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PubMed1987

Purification of the glutamine synthetase II isozyme of Drosophila melanogaster and structural and functional comparison of glutamine synthetases I and II.

Glutamine synthetase II was purified from Drosophila melanogaster adults. It was completely separable from the isozyme glutamine synthetase I by means of DEAE chromatography. The complete enzyme has an apparent molecular weight of 360,000. After two-dimensional electrophoresis it gave a single molecular species with an apparent molecular weight of 42,000. Structural analysis of the two isozymes showed that they are different both in subunit molecular weight and in isoelectric point. Peptide maps of the purified subunits showed considerable dissimilarity. Glutamine synthetase II is more active than glutamine synthetase I in the transferase assay, while the opposite is true in the biosynthetic assay. The kinetic parameters were determined, showing again noteworthy differences between the two isozymes. We therefore conclude that two forms of glutamine synthetase are present in Drosophila, with different primary structures, different kinetic behavior, and the possibility of different functional properties.

Animals↗
PubMed1991

Coordinated regulation of glutamine:fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine. Role of hexosamine biosynthesis in enzyme regulation.

We reported previously that glutamine:F-6-P amidotransferase (GFAT) plays an integral role in the development of insulin resistance by directing the flow of incoming glucose into the hexosamine biosynthesis pathway. To determine whether the enzymatic activity of GFAT is altered during desensitization of the glucose transport system, we treated isolated rat adipocytes with various combinations of insulin, glucose, and glutamine. Treatment with insulin or glucose alone (or in combination) failed to reduce cytosolic GFAT activity after 4 h, whereas combined treatment with all three components elicited a progressive loss of GFAT activity that was rapid (t1/2 of 2 h) and extensive (70% loss). A pronounced loss of GFAT activity was also seen in cells exposed to glucosamine, an agent known to directly enter the hexosamine pathway (55% loss at 4 h, ED50 of 360 microM). Moreover, a close correlation was observed between the induction of desensitization and the loss of GFAT activity as a function of glucose, insulin, glutamine, and glucosamine concentrations. When total intracellular hexosamine products were measured, we found that hexosamine formation was unaltered by insulin or glucose (or a combination) but was elevated by greater than 4-fold in the presence of insulin, glucose, and glutamine (t1/2 of 22 min), a condition known to cause both desensitization and loss of GFAT activity. Additional studies indicated that the loss of GFAT activity under desensitizing conditions is not due to allosteric regulation since removal of potential allosteric factors from the cytosol of desensitized cells by G-25 column chromatography failed to restore enzyme activity. Overall, these studies indicate that 1) GFAT is an insulin-regulated enzyme; however, control of enzyme activity is not due to a direct action of insulin, but rather is mediated by insulin-induced enhancement of glucose uptake; 2) the routing of incoming glucose through the hexosamine pathway and the formation of hexosamine products appears to regulate GFAT activity; and 3) the progressive loss of GFAT activity over several hours is probably not due to allosteric regulation.

Adipose Tissue↗
PubMed2001

Inhibition of oligo(glutamine) precipitation by glutamine-containing peptides.

When a solution of glutamine is added to solid 1,1'-carbonyldiimidazole, a mixture of oligo(glutamine)s up to about the 11-mer is formed rapidly. On standing overnight, the solution deposits a precipitate that does not easily redissolve. We have studied the inhibition of this precipitation by glutamine-containing peptides. We find that the alternating peptides (arg.gln)(4) and (arg.gln)(8) are efficient inhibitors of precipitation while arg(5), (glu.gln)(4), and a nonalternating octapeptide of the same composition as (arg.gln)(4) do not inhibit precipitation even though all the arg-containing peptides readily adsorb to oligo(glutamine) precipitates. A possible structural basis for this difference is discussed.

Adsorption↗
PubMed1998

The structure of glutamine-binding protein complexed with glutamine at 1.94 A resolution: comparisons with other amino acid binding proteins.

