Glutamine Synthetase Specific Activity Research Articles

Regulation of Glutamine Synthetase (GS) Activity and Root Morphological Plasticity in the Nitrogen Fixing Azolla- Anabaena Symbiont under Nitrogen Limiting Conditions

The present study was conducted to identify the role of the endosymbiont, Anabaena azollae in fixing atmospheric N2 in the Azolla-Anabaena symbiosis. The Azolla-Anabaena azollae (association) grown in nitrogen-free modified Hoagland’s medium supplemented with 10% (v/v) N2 using an artificial gas mix, resulted in the increase in root number and root length. The GS specific activity of the association was 75 ± 6 nmole γ-glutamate mg protein-1 min-1 and the total N content was lesser in cultures grown in 10% (v/v) N2. Similarly, the artificially produced Anabaena free Azolla (endophyte-free), developed increased root length, root number and reduced GS specific activity (30 ± 10 nmole γ-glutamate mg protein-1 min-1), which on supplementation with 2.5 mM NH4NO3 did not show any change in the root length, root number and GS activity. However, the association cultured in 10% (v/v) N2+2.5 mM NH4NO3, reversed the effect of 10% (v/v) N2 treatment exhibited a GS specific activity of 295 ± 120 nmole γ-glutamate mg protein-1 min-1 and the morphological features were similar to the control plants. The western blot analysis of the GS protein from the association showed two isoforms of GS (GS1 and GS2) of equal abundance under all growth conditions, whereas, one diffused band of intermediate size between GS1 and GS2 was observed in the endophyte-free fern.

Influence of N nutrients on GS activity and putative ammonium transporter1;2 (SaAMT1;2) expression in sandal plants (Santalum album L.)

Ammonium acts as better N source for sandal growth. The ammonium assimilation and transport is mediated by the up-regulation of putative leaf and root SaAMT1;2 in Santalum album. Nitrogen compounds play a major role in the concentration of macromolecules and expression of putative ammonium transporter1;2 (SaAMT1;2) in Santalum album (S. album) hydroponic cultures. Specific activity of glutamine synthetase (GS) and putative SaAMT1;2 expression levels were determined on the 7th, 14th and 21st day in S. album grown in nitrogen-free Hoagland’s solution supplemented with different concentrations (0-10 mM) of ammonium nitrate (NH4NO3), ammonium sulphate [NH4(SO4)2] and potassium nitrate (KNO3). Highest glutamine synthetase (GS) specific activity was observed in 0.5 mM KNO3 on the 14th day and chlorophyll content in 0.5 mM NH4NO3 on the 7th and 21st day. Highest quantity of total sugar and soluble sugar was detected in 0.5 mM NH4NO3 on the 14th day. The putative leaf and root SaAMT1;2 expression levels were positively correlated to GS specific activity, however, NH4(SO4)2 did not appreciably influence the GS specific activity and putative SaAMT1;2 expressions during the initial days. The relative expression of putative SaAMT1;2 followed an identical pattern in the NH4NO3/NH4(SO4)2 treated plants on the 7th and 21st day. The biochemical and molecular parameters indicated improved NH3 assimilation in hydroponic S. album cultures supplemented with different N compounds; however, it is time and concentration dependent.

Critical Evaluation of the Changes in Glutamine Synthetase Activity in Models of Cerebral Stroke.

The following article addresses some seemingly paradoxical observations concerning cerebral glutamine synthetase in ischemia-reperfusion injury. In the brain, this enzyme is predominantly found in astrocytes and catalyzespart of the glutamine-glutamate cycle. Glutamine synthetase is also thought to be especially sensitive to inactivation by the oxygen- and nitrogen-centered radicals generated during strokes. Despite this apparent sensitivity, glutamine synthetase specific activity is elevated in the affected tissues during reperfusion. Given the central role of the glutamine-glutamate cycle in the brain, we sought to resolve these conflicting observations with the view of providing an alternative perspective for therapeutic intervention in stroke.

