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Glutamate dehydrogenase from two Antarctic organisms, the icefish Chaenocephalus aceratus and the bacterium Psychrobacter sp. TAD1

Glutamate dehydrogenase from two Antarctic organisms, the icefish Chaenocephalus aceratus and the... Ital. J. Zool., SUPPLEMENT 1: 27-32 (2000) INTRODUCTION Glutamate dehydrogenase from two Antarctic organisms, the icefish Environmental factors, such as temperature and pres- Chaenocephalus aceratus and the sure, may have profound effects on catalytic activity and enzyme regulation and structure. Antarctic organ- bacterium Psychrobacter sp. TAD1 isms are an excellent source for studying the biochemi- cal mechanisms, which allow adaptation to low ex- tremes of temperature. In particular, comparative stud- M. ANTONIETTA CIARDIELLO ies of enzymes with key metabolic roles from organisms RAFFAELA DI FRAIA living in Antarctica and in temperate environments may ANTONELLA ANTIGNANI highlight the features developed during the cold-adap- VITO CARRATORE tation process. LAURA CAMARDELLA Glutamate dehydrogenase (GDH) is a ubiquitous en- GUIDO DI PRISCO zyme in higher and lower organisms and plays an im- Istituto di Biochimica delle Proteine ed Enzimologia, C.N.R., portant role in maintaining the glutamate and ammonia via Marconi 10, I-80125 Napoli, (Italy) levels in the cell. Ammonia assimilation can utilise two major routes, the GDH reaction and the sequential reac- tions catalysed by glutamine synthetase and glutamate synthase. The former pathway does not require ATP, but needs an efficient nitrogen source, since the K of GDH for ammonia is high. The disadvantage of the lat- ter route is energy requirement, since it is ATP-depen- dent. GDH has an important role also in ammonia dis- similation and subsequent recycling of amino groups (necessary for biosynthesis of amino acids, purines, and ABSTRACT pyrimidines) or formation of nitrogenous excretory products (urea, ammonia). Therefore, the reaction catal- Glutamate dehydrogenase (GDH) was purified from the liver of ysed by GDH, i.e., reversible oxidative deamination of the teleost Chaenocephalus aceratus (Notothenioidei: Chan- L-glutamate to a-ketoglutarate through reduction of nichthyidae) and the microorganism Psychrobacter sp. TAD1, from Antarctic marine and terrestrial environments, respectively. NAD+ or NADP+, represents a key enzymatic link be- GDH isolated from C. aceratus liver had a hexameric molecular tween catabolic and biosynthetic pathways, and be- structure very similar to that of other vertebrates and displayed tween carbohydrate and nitrogen metabolism. preference for NAD+, a feature shared with other fish enzymes. GDHs are classified in two classes depending on their The bovine and fish GDH activity and stability were differently af- fected by temperature and hydrostatic pressure. At low tempera- quaternary structure. Hexameric GDHs have been tures, the specific activity of fish GDH was higher than that mea- found in organisms coming from all domains of life, sured with the homologous bovine enzyme. Psychrobacter sp. whereas tetrameric GDHs have been found only in low TAD1 showed a feature quite unusual in bacteria, i.e. the pres- eukaria. Tetrameric GDHs are NAD+-dependent, while ence of two distinct GDHs specific either for NADP+ or for NAD+. NADP+ -dependent GDH was purified and characterised. It has a hexameric GDHs can be either specific for NAD+, or for hexameric structure with a subunit molecular weight similar to NADP+, or dual-coenzyme specific when they can use that described for this class of GDHs and a specific activity at low both coenzymes. Vertebrate GDHs are dual-coenzyme and moderate temperatures similar to that measured with the ho- specific, whereas the enzymes from microorganisms mologous enzyme from Escherichia coli. The kinetic properties of NADP+ -dependent GDH of Psychrobacter sp. TAD1 and the pres- and low eukarya are specific for either NAD+ or ence of another NAD+-dependent GDH suggest that, during the NADP+, although dual specific GDHs have also been cold-adaptation process, this enzymatic function acquired a pat- reported. A biosynthetic and a catabolic role have been tern of changes different from that of C. aceratus. attributed to NADP+- and NAD+-dependent GDHs, re- spectively, and an amphibolic role to the dual-coen- KEY WORDS: Antarctica - Glutamate dehydrogenase - Cold zyme specific enzymes. However, due to some excep- adaptation - Pressure adaptation - Chaenocephalus aceratus - Psy- tions (some fungal and bacterial GDHs) and to the chrobacter sp. TAD1 - Psychrophily. complexity of GDH function, the data available to date are not sufficient to generalise their metabolic role (for ACKNOWLEDGEMENTS a review, see Hudson & Daniel, 1993). A wealth of data on structural and functional proper- This research was supported by the Italian National Programme ties of hexameric GDH from mesophilic species (mi- for Antarctic Research, by the "CNR Target Project on Biotechnol- ogy", and by EU contract BIO4-CT96-0051. We would like to croorganisms, low and high eukarya) and from ther- thank the following colleagues for their collaboration: B. Schmitt mophilic and hyperthermophilic species is available in and G. Hervé, University P. et M. Curie, Paris, France (hydrostatic the literature, whereas GDH from organisms adapted to pressure studies); N. Russell, Wye college, Wye, UK (taxonomical the low extreme of temperature has so far received little classification); V. Wilquet and N. Glansdorff, University of Brus- sels, Belgium (gene cloning). (if any) attention. We are currently studying a vertebrate M. A. CIARDIELLO, R. DI FRAIA, A. ANTIGIANI, V. CARRATORE, L. CAMARDEIXA, G. DI PRISCO GDH, isolated from the liver of the icefish Chaeno- of C. aceratus and bovine GDHs, calculated after 10- cephalus aceratus and NAD+- and NADP+-dependent min incubation, were 50° C and 53° C, respectively GDHs, purified from the Antarctic bacterium Psychro- (Ciardiello et al, 1999a). bacter