Light dependent reduction of nitrate by pea chloroplasts in the presence of nitrate reductase and C4-dicarboxylic acids

In the presence of purified nitrate reductase (NR) and 1 mM NADH, illuminated pea chloroplasts catalysed reduction of NO₃^? to NH₃ with the concomitant evolution of O₂. The rates were slightly less than those for reduction of NO₂^? to NH₃ and O₂, evolution by chloroplasts in the absence of NR and NADH (ca 6 μg atoms N/mg Chl/hr). Illuminated chloroplasts quantitatively reduced 0.2 mM oxaloacetate (OAA) to malate. In the presence of an extrachloroplast malate-oxidizing system comprised of NAD-specific malate dehydrogenase (NAD-MDH), NAD, NR and NO₃^?, illuminated chloroplasts supported OAA-dependent reduction of NO₃^? to NH₃ with the evolution of O₂. The reaction did not proceed in the absence of any of these supplements or in the dark but malate could replace OAA. The results are consistent with the reduction of NO₃^?by reducing equivalents from H₂O involving a malate/OAA shuttle. The ratios for O₂, evolved: C₄-acid supplied and N reduced: C₄-acid supplied in certain experiments imply recycling of the C₄-acids.     

Light-dependent reduction of selenite by sonicated pea chloroplasts

Sonicated chloroplasts in the presence of catalytic concentrations of NADP(H) and GSSG supported light-dependent reduction of SeO₃^2? with the     

Light-dependent incorporation of selenite and sulphite into selenocysteine and cysteine by isolated pea chloroplasts

Illuminated intact pea chloroplasts in the presence of O-acetylserine (OAS) catalysed incorporation of SeO₃^2- and SO₃^2- into selenocysteine and cysteine at rates of ca 0.36 and 6 μmol/mg Chl per hr respectively. Sonicated chloroplasts catalysed SeO₃^2- and SO₃^2- incorporation at ca 3.9 and 32% respectively of the rates of intact chloroplasts. Addition of GSH and NADPH increased the rates to ca 91 and 98% of the intact rates, but SeO₃^2- incorporation under these conditions was essentially light-independent. In the absence of OAS, intact chloroplasts catalysed reduction of SO₃^2- to S^2- at rates of ca 5.8 μmol/mg Chl per hr. In the presence of OAS, S^2- did not accumulate. Glutathione (GSH) reductase was purified from peas and was inhibited by ZnCl₂. This enzyme, in the presence of purified clover cysteine synthase, OAS, GSH and NADPH, catalysed incorporation of SeO₃^2- into selenocysteine (but not SO₃^2- into cysteine). The reaction was inhibited by ZnCl₂. Incorporation of SeO₃^2- into selenocysteine by illuminated intact chloroplasts and sonicated chloroplasts (with NADPH and GSH) was also inhibited by ZnCl₂ but not by KCN. Conversely, incorporation of SO₃^2- into cysteine was inhibited by KCN but not by ZnCl₂. It was concluded that SeO₃^2- and SO₃^2- are reduced in chloroplasts by independent light-requiring mechanisms. It is proposed that SeO₃^2- is reduced by light-coupled GSH reductase and that the Se^2- produced is incorporated into selenocysteine by cysteine synthase.     

Ammonia assimilation in Corynebacterium glutamicum and a glutamate dehydrogenase-deficient mutant

In the wild-type of Corynebacterium glutamicum, the specific activity of glutamate dehydrogenase (GDH) remained constant at 1.3 U (mg protein)–1 when raising the ammonia (NH4) concentration in the growth medium from 1 to 90 mM. In contrast, the glutamine synthetase (GS) and glutamate synthase (GOGAT) activities decreased from 1.1 U (mg protein)–1 and 42 mU (mg protein)–1, respectively, to less than 10 % of these values at NH4 concentrations > 10 mM suggesting that under these conditions the GDH reaction is the primary NH4 assimilation pathway. Consistent with this suggestion, a GDH-deficient C. glutamicum mutant showed slower growth at NH4 concentrations 10 mM and, in contrast to the wild-type, did not grow in the presence of the GS inhibitor methionine sulfoximine. © Rapid Science Ltd. 1998     

Glutamine synthetase and glutamate dehydrogenase in cadmium-stressed triticale seedlings

