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PubMed1984

Glutamine amidotransferase function. Replacement of the active-site cysteine in glutamine phosphoribosylpyrophosphate amidotransferase by site-directed mutagenesis.

Site-directed mutagenesis was employed to replace cysteine 12 with phenylalanine in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase). Glutamine-dependent amidophosphoribosyltransferase activity was abolished as a consequence of the mutation. The mutant enzyme, however, exhibited NH3-dependent activity, contained Fe-S, and was normally regulated by AMP. These results document the role of the active site cysteine in activation of glutamine for amide transfer. NH3-dependent amidophosphoribosyltransferase was utilized for de novo purine nucleotide synthesis. Cells containing the mutant enzyme grew at nearly the wild-type rate in media containing a high concentration of NH4Cl. The Phe-12 mutation was used to study NH2-terminal processing. Whereas the wild-type Cys-12 enzyme is processed correctly in Escherichia coli by removal of 11 amino acid residues from the NH2 terminus, the Phe-12 mutant enzyme was not subject to undecapeptide processing. Neither the mutant nor wild-type enzyme made in vitro was correctly processed. Alternative enzymatic and autocatalytic processing mechanisms were considered. The available evidence favors autocatalytic NH2-terminal undecapeptide processing.

Adenosine Monophosphate↗
PubMed1983

Subcellular location of chicken brain glutamine synthetase and comparison with chicken liver mitochondrial glutamine synthetase.

Chicken brain glutamine synthetase has been found to be localized in the cytosolic fraction of this tissue in contrast to its mitochondrial location in chicken liver. Despite this difference in subcellular distribution, the enzyme from brain exhibits the same molecular weight during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has the same isoelectric point, and is immunochemically cross-reactive. In addition, the immunochemically isolated primary translation products for glutamine synthetase from brain and liver mRNA are the same size. These data suggest that cytosolic brain and mitochondrial liver glutamine synthetase may be derived from the same gene.

Animals↗
PubMed2001

Glutamine and alpha-ketoglutarate are metabolite signals involved in nitrate reductase gene transcription in untransformed and transformed tobacco plants deficient in ferredoxin-glutamine-alpha-ketoglutarate aminotransferase.

Transformed tobacco (Nicotiana tabacum L.) plants with varying activities of the key enzyme of ammonia assimilation, ferredoxin-glutamine-alpha-ketoglutarate aminotransferase (Fd-GOGAT; EC 1.4.7.1), were used to examine the roles of ammonium, glutamine (Gln) and alpha-ketoglutarate (alpha-KG) in the regulation of nitrate reductase (NR; EC 1.6.6.1) transcript abundance. In wild-type leaf discs, NR mRNA abundance was increased following feeding with NO3-, sucrose and alpha-KG and decreased by feeding Gln. In air, leaves with decreased GOGAT accumulated Gln and alpha-KG simultaneously; this was accompanied by increased NR transcripts. The inhibition of NR transcription by Gln observed in leaf-disc experiments was therefore not observed in the low-Fd-GOGAT plants that accumulate Gln in vivo. The results suggest that the negative effect of Gln on NR transcript abundance was offset by high alpha-KG and that the relative amounts of alpha-KG and Gln are more important in controlling NR gene transcription than the concentration of either metabolite alone.

Amino Acid Oxidoreductases↗
PubMed1985

Bovine retinal glutamine synthetase 2. Regulation and properties on the basis of glutamine synthetase and glutamyl transferase reactions.

Glutamine, the end product formed by the glutamine synthetase (GS) reaction, inhibits retinal GS activity in the presence of Mn2+, but not in the presence of Mg2+. In the presence of Mg2+, Mn2+ itself inhibits retinal GS activity. Other compounds which inhibit retinal GS activity significantly are methionine sulfoximine, D-alanine and carbamyl phosphate. Amino acids, such as L-alanine, L-serine and glycine, do not affect the enzyme activity. These amino acids, however, significantly inhibit the enzyme activity when measured on the basis of the glutamyl transferase (GT) reaction. GS isolated from neuronal tissues is regulated differently from that previously reported by others for non-neuronal tissues. The enzyme activity, as measured by GS activity, shows three-fold higher activity with Mg2+ over Mn2+ or Co2+ and on the basis of GT activity, shows about three-fold higher activity with Mn2+ over Mg2+ or Co2+. The optimum pH for the GS reaction lies in the range of 7.2-7.8 and for the GT reaction is 6.4-7.0. Both the GS and GT activities of the enzyme show similar heat stabilities.

Animals↗
PubMed1994

Effect of free glutamine and alanyl-glutamine dipeptide on mucosal proliferation of the human ileum and colon.