sp. TAD1. The characterisation comprises kinetic A comparative study of pressure-induced inactivation and thermodynamic analyses, thermostability experi- of C. aceratus and bovine GDHs was carried out (Cia- ments, effectors regulation, elucidation of primary and rdiello et al, 1997d, 1999b). At gradually increasing quaternary structures, and temperature effect on struc- pressure, fish GDH retained all activity up to 140 MPa, tural and functional properties. Recently, we have also whereas in the same pressure range the bovine enzyme undertaken a study of the hydrostatic pressure effect on was slightly inactivated. The kinetics of pressure-in- GDH from Antarctic organisms in order to collect infor- duced inactivation of Antarctic GDH displayed an expo- mation on the combined effect of temperature and nentially decreasing residual activity as a function of in- pressure on the structural and functional properties of cubation time, whereas the bovine enzyme showed a this enzyme. complex pattern of behaviour during the first 20 min of incubation. In fact, in bovine GDH a rapid decrease of activity during the early phase of incubation, followed by a slight increase and then a slow decrease, was ob- MATERIALS AND METHODS served at all pressures tested. The presence of NAD+ in Specimens of C. aceratus were collected by bottom trawling in the incubation mixture slightly stabilised both GDHs, an Dallman Bay and off Low Island, in the vicinity of Palmer Station, effect particularly evident in the initial phase of inactiva- Antarctica. Psychrobacter sp. TAD1 was isolated from frozen con- tion of the bovine enzyme, in which changed the shape tinental water near the French Station of Dumont d'Urville, of the curve (Fig. 1). The stability of C. aceratus GDH Antarctica. Purification of GDH from C. aceratus liver and from Psy- chrobacter sp. TAD1 was carried out following standard proce- dures as previously described (Camardella et al., 1976; Di Fraia et al., 2000). Enzymatic assays of C. aceratus and Psychrobacter sp. TAD1 GDH were carried out according to Ciardiello et al. (1997) and Di Fraia et al (2000). 10 0 RESULTS GDH from Chaenocephalus aceratus GDH was purified to homogeneity from frozen livers and characterised. The subunit molecular weight calcu- lated by SDS-PAGE was 57.9 kDa and the molecular weight of the native protein was compatible with a hexameric structure. These values were very similar to those reported for other vertebrate GDHs. The com- plete amino acid sequence of the subunit was estab- lished and showed very close identity with all verte- brate GDHs so far known (di Ciardiello et al, 1997b; Prisco et al, 1997). Similar to GDH of other vertebrates, the C. aceratus enzyme showed dual-coenzyme specificity, but had higher catalytic rates with NAD(H) (Ciardiello et al., 1997a). The preference for the unphosphorylated coen- zyme is shared with tuna and dogfish GDHs. The spe- cific activity of the pure enzyme, measured at 5° C, was four-fold higher than that of the bovine homologous enzyme, used as a reference. The effect of temperature on the catalytic rate of the reverse reaction (reductive amination of a-ketoglutarate), investigated in the range 5-55° C, indicated that at temperatures lower than 30° C 10 20 30 40 50 60 C. aceratus GDH was a better catalyst than the bovine Time (min) counterpart. Thermostability experiments indicated that C. aceratus and bovine GDHs were inactivated at simi- Fig. 1 - Stability under pressure as a function of time measured according to Ciardiello et al. (1999b). Chaenocephalus aceratus lar temperatures, however the Antarctic enzyme was in- and bovine GDHs were incubated at 200 (A) and 240 MPa (B), re- activated in a much narrower temperature range (Cia- spectively, in phosphate buffer (filled dot) and in the presence of rdiello et al, 1997c). The half-inactivation temperatures NAD+ (filled triangle) and BSA (filled square). GDH FROM C. ACERATUS AND PSYCHROBACTER SP. TAD1 was unaffected by bovine serum albumin (BSA) in the In Tris/Cl and in phosphate buffer, at 20° C the pH incubation mixture. In contrast, BSA had a protective ef- optimum values for the reverse and forward reactions fect on bovine GDH. were around pH 8.0 and pH 9.0, respectively, similar to those of the E. coli enzyme. GDH from Psychrobacter sp. TAD1 Apparent temperature optima for the reverse and for- ward reactions were observed at 25° C and 35° C, re- This strain was chosen after screening several Antarc- spectively. Above these temperatures the enzyme activi- tic bacterial isolates from the collection of C. Gerday ty decreased, although irreversible inactivation occurred (University of Liège, Belgium). It was originally isolated at higher temperatures. Activity as a function of temper- from frozen continental water near the French Antarctic ature of E. coli GDH, measured as a reference meso- Station of Dumont d'Urville (66°40'S, 14O°O1'E) and was philic enzyme, showed apparent maxima at 45° C for capable of aerobic growth in rich liquid medium. The both the reverse and forward reaction; irreversible inac- growth rate increased linearly in the temperature range tivation took place above this temperature. 