Immunocharacterization of Vitis vinifera L. ferredoxin-dependent glutamate synthase, and its spatial and temporal changes during leaf development.

The grapevine (Vitis vinifera L.) partial fragment of cDNA clone pGOGAT1 [Loulakakis and Roubelakis-Angelakis (1997) Physiol Plant 101:220-228], encoding the ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1), was overexpressed in Escherichia coli cells. A hybrid between the Fd-GOGAT fragment and maltose-binding protein was purified and used to raise a polyclonal antibody in a rabbit. The prepared antibody appeared to be specific towards Fd-GOGAT; it recognized a protein band of approximately 160 kDa on nitrocellulose blots after SDS-PAGE of total proteins from leaves, internodes, roots and calluses, and precipitated most of the enzyme activity present in grapevine protein extracts. The quantity of Fd-GOGAT protein was substantially higher in leaves than in other grapevine tissues tested, coincident with a similar distribution of the enzyme specific activity. Intracellular localization studies revealed that both the enzyme activity and the 160-kDa immunoreactive protein were associated with the chloroplastic fraction. Furthermore, the accumulation of Fd-GOGAT, glutamine synthetase (GS) and glutamate dehydrogenase (GDH), at the activity and protein levels, was monitored during leaf development of field-grown plants, from the stage of the newly expanding leaf to the senescing old leaf. Both the specific activity and quantity of the 160-kDa polypeptide of Fd-GOGAT were higher in the mature, full sized leaves and substantially lower in young and senescing leaves. GS specific activity and immunoreactive protein followed the same trend as Fd-GOGAT, while GDH showed opposite developmental patterns of accumulation. The biological significance of the presence of Fd-GOGAT in the various grapevine tissues and its physiological role during early development and natural senescence of the leaves are discussed.

Induction of muscle glutamine synthetase gene expression during endotoxemia is adrenal gland dependent.

Skeletal muscle plays a crucial role in maintaining nitrogen homeostasis during health and critical illness by exporting glutamine, the most abundant amino acid in the blood. We hypothesized that induction of glutamine synthetase (GS) expression, the principal enzyme of de novo glutamine biosynthesis, in skeletal muscle after endotoxin administration was adrenal gland dependent. We studied the expression of GS in normal and adrenalectomized rats after intraperitoneal administration of Escherichia coli lipopolysaccharide (LPS). Treatment of normal rats with LPS resulted in a marked increase in GS mRNA that was dose and time dependent, and preceded the increase in GS protein and specific activity. The increase in muscle GS mRNA observed in normal rats in response to LPS was abrogated in adrenalectomized rats at 3 h after high dose LPS treatment and markedly attenuated at 5.5 h after low dose LPS treatment. These and other studies implicate glucocorticoid hormones as a key, but not exclusive, regulator of skeletal muscle GS expression after a catabolic insult.

A time-dependent increase in glial fibrillary acidic protein expression and glutamine synthetase activity in long-term subculture of the GL15 glioma cell line.

1. Astrocytes are the most numerous cellular elements in the central nervous tissue, where they play a critical role in physiological and pathological events. The biological signals regulating astrocyte growth and differentiation are relevant for both physiology and pathology, but they are still little understood. 2. Using a poorly differentiated glioma cell line, GL15, we investigated whether, in long-term subculture, this could upregulate the expression of glial fibrillary acidic protein (GFAP), as described in some rodent astrocyte cell lines. Under the same culture conditions, we investigated glutamine synthetase (GS) activity, growth-associated protein (GAP)-43 expression, and expression of several neutrotrophic factors. 3. A dramatic increase in GFAP expression was evidenced by Western blotting during progressive in vitro growth of GL15 cells. GS specific activity was also upregulated in long-term culture. The time spent in vitro by GL15 cells did not affect GAP-43 and neutrophic factor BDNF and NT3 expression as revealed by RT-PCR analysis. 4. Our results suggest that, in GL15, GFAP and GS genes may have common or integrated regulatory mechanisms elicited at the cell confluency which could be relevant for both astrocyte physiology and astrocyte pathology. These mechanisms are not involved in GAP-43 and neutrophic factor BDNF and NT3 expression.

