Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD⁺/H or NADP⁺/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD⁺/H than with NADP⁺/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.     

Alanine dehydrogenase of the N2-fixing blue-green alga,Anabaena cylindrica

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane     

NADP-Specific Glutamate Dehydrogenase from Alkaliphilic Bacillus sp. KSM-635: Purification and Enzymatic Properties

An NADP-specific glutamate dehydrogenase [L-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (K_m 44 μM), 2-oxoglutarate (K_m 3.13 mM), NADP⁺ (K_m 29 μM), and L-glutamate (K_m 6.06 mM). The K_m for NH₄Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C.     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

Glutamate dehydrogenase of lupin nodules: Purification and properties

Glutamate dehydrogenase, GDH (l-glutamate: NAD⁺ oxidoreductase (deaminating) EC 1.4.1.2) was purified from the plant fraction of lupin nodules and the purity of the preparation established by gel electrophoresis and electrofocusing. The purified enzyme existed as 4 charge isozymes with a MW of 270000. The subunit MW, as determined by dodecyl sulphate electrophoresis, was 45 000. On the basis of the results of the MW determinations a hexameric structure is proposed for lupin-nodule GDH. The pH optima for the enzyme were pH 8.2 for the amination reaction and pH 8.8 for the deamination reaction. GDH from lupin nodules showed a marked preference for NADH over NADPH in the amination reaction and used only NAD⁺ for the deamination reaction. Pyridoxal-5′-P and EDTA inhibited activity. The enzyme displayed Michaelis-Menten kinetics with respect to all substrates except NAD⁺. When NAD⁺ was the varied substrate, there was a deviation from Michaelis-Menten behaviour towards higher activity at high concentrations of NAD⁺.     

The tricarboxylic acid cycle in L₃ Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L₃Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD⁺ and NADP⁺ specific) and α-ketoglutarate dehydrogenase in homogenates of L₃T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD⁺ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD⁺] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L₃T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L₃T. circumcincta are probably similar to those in the tissues of their host species.     

Enzymes of the ornithine-glutamate-proline pathway in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

A fully functional ornithine–glutamate–proline pathway was detected in L3 and adult Haemonchus contortus and Teladorsagia circumcincta, making the parasites capable of interconversion of these amino acids. Ornithine aminotransferase (OAT) (E.C. 2.6.1.13) was a reversible pyridoxal-5-phosphate (PLP)-dependent enzyme with an optimum pH 8.5. Hydroxylamine completely inhibited OAT activity in both parasites. For all five enzymes, substrate affinity was similar for each species and life cycle stage, the notable exceptions being the nearly 10-fold lower affinity for Δ¹-pyrroline-5-carboxylate (P5C) of P5C reductase (E.C. 1.5.1.2) in adult T. circumcincta and about half for P5C for L3 H. contortus P5C dehydrogenase (E.C. 1.5.1.12). P5C synthase (E.C. 1.2.1.41) activity was similar with either NADPH or NADH as co-factor. Proline oxidase (E.C. 1.5.99.8) was a co-factor independent enzyme with an optimal pH 8.5. Despite similarities to those in the host, enzymes of this pathway may still be useful as control targets if they differ antigenically, as a supply of proline is necessary for cuticle formation.     

Glutamate dehydrogenase of the germinating triticale seeds: gene expression,activity distribution and kinetic characteristics

On the cross-roads of main carbon and nitrogen metabolic pathways, glutamate dehydrogenase (GDH, E.C. 1.4.1.2) carries out the reaction of reductive amination of 2-oxoglutarate to glutamate (the anabolic activity; NAD(P)H–GDH), and the reverse reaction of oxidative deamination of glutamic acid (the catabolic activity; NAD(P)⁺–GDH). To date, there have been no reports on identification of GDH genes in cereals. Here, we report cloning and biochemical characterization of the GDH from germinating triticale seeds, a common Polish cereal. A single TsGDH1 gene is 1,620 bp long, while its 1,236 bp long open reading frame encodes a protein of 411 amino acids of high homology with the published GDH protein sequences from other plants. Phylogenetic analyses locate the TsGDH1 among other monocotyledonous proteins and among the sequences of the β-type subunit of plant GDHs. Changes in TsGDH1 expression and the dynamics of enzyme activity in germinating seeds confirm the existence of one TsGDH isoform with varying expression and activity patterns, depending on the tissue localization and stage of germination. The four-step purification method (including the anionite chromatography using HPLC) resulted in a protein preparation with a high-specific activity and purification factor of approx. 230. The purified enzyme exhibited an absolute specificity towards 2-oxoglutarate (NAD(P)H–GDH), or towards l-glutamate in the reverse reaction (NAD(P)⁺–GDH), while its low K _m constants towards all substrates and co-enzymes may suggest its aminating activity during germination, or, alternatively, its capability to adjust the direction of the catalyzed reaction according to the metabolic necessity.     