0-20° C; no growth was observed above 25° C. The The saturation curves towards a-ketoglutarate and characterisation and taxonomical classification of the NADPH, respectively substrate and cofactor of the re- bacterium is under way in collaboration with N. Russell verse reaction, showed a sigmoidal behaviour, indica- (Wye College, University of London, UK). tive of positive cooperativity in the low concentration The bacterial cells were lysed using a French pressure range, and inhibition at higher concentrations. cell press or ultrasonic treatment. In the first procedure, The amino acid sequence of Psychrobacter sp. TAD1 the cell extract contained comparable amounts of GDH was elucidated by direct Edman degradation of NAD+- and NADP+-dependent GDH activity, while in the protein and by sequencing peptides derived from the second procedure the NAD+-dependent activity was endoproteinase Asp-N and Lys-C digestion and from recovered in low and variable amounts, indicating that cyanogen bromide cleavage (Camardella et al., 1998b). it was partially destroyed during ultrasonic treatment. The complete sequence comprised 447 residues yield- Subsequent purification revealed that the two activities ing a molecular mass of 49.285 kDa. Table I shows the were associated with two different enzymes, NAD+-de- similarity level with GDHs from the low eukaria Neu- pendent and NADP+-dependent GDHs being precipitat- rospora crassa and Saccharomyces cerevisiae, the ed at 35-50% and at 60-70% ammonium sulfate satura- mesophilic eubacteria E. coli and Clostridium symbio- tion, respectively (Camardella et al., 1997a, b). sum, and the thermophilic archaea Sulfolobus solfatari- NADP+-dependent GDH was purified by anion-ex- cus and Pyrococcus furiosus. The higher degree of simi- change and affinity chromatography on 2',5'-ADP- larity with GDHs from eubacteria and low eukaria indi- Sepharose. The enzyme was homogeneous, as demon- cated that the subunit of TAD1 GDH belongs to family I strated by SDS polyacrylamide gel electrophoresis. The of hexameric GDHs, whereas thermophilic GDHs be- specific activities measured at 20° C for the reverse and long to family II. The comparison of the overall amino forward reactions were 204 U/mg and 78 U/mg, respec- acid composition indicated a marked increase in hy- tively. Details on the purification and characterisation of drophilic character of TAD1 enzyme (Table II). The se- NADP+-dependent GDH are described in Di Fraia et al., quence of Antarctic GDH was aligned with the corre- (2000). The enzyme was eluted as a sharp peak from sponding sequences of E. coli and C. symbiosum (Fig. both Sephadex G200 and Superóse 6 HR 10/30 gel fil- 2); their identity was close to 50%, and the essential tration columns showing an apparent molecular mass of residues were conserved. The most striking difference 290 kDa. The monomer had a molecular mass of 47 was Ser instead of Ala at position 239 of the TAD1 se- kDa by SDS-PAGE indicating a hexameric molecular quence. This position takes part in the Gly motif typical structure, similar to Escherichia coli NADP+-dependent of the coenzyme binding fold, and consistently has Ala GDH, and absence of polymerisation processes. TABLE I - Similarity percentages among Psychrobacter sp. TAD1 and mesophilic (N. crassa, S. cerevisiae, E. coli and C. symbiosurrt) and thermophilic (5. solfataricus, P. furiosus,) GDHs. Sequence data are from Di Fraia et al. (2000). S. cerevisiae E. coli C. symbiosum TAD1 S. solfataricus P. furiosus N. crassa 11 69 66 67 48 50 S. cerevisiae 67 64 46 50 E. coli 71 70 49 49 C. symbiosum 70 54 TAD1 50 50 S. solfataricus 61 M. A. CIARDIELLO, R. DI FRAIA, A. ANTIGIANI, V. CARRATORE, L. CAMARDELLA, G. DI PRISCO TABLE II - Amino acid composition o/'Psychrobacter φ. TAD1, E. coli, C. symbiosum, N. crassa, S. cerevisiae, S. solfataricus and P. fu- riosus GDHs and hydropathy index (calculated according to Kyte & Doolittle, 1982). Amino acid TAD1 E. coli C. symb. N. era. S. cer. S. soif. P. fur. Ala 42 47 35 33 43 39 Cys 1 4 4 6 6 1 1 Asp 27 19 24 15 16 22 24 Glu 34 36 . 36 36 31 31 33 Ph e 19 22 19 18 18 9 10 Gly 45 48 54 34 49 49 43 His 10 10 6 9 4 4 5 Ile 28 17 20 18 29 35 Lys 22 28 32 31 27 29 37 Leu 29 35 27 38 30 38 24 Met 18 13 17 9 8 11 13 Asn 20 17 20 23 20 20 15 Pro 14 20 15 15 13 13 19 Gin 20 23 16 15 21 13 11 Arg 18 22 18 17 17 20 Ser 22 23 20 29 37 20 14 Th r 16 22 17 16 22 18 Val 36 36 37 45 33 36 35 Trp 5 5 5 8 6 4 10 Tyr 10 21 15 16 20 19 19 Hydropathy index -0.158 -0.214 -0.206 -0.297 -0.264 -0.191 -0.279 and Gly in all GDHs of the first and second family, re- quaternary structure could be deduced (Camardella & spectively. Ser239 in this sequence is unique among all di Prisco, 1999). Attempts to elucidate the N-terminal GDHs known so far. The alignment of TAD1 GDH with sequenc e were unsuccessful because the first residue this sequence is fairly good, and only few single-residue was blocked. At low temperature, the specific activity gaps were introduced to optimise the alignment. Inter- was similar to that measured in the NADP+-dependent estingly, a five residue insertion was observed in the se- enzyme, but thermal stability was remarkably lower quence between α-helices 16 and 17, as determined in (Camardella et al, 1997b, 1998a). the crystallographic structure of C. symbiosum GDH. The increase in the length of loops interconnecting a- helices has been suggested to be a factor of increased DISCUSSION molecular flexibility in cold-adapted enzymes. Indeed, in GDH this position is at the hinge between the two At low and moderate temperature, the specific activity domains which move towards each other during the of C. aceratus GDH was higher than that of bovine catalytic cycle, and could be involved in some acquired GDH, indicating that during evolution some modifica- enzymatic features, in particular the apparent tempera- tions were acquired in order to cope with the reduced ture optimum at 25° C, and cooperativity in the binding catalytic efficiency due to the Q effect. In contrast, the l o of substrate and coenzyme. specific activity of hexameric NADP+-dependent GDH from Psychrobacter sp. TAD1 was similar to that of E. The primary structure of the NADP+-dependent GDH coli GDH; howeve r cooperativity (not shown by the (Di Fraia et al., 2000), obtained by direct protein se- mesophilic enzyme) suggests structural flexibility, pro- quencing, was identical to that deduced from the se- viding the possibility of fine regulation of activity in re- quenc e of the gene (Wilquet et al., 1998, 1999) ob- sponse to various metabolic requirements. Moreover, it tained by the Brussels team (V. Wilquet & N. Glans- should be noted that compensation for reduced activity dorff). at low temperatures can be reached not only through a NAD +-dependent GDH was purified as described in higher intrinsic catalytic