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PubMed2002

Cell-specific expression of the glutamine transporter SN1 suggests differences in dependence on the glutamine cycle.

Glutamine is involved in a variety of metabolic processes, including recycling of the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). The system N transporter SN1 mediates efflux as well as influx of glutamine in glial cells [Chaudhry et al. (1999), Cell, 99, 769-780]. We here report qualitative and quantitative data on SN1 protein expression in rat. The total tissue concentrations of SN1 in brain and in kidney are half and one-quarter, respectively, of that in liver, but the average concentration of SN1 could be higher in astrocytes than in hepatocytes. Light and electron microscopic immunocytochemistry shows that glutamatergic, GABAergic and, surprisingly, purely glycinergic boutons are ensheathed by astrocytic SN1 laden processes, indicating a role of glutamine in the production of all three rapid transmitters. A dedication of SN1 to neurotransmitter recycling is further supported by the lack of SN1 immunoreactivity in oligodendrocytes (cells rich in glutamine but without perisynaptic processes). All neuronal structures appear unlabelled implying that a different protein mediates glutamine uptake into nerve endings. In several regions, SN1 immunoreactivity is higher in association with GABAergic than glutamatergic synapses, in agreement with observations that exogenous glutamine increases output of transmitter glutamate but not GABA. Nerve terminals with low transmitter reuptake or high prevailing firing frequency are associated with high SN1 immunoreactivity in adjacent glia. Bergmann glia and certain other astroglia contain very low levels of SN1 immunoreactivity compared to most astroglia, including retinal Müller cells, indicating the possible existence of SN isoforms and alternative mechanisms for transmitter recycling.

Amino Acid Transport Systems, Neutral↗
PubMed2005

Congenital glutamine deficiency with glutamine synthetase mutations.

Glutamine synthetase plays a major role in ammonia detoxification, interorgan nitrogen flux, acid-base homeostasis, and cell signaling. We report on two unrelated newborns who had congenital human glutamine synthetase deficiency with severe brain malformations resulting in multiorgan failure and neonatal death. Glutamine was largely absent from their serum, urine, and cerebrospinal fluid. Each infant had a homozygous mutation in the glutamine synthetase gene (R324C and R341C). Studies that used immortalized lymphocytes expressing R324C glutamine synthetase (R324C-GS) and COS7 cells expressing R341C-GS suggest that these mutations are associated with reduced glutamine synthetase activity.

Amino Acid Metabolism, Inborn Errors↗
PubMed2003

Molecular identification of human glutamine- and ammonia-dependent NAD synthetases. Carbon-nitrogen hydrolase domain confers glutamine dependency.

NAD synthetase catalyzes the final step in the biosynthesis of NAD. In the present study, we obtained cDNAs for two types of human NAD synthetase (referred as NADsyn1 and NADsyn2). Structural analysis revealed in both NADsyn1 and NADsyn2 a domain required for NAD synthesis from ammonia and in only NADsyn1 an additional carbon-nitrogen hydrolase domain shared with enzymes of the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia. Consistent with the domain structures, biochemical assays indicated (i) that both NADsyn1 and NADsyn2 have NAD synthetase activity, (ii) that NADsyn1 uses glutamine as well as ammonia as an amide donor, whereas NADsyn2 catalyzes only ammonia-dependent NAD synthesis, and (iii) that mutant NADsyn1 in which Cys-175 corresponding to the catalytic cysteine residue in nitrilases was replaced with Ser does not use glutamine. Kinetic studies suggested that glutamine and ammonia serve as physiological amide donors for NADsyn1 and NADsyn2, respectively. Both synthetases exerted catalytic activity in a multimeric form. In the mouse, NADsyn1 was seen to be abundantly expressed in the small intestine, liver, kidney, and testis but very weakly in the skeletal muscle and heart. In contrast, expression of NADsyn2 was observed in all tissues tested. Therefore, we conclude that humans have two types of NAD synthetase exhibiting different amide donor specificity and tissue distributions. The ammonia-dependent synthetase has not been found in eucaryotes until this study. Our results also indicate that the carbon-nitrogen hydrolase domain is the functional domain of NAD synthetase to make use of glutamine as an amide donor in NAD synthesis. Thus, glutamine-dependent NAD synthetase may be classified as a possible glutamine amidase in the nitrilase family. Our molecular identification of NAD synthetases may prove useful to learn more of mechanisms regulating cellular NAD metabolism.