BACKGROUND/AIMS: Glutamine (Gln) is considered a trophic factor for small intestinal epithelia, which is important during severe illness. Its use in parenteral nutrition is precluded by its instability, a problem that may be overcome by use of the stable dipeptide L-alanyl-L-glutamine (Ala-Gln). The hypothesis was tested that Gln or Ala-Gln may stimulate cell proliferation not only in the ileum but also in the proximal and distal colon and, thus, may contribute to the gut barrier function. METHODS: Biopsy samples from the normal human ileum, proximal colon, and rectosigmoid were incubated for 4 hours with Gln (2 mmol/L), Ala-Gln (2 mmol/L), and saline (control). Cells in S phase were labeled with bromodeoxyuridine. In longitudinal crypt sections labeled and quiescent cells were counted. RESULTS: Gln as well as Ala-Gln stimulated crypt cell proliferation in the ileum, proximal colon, and rectosigmoid colon. In ileal specimens, labeling was greater in the entire crypt, whereas in both colonic regions, the trophic effect was confined to the basal crypt compartments. CONCLUSIONS: Gln and Ala-Gln have trophic effects not only in the ileum, but also in the proximal and distal colon. This could be important during parenteral nutrition when mucosal atrophy may weaken the gut barrier.

Adult↗
PubMed2006

The 3'-UTR of the glutamine-synthetase gene interacts specifically with upstream regulatory elements, contains mRNA-instability elements and is involved in glutamine sensing.

Glutamine synthetase (GS) is expressed at various levels in a wide range of tissues, suggesting that a complex network of modules regulates its expression. We explored the interactions between the upstream enhancer, regulatory regions in the first intron, and the 3'-untranslated region and immediate downstream genomic sequences of the GS gene (the GS "tail"), and compared the results with those obtained previously in conjunction with the bovine growth hormone (bGH) tail. The statistical analysis of these interactions revealed that the GS tail was required for full enhancer activity of the combination of the upstream enhancer and either the middle or the 3'-intron element. The GS tail also prevented a productive interaction between the upstream enhancer and the 5'-intron element, whereas the bGH tail did not, suggesting that the 5'-intron element is a regulatory element that needs to be silenced for full GS expression. Using the CMV promoter/enhancer and transfection experiments, we established that the 2.8 kb GS mRNA polyadenylation signal is approximately 10-fold more efficient than the 1.4 kb mRNA signal. Because the steady-state levels of both mRNAs are similar, the intervening conserved elements destabilize the long mRNA. Indeed, one but not all constructs containing these elements had a shorter half life in FTO-2B cells. A construct containing only 300 bases before and 100 bases after the 2.8 kb mRNA polyadenylation site sufficed for maximal expression. A stretch of 21 adenines inside this fragment conferred, in conjunction with the upstream enhancer and the 3'-part of the first intron, sensitivity of GS expression to ambient glutamine.

3' Untranslated Regions↗
PubMed2005

Does acute glutamine depletion enhance the response of glutamine synthesis to fasting in muscle in adult and old rats?

BACKGROUND AND AIMS: In earlier studies, skeletal muscle glutamine synthetase (GS) activity was shown to be enhanced by fasting and glucocorticoids, and inhibited by exogenous glutamine (Gln) supplementation. The current study was designed to determine whether phenylbutyrate (PhiB), a Gln-chelating agent in humans, (1) could trap Gln and produce a decline in plasma Gln in rats, as it does in humans, and (2) if so, whether (Phi)B would further enhance the response of muscle GS activity to fasting in rats. METHODS: Adult (6-8 months) and aged (20-21 months) rats were fasted for 5 days and received two doses of 0.5 g(Phi)Bby orogastric route at times 0 and 4 h, and were then sacrificed at 5.5 h. Plasma Gln was measured by enzymatic methods, other amino acids were quantified by amino acid analysis. GS activity was measured in soleus (SO) and tibialis anterior (TA) muscles. RESULTS: (Phi)B treatment was associated with: (1) a 20% decline in plasma Gln concentration from 572+/-54 to 424+/-34 micromol/L (P<0.05) and from 476+/-49 to 360+/-80 micromol/L (P<0.05) in fasted adult and old rats, respectively; and (2) a preservation of GS up-regulation by fasting in TA and SO muscles in both adult and aged rats, with TA muscle GS activities of 198+/-65 vs. 203+/-68 ((Phi)B-treated vs. vehicle-treated, NS), and 244+/-81 vs. 274+/-59 (NS) nmol/h/mg protein in adult and aged rats, respectively. CONCLUSION: These data suggest that: (1) large doses of (Phi)B deplete plasma Gln in fasted rats, regardless of age, (2) Gln depletion induced by Phi)B does not alter GS activity.

