Regulation Of Glutamine Synthetase Research Articles

No evidence of covalent modification of glutamine synthetase in the thermophilic phototropic bacteriumChloroflexus aurantiacus

Regulation of glutamine synthetase (GS) in the thermophilic green phototrophic bacterium, Chloroflexus aurantiacus, was studied. The enzyme was partially purified from cells grown photosynthetically in media with limiting (1 mM) or non-limiting (10 mM) NH+4-concentrations. GS preparations from both cell types were indistinguishable in respect to pH-optimum of GS-transferase activity, sensitivity to feedback modifiers (AMP, L-alanine, glycine) and lack of Mg-inhibition of transferase activity. In contrast to results obtained with a GS preparation from the facultatively phototrophic bacterium, Rhodopseudomonas sphaeroides, the catalytic properties of Chloroflexus GS did not change during incubation with snake venom phosphodiesterase suggesting the absence of in vivo regulation of Chloroflexus GS by adenylylation/deadenylylation.

Effect of L-methionine-DL-sulfoximine on acetylene reduction and vesicle formation in derepressed cultures of Frankia strain D11

L-Methionine-DL-sulfoximine, a glutamate analog known to inhibit glutamine synthetase (GS) and glutamate synthase (GOGAT), allowed nitrogenase biosynthesis by Frankia strain D11 in presence of NH4+. GS activity, detected in cell-free extracts of D11, was higher in N2-fixing than in nonfixing conditions. These facts suggested that nitrogenase biosynthesis in Frankia was probably regulated by GS, GOGAT, or a product of their reaction, but regulation of GS by an adenylylation–unadenylylation system was probably not involved. Similarities between Frankia and Anabaena cylindrica were stressed.

Nitrogen assimilation in facultative methylotrophic bacteria

A study was done of the pathways of nitrogen assimilation in the facultative methylotrophsPseudomonas MA andPseudomonas AM1, with ammonia or methylamine as nitrogen sources and with methylamine or succinate as carbon sources. When methylamine was the sole carbon and/or nitrogen source, both organisms possessed enzymes of the glutamine synthetase/glutamate synthase pathway, but when ammonia was the nitrogen sourcePseudomonas AM1 also synthesized glutamate dehydrogenase with a pH optimum of 9.0, andPseudomonas MA elaborated both glutamate dehydrogenase (pH optimum 7.5) and alanine dehydrogenase (pH optimum 9.0). Glutamate dehydrogenase and glutamate synthase from both organisms were solely NADPH-dependent; alanine dehydrogenase was NADH-dependent. No evidence was obtained for regulation of glutamine synthetase by adenylylation in either organism, nor did glutamine synthetase appear to regulate glutamate dehydrogenase synthesis.

Role of glnA-linked genes in regulation of glutamine synthetase and histidase formation in Klebsiella aerogenes.

We isolated mutants of Klebsiella aerogenes with insertions of transposon Tn5 linked to the structural gene for glutamine synthetase, glnA. We found that K. aerogenes, like Escherichia coli and Salmonella typhimurium, contains a regulatory gene, glnG, which is closely linked to but distinct from glnA. The product of glnG is essential for normal regulation of the synthesis of glutamine synthetase and histidase. We analyzed two mutations which affected the regulation of the formation of these enzymes and appeared to be outside glnG. The results of our studies suggest the presence in K. aerogenes of at least two regulatory genes located in the glnA region, namely, glnG and another gene, the transcription of which can be initiated at the promoter of glnA.

Regulation of glutamine synthetase from Saccharomyces cerevisiae by repression, inactivation and proteolysis.

Glutamine synthetase activity is modulated by nitrogen repression and by two distinct inactivation processes. Addition of glutamine to exponentially grown yeast leads to enzyme inactivation. 50% of glutamine synthetase activity is lost after 30 min (a quarter of the generation time). Removing glutamine from the growth medium results in a rapid recovery of enzyme activity. A regulatory mutation (gdhCR mutation) suppresses this inactivation by glutamine in addition to its derepressing effect on enzymes involved in nitrogen catabolism. The gdhCR mutation also increases the level of proteinase B in exponentially grown yeast. Inactivation of glutamine synthetase is also observed during nitrogen starvation. This inactivation is irreversible and consists very probably of a proteolytic degradation. Indeed, strains bearing proteinase A, B and C mutations are no longer inactivated under nitrogen starvation.

