Osmoregulation in Rhizobium meliloti: characterization of enzymes involved in glutamate synthesis

1990 ◽  
Vol 36 (7) ◽  
pp. 469-474 ◽  
Author(s):  
Rigoberto Gonzalez-Gonzalez ◽  
James L. Botsford ◽  
Thomas Lewis
Keyword(s):  
Glutamine Synthetase ◽  
Osmotic Stress ◽  
Specific Activity ◽  
Rhizobium Meliloti ◽  
Glutamate Synthase ◽  
The Other ◽  
Glutamine Synthetase Activity ◽  
Biochemical Basis ◽  
Glutamic Dehydrogenase ◽  
Glutamate Synthesis

Rhizobium meliloti, like many bacteria, accumulates elevated levels of glutamate when osmotically stressed. The biochemical basis for this increase in glutamate production was investigated. Enzymes involved in glutamate synthesis, including glutamine synthetase, glutamate synthase, and glutamic dehydrogenase, were characterized in dialyzed crude cell-free extracts. A transaminase activity, which uses branched chain amino acids for the amination of 2-ketoglutaric acid, was also characterized. With the exception of glutamic dehydrogenase, the specific activity of the enzymes did not vary more than 4-fold in response to the available source of nitrogen or supplemental glutamate. Glutamic dehydrogenase activity was 13-fold greater when cells grew with 10 mM [Formula: see text] than when cells grew with 0.5 mM [Formula: see text]. Glutamate synthase was repressed 2-fold when cells grew with supplemental glutamate. Conversely, this enzyme was derepressed 2× when cells grew with 0.5 mM [Formula: see text] or nitrate. Growing cells in minimal defined medium with 400 mM NaCl to cause osmotic stress had little effect on the specific activity of any of the enzymes. The addition of K+ to the reactions stimulated heat-stable glutamine synthetase activity, but inhibited the other enzymes. Glutamate synthase was inhibited to a limited extent by several intermediates in the Krebs' cycle and very severely by glyoxylate. The addition of 10 mM glutamate to the reaction inhibited glutamate synthase 20%, but had no effect on the other enzymes. Key words: enzymes, glutamate synthesis, osmotic stress.

scholarly journals In Vivo Regulation of Glutamine Synthetase Activity in the Marine Chlorophyll b-Containing CyanobacteriumProchlorococcus sp. Strain PCC 9511 (Oxyphotobacteria)

2001 ◽  
Vol 67 (5) ◽  
pp. 2202-2207 ◽  
Author(s):  
Sabah El Alaoui ◽  
Jesús Diez ◽  
Lourdes Humanes ◽  
Fermı́n Toribio ◽  
Frédéric Partensky ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Nitrogen Starvation ◽  
Glutamate Synthase ◽  
Nitrogen Sources ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Protein Determination ◽  
Physiological Regulation ◽  
Gs Protein

ABSTRACT The physiological regulation of glutamine synthetase (GS; EC6.3.1.2 ) in the axenic Prochlorococcus sp. strain PCC 9511 was studied. GS activity and antigen concentration were measured using the transferase and biosynthetic assays and the electroimmunoassay, respectively. GS activity decreased when cells were subjected to nitrogen starvation or cultured with oxidized nitrogen sources, which proved to be nonusable forProchlorococcus growth. The GS activity in cultures subjected to long-term phosphorus starvation was lower than that in equivalent nitrogen-starved cultures. Azaserine, an inhibitor of glutamate synthase, provoked an increase in enzymatic activity, suggesting that glutamine is not involved in GS regulation. Darkness did not affect GS activity significantly, while the addition of diuron provoked GS inactivation. GS protein determination showed that azaserine induces an increase in the concentration of the enzyme. The unusual responses to darkness and nitrogen starvation could reflect adaptation mechanisms of Prochlorococcus for coping with a light- and nutrient-limited environment.

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

1985 ◽  
Vol 31 (3) ◽  
pp. 211-213
Author(s):  
Charlotte M. McCarthy ◽  
Maria E. Alvarez
Keyword(s):  
Glutamine Synthetase ◽  
Ammonium Chloride ◽  
Mycobacterium Avium ◽  
Cultured Cells ◽  
Specific Activity ◽  
Glutamate Synthase ◽  
Ammonia Concentration ◽  
Apparent Difference ◽  
Growth Status ◽  
Chloride Concentrations

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 μmol/mL or with glutamine at 0.1 or 1 μmol/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.

Corrigendum to: Ammonium metabolism in Selaginella bryopteris in response to dehydration-rehydration and characterisation of desiccation tolerant, thermostable, cytosolic glutamine synthetase from plant

2021 ◽  
Vol 48 (3) ◽  
pp. 358
Author(s):  
Kamal K. Singh ◽  
Shyamaprasad Saha ◽  
Ram C. Kadiravana ◽  
Deepika Mazumdar ◽  
Vijeta Rai ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Metabolic Regulation ◽  
Specific Activity ◽  
Higher Plants ◽  
Glutamate Synthase ◽  
Immunoblot Analysis ◽  
Primary Metabolism ◽  
Ammonium Metabolism ◽  
Free Ammonium ◽  
Selaginella Bryopteris

Water deficit (WD) has adverse effects on plant growth, and acclimation requires responses allowing primary metabolism to continue. Resurrection plants can serve as model system to gain insight into metabolic regulation during WD. We herein report the response of a resurrection lycophyte, Selaginella bryopteris, to dehydration-rehydration cycle with emphasis on ammonium metabolism. Dehydration of S. bryopteris fronds resulted in decrease of total protein and increase of free ammonium levels and the effect was reversed on rehydration. The proline content increased twice after 24 h of dehydration, which again recovered to background levels comparable to that at full turgor state. The specific activity of glutamine synthetase (GS) didn’t change significantly till 6 h and then declined by 21% after 24 h of dehydration, whereas specific activities of glutamate synthase (GOGAT) and aminating glutamate dehydrogenase (GDH) were enhanced significantly during dehydration. The deaminating activity of GDH also increased during dehydration albeit at a slower rate. Immunoblot analysis indicated overexpression of GS and GDH polypeptides during dehydration and their levels declined on rehydration. The results suggested significant role of GDH along with GS/GOGAT in production of nitrogen-rich amino acids for desiccation tolerance. Unlike higher plants S. bryopteris expressed GS only in cytosol. The enzyme had pH and temperature optima of 5.5 and 60°C, respectively, and it retained 96% activity on preincubation at 60°C for 30 min indicating thermostability. Hence, like higher plants the cytosolic GS from S. bryopteris has a conserved role in stress tolerance.

scholarly journals Effect of glucose deprivation on rat glutamine synthetase in cultured astrocytes

1996 ◽  
Vol 315 (2) ◽  
pp. 607-612 ◽  
Author(s):  
Françoise ROSIER ◽  
Dominique LAMBERT ◽  
Jeannine MERTENS-STRIJTHAGEN
Keyword(s):  
Glutamine Synthetase ◽  
Specific Activity ◽  
Glucose Deprivation ◽  
Synthesis Rate ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Sprague Dawley ◽  
Experimental Conditions ◽  
Adult Rats ◽  
Total Recovery

Glutamine synthetase was purified from the cerebral cortex of adult rats and characterized. Polyclonal rabbit antibodies were raised against the enzyme, purified and their specific anti-(glutamine synthetase) activity determined. A primary astroglial culture was prepared from newborn Sprague–Dawley rats. Astrocytes at different ages of development were incubated in the presence and absence of glucose. In glucose-deprived conditions the specific activity of glutamine synthetase decreased. This decrease was more pronounced in 8-day-old than in 21-day-old cultures. Kinetic analysis demonstrated that the reduction in activity was mainly related to a decrease in Vmax. By immunoprecipitation, it was shown that the number of enzyme molecules in astrocytes was decreased in glucose-deprived conditions. On addition of glucose, a total recovery of glutamine synthetase was obtained after 36 h in 8-day-old culture. Rates of degradation and synthesis were investigated. When compared with an incubation in the presence of glucose, glucose deprivation increased enzyme turnover, as estimated from the first-order disappearance of radioactivity from glutamine synthetase. Synthesis rate was estimated from the incorporation of [35S]methionine during a 2 h incubation period and was decreased in glucose-deprived conditions. Trichloroacetate-precipitable proteins changed only slightly in the experimental conditions, and total protein did not vary significantly during the experimental period. A mathematical model is presented which attempts to integrate degradation and synthesis in our experimental model.