The Effect of Nitrogen Nutrition on the Cellular Localization of Glutamine Synthetase Isoforms in Barley Roots.

Glutamine synthetase (GS) was detected by immunogold localization in the cytosol and plastids of roots of 7-d-old barley (Hordeum vulgare L. cv Klaxon) seedlings grown in the presence or absence of NO3- (15 mM) or NH4+ (30 mM). The number of GS polypeptides changed during root development, and this was affected by N nutrition. There was no evidence of a NO3--inducible root plastid GS.In apical 5- to 10-mm regions of the root the concentration of immunogold labeling of cytosolic GS was higher in the cortical parenchyma than in the vascular cells of the stele, irrespective of N nutrition. This labeling was at least 50% higher in both cell types in N-free compared with N-grown (either NO3- or NH4+) seedlings. In contrast, GS specific activity was highest in roots of NO3--grown seedlings. It is suggested that this indicates the presence of inactive GS in roots grown without N. This study has identified both cell- and development-specific responses of GS to N nutrition.

Regulation of Glutamate Dehydrogenase and Glutamine Synthetase in Avocado Fruit during Development and Ripening.

The activity, protein, and isoenzymic profiles of glutamate de-hydrogenase (GDH) and glutamine synthetase (GS) were studied during development and ripening of avocado (Percea americana Mill. cv Hass) fruit. During fruit development, the activity and protein content of both GDH and GS remained relatively constant. In contrast, considerable changes in these enzymes were observed during ripening of avocado fruit. The specific activity of GDH increased about 4-fold, coincident with a similar increase in GDH protein content and mRNA levels. On the other hand, GS specific activity showed a decline at the end of the ripening process. On the isoenzymic profile of GDH, changes in the prevalence of the seven isoenzymes were found, with a predominance of the more cathodal isoenzymes in the unripe and of the most anodal isoenzymes in the ripe fruit. Two-dimensional electrophoresis revealed that avocado fruit GDH consists of two subunits whose association gives rise to seven isoenzymes. The results support the view that the predominance of the more anodal isoenzymes in the overripe fruit was due to the accumulation of the [alpha]-polypeptide.

Dexamethasone regulates glutamine synthetase expression in rat lung

Since the lungs play a central role in maintaining glutamine homeostasis in normal and catabolic disease states, we studied the regulation of glutamine synthetase (GS) expression by dexamethasone in rat lung. Adult rats received saline (controls) or dexamethasone (0.5 mg/kg). Lung total RNA was extracted for Northern hybridization and labeled with an α- 32P rat GS cDNA probe. The mRNA of the constitutively expressed gene β-actin was the control for RNA loading. GS transcripts were measured by laser densitometry and normalized to actin, and GS specific activity was also determined. Following a single injection of dexamethasone (0.5 mg/kg), lung GS activity increased by 40% at 4 hours and by 75% at 8 hours. The dexamethasone-mediated increase in GS activity was associated with a marked increase in GS mRNA levels, which preceded the increase in enzyme activity by approximately 2 hours. Serial daily dexamethasone administration for 3 and 6 days caused an even greater increase in GS mRNA levels and specific activity. No effect was seen on β-actin levels, demonstrating that the expression of GS was not part of a global response to steroids. Therefore, glucocorticoids stimulate GS expression in rat lung. This regulation appears to be one mechanism by which lung glutamine release is augmented during critical illness.