Glutamate dehydrogenase activity in developing soybean seed: Isolation and characterization of three forms of the enzyme

Three forms of glutamate dehydrogenase have been isolated from developing soybean seed. Their intracellular locations could not be determined directly because organelles and marker enzymes showed abnormal distribution on sucrose density gradient fractionation. By analogy with enzymes from other parts of the plant, glutamate dehydrogenase 2 was shown to be located in chloroplasts and glutamate dehydrogenase 3 in mitochondria. Glutamate dehydrogenase 1 could not be located in this way because it is found only in the seed. The three enzymes are similar in pH optima, molecular weight and substrate specificity with respect to 2-oxoglutarate and l-glutamate. The mitochondrial enzyme is specific for NAD⁺. The chloroplast enzyme shows low activity with NADP⁺ relative to NAD⁺ but uses NADPH readily in the aminating direction. Glutamate dehydrogenase 1 is active with both nucleotides and is the only form to show substantial deaminating activity with NADP⁺. Glutamate dehydrogenases 1 and 2 are activated and stabilized by glutathione and 2-mercaptoethanol whereas enzyme 3 is unaffected. No significant metabolic control of any of the enzymes could be detected. Malate, citrate, adenine nucleotides and long-chain fatty acyl CoA derivatives gave slight inhibition at high concentrations. Amino acids had no effect on activity. A possible role for the enzyme characteristic of the developing seed is discussed in relation to nutrient supply during the accumulation of reserve materials in the seed.     

Malate: A Possible Source of Error in the NAD Glutamate Dehydrogenase Assay

The effects of externally induced metabolic perturbations areoften studied through changes of the enzyme activity patternsin crude plant extracts. From glutamate dehydrogenase (GDH)it is reported that environmental changes not only influencethe amount of the enzymatic activity, but also the ratio ofthe aminating to the deaminating activities (NADH/NAD⁺ ratio).Using crude cell extracts of suspension cultures of wheat (Triticumaestivum L. cv. Heines Koga II) we find evidence that the pretreatmentof the homogenate directly influences this ratio. Dialysis ofthese crude cell extracts resulted in a 70% loss of the NAD⁺activity, while the NADH activity remained unchanged. The deaminatingactivity in the dialysed extract could be completely restoredupon addition of a dialysable factor which was identified tobe malate. The interference of malate with the glutamate dehydrogenasereaction is caused through the action of malate dehydrogenaseand glutamate oxaloacetate transaminase which are both presentin high activities in the extracts. Only in exhaustively dialysedcell extracts can the proper deaminating GDH activity be determined.The results are discussed in the light of the controversialreports on the variable ratio of the NADH/NAD⁺ activity of GDH. Key words: Glutamate dehydrogenase, malate, NADH/NAD⁺, activity, Triticum aestivum     

Glutamate synthase, but not GABA shunt enzymes, contributes to nitrogen metabolism of the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

Glutamate synthase (E.C. 1.4.1.14) (GOGAT) activity was not detectable in L3 Haemonchus contortus, but was present in L3 Teladorsagia circumcincta and adult worms of both species. GOGAT activity was inhibited by 80% by azaserine. Activity (nmol min⁻¹ mg⁻¹ protein) was 33–59 in adult H. contortus, 51–91 in adult T. circumcincta and 24–41 in L3 T. circumcincta, probably depending on exposure to ammonia, as incubation with 1 mM NH₄Cl doubled GOGAT activity. The pH optimum was 7.5 in both species. Either NAD or NADP acted as co-factor. The mean apparent K_m for 2-oxoglutarate was 0.7 (0.5–0.9) mM and for glutamine was 1.0 (0.5–1.7) mM for different homogenates. There was no detectable activity in whole parasite homogenates of glutamate decarboxylase (E.C. 4.1.1.15) or succinic semialdehyde dehydrogenase (E.C. 1.2.1.24), the first and third enzymes of the GABA shunt, respectively, suggesting that the GABA shunt is not important in general metabolism in these species.     