The studies were performed on young triticale seedlings grown on a mineral medium containing 5 mM NO ₃ ⁻ as the nitrogen source, with the addition of 0.5 mM CdCl₂. It was determined that cadmium ions accumulated mainly in the plant roots. Decreases in nitrate concentrations both in the roots and shoots of seedlings, as well as decreases in soluble protein contents with simultaneous increases in endopeptidase activity were also observed. Both in roots and shoots significant decreases in glutamic acid were noted. Toxic cadmium ion accumulation in seedlings significantly modified activity of primary nitrogen assimilating enzymes, i.e. glutamine synthetase (GS, EC 6.3.1.2) and glutamate dehydrogenase (GDH, EC 1.4.1.2). There was a significant decrease in GS activity both in roots and in shoots of the stressed plants, in comparison to plants grown without cadmium. In shoots of the control plants and plants subjected to stress two GS isoforms were discovered: cytoplasmatic (GS₁) and chloroplastic (GS₂). Substantial decreases in total glutamine synthetase activity in green parts of seedlings, occurring under stress conditions, result from dramatic decrease in GS₂ activity (by 60 % in relation to the control plants); despite simultaneous increases in the cytoplasmatic isoform (GS₁) activity by approx. 96 %. Cadmium ions accumulating in roots and shoots of seedlings not only increased GDH activity, but also modified its coenzymatic specificity.     

The synthesis of glutamate and the control of glutamate dehydrogenase in pea mitochondria

The synthesis of glutamate from α-oxoglutarate and NH₄⁺ by pea seedling mitochondria has been demonstrated under certain defined but non-physiological conditions. Malate acts as a hydrogen donor for the synthesis of glutamate but isocitrate is more effective, whilst succinate, in the presence or absence of ATP, is a poor donor of hydrogen. Glutamate dehydrogenase has been purified from pea mitochondria and from the cytosol. The similarities between the two preparations are interpreted to mean that the soluble glutamate dehydrogenase is released from the mitochondria during isolation. The kinetics of the mitochondrial enzyme and the effect of various metabolites on its activity have been examined. The results are discussed in relation to the proposed role of this enzyme and it is suggested that the ratio NADH-NAD⁺ may play a role in the control of glutamate metabolism.     

Some properties of glutamine synthetase and glutamate synthase from Derxia gummosa

The incorporation of ¹⁵N into washed cells of Derxia gummosa from labelled-(NH₄)₂SO₄ and -KNO₃ respectively was inhibited by both L-methionine-DL-sulphoximine and azaserine. Glutamine synthetase purified to homogeneity from this bacterium had a molecular weight of 708 000 and was composed of 12 similar subunits each of 59 000. The enzyme assayed by γ-glutamyltransferase method had K_m values for L-glutamine and hydroxylamine of 12.5 and 1.2 mM, respectively. Optimal pH values for adenylylated and deadenylylated forms were pH 7.0 and pH 8.0, respectively. The adenylylated enzyme was deadenylylated by treatment with snake venom phosphodiesterase. The inhibitions by both glutamate and ammonia were competitive. The activity was markedly inhibited by L-methionine-DL-sulphoximine, alanine, glycine and serine and to a lesser extent by aspartate, phenylalanine and lysine. Various tri-, di- and mono-phosphate nucleotides, organic acids (pyruvate, oxalate and oxaloacetate) were also inhibitory. Glutamate synthase purified 167-fold had specific requirements for NADH, L-glutamine and 2-ketoglutarate. The K_m values for NADH, glutamine and 2-ketoglutarate were 9.6, 270 and 24 μM respectively. Optimal pH range was 7.2–8.2. The enzyme was inhibited by azaserine, methionine, aspartate, AMP, ADP and ATP.     

Inhibition of pea leaf glutamine synthetase by methionine sulphoximine,phosphinothricin and other glutamate analogues

The kinetics of the inhibition of glutamine synthetase from Pisum sativum leaves by l-methionine sulphoximine and dl-phosphinothricin were determined. Inhibition by both compounds was mixed-competitive, and apparent K_i values of 0.16 mM and 0.073 mM respectively were determined. dl-5-Hydroxylysine, dl-glutamate-4-tetrazole and l-4-methyleneglutamic acid were also strong inhibitors. Analogues of methionine sulphoximine, dl-ethionine sulphoximine and dl-prothionine sulphoximine were poor inhibitors of glutamine synthetase. Other glutamine and glutamate analogues e.g. azaserine, albizziine, asparagine and kainic acid had no inhibitory action.     