The crystal structure of the glutamine-binding protein (GlnBP) complexed with its ligand (Gln) was determined and refined to 1.94 A resolution. This ellipsoidal protein has two globular domains and is approximately 52 Ax40 Ax35 A in size. The glutamine ligand is located in the cleft between the two domains and stablized by hydrogen bondings and ionic interactions with Asp10, Gly68, Thr70, Ala67, Asp157, Arg75, Lys115, Gly119 and His156. The aliphatic portion of the glutamine ligand is sandwiched in a hydrophobic pocket formed between Phe13 and Phe50 and has 21 van der Waals contacts with GlnBP. Lys115 and His156, that are unique to GlnBP among amino acid binding proteins, apparently contribute to the ligand binding specificity of GlnBP. Asp10 is within 3 A of Lys115. These two residues are over 10 A apart in the ligand-free form of the GlnBP. In addition, GlnBP-Gln exhibits a large-scale movement of the two hinges connecting the two globular domains upon ligand binding. The most significant changes are 41.1 degrees in the phi angle of Gly89 and 34.3 degrees in the psi angle of Glu181 from the first and the second hinge of the protein, respectively. Besides the original six hydrogen bonds, three extra hydrogen bonds can be observed between the two hinge strands upon ligand binding. A hydrogen bond network connects the large domain to the second hinge and a second hydrogen bond network coalesces the small domain to the same strand, both via interaction with the glutamine ligand. Although the two strands of the hinge connecting the domains do not directly participate in the ligand binding, Gln183 and Tyr185 from the second hinge may be involved in the cascade of the conformational change that is induced by ligand binding.

Amino Acid Sequence↗
PubMed1980

The role fo glutamine synthetase and glutamine metabolism in nitrogen metabolite repression, a regulatory phenomenon in the lower eukaryote Neurospora crassa.

Growth of Neurospora crassa on media containing NH4+ leads to the repression of a variety of permeases and alternative pathways which would generate NH4+, so called "ammonium repression." The mutant am2 which lacks NADP-GDH is not subject to ammonium repression of nitrate reductase or urea permease, but like the wild type has repressed levels of these systems when grown in the presence of proline, glutamate or glutamine. The glutamine synthetase (GS) mutant gln-1a has derepressed levels of the aforementioned systems unless grown with glutamine. The oligomeric state of GS depends upon the nitrogen sufficiency of the cell, a tetrameric form predominates under conditions of nitrogen limitation and an octameric form under conditions of nitrogen sufficiency. We have found that the tetrameric form GS predominates in the mutants am2 and gln-1a when they are ammonium derepressed. Th mechanism of NH4+ repression in N. crassa is thought to entail a cessation of positive gene action by the product of the nit-2 regulatory gene. We propose that under conditions of NH4+ sufficiency, and hence glutamine sufficiency, the octameric form of GS represses nit-2 gene expression and thereby achieves ammonium repression.

Enzyme Repression↗
PubMed1989

Effect of parenteral glutamine peptide supplements on muscle glutamine loss and nitrogen balance after major surgery.

Twelve patients admitted for elective resection of carcinoma of colon or rectum were allocated at random to experimental and control groups (six in each) and received a total parenteral nutrition regimen providing 230 mg N/kg and 166 KJ/kg daily over the first 5 postoperative days. In the experimental group the parenteral fluid was supplemented with a synthetic glutamine-containing dipeptide, L-alanyl-L-glutamine (54 mg peptide-N/kg per day) and the control group received corresponding amounts of alanine-N and glycine-N. On each postoperative day nitrogen balance was better in the experimental group; mean daily nitrogen balance with alanyl-glutamine was -1.5 (SE 0.4) g N/day and with the control solution -3.6 (0.2) g N/day. The cumulative nitrogen balances on the fifth postoperative day were -7.1 (2.2) and -18.1 (1.7) g N, respectively. With the peptide-containing solution intramuscular glutamine concentration remained close to the preoperative value whereas with the control solution it decreased from 19.7 (SE 0.9) to 12.0 (0.6) mmol/l intracellular water.

Clinical Trials as Topic↗
PubMed1998

The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state.

The regulation of Escherichia coli glutamine synthetase (GS) by reversible adenylylation has provided one of the classical paradigms for signal transduction by cyclic cascades. Yet, many mechanistic features of this regulation remain to be elucidated. We examined the regulation of GS adenylylation state in a reconstituted system containing GS, adenylyltransferase (ATase), the PII signal transduction protein that controls ATase, and the uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which has a role in regulating PII. In this reconstituted bicyclic cascade system, the adenylylation state of GS was regulated reciprocally by the small molecule effectors 2-ketoglutarate and glutamine at physiological effector concentrations. By examination of the individual regulatory monocycles and comparison to the bicyclic system and existing data, we could deduce that the only sensors of 2-ketoglutarate were PII and PII-UMP. At physiological conditions, we observed that the main role of 2-ketoglutarate in bringing about the deadenylylation of GS was to inhibit GS adenylylation, and this was due to the allosteric regulation of PII activity. Glutamine acted as an allosteric regulator of both ATase and UTase/UR. We also compared the regulation of GS adenylylation state to the regulation of phosphorylation state of the transcription factor NRI (NtrC) in a reconstituted bicyclic system containing NRI, the bifunctional kinase/phosphatase NRII (NtrB), PII, and the UTase/UR. This comparison indicated that, at a fixed 2-ketoglutarate concentration, the regulation of GS adenylylation state by glutamine was sharper and occurred at a higher concentration than did the regulation of NRI phosphorylation. The possible biological implications of this regulatory arrangement are discussed.