Endogen und exogen ausgelöste Änderung des Isoenzymspektrums der NAD-spezifischen Glutamatdehydrogenase im Sproß von Pisum sativum

Summary The isoenzymes of NAD-specific glutamic dehydrogenase (GDH) of Pisum sativum, separated by polyacrylamide gel electrophoresis, constitute two patterns, each of which covers seven individual isoenzymes. One pattern (GDH-I) is found in the cotyledons and young shoots. The second one (GDH-II) occurs together with at least some GDH-I isoenzymes in pea roots. In the shoots of older pea plants GDH-II isoenzymes become visible in addition to the GDH-I pattern.Section of the cotyledons (but not of the roots) of young pea seedlings causes the formation of the complete GDH-II isoenzyme pattern in the shoots within a few hours. It has been verified that the cotyledons specifically suppress the formation of the GDH-II pattern in the young shoot. In older plants which no longer depend on the cotyledons this effect is maintained somewhat less obviously by the root system.In experiments with isolated shoot segments or shoot tips it has been shown that NH ₄ ⁺ reinforces the formation of the GDH-II whereas glucose shows the opposite effect.The formation of the GDH-II isoenzymes in the presence of NH ₄ ⁺ is accompanied by an increase of the specific activity of GDH. Simultaneously the ratio of aminating activity (anabolic reaction) to deaminating activity (catabolic reaction) changes in favor of the anabolic reaction.The results support the supposition that the GDH-I and GDH-II isoenzyme patterns correspond to different molecular forms of one enzyme, the GDH-II representing a form with predominantly anabolic function and the GDH-I a form which has merely metabolic or catabolic function.     

Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum

Glutamate dehydrogenase (GDH) was purified from rough endoplasmic reticulum (RER) in rat liver using anion‐exchange and affinity chromatography. As GDH has been known as an enzyme that exists mainly in the matrix of mitochondria, the properties of purified GDH were compared with those of mitochondrial GDH. The GDH activity in 0.1% Triton X‐100‐treated RER subcellular fraction was nearly the same as intact RER, whereas that of the mitochondrial fraction increased by 50% after the detergent treatment. In kinetic values, in addition, mitochondrial GDH had a higher K_m value for NADP⁺ than NAD⁺, whereas the K_m value for NAD⁺ was higher than that for NADP⁺ in the case of GDH of RER, which showed a difference in specificity to cofactors. Moreover, when two GDH isoproteins were incubated at 42°C or treated with trypsin, GDH from RER was more stable against heat inactivation and less susceptible to proteolysis than mitochondrial GDH in both cases. In addition, GDH of RER had at least five amino acids different from mitochondrial GDH when sequences of N‐terminal and several internal peptide fragments were analyzed. These results showed that GDH of RER is another isoprotein of GDH, of whose properties are different from those of mitochondrial GDH. J. Cell. Biochem. 76:244–253, 1999. © 1999 Wiley‐Liss, Inc.     

The seven NAD(H)-glutamate dehydrogenase isoenzymes exhibit similar anabolic and catabolic activities

The specific activities of aminating NADH- and deaminating NAD⁺-glutamate dehydrogenase (GDH, EC 1.4.1.2) varied considerably in crude extracts of grapevine ( Vitis vinifera L. cv. Sultanina) callus and were dependent on the nitrogen source of the culture medium. However, dialysis of the enzyme preparations resulted in a significant decrease in the deaminating GDH specific activity while the aminating activity was not affected. The presence of malate in the crude extract resulted in erroneous overestimation of the NAD⁺-GDH activity through the malate dehydrogenase reaction. Thus, in dialysed extracts, the ratio of the NADH-GDH/NAD⁺-GDH specific activities remained relatively constant irrespective of the nitrogen source. In view of this evidence, we now have modified methods for staining both the NADH-GDH and NAD⁺-GDH activities on gels in order to compare the aminating and deaminating activities of each of the 7 GDH isoenzymes. The results from the staining of NADH-GDH and NAD⁺-GDH activity of enzyme preparations from calluses revealed the same isoenzyme profile. Furthermore, separated leaf isoenzymes showed similar activity ratios and kinetic properties. These results may suggest that each one of the 7 isoenzymes have similar in vitro anabolic and catabolic activities.     