Regulation of glutamine synthetase I fromRhizobium meliloti

Glutamine synthetase I fromRhizobium meliloti was found to be inhibited by adenosine 5′-monophosphate, alanine, glycine, carbamyl phosphate, cytidine 5′-triphosphate, tryptophan, histidine, and glucosamine-6-phosphate. Each inhibitor was independent in its action and the effect was cumulative when more than one inhibitor was added.

Regulatory Properties of Glutamine Synthetase from the Nitrogen-Fixing Phototrophic Bacterium Rhodopseudomonas palustris

Abstract Rhodopseudomonas palustris, Glutamine Synthetase Regulation, Adenylylation Glutamine synthetase from Rhodopseudomonas palustris is regulated via an adenylylation/deadenylylation mechanism. The enzyme purified from ammonia-grown cells, released AMP upon treatment with phosphodiesterase, along with drastic changes in its pH and metal dependency. Kinetic parameters for enzyme-substrate interaction were also dependent on the adenylylation state of the enzyme, as was the influence of several nitrogenous feedback inhibitors on the catalytic activity. The adenylylation state of the enzyme was modified in vivo by the availability of ammonia.

Cellular localization and regulation of glutamine synthetase in primary cultures of brain cells from newborn mice.

The cellular distribution of glutamine synthetase was determined by indirect immunofluorescence in cultures of dissociated brain cells from newborn mice. The enzyme could be detected in about 40% of all cells, among which cells with astrocytic morphology were clearly identified. Treatment with the glucocorticoid dexamethasone led to a strong increase in the number of positivity stained cells. Enzyme induction by dexamethasone was maximal after 36 h and at a concentration of 0.1 micrometer. Under these conditions glutamine synthetase specific activity was elevated about six fold. Steroid hormones other than corticosteroids had no effects. The basal activity in these cultures was near that found in brains of newborn mice, but far below the activity in adult brains, showing that in culture the normal development of these cells is disturbed. A comparison of glial and neuronal cell lines showed that glutamine synthetase is present in both types of cell lines at a very low specific activity. Inducibility of this enzyme by dexamethasone was found in glial but not in neuronal cell lines.

Nitrogen Starvation and the Regulation of Glutamine Synthetase in Agmenellum quadruplicatum

The level of glutamine synthetase activity in Agmenellum quadruplicatum strain PR-6 was dependent on the nitrogen source used for growth and on the nutritional status of the cells. During exponential growth, glutamine synthetase activity was low in cells grown on ammonia, urea, or nitrate. During the transition from nitrogen replete to nitrogen starved growth, glutamine synthetase activity began to rise. With ammonia as a nitrogen source, glutamine synthetase activity as determined in whole cells increased from 1 nanomole per minute per milliliter during exponential growth to 22 nanomoles per minute per milliliter during severe nitrogen starvation. In cells grown on nitrate the increase was from 5 to 39 nanomoles per minute per milliliter, and in cells grown on urea the increase was from 4 to 31 nanomoles per minute per milliliter.The rise in glutamine synthetase activity corresponded with the rapid decline in the nitrogen and c-phycocyanin content of the cells. Prior to nitrogen starvation, the nitrogen content of the cells was 140, 90, and 83 micrograms nitrogen per milligram dry weight for ammonia, urea, and nitrate grown cells, respectively. During nitrogen starvation where glutamine synthetase activity was highest, the nitrogen content of cells had declined to 35 to 40 micrograms nitrogen per milligram dry weight of cells. At the same time, the c-phycocyanin content of cells dropped by 95%.

Regulation of glutamine synthetase by regulatory protein PII in Klebsiella aerogenes mutants lacking adenylyltransferase.

A mutation of Klebsiella aerogenes causing production of an altered PII regulatory protein which stimulates overadenylylation of glutamine synthetase and also prevents its derepression was combined with mutations abolishing the activity of adenylyltransferase. The results support the idea that PII plays a role in the regulation of the level of glutamine synthetase which is independent of its interaction with adenylyltransferase.

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31

In cell-free preparations of NH 4 +-grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N 2-grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH 4 +-mediated repression. Glutamine synthetase activity in both N 2-grown and NH 4 +-grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N 2-grown cultures, a drastic decrease in the enzyme activity by exogenous NH 4 + can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K m values for NH 4 +, glutamate Mg 2+, and ATP similar to those observed for the enzyme from N 2- and NH 4 +-grown Anabaena, but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH 4 +-mediated modification to a less active form may be a means of regulation of glutamine synthetase in N 2-fixing cultures of Anabaena.