Glutamine synthetase from Mycobacterium avium

1984 ◽  
Vol 30 (3) ◽  
pp. 353-359 ◽  
Author(s):  
Maria E. Alvarez ◽  
C. M. McCarthy
Keyword(s):  
Enzyme Activity ◽  
Glutamine Synthetase ◽  
Nitrogen Source ◽  
Mycobacterium Avium ◽  
Specific Activity ◽  
Sedimentation Coefficient ◽  
Ammonia Assimilation ◽  
Methionine Sulfoximine ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity

Mycobacterium avium was previously shown to be dependent upon ammonia or glutamine as a nitrogen source. In an effort to assess the physiology of ammonia assimilation by M. avium, a characterization of its glutamine synthetase was performed. The enzyme from M. avium was purified by streptomycin sulfate treatment, ammonium sulfate precipitation, and affinity chromatography. The enzyme was unusual in that it had a pH optimum of 6.4 and maximum enzyme activity was obtained between 50 and 60 °C as shown by the transferase assay. The glutamine synthetase activity from batch-cultured cells decreased with increasing concentration of ammonium chloride in the range of 0.25–5 μ mol/mL of medium, which demonstrated a response to environmental supply of a nitrogen source. The mycobacterial enzyme was similar to the other bacterial glutamine synthetases in terms of molecular weight and sedimentation coefficient which were 600 000 and 19.5 S, respectively, and enzyme activity was lost by treatment with a glutamate analog, methionine sulfoximine. The isoelectric point was, however, pH 4.5. Treatment of the enzyme with snake venom phosphodiesterase resulted in an increase in specific activity. AMP was released by the phosphodiesterase treatment, thus demonstrating that M. avium glutamine synthetase was regulated by adenylylation modification.

scholarly journals Distinctive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus

2003 ◽  
Vol 373 (2) ◽  
pp. 357-368 ◽  
Author(s):  
Barbara MONTANINI ◽  
Marco BETTI ◽  
Antonio J. MÁRQUEZ ◽  
Raffaella BALESTRINI ◽  
Paola BONFANTE ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Glutamate Dehydrogenase ◽  
Expression Profiles ◽  
Glutamate Synthase ◽  
Synthetase Activity ◽  
Mrna Levels ◽  
Glutamine Synthetase Activity ◽  
Tuber Borchii ◽  
Root Cells ◽  
N Assimilation

Nitrogen retrieval and assimilation by symbiotic ectomycorrhizal fungi is thought to play a central role in the mutualistic interaction between these organisms and their plant hosts. Here we report on the molecular characterization of the key N-assimilation enzyme glutamine synthetase from the mycorrhizal ascomycete Tuber borchii (TbGS). TbGS displayed a strong positive co-operativity (n=1.7±0.29) and an unusually high S0.5 value (54±16 mM; S0.5 is the substrate concentration value at which v=½Vmax) for glutamate, and a correspondingly low sensitivity towards inhibition by the glutamate analogue herbicide phosphinothricin. The TbGS mRNA, which is encoded by a single-copy gene in the Tuber genome, was up-regulated in N-starved mycelia and returned to basal levels upon resupplementation of various forms of N, the most effective of which was nitrate. Both responses were accompanied by parallel variations of TbGS protein amount and glutamine synthetase activity, thus indicating that TbGS levels are primarily controlled at the pre-translational level. As revealed by a comparative analysis of the TbGS mRNA and of the mRNAs for the metabolically related enzymes glutamate dehydrogenase and glutamate synthase, TbGS is not only the sole messenger that positively responds to N starvation, but also the most abundant under N-limiting conditions. A similar, but even more discriminating expression pattern, with practically undetectable glutamate dehydrogenase mRNA levels, was observed in fruitbodies. The TbGS mRNA was also found to be expressed in symbiosis-engaged hyphae, with distinctively higher hybridization signals in hyphae that were penetrating among and within root cells.

scholarly journals Glutamine and asparagine as nitrogen donors for reductant-dependent glutamate synthesis in pea roots

1975 ◽  
Vol 149 (2) ◽  
pp. 403-409 ◽  
Author(s):  
B J Miflin ◽  
P J Lea
Keyword(s):  
Glutamine Synthetase ◽  
Glutamate Synthase ◽  
Crude Extracts ◽  
Pea Root ◽  
Pea Roots ◽  
Nitrogen Donors ◽  
Glutamate Synthesis