Adenosine Triphosphate↗
PubMed2001

Mechanisms governing the expression of the enzymes of glutamine metabolism--glutaminase and glutamine synthetase.

Whether on the scale of a single cell, organ or organism, glutamine homeostasis is to a large extent determined by the activities of glutaminase (GA, EC 3.5.1.2) and glutamine synthetase (GS, EC 6.3.1.2), the two enzymes that are the focus of this report. GA and GS each provide examples of regulation of gene expression at many different levels. In the case of GA, two different genes (hepatic- and kidney-type GA) encode isoforms of this enzyme. The expression of hepatic GA mRNA is increased during starvation, diabetes and high protein diet through a mechanism involving increased gene transcription. In contrast, the expression of kidney GA mRNA is increased post-transcriptionally by a mechanism that increases mRNA stability during acidosis. We found recently that several isoforms of rat and human kidney-type GA are formed by tissue-specific alternative RNA splicing. Although the implications of this post-transcriptional processing mechanism for GA activity are not yet clear, it allows for the expression of different GA isoforms in different tissues and may limit the expression of GA activity in muscle tissues by diverting primary RNA transcripts to a spliceform that produces a nonfunctional translation product. The expression of GS enzyme is also regulated by both transcriptional and post-transcriptional mechanisms. For example, the GS gene is transcriptionally activated by glucocorticoid hormones in a tissue-specific fashion. This hormonal response allows GS mRNA levels to increase in selected organs during catabolic states. However, the ultimate level of GS enzyme expression is further governed by a post-transcriptional mechanism regulating GS protein stability. In a unique form of product feedback, GS protein turnover is increased by glutamine. This mechanism appears to provide a means to index the production of glutamine to its intracellular concentration and, therefore, to its systemic demand. Herein, we also provide experimental evidence that GS protein turnover is dependent upon the activity of the 26S proteosome.

Animals↗
PubMed1983

Genetic and biochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs and their revertants.

In this paper we present the isolation and characterization of glutamine auxotrophs of Neurospora crassa and their revertants. The results show that although various enrichment procedures were used, we found only two types of auxotrophs. Genetic crosses performed between the different mutants showed that the mutations responsible for their phenotypes were highly linked and probably affected the same gene. The biochemical characterization of the glutamine synthetase polypeptides of the different mutants showed that both types contained the alpha monomer. However, in place of the normal beta monomer, each type had a new polypeptide differing from normal beta either in its molecular weight or in its isoelectric point. On the other hand, the revertants had only the alpha monomer and were capable of growing without glutamine. On the basis of these data, we propose that the lack of glutamine synthetase activity in the auxotrophs is due to the interaction of the altered beta with the alpha monomer, and as a consequence the alpha monomer of the revertants regains its activity because of the absence of the altered beta.

Centrifugation, Density Gradient↗
PubMed1976

[Regulation of glutamine metabolism in Chlorella pyrenoidosa. Mechanisms of regulating the activity of glutamine synthetase during ammonia assimilation].

Glutamine synthetase (GS) (E.C.6.3.1.2) activity in Chlorella cells decreased when NH4+ was added to nitrogen-free growth medium. This GS inactivation had such a rate, that it could not be due to the repression of enzyme synthesis: the GS activity decreased by 20% within 5 minutes of NH4+ assimilation. Glutamine content in cell increased in 2.5 times for this period. In vitro experiments have shown that glutamine is a strong inhibitor of GS from Chlorella grown in the presence of NO3-, and in a less degree--an inhibitor of GS from cells grown in ammonium-containing medium. The data obtained are negative with respect to possible mechanisms of GS activity regulation via adenylation and ATP-dependent destruction of glutamine synthetase.

Ammonia↗
PubMed1976

[Glutamine metabolism regulation in Chlorella pyrenoidosa. Regulation of Chlorella glutamine synthetase activity by amino acids].