Amide Synthases↗
PubMed1989

Addition of glutamine to total parenteral nutrition after elective abdominal surgery spares free glutamine in muscle, counteracts the fall in muscle protein synthesis, and improves nitrogen balance.

Twenty-two patients undergoing elective abdominal surgery were given total parenteral nutrition (TPN) after the operation. The TPN contained either a conventional amino acid solution supplemented with glutamine or a conventional amino acid solution without supplementation. To study amino acid and protein metabolism, muscle biopsy specimens were taken before surgery and on the third postoperative day. The postoperative decrease in the intracellular concentration of free glutamine was less pronounced in the glutamine group (21.8 +/- 5.5%) than in the control group (38.7 +/- 5.1%; p less than 0.05). The protein synthesis was reflected in the concentration and size distribution of ribosomes. No significant changes in these parameters were seen in the glutamine group after the operation. In the control group, the total concentration of ribosomes fell by 27.2 +/- 8.5% (p less than 0.05), and the relative proportion of polyribosomes fell by 10.6 +/- 2.9% (p less than 0.01). Although there were significant changes in the control group, no significant differences in the changes of these parameters between the two groups were detected. The cumulative nitrogen loss was significantly less in the glutamine group as compared to the control group during the period studied--2.3 +/- 1.4 g versus 8.5 +/- 1.5 g, respectively (p less than 0.01). Administration of glutamine to catabolic patients is advocated.

Cholecystectomy↗
PubMed1987

Influence of Na+, K+, and Ca2+ on glutamine synthesis and distribution in rat brain cortex slices: a possible linkage of glutamine synthetase with cerebral transport processes and energetics in the astrocytes.

The ouabain-induced suppression of glutamine synthesis and retention in incubating rat brain cortex slices was found to be mimicked by changes in the cationic content of the incubation medium, which cause an increase in the intracellular [Na+] and a decrease in the intracellular [K+]. The suppression of glutamine synthesis (and fixation of ammonia) was also found to take place when Ca2+ was omitted from the incubation medium. This occurred whether endogenous or exogenous glutamate was the substrate for glutamine synthesis. The suppressions cannot be due solely to an effect on glutamate uptake, because the uptake is not markedly affected by these conditions. The results show that Na+, K+, and Ca2+ influence the synthesis and distribution of glutamine in the brain. They suggest that Ca2+ and the Na+, K+ pump may serve a role in regulating the activity of ATP-dependent glutamine synthetase, a key enzyme of the glutamate-glutamine cycle, located in the astrocytes. This may be mediated via a direct effect on the enzyme or through an effect on the production of ATP.

Adenosine Triphosphate↗
PubMed1993

Uptake of glutamate, not glutamine synthetase, regulates adaptation of mammalian cells to glutamine-free medium.

Two cell lines (McCoy and MDCK) were studied in an attempt to understand the metabolic changes associated with adaptation to glutamine-free medium (GMEM + gmate). McCoy cells assumed normal growth rates after 2-3 passages in this medium whereas MDCK cells showed no growth in GMEM + gmate. The glutamine synthetase (GS) activity of both cell lines was elevated (up to x 9) as glutamine was depleted from normal media (GMEM + gmine). The high activity of GS was maintained during McCoy cell growth in GMEM + gmate. However, there was no apparent significant difference between the two cell lines in the pattern of changes of GS activity in response to glutamine. The cellular uptake rates of glutamine and glutamate from the medium differed significantly between the two cell lines. During the adaptation of McCoy cells to GMEM + gmate, the rate of glutamate uptake doubled to a value of 0.54 nmol/min per mg cell protein whereas the maximum value for MDCK cells was considerably lower (0.04 nmol/min per mg cell protein). We propose that the difference in intrinsic ability for glutamate transport accounts for the difference in growth response between the two cell lines in the glutamine-free medium.

Amino Acid Transport System X-AG↗
PubMed1976

Rapid transfer of oxygens from inorganic phosphate to glutamine catalyzed by Escherichia coli glutamine synthetase.

