The effect of different carbon and nitrogen sources on the activity of glutamine synthetase and glutamate dehydrogenase in lupine embryonic axes

Embryos of yellow lupine ( Lupinus luteus L. cv. Jantar), deprived of cotyledons, were incubated for 72 h in media containing various combinations of saccharose, ammonia, nitrate, glutamine and asparagine. Induction of glutamine synthetase (GS) was observed in embryos in media containing saccharose while the activity of this enzyme was inhibited by glutamine, asparagine and ammonia in the absence of sugar. The above mentioned nutritional factors had an opposite effect on the activity of glutamate dehydrogenase (GDH). Changes in glutamate dehydrogenase activity were preceded by reverse changes in the activity of glutamine synthetase. The possibility of GDH repression by GS in lupine embryos is discussed.     

The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops

This short review outlines the central role of glutamine synthetase (GS) in plant nitrogen metabolism and discusses some possibilities for crop improvement. GS functions as the major assimilatory enzyme for ammonia produced from N fixation, and nitrate or ammonia nutrition. It also reassimilates ammonia released as a result of photorespiration and the breakdown of proteins and nitrogen transport compounds. GS is distributed in different subcellular locations (chloroplast and cytoplasm) and in different tissues and organs. This distribution probably changes as a function of the development of the tissue, for example, GS1 appears to play a key role in leaf senescence. The enzyme is the product of multiple genes with complex promoters that ensure the expression of the genes in an organ- and tissue-specific manner and in response to a number of environmental variables affecting the nutritional status of the cell. GS activity is also regulated post-translationally in a manner that involves 14-3-3 proteins and phosphorylation. GS and plant nitrogen metabolism is best viewed as a complex matrix continually changing during the development cycle of plants. Along with GS, a number of other enzymes play key roles in maintaining the balance of carbon and nitrogen. It is proposed that one of these is glutamate dehydrogenase (GDH). There is considerable evidence for a GDH shunt to return the carbon in amino acids back into reactions of carbon metabolism and the tri-carboxylic acid cycle. Results with transgenic plants containing transferred GS genes suggest that there may be ways in which it is possible to improve the efficiency with which crop plants use nitrogen. Marker-assisted breeding may also bring about such improvements.     

The role of glutamine synthetase in regulation of nitrogen metabolism within the soil microbial community

Recent advances in our understanding of the enzymology and regulatory systems involved in microbial metabolism of N hold promise to elucidate some of the underlying factors controlling metabolism of N in soil ecosystems. A review of recent work is used to construct a paradigm for N metabolism regulation in soil based on the central role of glutamine synthetase (GS) in such regulation within the soil microbial community. The studies involved use of GS inhibitors to elucidate the role of GS activity in regulation of soil N metabolism. Such studies have shown that the glutamine formed by microbial assimilation of NH₄ ⁺ via GS activity influences the regulatory mechanisms controlling both the production and activity of enzymes involved in N metabolism. For example, these studies showed that the inhibition of GS activity within the soil microbial community relieved the repression of urease production caused by microbial assimilation of inorganic N and blocked the short-term regulation of assimilatory nitrate reductase (ANR) by NH₄ ⁺ assimilation. Other studies have indicated that common environmental factors in soil may influence GS activity in microorganisms and thereby may influence metabolism of N within the soil microbial community. The paradigm for N metabolism regulation in soil that has emerged from such studies should lead to a better understanding of the mechanisms controlling fate of N in soil ecosystems.     

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     

Inorganic nitrogen assimilation in the non-N2-fixing cyanobacterium Phormidium laminosum. I. Cellular levels of glutamine synthetase and NADPH-dependent glutamate dehydrogenase

Cell-free extracts of nitrate-grown as well as of ammonium-grown cells of the filamentous non-nitrogen-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) showed detectable levels of both glutamine synthetase (GS, EC 6.3.1.2) and NADPH-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) activities. The GS level of nitrate-grown cells was higher than that of ammonium-grown cells, whereas the GDH level was higher in ammonium-grown cells and depended on the external ammonium concentration. When nitrate-grown cells were transferred to an ammonium-containing medium, a decrease of GS and an increase of GDH specific activities occurred, even in the presence of nitrate. Conversely, when ammonia-grown cells were transferred to a nitrate-containing medium, an increase of GS and a decrease of GDH-specific activities took place. Both these effects were inhibited by chloramphenicol and were probably mediated by de novo protein synthesis. When either cell type was transferred to a medium without nitrogen source, the specific activities of both enzymes increased. When nitrate-grown cells were transferred to nitrate medium with L-methionine-DL-sulphoximine (MSX) added, the specific activity of GDH also increased. Here we present some evidence that, under certain conditions of nitrogen availability, GDH would play a minor role in ammonium assimilation.     

The role of glutamate dehydrogenase in plant nitrogen metabolism

In vivo nuclear magnetic resonance spectroscopy, in vitro gas chromatography-mass spectrometry, and automated ¹⁵N/¹³C mass spectrometry have been used to demonstrate that glutamate dehydrogenase is active in the oxidation of glutamate, but not in the reductive amination of 2-oxogiutarate. In cell suspension cultures of carrot (Daucus carota L. cv Chantenay), primary assimilation of ammonium occurs via the glutamate synthase pathway. Glutamate dehydrogenase is derepressed in carbonlimited cells and in such cells the function of glutamate dehydrogenase appears to be the oxidation of glutamate, thus ensuring sufficient carbon skeletons for effective functioning of the tricarboxylic acid cycle. This catabolic role for glutamate dehydrogenase implies an important regulatory function in carbon and nitrogen metabolism.     

Changes in the cellular and subcellular localization of glutamine synthetase and glutamate dehydrogenase during flag leaf senescence in wheat (Triticum aestivum L.)

In order to improve our understanding of the regulation of nitrogen assimilation and recycling in wheat (Triticum aestivum L.), we studied the localization of plastidic (GS2) and cytosolic (GS1) glutamine synthetase isoenzymes and of glutamate dehydrogenase (GDH) during natural senescence of the flag leaf and in the stem. In mature flag leaves, large amounts of GS1 were detected in the connections between the mestome sheath cells and the vascular cells, suggesting an active transfer of nitrogen organic molecules within the vascular system in the mature flag leaf. Parallel to leaf senescence, an increase of a GS1 polypeptide (GS1b) was detected in the mesophyll cytosol of senescing leaves, while the GS protein content represented by another polypetide (GS1a) in the phloem companion cells remained practically constant in both leaves and stems. Both GDH aminating activity and protein content were strongly induced in senescing flag leaves. The induction occurred both in the mitochondria and in the cytosol of phloem companion cells, suggesting that the shift in GDH cellular compartmentation is important during leaf nitrogen remobilization although the metabolic or sensing role of the enzyme remains to be elucidated. Taken together, our results suggest that in wheat, nitrogen assimilation and recycling are compartmentalized between the mesophyll and the vasculature, and are shifted in different cellular compartments within these two tissues during the transition of sink leaves to source leaves.     

Distribution of glutamate dehydrogenase and glutamine synthetase activity in the sheep and chicken digestive tract.

Glutamate dehydrogenase (GLDH, EC 1.4.1.3) and glutamine synthetase (GS, EC 6.3.1.2) activity were determined in the contents and tissues of the various parts of the sheep and chicken digestive tract, GLDH activity in the tissues of the sheep omasum, duodenum, rumen, reticulum, colon, caecum, jejunum and ileum ranged from 3.25+/-0.7 U (mumol/g dry weight . min) to 5.94+/-2.28 U; in the abomasum it was 9.67+/-1.27 U. GLDH activity in the contents of the ileum, abomasum, jejunum and duodenum varied from 0.85+/-0.19 U to 3.29+/-0.53 U and in the colon, caecum, reticulum, omasum and rumen from 6.34+/-2.64 U to 16.96+/-3.83 U. GS activity in the tissues of these parts of the digestive tract varied from 2.8+/-0.59 U to 8.6+/-1.4 U and their contents from 2.49+/-0.85 U to 10.76+/-2 U. GS activity in the contents of the colon was very low (0.26+/-0.07 U). In the tissues of the chicken duodenum, caecum, jejunum and ileum we found GLDH activity of 4.68+/-1.64 U to 7.96+/-1.73 U; in their contents it was 3.31+/-1.06 U to 3.8+/-0.73, but in the caecum it attained up to 66.7+/-24.3 U. GS activity was high from 57.6+/-2.0 U to 231+/-84 U in the tissues and 357+/-53 U to 383+/-76 U in the contents (in the caecum up to 2,500+/-233 U). The results show that conditions for the utilization of ammonia are present in the tissues and the contents in the whole of the sheep and chicken digestive apparatus. The hypothesis is confirmed that the different ability of ruminants and fowls to utilize ammonia formed from urea added to their feed, including ammonia formed by hydrolysis of blood urea, is due to the different GLDH and GS activity in their digestive tract as well as in their liver.     

