.14(6) 0.87 + 0.02(2) 0.77+ 0.01 (3) 0.90+0.18(4) 0.94+0.06(2) 0.36 + 0.13 (4)

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The variation coefficient of spec. act. for a given sample at a given substrate concentration (2-6 replicates) was + 3 % for both enzymes. This is comparable to Outlaw and Springer's (1983) data for ME (~< + 5 % for an unknown enzyme source) and to those reported by Schmidt and Schmidt (1983) for G D H in human serum (+2.5%). Use of M E and GDH as stress indicators Obervations of some modifications of the activity of metabolic key-enzymes as a function of mercury accumulation have been used to obtain information on the impact of induced stress on the health of tested animals and on their acclimation to stress (Blackstock, 1984). For example, Sastry and Rao (1982) observed a reduction in the maximal activities of malate dehydrogenase and hexokinase in kidney and liver of Channa punctatus exposed to HgC12 in water (3/~g Hg/I) for a period of 60 days, as a consequence of the impairment of carbohydrate metabolism partially compensated by the utilization of amino acids, as indicated by the increase in the maximal activity of L-amino acid oxidase. The kinetic parameters (Km and Vm~) of ME and G D H in the abdominal muscle of P. borealis were measured to assess the health status of shrimps during mercury contamination of food. The kinetics of mercury bioaccumulation in the abdominal muscle of P. borealis are discussed in detail elsewhere (Rouleau, 1989). Briefly, no increase in mercury concentration was observed in experiment A, but in experiment B, a slow mercury accumulation was observed from days 15 to 26 up to 0.9#g Hg/g (wet wt), twice the concentration at the beginning of the experiment (Table 2). Selenium had no effect on the mercury accumulation and did not bioaccumulate in abdominal muscle of P. borealis (Rouleau, 1989). Standard errors calculated by the programme Enzyme-PC are important, reflecting the great variability between individuals for the kinetic parameters of ME and G D H in the abdominal muscle of P. borealis (Table 3). However, the mean values of the kinetic parameters of G D H are not different for shrimps at day 0 in the three groups of experiment B, demonstrating clearly the reproducibility of the method• The similarity of the mean values of the kinetic parameters of G D H in shrimps sampled at CBPB ~/4--M

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day 0 and in the controls for both experiments indicates the relative acclimation of shrimps to laboratory conditions (Blackstock, 1984). In experiment A, no changes in the Km and Vmaxof ME and G D H were observed for both groups, feeding on contaminated mussels and controls (Table 3). There were no significant changes in the kinetic parameters of G D H for the three groups of experiment B, even if Vmx seemed to be higher in group 2 at day 32, and if Vmax seemed to be lower for the controls at day 21 (Table 3). It may also be noted that the values of Km and Vmaxare similar at day 0 for the three groups, and that they are not different to those of experiment A at day 0. The Km values of ME for L-malate measured in the abdominal muscle of crayfish O. limosus (0.66 mM, Sk.orkowski et al., 1977), and in the liver of bass Dicentrarchus labrax L. (0.31 mM, Iniesta et al., 1985; 0.40 mM, Madero et al., 1986), were similar to the Km measured in P. borealis, while the K m of ME from C. crangon was three times lower (0.13mM, Biegniewska and Skorkowski, 1983). The Km values of G D H for 2-ketoglutarate were 1.43 mM for C. crangon (Regnault and Batrel, 1987), and 1.8 and 1.7 mM in the gills of A. gigas and O. bicirrhosum, respectively (Fields et al., 1978). These values are two to three times higher than those from P. borealis. The Km of G D H in M. edulis hepatopancreas was 0.38mM (Ruiz Ruano et al., 1985), while it was 0.082 mM in the liver of Salmo gairdneri (Walton and Cowey, 1977). Such differences in the kinetic parameters of enzymes from different sources may be due to the occurrence of isoenzymes, and further work concerning the characterization of the isolated enzyme is needed. Despite the increase in mercury concentration in P. borealis abdominal muscle during experiment B, the kinetic parameters of G D H were unchanged. Pellerin-Massicotte et al. (1988, 1989a) observed that enzymes are very sensitive indicators in some circumstances. An increase by a factor of four of the Km of malate dehydrogenase was observed in the mantle of the blue mussel M. edulis exposed to 0.3#g Hg/1 of methylmercury in water over 3 weeks. Bioaccumulated mercury reached approximately the same level as observed in shrimps. The authors also observed a response of malate dehydrogenase for lower concentrations of mercury in mussels (about 0.1/~g Hg/l), after exposure to 0.01/zg Hg/l of methylmercury, and the modification of Km was proportional to mercury concentration in the tested animals. Physiological differences in nutrition, transport, assimilation and complexation processes, as well as mercury speciation, may be responsible for the difference in the enzymatic response of mussels and shrimps. However, the malate dehydrogenase response of M. edulis was subject to large variations, with changes in the experimental conditions, and may be completely eliminated despite mercury accumulation (Pelletier and PellerinMassicotte, 1990). The lack of response of G D H for P. borealis could, in the first instance, be attributed to unidentified environmental changes induced by experimental conditions compared to natural conditions occurring in the Saguenay Oord.

The absence of modification of enzymatic activities observed with P. borealis may be evidence of some other phenomena impossible to identify clearly with the present set of data. For example, the binding sites of mercury on G D H could not be closely related to its catalytic capacity measured here (Chung and Maines, 1982). Among other possibilities, the occurrence of detoxification mechanisms involving metallothioneins should be mentioned (Brown and Parsons, 1978; Ridlington et al., 1981; Engel and Brouwer, 1984). Although the binding capacity of these proteins is limited (Roesijadi, 1982), their synthesis can be pre-induced by chronic exposition to metals (Harrison et al., 1983). Shrimps from the Saguenay fjord may have a sufficient detoxification capacity to neutralize any modification of G D H activity despite the relatively high mercury concentration in the abdominal muscle. Several cases of acclimation to mercurial pollution have been demonstrated in the laboratory (Barkay, 1987; Khan and Weis, 1987; Kraus et al., 1988). The lack of response of G D H may possibly reflect an acclimation of the Saguenay shrimps to the contamination of their environment (Blackstock, 1984). CONCLUSION The lack of response of ME and G D H of the abdominal muscle of P. borealis to mercury contamination reported in this work was observed in a limited number of experiments, and much more work is needed to understand factors which could influence enzymatic responses to metal contamination, such as routes of contamination, experimental conditions, possible acclimation to polluted environment and physiological processes specific to P. borealis. The response of these two enzymes to mercury contamination should be studied with organisms from an uncontaminated environment, in laboratory experiments or in an experimental transfer of animals from a pristine natural site to a contaminated one, a method currently used to evaluate the environmental quality of the St Lawrence estuary (PellerinMassicotte et al., 1989b). Acknowledgements--This work was supported by the Natural Sciences and Engineering Research Council of Canada (C.R., postgraduate fellowship and E.P., operating grant) and the Department of Fisheries and Oceans, Canada. The authors would like to thank Johanne NoSl and Jocelyne Desgagn6 for their technical assistance in this work. This publication is a contribution of the Oceanographic Centre of Rimouski, a partnership of INRS (Institut National de la Recherche Scientifique) and UQAR (Universit6 du Qurbec ~ Rimouski), operating under the auspices of the Universit6 du Qurbec. REFERENCES

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