Influence of Progressive Tumor Growth on Glutamine Metabolism in Skeletal Muscle and Kidney

The effects of progressive malignant growth on glutamine metabolism in skeletal muscle and in kidney were investigated. Fast-growing tumors consume considerable quantities of glutamine and lead to a decrease in circulating glutamine concentrations. Experiments were performed at various stages of tumor growth in rats implanted subcutaneously with the non-metastasizing methylcholanthrene-induced (MCA) fibrosarcoma and in pair-fed non tumor-bearing controls. Tumor growth stimulated a twofold increase in hindquarter (muscle) glutamine release, which was not due to an increase in blood flow, but rather to a doubling in the fractional release rate. Consequently, a progressive decrease in skeletal muscle glutamine concentrations was observed over time. Simultaneously, the activity of glutamine synthetase (GS), the principal enzyme of de novo glutamine biosynthesis, increased more than twofold. This increase in muscle GS activity was accompanied by an increase in GS mRNA but the augmentation in GS expression apparently could not match the increased rate of efflux since muscle depletion developed. In rats with large tumors and severe glutamine depletion, GS activity was not elevated. Glutamine feeding increased muscle glutamine concentrations and glutamine synthetase specific activity. Although tumor growth led to the development of mild systemic acidemia, the classic renal adaptations normally observed, i.e., elevated glutaminase activity and accelerated renal glutamine utilization, were not present in acidotic tumor-bearing rats. Instead, renal GS activity was increased in tumor-bearing animals and ammoniagenesis was enhanced, in spite of a reduction in net renal glutamine uptake. These data suggest that marked alterations in muscle and renal glutamine handling occur in the host with cancer; the enhanced muscle glutamine release in conjunction with no increase in renal consumption is consistent with increased glutamine uptake in other organs, most likely the tumor itself and the liver.

1-Aminocyclopentane-trans-1,3-dicarboxylic acid induces glutamine synthetase activity in cultured astrocytes.

In this study we have investigated the effect of excitatory amino acids on the activity of glutamine synthetase, a glial-specific enzyme that plays a key role in the regulation of glutamate concentration in the CNS. We found that of L-glutamate, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, kainate, and 1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), only the metabotropic glutamate receptor agonist trans-ACPD had an effect on glutamine synthetase specific activity in cultures of rat type I cortical astrocytes. Exposure of astrocytes to 1.0 mM trans-ACPD for 24 h resulted in an increase in glutamine synthetase activity to 149 +/- 11% of that in control cultures. This effect was concentration dependent, stereoselective, and blocked by cycloheximide. In addition, the increase in glutamine synthetase activity occurred at lower concentrations of trans-ACPD that did not produce morphological alterations or lysis of the astrocytes as measured by the lactate dehydrogenase content. These findings are consistent with the hypothesis that activation of the metabotropic excitatory amino acid receptor in astrocytes is coupled to the regulation of an enzyme essential to the metabolism and recycling of the excitatory transmitter L-glutamate.

Adaptive regulation in skeletal muscle glutamine metabolism in endotoxin-treated rats.

The effects of a single dose of endotoxin (7.5 mg/kg BW) on skeletal muscle glutamine metabolism were studied in vivo in rats to gain further understanding of the altered glutamine metabolism that characterizes sepsis and other catabolic diseases. In endotoxin-treated animals the arterial glutamine concentration fell early initially and then increased compared with control values. Twelve hours after treatment, the arteriovenous concentration difference for glutamine across the hindquarter doubled, resulting in a significant increase in net muscle glutamine release in endotoxin-treated rats. As a consequence, the muscle glutamine concentration fell in the endotoxin-treated animals by 25%-40%, an event that was apparent as early as two hours after endotoxin treatment. Skeletal muscle glutaminase activity, the major enzyme of glutamine breakdown, was unchanged by endotoxemia, but expression of glutamine synthetase mRNA and glutamine synthetase specific activity increased in a time-dependent fashion. The glutamine depletion that develops in skeletal muscle during endotoxemia is caused by accelerated muscle glutamine release rather than an increase in intracellular degradation or a fall in intracellular biosynthesis. The adaptive increase in glutamine synthetase expression that occurs requires de novo RNA and protein synthesis and may be designed to prevent complete depletion of the intracellular glutamine pool.