Age Factors↗
PubMed2002

Impact of oral L-glutamine on glutathione, glutamine, and glutamate blood levels in volunteers.

OBJECTIVE: We investigated the effect of glutamine supplementation on plasma glutamine (Gln), glutamate (Glu), and whole-blood glutathione (GSH) concentrations in human volunteers. METHODS: Subjects first adapted to a standard diet with known intakes of protein, total GSH, cysteine, methionine, and total Glu (Glu values include Glu and Gln) for 3 d. Plasma Gln, Glu, and whole-blood GSH levels were then measured at 4-h intervals over 24 h. Supplemental oral Gln (0.3 g x kg(-1) x d(-1)) was ingested for 10 d and then 24-h plasma levels of Gln, Glu, and whole-blood GSH were measured. RESULTS: The plasma concentrations of Glu (116%; P = 0.006) and Gln (20%; P = 0.046) were significantly higher, whereas concentrations of GSH were significantly lower (37%; P = 0.00091) after oral Gln supplementation. CONCLUSION: Oral Gln increases Glu and Gln levels in plasma of healthy subjects but does not increase GSH red cell (whole-blood) levels. Thus, GSH biosynthesis and preservation of GHS stores in red blood cells may involve rate-limiting substrates other than Gln.

Administration, Oral↗
PubMed2005

Unfolding and refolding of the glutamine-binding protein from Escherichia coli and its complex with glutamine induced by guanidine hydrochloride.

The aim of this work was to study the conformational changes of the Escherichia coli glutamine-binding protein (GlnBP) induced by GdnHCl and the effect of the binding of glutamine (Gln) on these processes. To this end, GdnHCl-induced unfolding of GlnBP alone and its GlnBP-Gln complex was studied by protein intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy. The obtained spectroscopic data were interpreted taking into the account the peculiarities of protein three-dimensional structure. In particular, the fact that formation of a complex of GlnBP and Gln, which essentially changes the global structure of protein, affects only insignificantly the microenvironments of tryptophan residues elucidates the similarity of the emission spectra of GlnBP and the GlnBP-Gln complex, and the existence of quenching groups near tyrosine residues and an effective nonradiative Tyr --> Trp and/or Tyr --> Tyr --> Trp energy transfer provide an explanation for the negligibly small contribution of tyrosine to the bulk fluorescence of the native protein and for its increase in protein unfolding. The use of the parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP-Gln unfolding are three-step processes (N --> I(1) --> I(2) --> U), though in the case of the GlnBP-Gln complex these stages essentially overlap. Despite the complex character, GlnBP unfolding is completely reversible. The dramatic shift of the N --> I(1) process to higher GdnHCl concentrations for the GlnBP-Gln complex in comparison with GlnBP was shown.

Anilino Naphthalenesulfonates↗
PubMedDate not supplied

Fluorescence properties of glutamine-binding protein from Escherichia coli and its complex with glutamine.

In this work, the fluorescence of glutamine-binding protein (GlnBP) and its complex with glutamine (GlnBP/Gln) in native and unfolded forms was studied. The experimental data were interpreted on the basis of the results of the analysis of Trp and Tyr microenvironments taking into the account the data for GlnBP mutated forms Trp32Phe(Tyr) and Trp220Phe(Tyr), which have been obtained by Axelsen et al. (Biophys. J. 1991, 60, 650-659). This allowed us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of the fluorescence characteristics of GlnBP and GlnBP/Gln, and the uncommon effect of the excess of the fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm respect to the excitation at 280 nm. The last effect is explained by the spectral dependence of the Trp 32 and Trp 220 contributions to the protein absorption. The protein Trp fluorescence dependence on the excitation wavelength must be taken into account for the evaluation of the Tyr residues contribution to the bulk fluorescence of protein, and in principle, it also may be used for the development of an approach for the decomposition of a multicomponent protein fluorescence spectrum.

Carrier Proteins↗
PubMed1991

Selective modification by transglutaminase of a glutamine side chain in the hinge region of the histidine-388----glutamine mutant of yeast phosphoglycerate kinase.

The transglutaminase-catalysed incorporation of putrescine and monodansylcadaverine into yeast phosphoglycerate kinase has been studied. There is little incorporation of the amines into wild-type enzyme, but nearly stoichiometric incorporation into the histidine-388----glutamine mutant enzyme. C.d. studies show that the overall structure of the mutant enzyme is very similar to that of the wild-type enzyme. Incorporation of the amines into the mutant enzyme causes no significant change in its activity. Glutamine-388 was shown, by isolation and sequencing of the modified peptide, to be the site of incorporation of monodansylcadaverine into the mutant enzyme. The specificity of the transglutaminase reaction is discussed in the light of available data.

Amino Acid Sequence↗
PubMed2006

Protection elicited by two glutamine auxotrophs of Mycobacterium tuberculosis and in vivo growth phenotypes of the four unique glutamine synthetase mutants in a murine model.

We generated four individual glutamine synthetase (GS) mutants (DeltaglnA1, DeltaglnA2, DeltaglnA3, and DeltaglnA4) and one triple mutant (DeltaglnA1EA2) of Mycobacterium tuberculosis to investigate the roles of GS enzymes. Subcutaneous immunization with the DeltaglnA1EA2 and DeltaglnA1 glutamine auxotrophic mutants conferred protection on C57BL/6 mice against an aerosol challenge with virulent M. tuberculosis, which was comparable to that provided by Mycobacterium bovis BCG vaccination.

Animals↗
PubMed1967

Regulation of glutamine synthetase. V. Partial purification and properties of glutamine synthetase from Bacillus licheniformis.

The glutamine synthetase of Bacillus licheniformis has been obtained at about 15% purity. Sucrose gradient centrifugation gave a molecular weight value of approximately 612,000. Both l- and d-glutamate can be utilized as substrates in the biosynthetic reaction, although the l isomer was five times more active. The requirement for adenosine triphosphate (ATP) can be partially replaced by guanosine or inosine triphosphates, but not by cytidine or uridine triphosphates. The Mn(++) was required for activity, and the requirement cannot be satisfied with Mg(++). Maximal activity of the biosynthetic reaction was observed when ATP and Mn(++) were present in equimolar amounts. An excess of either reactant gave less activity. However, other purine and pyrimidine nucleotides, when added in combination with ATP, can partially substitute for ATP in attaining the equimolar ratio of nucleotide to Mn(++). A complex of ATP and Mn(++) is the preferred form of substrate. The B. licheniformis enzyme catalyzes the glutamyl transfer reaction but at a much slower rate than the Escherichia coli glutamine synthetase. Either adenosine diphosphate (ADP) or ATP can activate the glutamotransferase, although ADP is more active.

Acyltransferases↗
PubMed1976

Effects of acetylsalicylic acid (ASA), ASA plus L-glutamine and L-glutamine on healing of chronic gastric ulcer in the rat.

A chronic gastric ulcer model was produced in rats by the subserosal injection of 20% acetic acid solution (0.015 ml) in order to examine whether (1) acetylsalicylic acid (ASA) irritates the chronic gastric ulcer in active or healed or diminished stage, (2) L-glutamine, given together with ASA, inhibits the adverse effect of ASA. Oral ASA 200 mg/kg/day, given in two divided doses for 10 consecutive days, apparently delayed the healing of the gastric ulcer and irritated the healed ulcer to reulcerate. L-Glutamine, 1,500 mg/kg/day, which was given together with ASA in two divided doses, markedly protected the gastric ulcer both in active and healed stages from the deleterious activity of ASA.

Animals↗
PubMed2005

[The structure and stability of the glutamine-binding protein from Escherichia coli and its complex with glutamine].

A study was made of the conformational changes in the Escherichia coli glutamine-binding potein (GlnBP) induced by GdnHCl, and of the effect of glutamine (Gln) binding on these processes. Intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy were used. The obtained experimental data were interpreted, taking into the account results of the analysis of tryptophan and tyrosine residues microenvironments. This enabled us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of fluorescence characteristics of GlnBP and GlnBP/Gln, and an uncommon effect of the excess of fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm compared to the excitation at 280 nm. The latter effect is explained by the spectral dependence of Trp 32 and Trp 220 contributions to protein absorption. The dependence of Trp fluorescence of protein on the excitation wavelength must be taken into account for the evaluation of Tyr residues contribution to the bulk fluorescence of protein, and in principle, it may also be used for the development of an approach to decomposition of multi-component protein fluorescence spectrum. The parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP/Gln unfolding are three-step processes (N-->I1-->I2-->U), though in the case of the GlnBP/Gln complex these stages essentially overlap. Despite its complex character, GlnBP unfolding is completely reversible. In comparison with GlnBP, in the case of GlnBP/Gln the dramatic shift of N-->I1 process to higher GdHCl concentrations is shown.

Escherichia coli Proteins↗
PubMed1977

[Regulation of glutamine metabolism in Chlorella pyrenoidosa. Regulation of glutamine synthetase activity by adenylic system components].