Effect of glutamine and its metabolites (amino acids) on Chlorella glutamine synthetase (GS) (E.C.6.3.1.2) in the presence of Mg or Mn was studied. Purified GS preparation was used, isolated from Chlorella grown in the presence of NH as a sole nitrogen source. Glutamate, aspartate, alanine and glycine inhibit GS activity in the presence of both Mg and Mn. Tryptophane and valine (up to 15 mM) activate GS in the presence of Mn. Tryptophane inhibits GS in the system with Mg. Sinergistic inhibition was observed under the combined effect of amino acids on GS in the presence of Mn and aspartate or alanine. The change of GS activity observed is supposed to be due to the inhibitory effect of glutamine and amino acids studied, since the glutamine content is increased (in 2.5 times for 5 min) and that of alanine and dicarbonic amino acids (for the following 15 min) under NH assimilation in Chlorella cells.

Amino Acids↗
PubMed1997

[Plasma glutamine level monitored with high performance liquid chromatography for patient infusing glutamine after small bowel transplantation].

To promote the restoration of the structure and functions of the graft intestine for a patient with small bowel transplantation, the authors gave intravenous infusion of 5% glutamine to the patient and the blood glutamine levels were determined with HPLC technique. The ultrafiltration technique was adapted to remove blood proteins. Good separation could be achieved when using micro-Bondapak C18 as separation cartridge, phthaldialdehyde as derivatization agent, gradient elution with methanol and phosphate buffer and detection at wavelength of 340 nm. The recovery rate was 90% to 105% and the lower limit of detection was 40 micromol/L with satisfactory separation. The results of this method are proved to be a timely and reliable indicator for glutamine level in blood for patients of small bowel transplantation with glutamine infusion.

Chromatography, High Pressure Liquid↗
PubMed1984

Glutamine homeostasis: role of pCO2 in regulating arterial glutamine in metabolic acidosis.

In chronic metabolic acidosis arterial plasma glutamine concentration is reduced 46%. This effect is attributable to a reduction in arterial pCO2 rather than acidemia since elevating pCO2 returns glutamine toward the control level. The extracellular glutamine precursors ammonia and glutamate exhibit a reciprocal decline with the elevation in pCO2. This reduction in metabolic acidosis appears to play an important role in sparing glutamine for renal base generation.

Acidosis↗
PubMed1982

Glutamine phosphoribosylpyrophosphate amidotransferase from cloned Escherichia coli purF. NH2-terminal amino acid sequence, identification of the glutamine site, and trace metal analysis.

Glutamine 5-phosphoribosylamine pyrophosphate phosphoribosyltransferase (amidophosphoribosyltransferase) was purified in large amounts from an Escherichia coli strain harboring a purF hybrid plasmid. Purified E. coli amidophosphoribosyltransferase lacks iron as well as other trace metals as determined by x-ray fluorescence spectrometry. The NH2-terminal amino acid sequence of the enzyme was determined and is in agreement with that deduced from the DNA sequence. [6-14C] Diazo-5-oxo-norleucine (DON), an active site-directed affinity analog of glutamine, selectively inactivated the glutamine-dependent amidophosphoribosyltransferase. Inactivation was accompanied by incorporation of 1 eq of [6-14C]DON per enzyme subunit. A 10-residue cyanogen bromide peptide labeled by [6-14C]DON was isolated and sequenced. The NH2-terminal cysteine of amidophosphoribosyltransferase was determined to be the residue alkylated by [6-14C]DON. These results establish that the NH2-terminal cysteine is the active site residue required for the glutamine amide transfer function of the enzyme. The experiments reported in this and the preceding article (Tso, J. Y., Zalkin, H., van Cleemput, M., Yanofsky, C., and Smith, J. M. (1982) 257, 3525-3531) demonstrate the application of affinity labeling, rapid peptide purification by high pressure liquid chromatography, and nucleotide sequence determination of a structural gene to localize an amino acid residue, peptide fragment, or functional domain in a long protein chain.

Amidophosphoribosyltransferase↗
PubMed1994

Functional roles for the glutamines within the glutamine-rich region of the transcription factor sigma 54.

