Effects of medium glutamine,glutamate, and ammonia on glutamine synthetase activity in cultured mouse astroglial cells

Mouse astroglial cells were grown during the last week of culture in either glutamine-free or glutamine-containing medium. The addition of cortisol to the glutamine-containing medium resulted in a doubling of astroglial glutamine synthetase (GS) activity. Withdrawal of glutamine from the medium resulted in a 50% elevation of GS and addition of cortisol to such a medium resulted in a further increase in GS which was not additive to glutamine withdrawal. Both in glutamine-free and glutamine-containing medium, the addition of glutamate resulted in a depression of both basal and cortisol induced GS activity. The simultaneous addition of ammonia plus glutamate to the culture medium ameliorated the glutamate mediated depressive effects on cortisol induced but not basal GS activity. Glutamine withdrawal from the culture medium resulted in an astroglial protein deficit. The addition of ammonia to the medium considerably reduced this deficit and the addition of glutamate completely eliminated this protein deficit.     

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration

The effect of tyrosine nitration on mammalian GS activity and stability was studied in vitro. Peroxynitrite at a concentration of 5 micro mol/l produced tyrosine nitration and inactivation of GS, whereas 50 micro mol/l peroxynitrite additionally increased S-nitrosylation and carbonylation and degradation of GS by the 20S proteasome. (-)Epicatechin completely prevented both, tyrosine nitration and inactivation of GS by peroxynitrite (5 micro mol/l). Further, a putative "denitrase" activity restored the activity of peroxynitrite (5 micro mol/l)-treated GS. The data point to a potential regulation of GS activity by a reversible tyrosine nitration. High levels of oxidative stress may irreversibly damage and predispose the enzyme to proteasomal degradation.     

Developmentally regulated primary glucocorticoid hormone induction of chick retinal glutamine synthetase mRNA

We have characterized the glucocorticoid hormone induction of glutamine synthetase mRNA in embryonic chick retinal organ cultures by quantitative dot hybridization using a cDNA clone derived from chick retinal RNA. Hydrocortisone (Kapp = 3-4 nM) and dexamethasone (Kapp = 1-2 nM) produce an approximate 30-fold increase in glutamine synthetase mRNA after incubation of organ cultures derived from embryonic day 12 retinae with either hormone for 3 hr. Progesterone is a poor inducer. The glucocorticoid-mediated rise is rapid (t1/2 = 2-3 hr) and occurs in the presence of either of the protein synthesis inhibitors cycloheximide or puromycin, indicating that the induction is a primary or direct response to the hormone. However, the magnitude of the hormonal response observed in culture increases markedly during retinal development. These observations, coupled with the previously reported absence of a hormonal induction in embryonic liver, raise the possibility of a synergistic mechanism, involving tissue-specific regulatory molecules in addition to the glucocorticoid hormone receptor, to explain the retinal-specific primary glucocorticoid hormone induction of glutamine synthetase mRNA.     

Effect of histamine on the development of astroglial cells in culture

The effect of histamine on different aspects of the growth of astrocytes was studied using primary cultures derived either from forebrain or from cerebellum of the rat. The influence on general growth and differentiation was monitored in terms of the activities of ornithine decarboxylase and glutamine synthetase enzymes, whereas [³H]thymidine incorporation into DNA was used as a specific index of cell proliferation. Treatment with 500 nM histamine of cells grown for 6 days in vitro, caused a time-dependent significant increase in ornithine decarboxylase activity of astrocytes from both sources. The maximum increase was observed at 4 h after histamine treatment, at that time the elevation in ornithine decarboxylase activity being about 80% and 300% over control values in the forebrain and the cerebellar astrocytes, respectively. Under similar experimental conditions, addition of histamine (500 nM) to medium resulted in a significant increase in [³H]thymidine incorporation into DNA in both types of cultures: in comparison with control, the elevation was about 45% at 48 h in forebrain astrocytes and at 24 h in cerebellar astrocytes. On the other hand, the specific activity of glutamine synthetase in cerebellar astrocytes was markedly enhanced (about 100%) by treatment with histamine (500 nM) for 4 days, but forebrain astrocytes were little affected. Addition of histamine to the culture medium produced no significant alteration in the activity of lactate dehydrogenase and protein content of either type of astroglial cells. The present findings, which support our earlier proposal that the biochemical properties of astrocytes differ between various brain regions, provide direct evidence for the involvement of histamine in the regulation of growth and development of astrocytes.     