Nickel and Al-excess inhibit nitrate reductase but upregulate activities of aminating glutamate dehydrogenase and aminotransferases in growing rice seedlings

The present study was undertaken to examine the influence of toxic levels of Ni and Al, on the activities of key nitrogen assimilatory enzymes in roots and shoots of growing rice seedlings. When seedlings of two inbred rice (Oryza sativa L.) cvs. Malviya-36 and Pant-12, sensitive to both Ni and Al, were raised in sand cultures containing 200 and 400 μM NiSO₄ or 80 and 160 μM Al₂(SO₄)₃, a marked inhibition in the activities of NO₃ ⁻ assimilatory enzymes NR and GS was observed in roots as well as shoots during a 5–20 day growth period. Both Ni and Al treatments, in growth medium, stimulated the activity of aminating glutamate dehydrogenase (NADH-GDH) whereas the activity of deaminating GDH (NAD⁺-GDH) decreased under metal toxicities. The activities of the aminotransferases studied; alanine aminotransferase (AlaAT) and aspartate amino transferase (AspAT) increased due to Ni and Al treatments. Results suggest that both Ni and Al treatments impair N assimilation in rice seedlings by inhibiting the activities of NR and GS and that GDH appears to play a role in assimilation of NH₄ ⁺ in metal stress conditions. Further, higher activity of aminotransferases in metal stressed seedlings might be helpful in meeting higher demand of amino acids under stressed conditions.     

Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum

The glutamate dehydrogenase (gdh) gene of Escherichia coli was transferred into an ammonium assimilation deficient mutant (Asm^-) of Rhizobium japonicum (CJ9) using plasmid pRP301, a broad host range derivative of RP₄. Exconjugants capable of growth on ammonia as sole N-source occurred at a frequency of 6.8×10^-6. Assimilatory GDH (NADP⁺) activity was detected in the strain carrying the E. coli gdh gene and the pattern of ammonia assimilation via GDH was similar to that of the Asm⁺ wild type strain. However, GDH mediated ammonia assimilation was not subject to regulation by l-glutamate. Nitrogenase activity was expressed ex planta in R. japonicum CJ9 harbouring the gdh gene, however, the presence of the gdh gene did not restore symbiotic effectiveness to the CJ9 Asm^- strain in nodules. The gdh plasmid was maintained in approximately 90% of the isolates recovered from soybean nodules.Abbreviations gdh glutamate dehydrogenase - Asm^- mutant ammonia assimilation deficient mutant     

Complete Reversal of Coenzyme Specificity of Isocitrate Dehydrogenase from <Emphasis Type="Italic">Haloferax volcanii</Emphasis>

Haloferax volcanii Ds-threo-isocitrate dehydrogenase (ICDH) was highly expressed in bacteria as inclusion bodies. The recombinant enzyme was refolded, purified and characterized, and was found to be NADP-dependent like the wild-type protein. Sequence alignment of several isocitrate dehydrogenases from evolutionarily divergent organisms including H. volcanii revealed that the amino acid residues involved in coenzyme specificity are highly conserved. Our objective was to switch the coenzyme specificity of halophilic ICDH by altering these conserved amino acids. We were able to switch coenzyme specificity from NADP⁺ to NAD⁺ by changing five amino acids by site-directed mutagenesis (Arg291, Lys343, Tyr344, Val350 and Tyr390). The five mutants of ICDH were overexpressed in Escherichia coli as inclusion bodies and each recombinant ICDH protein was refolded and purified, and its kinetic parameters were determined. Coenzyme specificity did not switch until all five amino acids were substituted.     

Application of an Electrochemical NAD+ Recycling System Involving a String-Like Carbon Fiber to an Enzyme Reactor

A string-like carbon fiber was found to be very suitable as a working electrode material for direct electrochemical oxidation of β-nicotinamide adenine dinucleotide reduced form (NADH), and direct use of it for an enzyme reactor was possible. The electrochemical NAD⁺ recycling system was applied to glucose dehydrogenase (GDH) and to the recombinant formate dehydrogenase (RFDH) reactors. The maximum oxidation current value increased to 3.9 mA in the case of the GDH reactor. The remaining GDH activity after the reaction for 10 h amounted to 57% of the initial level. The remaining NAD⁺ activity amounted to 78% of the initial level. The current efficiency was calculated to be 80%. Furthermore, RFDH, which was more stable than GDH, was applied to the system. The maximum current value reached 5.9 mA. The remaining RFDH activity after reaction for 10 h amounted to 81% of the initial level. The remaining NAD⁺ activity was 78% of the initial level. The current efficiency was calculated to be 73%. Based on these results, both the enzyme and NAD⁺ were found to be acceptably stable in the electrochemical NAD⁺ recycling system.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine