Loss Of Glutamine Synthetase Activity Research Articles

Glutamine synthetase mutations that cause glutamine deficiency: mechanistic insights from molecular dynamics simulations

Glutamine synthetase mutations that cause glutamine deficiency: mechanistic insights from molecular dynamics simulations

Glutamine synthetase is essential for proliferation of fetal skin fibroblasts

Background. Glutamine synthetase (GS) is ubiquitously expressed in the human and plays a major role for many metabolic pathways. However, little is known about its role during the fetal period. Methods. Cultured skin fibroblasts derived from an aborted fetus deficient in GS activity due to a R324C exchange as well as fetal and mature controls were used to determine the level of GS-expression, apoptosis, and proliferation in presence or absence of exogenous glutamine. Results. Glutamine synthetase can be found at early gestational stages. Loss of GS activity either inherited or induced through l-methionine sulfoximine leads to an upregulation of the GS protein but not of the GS mRNA and results in a significant drop in the proliferation rate but has no effect on apoptosis. Exogenous glutamine does not influence the rate of apoptosis but increases proliferation rates of the fetal but not the mature fibroblasts. Conclusion. GS can be found during early human fetal stages when it displays a significant effect on cell proliferation.

Isolation of the Corynebacterium glutamicum glnA gene encoding glutamine synthetase I.

The Corynebacterium glutamicum glutamine synthetase I (GSI) structural gene glnA was cloned by a PCR approach using oligonucleotide primers derived from conserved amino acid sequences of the GSI proteins from various bacteria. Disruption or deletion of this gene in C. glutamicum led to a glutamine auxotrophic phenotype and complete loss of glutamine synthetase activity, indicating the key role of this enzyme in nitrogen metabolism. Additionally, indications for a second glutamine synthetase, GSII, were found.

ORGANOTINS DISRUPT COMPONENTS OF GLUTAMATE HOMEOSTASIS IN RAT ASTROCYTE CULTURES

A rat cortical astrocyte preparation was used to investigate the effects of organotins on glutamate regulation by astrocytes. Exposure of astrocytes to low levels of organotins produced significant changes in two key components of glutamate homeostasis: glutamine synthetase (GS) activity and the high-affinity transport of L-glutamate. Trimethyltin (TMT), triethyltin (TET), and triphenyltin (TPT) exhibited differential abilities to reduce GS activity and glutamate uptake. Cultures incubated with 1 µM TET or TPT, but not TMT, exhibited a marked decrease in GS activity. Exposure to TET or TPT also produced a significant decrease in glutamate transport activity that was not observed with TMT. These declines in activity were not attributable to cell loss as measured by MTT reduction and lactate dehydrogenase (LDH) leakage. Since the loss of GS activity and transporter activity was not seen with acute organotin exposure, it is most likely attributable to a decreased presence of fully functioning protein. While the attenuation of GS and glutamate transporter activities by organotins does not match their pattern of neurotoxicity, the results indicate the potential for subtoxic concentrations of these compounds to increase extracellular glutamate and interact with other excitotoxic episodes.

Isolation of the Corynebacterium glutamicum glnA gene encoding glutamine synthetase I

The Corynebacterium glutamicum glutamine synthetase I (GSI) structural gene glnA was cloned by a PCR approach using oligonucleotide primers derived from conserved amino acid sequences of the GSI proteins from various bacteria. Disruption or deletion of this gene in C. glutamicum led to a glutamine auxotrophic phenotype and complete loss of glutamine synthetase activity, indicating the key role of this enzyme in nitrogen metabolism. Additionally, indications for a second glutamine synthetase, GSII, were found.

Ethanolic dehydration and its effect on membrane-bound enzymes of Chlorogloeopsis fritschii and Chlorella pyrenoidosa

Ethanolic dehydration (20% to 70%) of the thylakoid membranes of Chlorogloeopsis fritschii resulted in an 8% to 58% loss of glutamine synthetase activity. In Chlorella pyrenoidosa, hydroxypyruvate reductase and fumarase, marker enzymes of the peroxisomes and mitochondria, respectively, diffused from the organelles on dehydration.