A decrease of glutamine synthetase (E. C. 6.3.1.2.) activity was observed under the assimilation of ammonium nitrogen in Chlorella. At the same time a decrease of ATP content in Chlorella cells took place. The ATP content was 7-fold decreased, while ADP and AMP contents were 4-fold and 3-fold increased respectively, after 15 min. of Chlorella incubation on "ammonium" medium. Further incubation for 45 min, resulted in gradual increase of ATP content and in decrease of ADP and AMP contents. The value of energy charge in ammonium assimilating Chlorella cells sharply decreased for first 15 min. of incubation and then it normalized gradually. The experiments with glutamine synthetase preparation, isolated from ammonium assimilating cells, have shown that ADP and AMP are strong inhibitors of the enzyme in the presence of Mg2+, and only ADP produces the inhibitory effect in the presence of Mn2+. No enzyme reactivation was observed after the transfer of ammonium assimilating cells into nitrogen-free medium or nitrate medium, the enzyme activity increasing at the expense of enzyme protein synthesis denovo.

Adenine Nucleotides↗
PubMed1994

Molecular characterization of an NAD-specific glutamate dehydrogenase gene inducible by L-glutamine. Antisense gene pair arrangement with L-glutamine-inducible heat shock 70-like protein gene.

The gene for an NAD-specific glutamate dehydrogenase (NAD-GDH) that is allosterically activated by NADP+ (non-substrate) was cloned, and its physical structure and nucleotide sequence was determined. The gene consists of 9 introns and 10 exons; the 10th and largest exon, which is 1863 nucleotides long, is at the 3'-end of the gene. The shortest exon of 33 base pairs is the first and is located at the 5'-end of the gene. The large exon is in perfect register along the complementary strand with a heat shock 70 (HSP)-like protein gene. The NAD-GDH gene is inducible with L-glutamine, just as the HSP 70-like protein gene (LéJohn, H.B., Cameron, L.E., Yang, B., MacBeath, G., Barker, D.S., and Williams, S.A. (1994) J. Biol. Chem. 269, 4513-4522). The phenomenon of anti-parallel coupling of two genes is named antisense gene pair. By Northern and Western blotting techniques, we obtained indirect evidence that the gene is expressed in vivo. The gene encodes a protein of M(r) 118,740 which consists of 1063 amino acid residues. The 5' and 3' borders of the gene display typical but unproven promoter motifs of CCAAT, TATAAT, and AAATAAAA polyadenylation signal bounded by a pyrimidine-rich transcription termination-type format. Restriction endonuclease site mapping of all the genomic clones isolated that carry most or all of the gene, and of the genome itself, gave hybridization patterns that are consistent with the interpretation that the organism, Achlya klebsiana, has only one form of the gene. 3'-End-labeling of a 5.2-kb XbaI DNA fragment (carrying the antisense gene pair) that was then asymmetrically cleaved to produce two single 3'-end-labeled pieces that were used as probes on L-glutamine-induced cell poly(A)+ RNA, showed that the end-labeled DNA equivalent to the HSP 70-like protein mRNA hybridized to a 3.4-kb transcript and the end-labeled DNA equivalent to the NAD-GDH mRNA hybridized to a 2.4-kb transcript.

Amino Acid Sequence↗
PubMed1982

Rhythms in glutamine synthetase activity, energy charge, and glutamine in sunflower roots.

Roots of sunflower plants (Helianthus annuus L. var. Mammoth Russian) subjected to L12:D12, L18:D6, and L12:D12 followed by continuous light all display rhythms of about 12 hours for glutamine synthetase (GS) activity (transferase reaction) with one peak in the ;light phase' and one in the ;dark phase.' Root energy charge (EC = ATP+(1/2)ADP/ATP+ADP+AMP) is directly correlated with GS, but the GS rhythm is better explained as the result of a rhythmic adenine nucleotide ratio (ATP/ADP+AMP) that regulates enzyme activity through allosteric modification. When L12:D12 plants are subjected to free-running conditions in continuous darkness, only diurnal rhythms for GS and EC, with peaks in the dark phase, remain. The 12-hour root rhythms for GS and EC appear to be composed of two alternating rhythms, one a diurnal, light-dependent, incompletely circadian light phase rhythm and the other a light-independent, circadian dark phase rhythm.Only glutamine, of the root amino acids, displays cyclical changes in concentration, maintaining under all conditions a 12-hour rhythm that is consistently synchronized with, but nearly always inversely correlated with, GS and EC rhythms.

Journal Article↗