Regulation of Glutamine Synthetase by Light and during Nitrogen Deficiency in Synchronous Chlorella sorokiniana

A method is described to achieve density labeling of proteins in unicellular algae by using (13)CO(2). This is a satisfactory procedure especially for work on nitrogen metabolism. The increase in activity of glutamine synthetase (EC 6.3.1.2.) and glutamate synthase (EC 1.4.7.1.) in Chlorella sorokiniana mediated by a dark/light shift and by nitrogen starvation were investigated. Using the method of density labeling and isopycnic centrifugation, we demonstrated that the increase in enzyme activity after a dark/light shift is based on activation rather than de novo synthesis. The increase in enzyme activity after transfer to nitrogen-deficient medium is based both on activation and de novo synthesis.

Regulation of glutamine synthetase in the blue-green alga Anabaena flos-aquae

Glutamine synthetase (GS) was isolated from log phase cells and purified to a single protein as evidenced by gel electrophoresis. Protamine and ammonium sulfate precipitation and chromatography on DEAE-cellulose and Bio-Gel resulted in 380-fold purification. The enzyme was most sensitive to alanine (85% inhibition at 0.1 mM) but was also inhibited by glycine, arginine and serine. Combinations of inhibitory amino acids or nucleotides (AMP, ADP, ATP) exhibited cumulative inhibition. Cooperative inhibition was noted with CTP and any single nucleotide. Inhibition by CTP alone was uncompetitive with respect to glutamine. The enzyme was also regulated by the energy charge of the cell.

Regulation of nitrogen utilization of hisT mutants of Salmonella typhimurium.

Mutations in the hisT gene of Salmonella typhimurium alter pseudouridine synthetase I, the enzyme that modifies two uridines in the anticodon loop of numerous transfer ribonucleic acid species. We have examined two strains carrying different hisT mutations for their ability to grow on a variety of nitrogen sources. The hisT mutants grew more rapidly than did hisT+ strains with either arginine or proline as the nitrogen source and glucose as the carbon source. The hisT mutations were transduced into new strains to show that these growth properties were due to the hisT mutations. The hisT mutations did not influence the growth of mutants having altered glutamine synthetase regulation. Assays of the three primary ammonia-assimilatory enzymes, glutamate dehydrogenase, glutamine synthetase, and glutamate synthase, showed that glutamate synthase activities were lower in hisT mutants than in isogenic hisT+ controls; however, the glutamate dehydrogenase activity was about threefold higher in the hisT strains grown in glucose-arginine medium. The results suggest that the controls for enzyme synthesis for nitrogen utilization respond either directly or indirectly to transfer ribonucleic acid species affected by the hisT mutation.

Regulation of Neurospora crassa Glutamine Synthetase by the Carbon and Nitrogen Source

The effect of the carbon and nitrogen source on the regulation of glutamine synthetase (GS) has been studied by omitting one or both from mycelium which had been grown in sucrose/ glutamate medium, a condition in which GS is fully induced in its octameric form. The activity and antigen concentration of GS decreased when mycelium was deprived of the carbon source. Even more enzyme was degraded when glutamate was replaced by glutamine and an oligomeric form of GS lower than the octamer was also observed. When the carbon source was restored, some of the enzyme was resynthesized during incubation with glutamate but not with glutamine. In both cases the final predominant oligomeric form of GS was the tetramer. When the carbon and nitrogen sources were both omitted from the medium, degradation of GS again occurred and the restoration of both sources did not result in an increase in GS: no change in the oligomeric state of GS was observed during these shifts. The deprivation of the nitrogen source resulted in the replacement of the octamer by the highly active tetrameric form of GS. These changes were not reversed when either the carbon or the nitrogen source was again supplied.

Regulation of glutamine synthetase in the blue-green alga Anabaena L-31

In N 2-grown cultures of Anabaena L-31, in which protein synthesis was prevented by chloramphenicol, presence of NH 4 + caused a drastic decrease of glutamine synthetase ( l-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) activity indicating NH 4 +-mediated inactivation or degradation of the enzyme. The half-life of glutamine synthetase was more than 24 h, whereas that of nitrogenase (reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolysing), EC 1.18.2.1) was less than 4 h, suggesting that glutamine synthetase may not act as positive regulator of nitrogenase synthesis in Anabaena. Glutamine synthetase purified to homogeneity was subject to cumulative inhibition by alanine, serine and glycine. The amino acids, however, exhibited partial antagonism in this behaviour. Glyoxylate, an intermediate in photorespiration, virtually prevented the amino acid inhibition. Kinetic studies revealed inhibition of the enzyme activity by high Mg 2+ concentration under limiting glutamate level and by high glutamate in limiting Mg 2+. Maximum enzyme activity occurred when the ratio of glutamate to free Mg 2+ was 0.5 to 1.0. The results demonstrate that the enzyme is subject to multiple regulation by various metabolites involved in nitrogen assimilation.