rate, but also through a higher Camardella et al. (manuscript submitted). The enzyme, number of enzyme molecules. Relatively high levels of whe n loaded on a Sephadex G200 column, coeluted NADP +-dependent GDH wer e found in Psychrobacter with NADP+-dependent GDH, indicating a native mole- cular weight of 300 kDa. Since the subunit molecular sp. TAD1 cells grown on rich medium containing yeast weight obtained by SDS-PAGE was 160 kDa, a dimeric extract and casein hydrolysate. Under these conditions, GDH FROM C. ACERATUS AND PSYCHROBACTER SP. TAD1 C.symb SKY . VDR V I AEVEKKYADEPEFVQTVEEVLSSLGPVVDAHPEYEEVA L E.COli iMDQTYSLESFLNHVQKRDPNQTEFAQAVREVMTTLWPFLEQNPKYRQMS L TAD 1 S I SK A I EKVEARYAHQPE F I QAVKEV A Ι Τ Ι KP LYDAHPEYDKLK V pa pb C.sym b LERHV I PERV I EFRVPWEDDNGKVHVNTGYRVQFNGAIGPYKGGLRFAP S E.COli siLERLVEPER V I QFRVVWVDDRN Q I QVNRAWRVQFSS A I GPYKGGMRFHPS10 0 TAD 1 FERLVEPDRVFGFRVNWEDDNGEIQINRGWRVQFSNALGPYKGGLRFHPT α 7a C.symb VNLSIMKFLGFEQAFKDSLTTLPMGGAKGGSDFDPNGKSDREVMRFCQA F E.COli 101 V Ν L S I LKFLGFEQTFKNALTTLPMGGGKGGSDFDPKGKSEGEVMRFCQALis o TAD1 VNQSVLKFLGFEQIFKNALTGLPIGGGKGGSDFDPKGKTDSEIRRFCYAF pe C.sym b MTELYR H I GP D I DVPAGDLGVGAR E I GYMYGQYR K I VGGFYNGVLTGKAR S E.COli κ, MTELYRHLGADTDVPAG D IGVGGREVGFMAGMMKKLS N . NTACVFTGKGLSa » TAD 1 MR E LHHYVNKDMDVPAG D I GVGGREVSYMFAMYKNLT R . ESTG V I TGKGV G α 1 0 C.symb FGGSLVRPEATGYGSVYYVEAVMKHENDTLVGKTVALAGFGNVAWGAAKK E.COli 201FGGSL I RPEATGYGLVYFTEAMLKRHGMGFEGMRVSVSGSGNVAQY A I EK2S 0 TAD 1 FGGSLMRTEATGYGAVYFLQNMLAAQNES I EGKKVLVSGAGNVSLHAAE K α 1 1 α 1 2 ph PI C.sym b LAELGAKAVTLSGPDG Y I YDPEG I TTEE K I NYMLEMRASGRNKVQDYADK F E.COli 2!iAMEFGAR V I TASDSSGTVVDESG F . TKEKLAR L I E I KASRDGRVADYAKEF* » TAD 1 A T L I G A I VLTVSDSKG T I YDAKG L . NQE K I DWLKVQKDQHK P . LADYVEV F α 13 pk C.symb GVQFFPGEKPWGQKV D I I MPCATQNDVDLEQAK K I VANNVKY Y I EVANM P E.COli soiGLVYLEGQQPWSLPVDIALPCATQNELDVDAAHQLIANGVKAVAEGANMPsso TAD 1 GGEWMADQKPWS I K A D I A I PSATQN E I NEEDA K L LVDNGVK Y I VEGANM P α 14 α 15a o 15b C.symb TTNEALRFLMQQPNMVVAPSKAVNAGGVLVSGFEMSQNSERLSWTAEEVDS E.COli w Τ Τ I EATEL F . QQAGVLFAPGKAANAGGVATSGLEMAQNAARLGWKAEKVDA40 0 TAD1 LTAEA I D Y I . RLHRVHYAPGKAANAGGVAVSALEMSQNSVRQYQTFEQVD E α 16 α 17 4*3 C.symb KLHQVMTDIHDGSAAAAERYGl . . .GY.NLVAGANIVGFQKIADAMMAQGIA W Eco« «1RLHHIMLDIHHACVEHGGEGEQ T NYVQGANIAGFVKVADAMLAQGVItu TAD1 RLQ G I MK D I HDSSAQASEMYGQTDEGY I DYMSGANMVGFKRVADALVAF G I L N 400 440 Fig. 2 - Alignment of sequences of Psychrobacter sp. TAD1, Escherichia coli, and Clostridium symbiosum GDHs (Di Fraia et al., 2000). Lines above the sequences indicate the secondary structure elements (i. e., α-helices and ß-strands) elucidated by crystallographic analysis of C. symbiosum GDH. Asterisks indicate the coenzyme Gly pattern. the enzyme accounts for almost 0.2% of total soluble tures, developed high stenothermy, whereas microor- protein. This value is similar to that of NADP+-depen- ganisms living in continental frozen water have been dent GDH in E. coli, after induction obtained by grow- exposed to sharp variations of climatic and metabolic ing the cells in a minimal medium with glucose as the conditions. It is conceivable that the amplitude of the sole carbon source and a proper level of ammonia. The range of temperatures experienced by the organism can presence of a second, NAD+-dependent GDH, an un- modulate the pattern of modifications acquired during usual feature in bacteria, increases the activity in the evolution. Characterisation of other Antarctic microor- cell and may provide greater flexibility in adaptation to ganisms may shed light on the correlation between the environment. These results suggest that different physiological and biochemical properties and cold patterns of temperature adaptation could be developed. adaptation. Antarctic marine organisms, living at constant tempera- Chaenocephalus aceratus GDH is affected differently M. A. CIARDIEIXO, R. DI FRAIA, A. ANTIGIANI, V. CARRATORE, L. CAMARDELLA, G. DI PRISCO Ciardiello Μ. Α., Camardella L., di Prisco G., 1997c - Enzymes of by hydrostatic pressure with respect to its bovine coun- Antarctic fishes: effect of temperature on catalysis. Cybium, 21: terpart (Ciardiello et al., 1999b). NAD+ stabilised both 443-450. enzymes, whereas BSA exerted slight protection only Ciardiello Μ. Α., Schmitt Β., di Prisco G., Hervé G., 1997d - Pres- for bovine GDH. Under certain conditions, bovine GDH sure-induced inactivation of L-glutamate dehydrogenase from easily undergoes reversible aggregation (Smith et al., Antarctic fish: comparison with the homologoues enzyme from ox. Abstracts of the Final conference of the European Science 1975). In contrast, similar to tuna (Veronese et al., 1976) Foundation Network "Fishes of the Antarctic Ocean", 22-24 and dogfish (Corman et al, 1967).GDHs, the C. acera- May, Pontignano, Siena, Italy. tus enzyme does not aggregate. Rapid application of Ciardiello Μ. Α., Camardella L., di Prisco G., 1999a - Temperature high pressures drives reversible aggregation of bovine adaptation in enzymes of Antarctic fish. In: R. Margesin & F. GDH. The low propensity to aggregate displayed by the Schinner (eds), Cold-adapted organisms: Ecology, physiology, enzymology and molecular biology. Springer Verlag, Berlin, fish enzymes could be an adaptive feature, related to Heidelberg, New York, pp. 297-304. the life style of an organism which needs to swim at dif- Ciardiello Μ. Α., Schmitt Β., di Prisco G., Hervé G., 1999b - Hy- ferent depths. drostatic pressure influence on L-glutamate dehydrogenase from the Antarctic fish Chaenocephalus aceratus. Mar. Biol., 134: 631-636. Corman L., Prescott L. M., Kaplan N. O., 1967 - Purification and REFERENCES kinetic characteristics of dogfish liver glutamate dehydrogenase. J. Biol. Chem., 242: 1383-1390. Camardella L., Antignani Α., Di Fraia R., Ciardiello Μ. Α., di Prisco Di Fraia R., Wilquet V., Ciardiello Μ. Α., Carratore V., Antignani Α., G., 1998a - A novel glutamate dehydrogenase in the Antarctic Camardella L., Glansdorff N., di Prisco G., 2000 - NADP+-depen- bacterial strain TAD1. Abstracts of the Second Meeting on Ex- dent glutamate dehydrogenase in the Antarctic psychrotolerant tremophiles as Cell Factories, 18-21 April, Dublin, Ireland, p. bacterium Psychrobacter sp. TAD1. Eur. J. Biochem., 267: 121-131. di Prisco G., Camardella L., Carratore V., Ciardiello Μ. Α., Cocea Camardella L., Ciardiello Μ. Α., Di Fraia R., di Prisco G., 1997a - Ε., D'Avino R., Romano M., Tamburrini M., 1997 - Structure and Enzymes in Antarctic extremophilic micro-organisms. In: G. di function of hemoglobins and enzymes from Antarctic organ- Prisco, S. Focardi & P. Luporini (eds), Proceedings of the 3nd isms. The search for correlations with adaptive evolution. In: G. Meeting on Antarctic Biology, Camerino University Press, di Prisco, S. Focardi & P. 