Physiological and biochemical analysis of the glutamine synthetase-impaired mutants of the nitrogen-fixing cyanobacteriumNostoc muscorum

Three types of glutamine synthetase (GS)-impaired mutants (gln) ofNostoc muscorum were isolated as ethylenediamine (EDA)-resistant phenotypes and characterized with respect to heterocyst development, nitrogen fixation, ammonium metabolism, photosynthetic characteristics, and glutamine synthetase activity. The criterion for categorizing the mutants was the extent of loss of GS activity (both in transferase and biosynthetic assays) compared with wild type; it was 70% in EDA-1, 30% in EDA-2, and more than 90% in EDA-3 strains. The level of nitrogenase activity in mutant strains was proportionate to heterocyst frequency and was found refractory to ammonium and EDA repression. In EDA-resistant strains, development of heterocysts and their spacing pattern remained unaffected and did not respond to treatment of amino acid analogues, drugs, and ammoniacal compounds which otherwise either stimulated or suppressed the number and altered the spacing pattern in wild type. A biphasic pattern of ammonium uptake indicating two transport systems was observed in all the strains except that the Km values for both high- and low-affinity systems were altered in mutant strains. In vivo treatment with MSX or EDA significantly inhibited the GS activity in wild type, whereas mutant strains did not respond to these treatments and were able to liberate NH 4 + continuously into the medium without MSX treatment. During NH 4 + uptake, percentage inhibition of O2 evolution and changes in increase of fluorescence intensity were low in EDA strains compared with wild type. Assessment of GS protein with antibodies against GS and quantitative polyacrylamide gel electrophoresis (PAGE) suggested that loss in specific activity of GS per milligram of extractable protein in EDA mutants was owing to low production of GS-specific protein. SDS-PAGE of purified GS enzyme from all the strains revealed only one polypeptide band of molecular weight of about 51.28 kDa.

Effect of portacaval anastomosis on glutamine synthetase activities in liver, brain, and skeletal muscle.

Glutamine synthetase is responsible for the ATP-dependent amidation of glutamate to glutamine. In liver the enzyme is highly localized in perivenous hepatocytes; in brain the enzyme is localized in astrocytes. Portacaval anastomosis resulted in liver atrophy, hyperammonemia, and up to 90% loss of glutamine synthetase activity in liver homogenates. This effect, which appears to be irreversible, probably reflects the selective loss of perivenous hepatocytes following portacaval anastomosis. Glutamine synthetase activities in brain were unaffected by portacaval anastomosis of up to 12 weeks' duration. Enzyme activities in homogenates of skeletal muscle, on the other hand, were significantly increased at one and four weeks after shunt surgery. These effects were not the result of decreased food intake in shunted animals. These findings suggest fundamentally different regulatory mechanisms for glutamine synthetase in these tissues. Skeletal muscle may thus provide an important alternative site for ammonia detoxification after portal-systemic shunting.

In vitro reactivation of in vivo ammonium-inactivated glutamine synthetase from Synechocystis sp. PCC 6803

Glutamine synthetase from Synechocystis sp. strain PCC 6803 is inactivated by ammonium addition to cells growing with nitrate as the nitrogen source. The enzyme can be reactivated in vitro by different methods such as alkaline phosphatase treatment, but not phosphodiesterase, by raising the pH of the crude extract to values higher than 8, by increasing the ionic strength of the cell-free extract, or by preincubation with organic solvents, such as 2-propanol and ethanol. These results suggest that the loss of glutamine synthetase activity promoted by ammonium involves the non-covalent binding of a phosphorylated compound to the enzyme and support previous results that rule out the existence of an adenylylation/deadenylylation system functioning in the regulation of cyanobacterial glutamine synthetase.