The role of growth hormone, dexamethasone and triiodothyronine in the regulation of glutamine synthetase in primary cultures of rat hepatocytes.

Parenchymal cells from adult rat liver, maintained in monolayer culture, showed a rapid decay of the specific activity of glutamine synthetase between 12 and 20 h after plating, and a subsequent stabilization at about 30% of initial levels. Single hormone treatment with dexamethasone (2 μM) or human growth hormone (4 μg/ml) did not significantly alter the enzyme activity. However, simultaneous addition of both hormones during the second day of cultivation increased the level of glutamine synthetase 60–70% above control within 24 h, and more than 100%, if hormone addition was repeated after 10 h. This stimulation occurred after a lag phase of 6–8 h, and could be reinforced by the presence of triiodothyronine, which neither by itself nor in combination with dexamethasone or with growth hormone affected the enzyme activity. The concerted action of growth hormone and dexamethasone depended strongly on time, probably due to an insensitivity of the cultured hepatocytes against growth hormone during the first and after the third day of cultivation, while dexamethasone was found to act on the hepatocytes throughout the cultivation causing induction of tyrosine aminotransferase. The study of the dose-response of either dexamethasone or growth hormone revealed that the glucocorticoid had to be present in effective concentrations throughout to yield a continuous increase in enzyme activity. Human growth hormone was maximally effective in concentrations 50–100 times that of normal rat plasma. The effect of this hormone could be attributed to a somatogenic action on the hepatocytes, because its effect was mimicked by porcine growth hormone, whereas prolactin failed to stimulate glutamine synthetase. Preincubation of the hepatocytes with dexamethasone resulted in a shortening of the lag phase, while preincubation with growth hormone enhanced the rate of stimulation. Presence of cycloheximide or puromycin during the preincubation period completely abolished the effect of dexamethasone, whereas the increment in response to growth hormone was only partially affected, particularly during the first part of the preincubation period. It is suggested that dexamethasone exerts a permissive action, and growth hormone is responsible for the onset of the stimulatory process. Several presumptive characteristics of the hormonal action are outlined in the discussion.

Nitrogen and ammonia assimilation in the cyanobacteria: Regulation of glutamine synthetase

Glutamine synthetase, the first enzyme of the ammonia assimilatory pathway, has been purified from Anabaena sp. CA by use of established procedures and by affinity chromatography as a final step. No adenylylation system controlling glutamine synthetase activity was found. The enzyme shows a marked specificity for Mg 2+ in the biosynthetic assay and Mn 2+ in the transferase assay. Under physiological conditions, Co 2+ produces a large stimulatory effect on the Mg 2+-dependent biosynthetic activity. The enzyme is inhibited by the feedback modifiers l-alanine, glycine, l-serine, l-aspartate, and 5′-AMP. Inhibition by l-serine and l-aspartate is linear, noncompetitive with respect to l-glutamate with apparent K i values of 3 and 13 m m, respectively. Cumulative inhibition is seen with mixtures of l-serine, l-aspartate, and 5′-AMP. The results indicate that, in vivo, divalent cation availability and the presence of feedback inhibitors may play the dominant role in regulating glutamine synthetase activity and hence ammonia assimilation in nitrogen-fixing cyanobacteria.

Glutamine synthetase regulation, adenylylation state, and strain specificity analyzed by polyacrylamide gel electrophoresis.

We used polyacrylamide gel electrophoresis to examine the regulation and adenylylation states of glutamine synthetases (GSs) from Escherichia coli (GS(E)) and Klebsiella aerogenes (GS(K)). In gels containing sodium dodecyl sulfate (SDS), we found that GS(K) had a mobility which differed significantly from that of GS(E). In addition, for both GS(K) and GS(E), adenylylated subunits (GS(K)-adenosine 5'-monophosphate [AMP] and GS(E)-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GS(K)-AMP < GS(K) < GS(E)-AMP < GS(E). We were able to detect these mobility differences with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels, we showed that: (i) the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, (ii) the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GS(K), and (iii) strains carrying the glnA10 allele contained no polypeptide having the mobility of GS(K) or GS(K)-AMP. Using native polyacrylamide gels, we detected the increased amount of dodecameric GS present in cells grown under nitrogen limitation compared with cells grown under conditions of nitrogen excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GSs nor a GS-like protein in cells carrying the glnA10 allele.

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.