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Glutamate dehydrogenase from two Antarctic organisms, the icefish Chaenocephalus aceratus and the bacterium Psychrobacter sp. TAD1

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Abstract

Ital. J. Zool., SUPPLEMENT 1: 27-32 (2000) INTRODUCTION Glutamate dehydrogenase from two Antarctic organisms, the icefish Environmental factors, such as temperature and pres- Chaenocephalus aceratus and the sure, may have profound effects on catalytic activity and enzyme regulation and structure. Antarctic organ- bacterium Psychrobacter sp. TAD1 isms are an excellent source for studying the biochemi- cal mechanisms, which allow adaptation to low ex- tremes of temperature. In particular, comparative stud- M. ANTONIETTA CIARDIELLO ies of enzymes with key metabolic roles from organisms RAFFAELA DI FRAIA living in Antarctica and in temperate environments may ANTONELLA ANTIGNANI highlight the features developed during the cold-adap- VITO CARRATORE tation process. LAURA CAMARDELLA Glutamate dehydrogenase (GDH) is a ubiquitous en- GUIDO DI PRISCO zyme in higher and lower organisms and plays an im- Istituto di Biochimica delle Proteine ed Enzimologia, C.N.R., portant role in maintaining the glutamate and ammonia via Marconi 10, I-80125 Napoli, (Italy) levels in the cell. Ammonia assimilation can utilise two major routes, the GDH reaction and the sequential reac- tions catalysed by glutamine synthetase and glutamate synthase. The former pathway does not require ATP, but needs an efficient nitrogen source, since the K of GDH for ammonia is high. The disadvantage of the lat- ter route is energy requirement, since it is ATP-depen- dent. GDH has an important role also in ammonia dis- similation and subsequent recycling of amino groups (necessary for biosynthesis of amino acids, purines, and ABSTRACT pyrimidines) or formation of nitrogenous excretory products (urea, ammonia). Therefore, the reaction catal- Glutamate dehydrogenase (GDH) was purified from the liver of ysed by GDH, i.e., reversible oxidative deamination of the teleost Chaenocephalus aceratus (Notothenioidei: Chan- L-glutamate to a-ketoglutarate through reduction of nichthyidae) and the microorganism Psychrobacter sp. TAD1, from Antarctic marine and terrestrial environments, respectively. NAD+ or NADP+, represents a key enzymatic link be- GDH isolated from C. aceratus liver had a hexameric molecular tween catabolic and biosynthetic pathways, and be- structure very similar to that of other vertebrates and displayed tween carbohydrate and nitrogen metabolism. preference for NAD+, a feature shared with other fish enzymes. GDHs are classified in two classes depending on their The bovine and fish GDH activity and stability were differently af- fected by temperature and hydrostatic pressure. At low tempera- quaternary structure. Hexameric GDHs have been tures, the specific activity of fish GDH was higher than that mea- found in organisms coming from all domains of life, sured with the homologous bovine enzyme. Psychrobacter sp. whereas tetrameric GDHs have been found only in low TAD1 showed a feature quite unusual in bacteria, i.e. the pres- eukaria. Tetrameric GDHs are NAD+-dependent, while ence of two distinct GDHs specific either for NADP+ or for NAD+. NADP+ -dependent GDH was purified and characterised. It has a hexameric GDHs can be either specific for NAD+, or for hexameric structure with a subunit molecular weight similar to NADP+, or dual-coenzyme specific when they can use that described for this class of GDHs and a specific activity at low both coenzymes. Vertebrate GDHs are dual-coenzyme and moderate temperatures similar to that measured with the ho- specific, whereas the enzymes from microorganisms mologous enzyme from Escherichia coli. The kinetic properties of NADP+ -dependent GDH of Psychrobacter sp. TAD1 and the pres- and low eukarya are specific for either NAD+ or ence of another NAD+-dependent GDH suggest that, during the NADP+, although dual specific GDHs have also been cold-adaptation process, this enzymatic function acquired a pat- reported. A biosynthetic and a catabolic role have been tern of changes different from that of C. aceratus. attributed to NADP+- and NAD+-dependent GDHs, re- spectively, and an amphibolic role to the dual-coen- KEY WORDS: Antarctica - Glutamate dehydrogenase - Cold zyme specific enzymes. However, due to some excep- adaptation - Pressure adaptation - Chaenocephalus aceratus - Psy- tions (some fungal and bacterial GDHs) and to the chrobacter sp. TAD1 - Psychrophily. complexity of GDH function, the data available to date are not sufficient to generalise their metabolic role (for ACKNOWLEDGEMENTS a review, see Hudson & Daniel, 1993). A wealth of data on structural and functional proper- This research was supported by the Italian National Programme ties of hexameric GDH from mesophilic species (mi- for Antarctic Research, by the "CNR Target Project on Biotechnol- ogy", and by EU contract BIO4-CT96-0051. We would like to croorganisms, low and high eukarya) and from ther- thank the following colleagues for their collaboration: B. Schmitt mophilic and hyperthermophilic species is available in and