The pathway of nitrogen assimilation in plants

The major route of nitrogen assimilation has been considered for many years to occur via the reductive amination of α-oxoglutarate, catalysed by glutamate dehydrogenase. However, recent work has shown that in most bacteria an alternative route via glutamine synthetase and glutamine: 2-oxoglutarate aminotransferase (glutamate synthase) operates under conditions of ammonia limitation. Subsequently the presence of a ferredoxin-dependent glutamate synthase in green leaves and green and blue-green algae, and a NAD(P)H and ferredoxin-dependent enzyme in roots and other non-green plant tissues, has suggested that this route may also function in most members of the plant kingdom. The only exceptions are probably the majority of the fungi, where so far most organisms studied do not appear to contain glutamate synthase. Besides the presence of the necessary enzymes there is other evidence to support the contention that the assimilation of ammonia into amino acids occurs via glutamine synthetase and glutamate synthase, and that it is unlikely that glutamate dehydrogenase plays a major role in nitrogen assimilation in bacteria or higher plants except in circumstances of ammonia excess.     

Pathway of ammonia assimilation in illuminated and darkened Chlamydomonas reinhardii

Evidence is presented which shows that NH₃ assimilation in Chlamydomonas occurs exclusively via the glutamate synthase cycle in illuminated and darkened cells and those in which the internal level of NH₃ is elevated. This result indicates that glutamate dehydrogenase probably plays a catabolic rather than anabolic role in the N nutrition of the alga. Glutamine synthetase and glutamate dehydrogenase were characterized and their kinetic properties shown to be consistent with these proposals. It is suggested that reversible activity modulations of glutamine synthetase regulate the operation of the glutamate synthase cycle in the light but the availability of reductant and ATP limits its activity in darkened cells. The possible involvement of the two glutamate synthase enzymes in both light and dark assimilation is discussed.     

Changes in the levels of enzymes involved in ammonia assimilation during the development of Phaseolus vulgaris seedlings.Effects of exogenous ammonia

Seeds of Phaseolus vulgaris L. cv. White Kidney were germinated and grown either in a nitrogen-free or in an ammonia-supplied medium. The changes in the soluble protein concentration and in the levels of glutamine synthetase (GS, EC 6.3.1.2), NADH–glutamate synthase (NADH-GOGAT, EC 1.4.1.14), ferredoxin-glutamate synthase (Fd-GOGAT, EC 1.4.7.1) and glutamate dehydrogenase (GDH, EC 1.4.1.2), both NADH- and NAD⁺-dependent, were examined in cotyledons and roots during the first 10 days after sowing. Soluble protein declined rapidly in the cotyledons and increased slightly in the roots. GS activity was initially high both in cotyledons and roots but subsequently decreased during seedling growth. Exogenous ammonia hardly affected GS activity. High levels of NADH-GOGAT were present both in cotyledons and roots during the first days of germination. The activity then gradually declined in both organs. In contrast, Fd-GOGAT in cotyledons was initially low and progressively increased with seedling development. In roots, the levels of Fd-GOGAT were higher in young than in old seedlings. Supply of ammonia to the seedlings increased the levels of NADH-GOGAT and Fd-GOGAT both in cotyledons and roots. NADH-GDH (aminating) activity gradually increased during germination. In contrast, the levels of NAD⁺-GDH (deaminating) activity were highest during the first days of germination. Exogenous ammonia did not significantly affect the activities of GDH.     

Activity profiles of enzymes involved in glutamine and glutamate metabolism in the apple during autumnal senescence

Seasonal changes in glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) were measured in both senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). From the measured enzyme activities we attempted to estimate the in vivo catalytic potentials of the enzymes with special reference to nitrogen mobilization and conservation of senescing apple trees. The cumulative glutamine synthetase activity of leaf tissue was about three times higher than that of bark. The estimated catalytic potential of leaf glutamine synthetase was 800-fold higher than the actual protein nitrogen loss of senescing leaves. The cumulative glutamate synthase activity of bark was about six times higher than that of leaf. The estimated catalytic potential of bark glutamate synthase was 160-times higher than the actual protein nitrogen gain in that tissue. The cumulative glutamate dehydrogenase activities in leaf and bark tissue were approximately the same. However, the catalytic potential of leaf glutamate dehydrogenase was twice that of leaf glutamate synthase. It is thus concluded that the physiological role of glutamine synthetase in senescing leaf tissue is to furnish the amide(s) prior to mobilization of nitrogen to storage tissue. The higher activity of glutamate synthase in bark tissue could provide a mechanism to transform the imported amide nitrogen to amino nitrogen of glutamate for storage protein synthesis. The possible regulatory factors upon the activity of these enzymes in the tissues of senescing apple trees are discussed.     