Glutamine, in the presence of α-oxoglutarate, stimulates nicotinamide nucleotide oxidation by crude extracts of pea roots and leads to a reductant-dependent formation of glutamate. Commercially available asparagine also stimulates nicotinamide nucleotide oxidation in the presence of α-oxoglutarate, but the reaction causing the stimulation can occur in the absence of a reductant, is inhibited by transaminase inhibitors, and is additive to the glutamine reaction. The asparagine used was found to be contaminated with aspartate. Repurified asparagine, chromatographically free of aspartate, did not stimulate the rate of nicotinamide nucleotide oxidation, and it is probable that the original stimulation was due to aspartate contamination. It is concluded that pea-root glutamine (amide)-α-oxoglutarate aminotransferase (glutamate synthase), in common with the enzyme in leaves, is specific for glutamine as the N donor and α-oxoglutarate as the N acceptor. The significance of the enzyme in conjunction with glutamine synthetase in the assimilation of nitrate by roots is discussed.

scholarly journals Physiological and Molecular Osmotic Stress Responses in Three Durum Wheat (Triticum Turgidum ssp Durum) Genotypes

Agronomy ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 550 ◽  
Author(s):  
Salma Jallouli ◽  
Sawsen Ayadi ◽  
Simone Landi ◽  
Giorgia Capasso ◽  
Giorgia Santini ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Osmotic Stress ◽  
Durum Wheat ◽  
Stress Responses ◽  
Triticum Turgidum ◽  
Growth Traits ◽  
Glutamate Synthase ◽  
Dry Weight ◽  
Transcript Abundance ◽  
Primary Metabolism

This study aims to investigate the activities and expression of enzymes of primary metabolism and relate these data with the growth performance of three different durum wheat genotypes (Maali; YT13; and ON66) under osmotic stress. Growth traits—including plant height, dry weight (DW) and relative water content (RWC)—were measured to classify genotypes depending on their tolerance to stress. Several enzymes were investigated: Ascorbate peroxidase (APX), Glutamine Synthetase (GS), Glutamine dehydrogenase (GDH), Glutamate synthase (GOGAT), Glucose 6-phosphate dehydrogenase (G6PDH), and Phosphoenolpyruvate Carboxylase (PEPC). The expression of the cytosolic and plastidic glutamine synthetase (TaGS1 and TaGS2), high affinity nitrate transporters (TaNRT2.3) and Glutamate dehydrogenase (TaGDH) were also detected by qRT-PCR. The results indicated different growth performances among genotypes, indicating Maali and YT13 as tolerant genotypes and ON66 as a drought-susceptible variety. Data showed a decrease in PEPC and increase in APX activities under osmotic stress; a slight decrease in GS activity was observed, together with an increase in G6PDH in all genotypes; GS and NRT2 expressions changed in a similar pattern in the different genotypes. Interestingly, Maali and YT13 showed higher transcript abundance for GDH under stress compared to ON66, suggesting the implication of GDH in protective phenomena upon osmotic stress.

Regulation of ammonia-assimilating enzymes by various nitrogen sources in cultured Ipomoea spp.

1989 ◽  
Vol 67 (11) ◽  
pp. 3127-3133 ◽  
Author(s):  
M. W. Zink
Keyword(s):  
Glutamine Synthetase ◽  
Glutamate Dehydrogenase ◽  
Growth Medium ◽  
Glutamate Synthase ◽  
Nitrogen Sources ◽  
Defined Medium ◽  
Morning Glory ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Chemically Defined Medium

The levels of ammonia-assimilating enzymes of Ipomoea spp. (morning glory) grown in suspension culture on a chemically defined medium are affected by various nitrogen sources. An increase in nitrate or ammonium concentrations in the growth medium elevated the levels of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. The levels of the enzymes varied in a manner similar to the growth pattern of the cells. When the growth rate of Ipomoea was maximal, high levels of enzymes were obtained; when cell growth was poor, a low yield of enzymes was obtained. Glutamine, whether used as the sole nitrogen source or as a supplement in media containing ammonium plus nitrate, increased the level of glutamate dehydrogenase and decreased the level of the other two enzymes. The developmental pattern of glutamine synthetase activity with culture age in Ipomoea differed with different nitrogen sources in the growth medium.

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