G. Hervé, University P. et M. Curie, Paris, France (hydrostatic the literature, whereas GDH from organisms adapted to pressure studies); N. Russell, Wye college, Wye, UK (taxonomical the low extreme of temperature has so far received little classification); V. Wilquet and N. Glansdorff, University of Brus- sels, Belgium (gene cloning). (if any) attention. We are currently studying a vertebrate M. A. CIARDIELLO, R. DI FRAIA, A. ANTIGIANI, V. CARRATORE, L. CAMARDEIXA, G. DI PRISCO GDH, isolated from the liver of the icefish Chaeno- of C. aceratus and bovine GDHs, calculated after 10- cephalus aceratus and NAD+- and NADP+-dependent min incubation, were 50° C and 53° C, respectively GDHs, purified from the Antarctic bacterium Psychro- (Ciardiello et al, 1999a). bacter sp. TAD1. The characterisation comprises kinetic A comparative study of pressure-induced inactivation and thermodynamic analyses, thermostability experi- of C. aceratus and bovine GDHs was carried out (Cia- ments, effectors regulation, elucidation of primary and rdiello et al, 1997d, 1999b). At gradually increasing quaternary structures, and temperature effect on struc- pressure, fish GDH retained all activity up to 140 MPa, tural and functional properties. Recently, we have also whereas in the same pressure range the bovine enzyme undertaken a study of the hydrostatic pressure effect on was slightly inactivated. The kinetics of pressure-in- GDH from Antarctic organisms in order to collect infor- duced inactivation of Antarctic GDH displayed an expo- mation on the combined effect of temperature and nentially decreasing residual activity as a function of in- pressure on the structural and functional properties of cubation time, whereas the bovine enzyme showed a this enzyme. complex pattern of behaviour during the first 20 min of incubation. In fact, in bovine GDH a rapid decrease of activity during the early phase of incubation, followed by a slight increase and then a slow decrease, was ob- MATERIALS AND METHODS served at all pressures tested. The presence of NAD+ in Specimens of C. aceratus were collected by bottom trawling in the incubation mixture slightly stabilised both GDHs, an Dallman Bay and off Low Island, in the vicinity of Palmer Station, effect particularly evident in the initial phase of inactiva- Antarctica. Psychrobacter sp. TAD1 was isolated from frozen con- tion of the bovine enzyme, in which changed the shape tinental water near the French Station of Dumont d'Urville, of the curve (Fig. 1). The stability of C. aceratus GDH Antarctica. Purification of GDH from C. aceratus liver and from Psy- chrobacter sp. TAD1 was carried out following standard proce- dures as previously described (Camardella et al., 1976; Di Fraia et al., 2000). Enzymatic assays of C. aceratus and Psychrobacter sp. TAD1 GDH were carried out according to Ciardiello et al. (1997) and Di Fraia et al (2000). 10 0 RESULTS GDH from Chaenocephalus aceratus GDH was purified to homogeneity from frozen livers and characterised. The subunit molecular weight calcu- lated by SDS-PAGE was 57.9 kDa and the molecular weight of the native protein was compatible with a hexameric structure. These values were very similar to those reported for other vertebrate GDHs. The com- plete amino acid sequence of the subunit was estab- lished and showed very close identity with all verte- brate GDHs so far known (di Ciardiello et al, 1997b; Prisco et al, 1997). Similar to GDH of other vertebrates, the C. aceratus enzyme showed dual-coenzyme specificity, but had higher catalytic rates with NAD(H) (Ciardiello et al., 1997a). The preference for the unphosphorylated coen- zyme is shared with tuna and dogfish GDHs. The spe- cific activity of the pure enzyme, measured at 5° C, was four-fold higher than that of the bovine homologous enzyme, used as a reference. The effect of temperature on the catalytic rate of the reverse reaction (reductive amination of a-ketoglutarate), investigated in the range 5-55° C, indicated that at temperatures lower than 30° C 10 20 30 40 50 60 C. aceratus GDH was a better catalyst than the bovine Time (min) counterpart. Thermostability experiments indicated that C. aceratus and bovine GDHs were inactivated at simi- Fig. 1 - Stability under pressure as a function of time measured according to Ciardiello et al. (1999b). Chaenocephalus aceratus lar temperatures, however the Antarctic enzyme was in- and bovine GDHs were incubated at 200 (A) and 240 MPa (B), re- activated in a much narrower temperature range (Cia- spectively, in phosphate buffer (filled dot) and in the presence of rdiello et al, 1997c). The half-inactivation temperatures NAD+ (filled triangle) and BSA (filled square). GDH FROM C. ACERATUS AND PSYCHROBACTER SP. TAD1 was unaffected by bovine serum albumin (BSA) in the In Tris/Cl and in phosphate buffer, at 20° C the pH incubation mixture. In contrast, BSA had a protective ef- optimum values for the reverse and forward reactions fect on bovine GDH. were around pH 8.0 and pH 9.0, respectively, similar to those of the E. coli enzyme. GDH from Psychrobacter sp. TAD1 Apparent temperature optima for the reverse and for- ward reactions were observed at 25° C and 35° C, re- This strain was chosen after screening several Antarc- spectively. Above these temperatures the enzyme activi- tic bacterial isolates from the collection of C. Gerday ty decreased, although irreversible inactivation occurred (University of Liège, Belgium). It was originally isolated at higher temperatures. Activity as a function of temper- from frozen continental water near the French Antarctic ature of E. coli GDH, measured as a reference meso- Station of Dumont d'Urville (66°40'S, 14O°O1'E) and was philic enzyme, showed apparent maxima at 45° C for capable of aerobic growth in rich liquid medium. The both the reverse and forward reaction; irreversible inac- growth rate increased linearly in the temperature range tivation took place above this temperature. 