Effect of some disaccharides, hexoses and pentoses on nitrate reductase, glutamine synthetase and glutamate dehydrogenase in excised pea roots

The effects of exogenous sucrose, lactose, d -glucose, d (-)fructose, d -galactose, d -mannose, l -sorbose, l -arabinose and d -xylose on nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) levels, on anaerobic nitrite production and on respiratory O₂ consumption were studied in excised roots of pea (Pisum sativum L. cv. Raman). Sucrose, glucose and fructose increase NR and GS levels and decrease GDH level (when compared with roots cultures without any sugar) at all concentrations used, but the extent of this effect varies. NR induction is enhanced by all sugars within the concentration range studied. Precultivation of roots with mannose and galactose results in an increase in anaerobic nitrite production in a medium consisting of phosphate buffer and KNO₃. GS reaches its maximum at lower sugar concentrations, this fact being especially clear-cut with galactose. The decrease in GS level observed in roots cultured without sucrose is enhanced by higher sorbose concentrations. The increase in GDH level occurring in roots cultured without sucrose is depressed by low galactose and mannose concentrations but enhanced by high galactose, mannose, xylose and a wide range of sorbose concentrations. Lactose exerts only slight influence on the enzymes. The effects of sugars are in no case consistent with their effect on respiratory O₂ consumption which is most pronounced with NR. The above results show that the effects of sugars on NR, GS and GDH are not mediated by one universal mechanism.     

Effect of NaCl on the activities of glutamate synthase from a halophyte Suaeda maritima and from a glycophyte Phaseolus vulgaris

The action of NaCl on the activity of root and leaf glutamate synthase is compared in a halophyte, Suaeda maritima var. macrocarpa and in a glycophyte Phaseolus vulgaris. The addition of salt in the nutrient medium lowers the activity of glutamate synthase from Phaseolus without affecting that of Suaeda. This result, attributed to the fact that glutamate synthase is stimulated while glutamate dehydrogenase is partly inhibited in the halophyte grown in presence of high NaCl concentrations, suggests that the GS-GOGAT pathway is the primary route for ammonia assimilation. This pathway is especially active in the leaves. In vitro, NaCl (25–300mM) reduces the activity of glutamate synthase in Phaseolus as well as in Suaeda. Comparison with results obtained in situ suggests that there are differences in intracellular compartmentalization between the two types of plant.     

Involvement of NAD(P)+-glutamate dehydrogenase isoenzymes in carbon and nitrogen metabolism in Chlamydomonas reinhardtü

The involvement of three NAD(P)₊-L-glutamate dehydrogenase (GDH; EC 1.4.1.3) isoenzymes, named GDH₁, GDH₂ and GDH₃, in the carbon and nitrogen metabolism of the green alga Chlamydomonas reinhardtü 6145c has been investigated under different environmental and stress conditions. GDH₁ activity decreased, but GDH₂ and GDH₃ activities increased with the age of cultures. When the extracellular ammonium concentration was high, only GDH₁ activity increased with growth whereas GDH₂ and GDH₃ remained unchanged. In the presence of L-methionine-D,L-sulfoximine (MSX), an inhibitor of L-glutamine synthetase (GS), a significant increase of GDH₁ and a slight decrease in GDH₃ activity was observed, whereas GDH₂ did not change. A significant increase in the intracellular 2-oxoglutarate was also found upon addition of azaserine, an inhibitor of L-glutamate synthase (GOGAT) activity. However, no significant changes in GDH isoenzyme activities were observed after addition of azaserine or azaserine plus MSX, except an induction of GDH₃ in the latter case. Moreover, in the presence of ethoxyzolamide (ETZ), an inhibitor of carbonic anhydrase activity, an induction of total GDH activity, mainly due to an increase in GDH₁ and to a minor extent in GDH₂, was observed in cells under low CO₂ (0.03%). In the dark, cells showed an increase in GDH₁ activity, but when acetate was present GDH₁ activity was repressed. All these results taken together suggest a relationship between GDH₁ and nitrogen assimilation, whereas GDH₂ and GDH₃ seem to be involved in the production of 2-oxoglutarate to fuel the tricarboxylic acid (TCA) cycle.     