Early events in the induction of glutamine synthetase activity by hydrocortisone in embryonic chick neural retina

Summary Primary cultures of 10-day embryonic chick neural retinas were used to investigate early aspects of the mechanism of hydrocortisone action on glutamine synthetase activity. As little as 2 hr of hydrocortisone exposure served to initiate significant increases in the glutamine synthetase activity levels assayed after 24 hr culture. Time course studies indicated that the increase in glutamine synthetase activity observed after 24 hr in culture resulted from a two-phase rise in activity and that cycloheximide was effective in suppressing the second-phase rise. Additional inhibition studies demonstrated that the second-phase increase in enzyme activity required continuous protein synthesis during the initial 6 hr. The evidence suggests a mechanism of hydrocortisone action involving the production of a protein which is important for the induction of glutamine synthetase activity by hydrocortisone. This work was supported by a National Science Foundation (U.S.A.) Training Grant.     

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.     

Purification and characterisation of glutamine synthetase fromNocardia corallina

Glutamine synthetase (GS) (EC 6.3.1.2) has been purified 67-fold fromNocardia corallina. The apparentM _r of the GS subunit was approximately 56,000. Assuming the enzyme is a typical dodecamer this indicates a particle mass for the undissociated enzyme of 672,000. The GS is regulated by adenylylation and deadenylylation, and subject to feedback inhibition by alanine and glycine. The pH profiles assayed by the -glutamyl transferase method were similar for NH₄ ⁺-treated and untreated cell extracts and an isoactivity point was not obtained from these curves. GS activity was repressed by (NH₄)₂SO₄ and glutamate. Cells grown in the presence of glutamine, alanine, proline and histidine had enhanced levels of GS activity. The GS ofN. corallina cross-reacted with antisera prepared against GS from a Gram-negativeThiobacillus ferrooxidans strain but not with antisera raised against GS from a Gram-positiveClostridium acetobutylicum strain.     

Effect of fructose-1,6-bisphosphate on glutamate uptake and glutamine synthetase activity in hypotix astrocyte cultures

Astrocytes are important in regulating the microencironment of neurons both by catabolic and synthetic pathways. The glutamine synthetase (GS) activity observed in astrocytes affects neurons by removing toxic substances, NH₃ and glutamate; and by providing an important neuronal substrate, glutamine. This glutamate cycle might play a critical role during periods of hypoxia and ischemia, when an increase in extracellular excitatory amino acids is observed. It was previously shown in our laboratory that fructose-1,6-bisphosphate (FBP) protected cortical astrocyte cultures from hypoxic insult and reduced ATP loss following a prolonged (18–30 hrs) hypoxia. In the present study we established the effects of FBP on the level of glutamate uptake and GS activity under normoxic and hypoxic conditions. Under normoxic conditions, [U-¹⁴C]glutamate uptake and glutamine production were independent of FBP treatment; whereas under hypoxic conditions, the initial increase in glutamate uptake and an overall increase in glutamine production in astrocytes were FBP-dependent. Glutamine synthetase activity was dependent on FBP added during the 22 hours of either normoxic- or hypoxic-treatment, hence significant increases in activity were observed due to FBP regardless of the oxygen/ATP levels in situ. These studies suggest that activation of GS by FBP may provide astrocytic protection against hypoxic injury.     

Regulation of ammonium assimilation in Haloferax mediterranei: Interaction between glutamine synthetase and two GlnK proteins

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK₁ and glnK₂. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK₁ and GlnK₂. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK₁ and GlnK₂ produced equivalent results.     