0-20° C; no growth was observed above 25° C. The The saturation curves towards a-ketoglutarate and characterisation and taxonomical classification of the NADPH, respectively substrate and cofactor of the re- bacterium is under way in collaboration with N. Russell verse reaction, showed a sigmoidal behaviour, indica- (Wye College, University of London, UK). tive of positive cooperativity in the low concentration The bacterial cells were lysed using a French pressure range, and inhibition at higher concentrations. cell press or ultrasonic treatment. In the first procedure, The amino acid sequence of Psychrobacter sp. TAD1 the cell extract contained comparable amounts of GDH was elucidated by direct Edman degradation of NAD+- and NADP+-dependent GDH activity, while in the protein and by sequencing peptides derived from the second procedure the NAD+-dependent activity was endoproteinase Asp-N and Lys-C digestion and from recovered in low and variable amounts, indicating that cyanogen bromide cleavage (Camardella et al., 1998b). it was partially destroyed during ultrasonic treatment. The complete sequence comprised 447 residues yield- Subsequent purification revealed that the two activities ing a molecular mass of 49.285 kDa. Table I shows the were associated with two different enzymes, NAD+-de- similarity level with GDHs from the low eukaria Neu- pendent and NADP+-dependent GDHs being precipitat- rospora crassa and Saccharomyces cerevisiae, the ed at 35-50% and at 60-70% ammonium sulfate satura- mesophilic eubacteria E. coli and Clostridium symbio- tion, respectively (Camardella et al., 1997a, b). sum, and the thermophilic archaea Sulfolobus solfatari- NADP+-dependent GDH was purified by anion-ex- cus and Pyrococcus furiosus. The higher degree of simi- change and affinity chromatography on 2',5'-ADP- larity with GDHs from eubacteria and low eukaria indi- Sepharose. The enzyme was homogeneous, as demon- cated that the subunit of TAD1 GDH belongs to family I strated by SDS polyacrylamide gel electrophoresis. The of hexameric GDHs, whereas thermophilic GDHs be- specific activities measured at 20° C for the reverse and long to family II. The comparison of the overall amino forward reactions were 204 U/mg and 78 U/mg, respec- acid composition indicated a marked increase in hy- tively. Details on the purification and characterisation of drophilic character of TAD1 enzyme (Table II). The se- NADP+-dependent GDH are described in Di Fraia et al., quence of Antarctic GDH was aligned with the corre- (2000). The enzyme was eluted as a sharp peak from sponding sequences of E. coli and C. symbiosum (Fig. both Sephadex G200 and Superóse 6 HR 10/30 gel fil- 2); their identity was close to 50%, and the essential tration columns showing an apparent molecular mass of residues were conserved. The most striking difference 290 kDa. The monomer had a molecular mass of 47 was Ser instead of Ala at position 239 of the TAD1 se- kDa by SDS-PAGE indicating a hexameric molecular quence. This position takes part in the Gly motif typical structure, similar to Escherichia coli NADP+-dependent of the coenzyme binding fold, and consistently has Ala GDH, and absence of polymerisation processes. TABLE I - Similarity percentages among Psychrobacter sp. TAD1 and mesophilic (N. crassa, S. cerevisiae, E. coli and C. symbiosurrt) and thermophilic (5. solfataricus, P. furiosus,) GDHs. Sequence data are from Di Fraia et al. (2000). S. cerevisiae E. coli C. symbiosum TAD1 S. solfataricus P. furiosus N. crassa 11 69 66 67 48 50 S. cerevisiae 67 64 46 50 E. coli 71 70 49 49 C. symbiosum 70 54 TAD1 50 50 S. solfataricus 61 M. A. CIARDIELLO, R. DI FRAIA, A. ANTIGIANI, V. CARRATORE, L. CAMARDELLA, G. DI PRISCO TABLE II - Amino acid composition o/'Psychrobacter φ. TAD1, E. coli, C. symbiosum, N. crassa, S. cerevisiae, S. solfataricus and P. fu- riosus GDHs and hydropathy index (calculated according to Kyte & Doolittle, 1982). Amino acid TAD1 E. coli C. symb. N. era. S. cer. S. soif. P. fur. Ala 42 47 35 33 43 39 Cys 1 4 4 6 6 1 1 Asp 27 19 24 15 16 22 24 Glu 34 36 . 36 36 31 31 33 Ph e 19 22 19 18 18 9 10 Gly 45 48 54 34 49 49 43 His 10 10 6 9 4 4 5 Ile 28 17 20 18 29 35 Lys 22 28 32 31 27 29 37 Leu 29 35 27 38 30 38 24 Met 18 13 17 9 8 11 13 Asn 20 17 20 23 20 20 15 Pro 14 20 15 15 13 13 19 Gin 20 23 16 15 21 13 11 Arg 18 22 18 17 17 20 Ser 22 23 20 29 37 20 14 Th r 16 22 17 16 22 18 Val 36 36 37 45 33 36 35 Trp 5 5 5 8 6 4 10 Tyr 10 21 15 16 20 19 19 Hydropathy index -0.158 -0.214 -0.206 -0.297 -0.264 -0.191 -0.279 and Gly in all GDHs of the first and second family, re- quaternary structure could be deduced (Camardella & spectively. Ser239 in this sequence is unique among all di Prisco, 1999). Attempts to elucidate the N-terminal GDHs known so far. The alignment of TAD1 GDH with sequenc e were unsuccessful because the first residue this sequence is fairly good, and only few single-residue was blocked. At low temperature, the specific activity gaps were introduced to optimise the alignment. Inter- was similar to that measured in the NADP+-dependent estingly, a five residue insertion was observed in the se- enzyme, but thermal stability was remarkably lower quence between α-helices 16 and 17, as determined in (Camardella et al, 1997b, 1998a). the crystallographic structure of C. symbiosum GDH. The increase in the length of loops interconnecting a- helices has been suggested to be a factor of increased DISCUSSION molecular flexibility in cold-adapted enzymes. Indeed, in GDH this position is at the hinge between the two At low and moderate temperature, the specific activity domains which move towards each other during the of C. aceratus GDH was higher than that of bovine catalytic cycle, and could be involved in some acquired GDH, indicating that during evolution some modifica- enzymatic features, in particular the apparent tempera- tions were acquired in order to cope with the reduced ture optimum at 25° C, and cooperativity in the binding catalytic efficiency due to the Q effect. In contrast, the l o of substrate and coenzyme. specific