Single and multiple point mutations were introduced to change the 12 glutamine residues within a 37-amino acid region of sigma 54. Multiple changes are shown to be required in order to interfere significantly with the function of this protein which is associated with enhancer-dependent bacterial transcription. Mutation of the central 4 glutamines leads to the production of less m-RNA, caused by an inability to fully open the promoter start site. DNA binding, however, is normal. Mutation of 4 other adjacent glutamines causes the promoter start site to open more readily than wild type, although this enhanced opening is not accompanied by more mRNA. The enhanced DNA melting is not caused by enhanced promoter binding, as indicated by normal protection of the polymerase-bound promoter against dimethyl sulfate attack. The results suggest that multiple glutamines play a role in transducing the melting signal from the enhancer protein to the polymerase.

Amino Acid Sequence↗
PubMed1999

Macrophage-mediated lysis of a beta-cell line, tumour necrosis factor-alpha release from bacillus Calmette-Guérin (BCG)-activated murine macrophages and interleukin-8 release from human monocytes are dependent on extracellular glutamine concentration and glutamine metabolism.

Macrophages and monocytes are cells with a large capacity for cytokine production. Cytokines produced by these cells are not preformed and released upon stimulation, but must be transcribed and translated. Although much is known concerning the regulation of the latter processes at the molecular level, the role of exogenous amino acids in the secretory process has not been actively investigated. Glutamine is utilized by macrophages at a much faster rate than any other amino acid. The role for high rates of glutamine utilization in macrophages or monocytes is not fully understood. We demonstrate here that the rates of lipopolysaccharide-stimulated tumour necrosis factor-alpha secretion from bacillus Calmette-Guérin (BCG)-activated murine peritoneal macrophages and lipopolysaccharide-stimulated interleukin-8 production from human monocytes are dependent upon extracellular glutamine concentration. We also demonstrate that potent inhibition of cytokine production can be achieved by incubating macrophages or monocytes in the presence of the glutaminase inhibitor 6-diazo-5-oxo-norleucine. On co-culture of BCG-activated macrophages and the clonal pancreatic beta-cell line BRIN-BD11, macrophage-specific beta-cell death was significantly reduced on prior exposure of macrophages to 6-diazo-5-oxo-norleucine. Thus glutamine metabolism may be essential for generation of cytotoxic products from macrophages, including tumour necrosis factor-alpha.

Animals↗
PubMed1989

Role of glutamine as a direct co-repressor of glutamine synthetase in Rhodobacter capsulatus E1F1.

High performance liquid chromatography (HPLC) has been used to determine the internal levels of amino acids in Rhodobacter capsulatus E1F1 cells, subjected to different treatments and nutritional conditions. Glutamine synthetase activity and enzyme concentration correlated negatively with the level of glutamine, suggesting that glutamine per se acts as a co-repressor in the enzyme synthesis. Moreover, addition of the specific inhibitor L-methionine-D,L-sulfoximine, that produced an increase in enzyme concentration, specifically promoted a depletion of intracellular glutamine.

Enzyme Repression↗
PubMed2000

Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-L-glutamate/glutamine cell wall structure, and bacterial replication.

New antibiotics to combat the emerging pandemic of drug-resistant strains of Mycobacterium tuberculosis are urgently needed. We have investigated the effects on M. tuberculosis of phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODNs) against the mRNA of glutamine synthetase, an enzyme whose export is associated with pathogenicity and with the formation of a poly-L-glutamate/glutamine cell wall structure. Treatment of virulent M. tuberculosis with 10 microM antisense PS-ODNs reduced glutamine synthetase activity and expression by 25-50% depending on whether one, two, or three different PS-ODNs were used and the PS-ODNs' specific target sites on the mRNA. Treatment with PS-ODNs of a recombinant strain of Mycobacterium smegmatis expressing M. tuberculosis glutamine synthetase selectively inhibited the recombinant enzyme but not the endogenous enzyme for which the mRNA transcript was mismatched by 2-4 nt. Treatment of M. tuberculosis with the antisense PS-ODNs also reduced the amount of poly-L-glutamate/glutamine in the cell wall by 24%. Finally, treatment with antisense PS-ODNs reduced M. tuberculosis growth by 0. 7 logs (1 PS-ODN) to 1.25 logs (3 PS-ODNs) but had no effect on the growth of M. smegmatis, which does not export glutamine synthetase nor possess the poly-L-glutamate/glutamine (P-L-glx) cell wall structure. The experiments indicate that the antisense PS-ODNs enter the cytoplasm of M. tuberculosis and bind to their cognate targets. Although more potent ODN technology is needed, this study demonstrates the feasibility of using antisense ODNs in the antibiotic armamentarium against M. tuberculosis.

Antitubercular Agents↗