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

Role of glutamine synthetase in citric acid fermentation byAspergillus niger

The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6·0–6·5, when the pH of the external medium was varied between 2·3–7·0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn²⁺ ions partially protected the enzyme against this inactivation. Mn²⁺-dependent glutamine synthetase activity was higher at acidic pH (6·0) compared to Mg²⁺-supported activity. While the concentration of Mg²⁺ required to optimally activate glutamine synthetase at pH 6·0 was very high (≥ 50 mM), Mn²⁺ was effective at 4 mM. Higher concentrations of Mn²⁺ were inhibitory. The inhibition of both Mn²⁺ and Mg²⁺-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn²⁺ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis     

Glutamate dehydrogenase and glutamine synthetase activities during the development of triticale grains

Glutamate dehydrogenase (GDH, EC 1.4.1.2–4) and glutamine synthetase (GS, EC 6.3.1.2) activities as well as protein content and dry matter in developing kernels of winter Triticale were determined. The relatively low level of GS activity compared to high level of NAD(P)H-dependent GDH activity during intensive filling of grains with storage compounds may indicate the participation of GDH in reductive amination of 2-oxoglutarate. The amination activity of this enzyme in all grain development phases exceeded the deaminating activity several fold. Moreover, the dynamics in the change of NAD(P)H-GDH and NAD(P)⁺-GDH activities were analysed in various tissues of the developing grains. The high amination activity of the enzyme in the seed coat, where the intensive protein synthesis occurs would also be an indication of the anabolic function of this enzyme.     

Induction of glutamine synthetase by 8-bromo cyclic AMP in primary cultures of rat brain astrocytes

Glutamine synthetase (GS) is the key enzyme in cerebral glutamine production. Understanding the regulation of the expression of GS is important for definition of the control of glutamine metabolism in brain. Therefore, we studied the control of GS expression by 8-bromo cyclic AMP in primary cultures of astrocytes prepared from brains of neonatal rats. GS activity was increased by 8-bromo cyclic AMP in a dose- and time-dependent manner. This increase was associated with a corresponding increase in the steady-state level of GS mRNA.     

Bacterial type I glutamine synthetase of the rifamycin SV producing actinomycete, Amycolatopsis mediterranei U32, is the only enzyme responsible for glutamine synthesis under physiological conditions

The structural gene for glutamine synthetase, glnA, from Amycolatopsis mediterranei U32 was cloned via screening a genomic library using the analog gene from Streptomyces coelicolor. The clone was functionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutant under the control of a lac promoter. Sequence analysis showed an open reading frame encoding a protein of 466 amino acid residues. The deduced amino acid sequence bears significant homologies to other bacterial type I glutamine synthetases, specifically, 71% and 72% identical to the enzymes of S. coelicolor and Mycobacterium tuberculosis, respectively. Disruption of this glnA gene in A. mediterranei U32 led to glutamine auxotrophy with no detectable glutamine synthetase activity in vivo. In contrast, the cloned glnA^＋ gene can complement for both phenotypes in trans. It thus suggested that in A. mediterranei U32, the glnA gene encoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratory defined physiological conditions.     

Intracellular localization of glutamine synthetase in Chlorogloeopsis fritschii

After differential centrifugation of cell-free extracts of Chlorogloeopsis fritschii, 71% of the original glutamine synthetase (GS) activity was associated with the thylakoids, while little activity was detected in the cytoplasmic membranes. Monospecific antiserum to a purified GS inhibited 88% of the enzyme activity in solubilized thylakoid membranes. An antiserum raised against thylakoids gave 81% inhibition. However, using intact thylakoid membranes, only 7% inhibition was obtained with the GS antiserum, indicating that GS is located inside the thylakoid membranes.The author is with the Department of Biological Sciences, University of Science and Technology, Irbid, Jordan     

Distribution of the glutamine synthetase isozyme GSp1 in maize (Zea mays)

In maize (Zea mays L.), GSp1, the predominant GS isozyme of the developing kernel, is abundant in the pedicel and pericarp, but absent from the endosperm and embryo. Determinations of GSp1 tissue distribution in vegetative tissues have been limited thus far to root and leaves, where the isozyme is absent. However, the promoter from the gene encoding GSp1 has been shown to drive reporter gene expression not only in the maternal seed-associated tissues in transgenic maize plants, but also in the anthers, husks and pollen (Muhitch et al. 2002, Plant Sci 163: 865-872). Here we report chromatographic evidence that GSp1 resides in immature tassels, dehiscing anthers, kernel glumes, ear husks, cobs and stalks of maize plants, but not in mature, shedding pollen grains. RNA blot analysis confirmed these biochemical data. In stalks, GSp1 increased in the later stages of ear development, suggesting that it plays a role in nitrogen remobilization during grain fill.