activity of hexameric NADP+-dependent GDH from Psychrobacter sp. TAD1 was similar to that of E. The primary structure of the NADP+-dependent GDH coli GDH; howeve r cooperativity (not shown by the (Di Fraia et al., 2000), obtained by direct protein se- mesophilic enzyme) suggests structural flexibility, pro- quencing, was identical to that deduced from the se- viding the possibility of fine regulation of activity in re- quenc e of the gene (Wilquet et al., 1998, 1999) ob- sponse to various metabolic requirements. Moreover, it tained by the Brussels team (V. Wilquet & N. Glans- should be noted that compensation for reduced activity dorff). at low temperatures can be reached not only through a NAD +-dependent GDH was purified as described in higher intrinsic catalytic rate, but also through a higher Camardella et al. (manuscript submitted). The enzyme, number of enzyme molecules. Relatively high levels of whe n loaded on a Sephadex G200 column, coeluted NADP +-dependent GDH wer e found in Psychrobacter with NADP+-dependent GDH, indicating a native mole- cular weight of 300 kDa. Since the subunit molecular sp. TAD1 cells grown on rich medium containing yeast weight obtained by SDS-PAGE was 160 kDa, a dimeric extract and casein hydrolysate. Under these conditions, GDH FROM C. ACERATUS AND PSYCHROBACTER SP. TAD1 C.symb SKY . VDR V I AEVEKKYADEPEFVQTVEEVLSSLGPVVDAHPEYEEVA L E.COli iMDQTYSLESFLNHVQKRDPNQTEFAQAVREVMTTLWPFLEQNPKYRQMS L TAD 1 S I SK A I EKVEARYAHQPE F I QAVKEV A Ι Τ Ι KP LYDAHPEYDKLK V pa pb C.sym b LERHV I PERV I EFRVPWEDDNGKVHVNTGYRVQFNGAIGPYKGGLRFAP S E.COli siLERLVEPER V I QFRVVWVDDRN Q I QVNRAWRVQFSS A I GPYKGGMRFHPS10 0 TAD 1 FERLVEPDRVFGFRVNWEDDNGEIQINRGWRVQFSNALGPYKGGLRFHPT α 7a C.symb VNLSIMKFLGFEQAFKDSLTTLPMGGAKGGSDFDPNGKSDREVMRFCQA F E.COli 101 V Ν L S I LKFLGFEQTFKNALTTLPMGGGKGGSDFDPKGKSEGEVMRFCQALis o TAD1 VNQSVLKFLGFEQIFKNALTGLPIGGGKGGSDFDPKGKTDSEIRRFCYAF pe C.sym b MTELYR H I GP D I DVPAGDLGVGAR E I GYMYGQYR K I VGGFYNGVLTGKAR S E.COli κ, MTELYRHLGADTDVPAG D IGVGGREVGFMAGMMKKLS N . NTACVFTGKGLSa » TAD 1 MR E LHHYVNKDMDVPAG D I GVGGREVSYMFAMYKNLT R . ESTG V I TGKGV G α 1 0 C.symb FGGSLVRPEATGYGSVYYVEAVMKHENDTLVGKTVALAGFGNVAWGAAKK E.COli 201FGGSL I RPEATGYGLVYFTEAMLKRHGMGFEGMRVSVSGSGNVAQY A I EK2S 0 TAD 1 FGGSLMRTEATGYGAVYFLQNMLAAQNES I EGKKVLVSGAGNVSLHAAE K α 1 1 α 1 2 ph PI C.sym b LAELGAKAVTLSGPDG Y I YDPEG I TTEE K I NYMLEMRASGRNKVQDYADK F E.COli 2!iAMEFGAR V I TASDSSGTVVDESG F . TKEKLAR L I E I KASRDGRVADYAKEF* » TAD 1 A T L I G A I VLTVSDSKG T I YDAKG L . NQE K I DWLKVQKDQHK P . LADYVEV F α 13 pk C.symb GVQFFPGEKPWGQKV D I I MPCATQNDVDLEQAK K I VANNVKY Y I EVANM P E.COli soiGLVYLEGQQPWSLPVDIALPCATQNELDVDAAHQLIANGVKAVAEGANMPsso TAD 1 GGEWMADQKPWS I K A D I A I PSATQN E I NEEDA K L LVDNGVK Y I VEGANM P α 14 α 15a o 15b C.symb TTNEALRFLMQQPNMVVAPSKAVNAGGVLVSGFEMSQNSERLSWTAEEVDS E.COli w Τ Τ I EATEL F . QQAGVLFAPGKAANAGGVATSGLEMAQNAARLGWKAEKVDA40 0 TAD1 LTAEA I D Y I . RLHRVHYAPGKAANAGGVAVSALEMSQNSVRQYQTFEQVD E α 16 α 17 4*3 C.symb KLHQVMTDIHDGSAAAAERYGl . . .GY.NLVAGANIVGFQKIADAMMAQGIA W Eco« «1RLHHIMLDIHHACVEHGGEGEQ T NYVQGANIAGFVKVADAMLAQGVItu TAD1 RLQ G I MK D I HDSSAQASEMYGQTDEGY I DYMSGANMVGFKRVADALVAF G I L N 400 440 Fig. 2 - Alignment of sequences of Psychrobacter sp. TAD1, Escherichia coli, and Clostridium symbiosum GDHs (Di Fraia et al., 2000). Lines above the sequences indicate the secondary structure elements (i. e., α-helices and ß-strands) elucidated by crystallographic analysis of C. symbiosum GDH. Asterisks indicate the coenzyme Gly pattern. the enzyme accounts for almost 0.2% of total soluble tures, developed high stenothermy, whereas microor- protein. This value is similar to that of NADP+-depen- ganisms living in continental frozen water have been dent GDH in E. coli, after induction obtained by grow- exposed to sharp variations of climatic and metabolic ing the cells in a minimal medium with glucose as the conditions. It is conceivable that the amplitude of the sole carbon source and a proper level of ammonia. The range of temperatures experienced by the organism can presence of a second, NAD+-dependent GDH, an un- modulate the pattern of modifications acquired during usual feature in bacteria, increases the activity in the evolution. Characterisation of other Antarctic microor- cell and may provide greater flexibility in adaptation to ganisms may shed light on the correlation between the environment. These results suggest that different physiological and biochemical properties and cold patterns of temperature adaptation could be developed. adaptation. Antarctic marine organisms, living at constant tempera- Chaenocephalus aceratus GDH is affected differently M. A. CIARDIEIXO, R. DI FRAIA, A. ANTIGIANI, V. CARRATORE, L. CAMARDELLA, G. DI PRISCO Ciardiello Μ. Α., Camardella L., di Prisco G., 1997c - Enzymes of by hydrostatic pressure with respect to its bovine coun- Antarctic fishes: effect of temperature on catalysis. Cybium, 21: terpart (Ciardiello et al., 1999b). NAD+ stabilised both 443-450. enzymes, whereas BSA exerted slight protection only Ciardiello Μ. Α., Schmitt Β., di Prisco G., Hervé G., 1997d - Pres- for bovine GDH. Under certain conditions, bovine GDH sure-induced inactivation of L-glutamate dehydrogenase from easily undergoes reversible aggregation (Smith et al., Antarctic fish: comparison with the homologoues enzyme from ox. Abstracts of the Final conference of the European Science 1975). In contrast, similar to tuna (Veronese et al., 1976) Foundation Network "Fishes of the Antarctic Ocean", 22-24 and dogfish (Corman et al, 1967).GDHs, the C. acera- May, Pontignano, Siena, Italy. tus enzyme does not aggregate. Rapid application of Ciardiello Μ. 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Journal

Italian Journal of Zoology – Taylor & Francis

Published: Jan 1, 2000

Keywords: Antarctica; Glutamate dehydrogenase; Cold adaptation; Pressure adaptation; Chaenocephalus aceratus; Psychrobacter sp. TAD1; Psychrophily

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