Regulation of asparaginase, glutamine synthetase, and glutamate dehydrogenase in response to medium nitrogen concentrations in a euryhaline chlamydomonas species

The ammonium assimilatory enzymes glutamine synthetase (EC 6.3.1.2) and glutamate dehydrogenase (EC 1.4.1.3) were investigated for a possible role in the regulation of asparaginase (EC 3.5.1.1) in a Chlamydomonas species isolated from a marine environment. Cells grown under nitrogen limitation (0.1 millimolar NH(4) (+), NO(3) (-), or l-asparagine) possessed 6 times the asparaginase activity and approximately one-half the protein of cells grown at high nitrogen levels (1.5 to 2.5 millimolar). Biosynthetic glutamine synthetase activity was 1.5 to 1.8 times greater in nitrogen-limited cells than cells grown at high levels of the three nitrogen sources.Conversely, glutamate dehydrogenase (both NADH- and NADPH-dependent activities) was greatest in cells grown at high levels of asparagine or ammonium, while nitrate-grown cells possessed little activity at all concentrations employed. For all three nitrogen sources, glutamate dehydrogenase activity was correlated to the residual ammonium concentration of the media after growth (r = 0.88 and 0.94 for NADH- and NADPH-dependent activities, respectively).These results suggest that glutamate dehydrogenase is regulated in response to ambient ammonium levels via a mechanism distinct from asparaginase or glutamine synthetase. Glutamine synthetase and asparaginase, apparently repressed by high levels of all three nitrogen sources, are perhaps regulated by a common mechanism responding to intracellular nitrogen depletion, as evidenced by low cellular protein content.     

Arginase, glutamine synthetase and glutamate dehydrogenase activities in moist chilled and warm-incubated walnut kernels

The activities of arginase, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were studied in both moist chilled (5°C) and warm (27°C) incubated walnut (Juglans regia. L) kernels to asses whether the non-germinability of dormant kernels is associated with failure in amino acid metabolism. Warm-incubated kernels showed low germination (25%), whereas cold-stratified kernels displayed germination up to 61%. Arginase activity increased about twofold in imbibed kernels. It remained at a high level in cold-stratified kernels from mid-period of incubation onwards; however, in warm-incubated kernels the activity declined after an initial increase so that by 20 days, it was negligible. No significant differences in GS activity occurred between cold-stratified and warm-incubated kernels, but the activity of GDH was significantly more in kernels incubated at warm conditions. Thin-layer chromatographic separation of polyamines revealed greater ammonia, spermidine and an unknown polyamine accumulation in warm-incubated kernels. Thus, the declined rate of walnut kernel germination under warm conditions is mainly correlated with rapid inactivation of arginase, greater levels of ammonia and alterations in kernel polyamine composition. The enhanced activity of GDH in warm-incubated kernels implies that catabolic deamination of amino acids and their subsequent respiration is the favored pathway ongoing under warm conditions. This situation compromises germination-specific metabolism of amino acids which likely to operate better at lower temperatures during cold stratification of kernels.     

Absence of involvement of glutamine synthetase and of NAD-linked glutamate dehydrogenase in the nitrogen catabolite repression of arginase and other enzymes in Saccharomyces cerevisiae

The escape of several enzymes from “ammonia catabolite repression” in gdhA^? (NADP-linked glutamate-dehydrogenase-less) mutants, as well as in gdhCR mutants of Saccharomyces cerevisiae, does not involve glutamine synthetase, either as a positive or as a negative control element. A glutamine-synthetase-less mutant (gln^?) was used in this demonstration.In addition to its derepressing effect on the NAD-linked glutamate dehydrogenase, the gdhCR mutation releases “nitrogen catabolite repression” on arginase and allatoinase, as well as glutamine repression on glutamine synthetase. A gdhCS mutation was used to demonstrate that these effects are not mediated through the NAD-linked glutamate dehydrogenase.     