Age influence on oxidative events during brain ischemia/reperfusion

Brain readily undergoes oxidative damage if it is organizationally disrupted. Iron plays a catalytic role in mediating oxidative damage in brain homogenate. Utilizing several methods to assess oxidative events, including salicylate trapping of hydroxyl free radicals, spin-trapping of free radicals and quantitation of protein oxidation, we have clearly demonstrated that oxidative events do occur in the ischemia/ reperfusion lesioned Mongolian gerbil brain. In the gerbil model, ischemia/reperfusion insult (IRI) mediated protein oxidative damage is accompanied by a concomitant loss of glutamine synthetase activity. The loss of glutamine synthetase activity may possibly lead to the build-up of glutamate, an excitotoxic amino acid which has been strongly implicated in the neuronal death observed in hippocampus following an IRI. The brain homogenate from an IRI-lesioned animal is more susceptible to peroxidation than comparable sham-operated control animals. We have discovered that an IRI causes lethality in older gerbils more readily than in younger gerbils. We have also found that the spin-trapping agent α-phenyl-tert-butyl nitrone (PBN) protects gerbils from an IRI. Data are presented which implicate that an IRI may seriously impair the energy production capacity of older gerbils more than comparable oxidative stresses in younger animals. We hypothesize that energy production capacity and its utilization for the synthesis of new gene products may be key events which help mediate survival from an IRI. In addition we hypothesize that higher brain functions, including memory and learning are inversely correlated with oxidative damage in the brain. The elucidation of the mechanisms involved in this fascinating area of research and the design of new protective agents to prevent oxidative damage in brain is a most exciting challenge.

Oxidative damage to brain proteins, loss of glutamine synthetase activity, and production of free radicals during ischemia/reperfusion-induced injury to gerbil brain.

Free radical-mediated oxidative damage has been implicated in tissue injury resulting from ischemia/reperfusion events. Global cortical ischemia/reperfusion injury to Mongolian gerbil brains was produced by transient occlusion of both common carotid arteries. Protein oxidation, as measured by protein carbonyl content, increased significantly during the reperfusion phase that followed 10 min of ischemia. The activity of glutamine synthetase, an enzyme known to be inactivated by metal-catalyzed oxidation reactions, decreased to 65% of control levels after 2 hr of reperfusion that followed 10 min of ischemia. We also report that the free radical spin trap N-tert-butyl-alpha-phenylnitrone [300 mg/kg (body weight)] administered 60 min before ischemia/reperfusion is initiated, partially prevents protein oxidation and protects from loss of glutamine synthetase activity. In addition, we report a N-tert-butyl-alpha-phenylnitrone-dependent nitroxide radical obtained in the lipid fraction of the ischemia/reperfusion-lesioned brains, but there was very little radical present in the comparable sham-operated control brains. These data strengthen the previous observation utilizing in vivo-trapping methods, that free radical flux is increased during the reperfusion phase of the ischemia-lesioned gerbil brain. The loss of glutamine synthetase would be expected to increase the levels of brain L-glutamate. Thus, the oxidative inactivation of glutamine synthetase may be a critical factor in the neurotoxicity produced after cerebral ischemia/reperfusion injury.

Role of oxygen free radicals in carcinogenesis and brain ischemia

Even though oxygen is necessary for aerobic life, it can also participate in potentially toxic reactions involving oxygen free radicals and transition metals such as Fe that damage membranes, proteins, and nucleic acids. Oxygen free radical reactions and oxidative damage are in most cases held in check by antioxidant defense mechanisms, but where an excessive amount of oxygen free radicals are produced or defense mechanisms are impaired, oxidative damage may occur and this appears to be important in contributing to several pathological conditions including aging, carcinogenesis, and stroke. Several newer methods, such as in vivo spin-trapping, have become available to monitor oxygen free radical flux and quantitate oxidative damage. Using a combination of these newer methods collectively focused on one model, recent results show that oxidative damage plays a key role in brain injury that occurs in stroke. Subtle changes, such as oxidative damage-induced loss of glutamine synthetase activity, may be a key event in stroke-induced brain injury. Oxygen free radicals may play a key role in carcinogenesis by mediating formation of base adducts, such as 8-hydroxyguanine, which can now be quantitated to very low levels. Evidence is presented that a new class of free radical blocking agents, nitrone spin-traps, may help not only to clarify if free radical events are involved, but may help prevent the development of injury in certain pathological conditions.

Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes

When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged (a) by the incorporation of [ 14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, (b) by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incroporation of [ 14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and (c) by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogeneously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn 2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe 2+ (or Ni 2+ together with ATP (or other nucleoside di- and triphosphates. After 40–60 h dialysis Fe 3+ together with NADH (but not ATP) are required for glutamine synthetase inactivation. The results suggest that accelerated protein degradation in cells exposed to nitrogen-limited conditions reflects the differential destruction of some proteins, including aspartokinases I and III, in order to sustain the biosynthesis of others such as glutamine synthetase. The loss of glutamine synthetase activity in cell-free extracts is likely mediated in part by mixed-function oxidation systems and could represent a ‘marking’ step in protein turnover.

Benzyl viologen-mediated in vivo and in vitro inactivation of glutamine synthetase in Azotobacter chroococcum

Addition of benzyl viologen to a cell suspension of the aerobic bacterium Azotobacter chroococcum growing on nitrate resulted in a rapid loss of glutamine synthetase activity as assayed in situ. When a glutamine synthetase preparation which exhibited NADH-benzyl viologen oxidoreductase activity was incubated, under air, with NADH and benzyl viologen, glutamine synthetase was inactivated in a short period of time. This in-vitro inactivation process could be prevented in the presence of added catalase, thus indicating that hydrogen peroxide was involved in the process, and by EDTA, suggesting that metal ions are also involved. The characteristics of the benzyl viologen-dependent glutamine synthetase inactivation observed with externally added H2O2 and a preincubated sample are similar. Inhibition of glutamine synthetase inactivation by histidine suggests that hydroxyl radicals, or something with similar reactivity, is the inactivating agent. The fact that inactivation can also be catalyzed by a model system consisting of Fe2+ and H2O2 leads to the conclusion that hydroxyl radicals are most likely produced in a Fenton reaction in which hydrogen peroxide reacts with adventitious iron ions. Since A. chroococcum contained a high level of catalase it may be concluded that cellular compartmentation plays an important role in the in-vivo inactivation of glutamine synthetase.

Tabtoxin, produced by Pseudomonas tabaci, decreases Nicotiana tabacum glutamine synthetase in vivo and causes accumulation of ammonia

Tabtoxin caused the rapid loss of activity of Nicotiana tabacum glutamine synthetase in vivo . Following infiltration of leaves with a solution of tabtoxin (0·5 mg ml −1 ) glutamine synthetase was detectably reduced after 1 h and was 5% of control levels after 4 h. No recovery of activity was detected. Ammonia started to accumulate in leaves between 3 and 4 h after treatment with tabtoxin. An inverse relation was demonstrated between glutamine synthetase activity and ammonia levels in leaves infiltrated with different concentrations of tabtoxin. The smallest dose of tabtoxin, 85 ng cm −2 leaf, which caused accumulation of ammonia was also the smallest dose causing chlorosis. Doses of tabtoxin greater than 1·35 mg cm −2 leaf caused necrosis. The ammonia accumulating in tabtoxin-treated leaves was accounted for quantitatively by the inhibition, of ammonia assimilation resulting from the loss of glutamine synthetase activity. Glutamate dehydrogenase was unaffected by tabtoxin. Necrosis occurring in tissues infiltrated with P. tabaci was not preceded by a reduction in glutamine synthetase activity or by accumulation of ammonia and it is doubtful therefore that tabtoxin is causally involved in this symptom. However, the chlorosis which is characteristic of P. tabaci infections was associated with loss of glutamine synthetase activity and accumulation of ammonia, confirming the causal role of tabtoxin in this symptom.

Glutamine synthetase activity, ammonia assimilation and control of nitrate reduction in the unicellular red algaCyanidium caldarium

Addition ofl-methionine-dl-sulphoximine to cells ofCyanidium caldarium brings about a loss of glutamine synthetase activity. Concomitantly ammonia assimilation is prevented.