Regulation of Nodule Glutamine Synthetase by CO2 Levels in Bean (Phaseolus vulgaris L.)

Nodulated bean (Phaseolus vulgaris) plants were grown for 17 days after infection in normal (0.02%) CO(2) and from day 8 to 17 in high (0.1%) CO(2) in order to increase nitrogen fixation and define how nodule glutamine synthetase (GS) isoforms are regulated by the ammonia derived from the bacteroid. Nitrogenase activity was detected by day 10, and by day 17 activity was over twofold higher in 0.1% of CO(2) compared with plants grown in 0.02% CO(2) and inoculated with Rhizobium wild-type strain CE3. Likewise, plant fresh weight increased in response to increased CO(2), particularly in plants inoculated with the Rhizobium phaseoli mutant strain CFN037. Glutamine synthetase specific activity increased 2.5- to 6.5-fold from day 11 to 17. However, increased CO(2) did not appear to have an effect on GS specific activity. Analysis of the nodule GS polypeptide composition revealed that the gamma polypeptide was significantly reduced in response to high CO(2), whereas the beta polypeptide was not affected. The significance of this result in relation to the regulation of GS isoforms and their role in the assimilation of ammonia in the nodule is discussed in this paper.

Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Intoxication of rats with CCl4 (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS+) hepatocytes were reestablished. However, while the GS+ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS+ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS+ and GS- hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS+ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS- hepatocytes. Immediately after liver damage by CCl4 and during regeneration small but significant changes in the nuclear pattern were noted for GS- hepatocytes. However, the first GS+ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS- hepatocytes. These results indicate that new GS+ hepatocytes may be derived from formerly GS- cells which are induced to express GS if they have reached the border of the central veins.

Glutamine synthetase modulation in astrocyte cultures of different mouse brain areas

Astroglial cells from mouse cerebral hemispheres, cerebellum, olfactory bulbs, and medulla oblongata were grown in the presence of either hormones (hydrocortisone, insulin) or cell second messengers (dBcAMP, dBcGMP). Glutamine synthetase (GS) specific activity, GS protein level, and GS translation were investigated under the effect of these factors. Hydrocortisone produced a simultaneous increase in GS translation, GS level, and activity. This increase was observed in the astrocytes cultured from the four brain areas but at a variable magnitude depending on the area. The hydrocortisone effect appeared at the transcriptional level. Inversely, insulin decreased both the GS activity and the in vitro translated GS. This effect was seen only in the olfactory bulbs and the medulla. DBcAMP increased the GS biological activity only in the cerebral hemisphere cultures. It raised, however, the level of translated GS and GS protein in astrocytes from all the areas, suggesting a post-translational effect for intracellular cAMP. DBcGMP only affected GS in the astrocytes from cerebral hemispheres and the medulla modulating either the GS transcription or the messenger RNA stability. These results suggest specific regulation for GS expression, depending on the brain area from which the cells were dissociated or on the astroglial cell population present in these cultures affecting either the transcription, the mRNA stability, or the biological activity of the protein.

Reduction of glutamine synthetase specific activity in cultured astroglia by ferrous chloride

Immature and mature rat astroglia in culture were assayed for glutamine synthetase (GS) activity after a single exposure to the epileptogen FeCl2. Cells were cultured with both standard and elevated extracellular potassium or glutamate (Glu) concentrations. FeCl2 reduced GS activity below control levels, whereas high Glu increased GS activity. However, stimulation by high Glu was significantly attenuated in cultures given both FeCl2 and high Glu, indicating that cells treated with FeCl2 were not able to respond as effectively to increased extracellular glutamate by increasing their GS activity. The significance of these findings is that glial regulation of the neuronal environment may be impaired, based on the proposed importance of GS in ammonia detoxification in the brain.