Influence of nitrogen source and growth status on glutamine synthetase and glutamate synthase activity in Mycobacterium avium

An investigation was made of the activity of glutamine synthetase and glutamate synthase from batch-cultured cells of Mycobacterium avium. The bacteria were grown in medium with ammonium chloride concentrations of 0, 0.1, 0.25, 1, 5, or 25 mumol/mL or with glutamine at 0.1 or 1 mumol/mL. The specific activity of the two enzymes was determined at 0, 22, 45, and 70 h of incubation. Regardless of the ammonia concentration in the medium, glutamate synthase specific activity was two to five times higher in extracts from elongating cells, incubated 22 h, than in those from shortened cells, incubated 45 or 70 h. In contrast, there was no apparent difference in glutamine synthetase specific activity with regard to culture age; however, glutamine synthetase specific activity varied inversely with the concentration of ammonium chloride in the medium. Cells grown in glutamine had high activity of glutamine synthetase.     

The influence of exogenously supplied sucrose on glutamine synthetase and glutamate dehydrogenase levels in excisedPisum sativum roots

Glutamine synthetase (GS) level is positively influenced by exogenously supplied sucrose in isolated pea roots (similarly as nitrate reductase - NR), glutamate dehydrogenase (GDH) level negatively. Comparison with previous results shows that GS level decreases more slowly than NR level when sucrose is omitted from the medium; the rate of changes in GS level corresponds rather to that in GDH level. The increase in GDH level in the tips of isolated roots cultivated in the medium lacking sucrose stops after approx. 24 h, but continues for at least 72 h in more mature root parts. GS level decreases during the first 24 h in the tips of isolated roots (compared with roots of intact seedlings) cultivated both with sucrose and without it (without sucrose more), however it again rises in the course of further cultivation with sucrose. The differences in GS and GDH levels caused by omission of sucrose are small in isolated roots from which root tips were removed, the difference in NR level in decapitated roots is similar to that found in isolated roots with root tips left. Decapitated isolated roots cultivated without sucrose contain higher amounts of soluble sugars than corresponding roots with root tips left. These facts are dismissed with regard to sugar consumption, transport, and compartmentalisation, and with respect to production in root tips and other plant parts of unknown compounds involved in GS and GDH regulation. The results obtained suggest that GDH functions in pea roots in the deaminating direction.     

Tissue glutamine synthetase associated with ammonia detoxication and nitrogen metabolism in Clarias batrachus

The composition of reaction mixture of glutamine synthetase (GS) assay system was perfected and utilized to determine the activity of this enzyme spectrophotometrically in selected tissues of the freshwater teleostean fish, Clarias batrachus. Of these tissues, brain was found to contain comparatively a very high activity representing a rapid role of GS in ammonia detoxication and synthesis of essential neurotransmitter substance in this tissue. Of other tissues, liver, kidney and gill were found to contain significant activities in the order representing their relative metabolic activities. The study was extended by examining the brain (neural) and liver (non-neural) GS system in more detail with a view to see the alterations (if any). GS activity of both, neural and non-neural tissues was found to be the same and also in the range reported for other Vertebrates. Observations regarding kinetic, physical and chemical properties of the enzyme are reported. Maximum enzyme activity was observed at pH 7.2 to 7.4 and temperature 35 degrees C. The enzyme was found to be more stable at 25 degrees C while activity decreased at higher temperatures (above 40 degrees C) and showed no activity at 60 degrees C (liver) and 70 degrees C (brain). A comparison and possible physiological roles of the enzyme for the concept of ammonia excretion, protein synthesis and nitrogen metabolism in teleostean fish tissues are discussed.     

Differential role of glutamate dehydrogenase in nitrogen metabolism of maize tissues

Both calli and plantlets of maize (Zea mays L. var Tuxpeño 1) were exposed to specific nitrogen sources, and the aminative (NADH) and deaminative (NAD⁺) glutamate dehydrogenase activities were measured at various periods of time in homogenates of calli, roots, and leaves. A differential effect of the nitrogen sources on the tissues tested was observed. In callus tissue, glutamate, ammonium, and urea inhibited glutamate dehydrogenase (GDH) activity. The amination and deamination reactions also showed different ratios of activity under different nitrogen sources. In roots, ammonium and glutamine produced an increase in GDH-NADH activity whereas the same metabolites were inhibitory of this activity in leaves. These data suggest the presence of isoenzymes or conformers of GDH, specific for each tissue, whose activities vary depending on the nutritional requirements of the tissue and the state of differentiation.