Enzymes of glutamine metabolism in testa-pericarp and endosperm of developing wheat grain

Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate puruvate transaminase and glutamate oxaloacetate transaminase have been assayed in developing testa-pericarp and endosperm of two wheat varieties, namely Shera (11.6% protein) and C-306 (9.8% protein). On per organ basis, activities of all the enzymes studied, except glutamine synthetase, increased during development. Glutamine synthetase activity decreased during development in the testa-pericarp, whereas, no glutamine synthetase activity could be detected in endosperm of either variety at any stage of development. Compared to testa-pericarp, endosperm had higher activities of glutamate synthase and glutamate pyruvate transaminase. On the whole, enzyme activities in Shera were higher, as compared to C-306. Developmental patterns and relative levels of enzyme activities in the two varieties were more or less the same, when expressed on dry weight basis or as specific activities. The results suggest that ammonia assimilation in developing wheat grain takes place by the glutamate dehydrogenase pathway in the endosperm; and both by the glutamate dehydrogenase and glutamine synthetase—glutamate synthase pathways in the testa-pericarp.     

Betaxanthin formation and free amino acids in hairy roots of Beta vulgaris var. lutea depending on nutrient medium and glutamate or glutamine feeding

Feeding of amino acids to hairy roots of the yellow beet (Beta vulgaris var. lutea) usually results in the formation of the respective betaxanthins. One exception is (S)-glutamate whose feeding leads to an increase in the betaxanthin vulgaxanthin I (glutamine as amino-acid moiety) instead of vulgaxanthin II (glutamate as amino-acid moiety). To elucidate this phenomenon, hairy roots were cultivated in modified standard medium and (S)-glutamate was fed. Under most nutrient conditions tested, glutamine and vulgaxanthin I in the tissue dominated over glutamate and vulgaxanthin II. Glutamate, opposed to glutamine, was readily metabolized so that its concentration was lower than that of glutamine. Maximum concentrations of glutamate were reached when the activity of glutamine synthetase was low. Even then, however, vulgaxanthin II stayed on a low level. In contrast, the level of vulgaxanthin I increased with increasing concentrations of glutamine in the tissue. Also 4-aminobutyric acid (GABA) was a major amino acid in the hairy roots. Its concentration reached maximum levels when (S)-glutamate, a GABA precursor, was fed, or when sucrose, the C source of the roots, was replaced by glucose. The respective GABA-betaxanthin, however, was hardly detectable. When both (S)-glutamate and glucose were supplied, the GABA concentration exceeded that of all other amino acids. Only then the GABA-betaxanthin could be characterized in small amounts. Interestingly, the level of the main betaxanthin, miraxanthin V, consisting of betalamic acid and dopamine, was most markedly reduced by a replacement of sucrose with glucose. We conclude that the reaction of betalamic acid with glutamate and GABA was considerably lower than with glutamine and dopamine, irrespective of the concentration of the amino acid in the tissue. Possible reasons will be discussed, also with respect to the occurrence of species-specific patterns of betaxanthins.     

Cortical metabolism in pyruvate dehydrogenase deficiency revealed by ex vivo multiplet C NMR of the adult mouse brain

The pyruvate dehydrogenase complex (PDC), required for complete glucose oxidation, is essential for brain development. Although PDC deficiency is associated with a severe clinical syndrome, little is known about its effects on either substrate oxidation or synthesis of key metabolites such as glutamate and glutamine. Computational simulations of brain metabolism indicated that a 25% reduction in flux through PDC and a corresponding increase in flux from an alternative source of acetyl-CoA would substantially alter the ¹³C NMR spectrum obtained from brain tissue. Therefore, we evaluated metabolism of [1,6-¹³C₂]glucose (oxidized by both neurons and glia) and [1,2-¹³C₂]acetate (an energy source that bypasses PDC) in the cerebral cortex of adult mice mildly and selectively deficient in brain PDC activity, a viable model that recapitulates the human disorder. Intravenous infusions were performed in conscious mice and extracts of brain tissue were studied by ¹³C NMR. We hypothesized that mice deficient in PDC must increase the proportion of energy derived from acetate metabolism in the brain. Unexpectedly, the distribution of ¹³C in glutamate and glutamine, a measure of the relative flux of acetate and glucose into the citric acid cycle, was not altered. The ¹³C labeling pattern in glutamate differed significantly from glutamine, indicating preferential oxidation of [1,2-¹³C]acetate relative to [1,6-¹³C]glucose by a readily discernible metabolic domain of the brain of both normal and mutant mice, presumably glia. These findings illustrate that metabolic compartmentation is preserved in the PDC-deficient cerebral cortex, probably reflecting intact neuron–glia metabolic interactions, and that a reduction in brain PDC activity sufficient to induce cerebral dysgenesis during development does not appreciably disrupt energy metabolism in the mature brain.