Differential phenotypic expression induced in cultured rat astroblasts by acidic fibroblast growth factor, epidermal growth factor, and thrombin

We compared the effects of three growth factors, acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), and thrombin, on rat astroblast proliferation, morphology, glutamine synthetase-specific activity, and phenotypic expression of proteins. In vitro experiments were made on 20-day-old primary cultures. Astroblast proliferation was stimulated transiently (after 48 h treatment) by the three growth factors, while the cell glutamine synthetase activity began to increase significantly only after 3 days of treatment. Acidic FGF and EGF, but not thrombin, modified the cell morphology. The effects on phenotypic expression were first determined after 5 days of treatment to minimize the mitogenic effect of the factors. Proteins synthesized during the last 18 h of the treatments were separated by two-dimensional polyacrylamide gel electrophoresis. About 600 spots were compared, 54 were modulated by the various treatments, 13 were altered similarly by all three factors, 28 by aFGF and EGF, 7 by only aFGF, 3 by only EGF, and 3 by only thrombin. These results indicate a large similarity of effects between aFGF and EGF (41 proteins) and show that these factors elicit a more extended modulation of the phenotypic expression than thrombin (13 proteins). Each of the three factors has a few specific effects, which suggests that even for aFGF and EGF, which are supposed to elicit their effects through membrane receptor-associated tyrosine kinase activity, some specificity appears in their mechanism of action. A model is proposed to suggest that cell maturation is characterized by the modulation of the synthesis of many proteins which can be grouped into classes. Each class appears to be under the control of one regulatory element. The specificity of the effect of a growth factor should result from the activation of a specific combination of such regulatory elements. Analysis of the proteins after only 18 h of treatment, when neither proliferation nor maturation were significantly affected, showed that 11 proteins were regulated only at that time. These proteins could be related to intermediate steps of the growth factor signal transduction.

Glutamine synthetase specific activity and protein concentration in symbiotic Anabaena associated with Azolla caroliniana.

Glutamine synthetase (GS) is the primary NH4+ assimilating enzyme of cyanobacteria. The specific activities and cellular protein concentration of GS in symbiotic cyanobacteria associated with the water fern Azolla caroliniana were determined and compared to free-living cultures of Nostoc sp. strain 7801, a strain originally isolated from symbiotic association with the bryophyte Anthoceros punctatus. Both the in vitro specific activity and concentration of GS in symbiotic cyanobacteria separated from A. caroliniana were approximately 3-fold lower than the free-living Nostoc sp. strain 7801 culture. These results imply depressed synthesis of GS by the symbiont associated with A. caroliniana.

Alteration in morphology and induction of glutamine synthetase in rat glioma C6BU-1 cells cultured with prostaglandin D2-supplemented media

We evaluated the effects of prostaglandins (PGs) on rat glioma C6BU-1 cells by supplementing the culture media with PGs. In the medium containing PGD(2) (15 or 20 ?M), the glial cells showed altered morphology from an elongated fibroblastic form to a spreading multipolar one within 24 h, and their growth rate was suppressed to half of that of control cultures. In these cultures, the specific activity of glutamine synthetase (GS) increased approximately twofold within 48 h in comparison to the value for vehicle-treated controls. Simultaneous treatment with actinomycin D or cycloheximide completely blocked the PGD(2)-elicited increase in GS specific activity, suggesting that the increase was due to de novo synthesis of the enzyme. PGD(2)-like prostanoids such as PGD(1) and 9-deoxy-?(9), ?(12)-13,14-dihydro-PGD(2) (?(12)-PGJ(2)), when added to the culture medium, mimicked the actions of PGD(2) on the C6BU-I cells, though their effective concentrations were not necessarily identical. PGs of the E- and F-series had almost no discernible effect on the glioma. These results might imply a possibility that PGD(2) plays a regulatory effect in growth and/or differentiation of rat glioma C6BU-1 cells.

Temperature and oxygen regulated expression of a glutamine synthetase gene from Vibrio alginolyticus cloned in Escherichia coli.

Glutamine synthetase (GS) synthesis in Vibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene, glnA from V. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabled Escherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. The V. 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 an E. 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 in E. coli.