Measurements are reported on certain isotopic fluxes during the net conversion of glutamine, ADP and Pi to glutamate, NH3, and ATP by Escherichia coli glutamine synthetase (adenylylated form, Mn2+ activated) in presence of a hexokinase/glucose trap to remove the ATP formed during the reaction. The results show that the transfer of oxygens from Pi to glutamine is the most rapid of the measured isotopic interchanges, over five oxygens from Pi being transferred to glutamine for each glutamate formed by net reaction. Under similar conditions, the oxygen transfer from Pi to glutamate, was stimulated somewhat by an increase in the glutamate concentration but inhibited by an increase in the ammonia concentration. The enzyme from brain or peas did not show the rapid transfer of 18O from Pi to glutamine shown by the E. coli enzyme. Deductions are also made from the data about the availability of the oxygens of gamma-carboxyl of bound glutamate for reaction. The most logical explanation of the results with the E. coli enzyme is that the gamma-carboxyl group of bound glutamate has sufficient rotational freedom so that under conditions of rapid substrate interconversion either carboxylate oxygen can participate in the reaction. The results with the pea enzyme are consistent with hindered rotation of the gamma-care additional findings make likely a relative order of certain catalytic steps for the E. coli enzyme as follows: ATP release less than NH3 release less than glutamate release less than substrate interconversion less than glutamine release and Pi release and glutamate release less than ADP release.

Ammonia↗
PubMed1985

Primary structure of peptides from bovine brain glutamine synthetase. Comparison with sequences of glutamine synthetases from other organisms.

An analysis of the covalent structure of bovine brain glutamine synthetase has been initiated. Cyanogen bromide and tryptic digests have yielded peptides accounting for most of the polypeptide subunit, and sequence analysis has placed in order over half of the amino acids within these peptides. The amino terminus is acetylated and has the following partial sequence: Ac(H, S3, A2, T)-L-B-K-G-I-K-Z-V-Y-M. The carboxyl-terminal sequence is: A-L-P-Q-G-D-K-V-Q-A-M. The peptides isolated from bovine glutamine synthetase show a high degree of homology with peptides isolated from ovine and porcine brain glutamine synthetases. In contrast to the sequence homologies of the proteins from eukaryotic sources, there are no obvious amino acid sequence homologies between bovine brain glutamine synthetase and any prokaryotic glutamine synthetase. Bovine brain glutamine synthetase is inactivated by phenylglyoxal and N-ethylmaleimide. In both cases catalytic activity is protected by the presence of ATP, suggesting the presence of arginine and cysteine residues at or near the ATP binding site.

Amino Acid Sequence↗
PubMed1975

Regulation of synthesis of glutamine synthetase by adenylylated glutamine synthetase.

We have examined three mutants of Klebsiella aerogenes whose genetic lesions (glnB, glnD, and glnE) are in loci unlinked to the structural gene for glutamine sythetase (glnA) and in which the control of both the level and state of adenylylation of glutamine synthetase is altered. Each mutation alters a different component of the adenylylation system of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. Inability of the cell to deadenylylate glutamine synthetase (glnB and glnD) greatly decreases its production, while inability to adenylylate glutamine sythetase (glnE) results in its constitutively high production. These results together with our previous results indicate that adenylylated glutamine synthetase inhibits the transcription of glnA.

Adenosine Monophosphate↗
PubMed1981

Mechanism of l-Glutamine Production by an l-Glutamine-Producing Mutant of Flavobacterium rigense.

Properties of some enzymes involved in l-glutamine biosynthesis in an l-glutamine-producing mutant of Flavobacterium rigense were examined. Glutamate-oxaloacetate transaminase in the mutant was nearly at the same level as that in the parent strain and was the most active among the enzymes participating in glutamate biosynthesis from alpha-ketoglutarate. Glutamine synthetase formation in the mutant was enhanced by increasing the concentration of (NH(4))(2)-fumarate in the medium, but the activity of this enzyme in the parent strain was very low, and its formation was not influenced by the concentration of (NH(4))(2)-fumarate. Glutaminase formation by both strains was similar and was not influenced by the levels of (NH(4))(2)-fumarate. Glutaminase activity of the mutant was inhibited by ammonia and fumarate. Intracellular amino acids and extracellular free amino acids in the mutant were compared with those of the parent strain. It seems reasonable to conclude that l-glutamine leaks out specifically through the cell membrane of strain 703 and that this specific excretion of l-glutamine probably allows a continuous conversion of l-glutamate to l-glutamine inside the cell.

Journal Article↗
PubMed2005

Binding of glutamine to glutamine-binding protein from Escherichia coli induces changes in protein structure and increases protein stability.

Glutamine-binding protein (GlnBP) from Escherichia coli is a monomeric protein localized in the periplasmic space of the bacterium. It is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. The protein consists of two similar globular domains linked by two peptide hinges, and X-ray crystallographic data indicate that the two domains undergo large movements upon ligand binding. Fourier transform infrared spectroscopy (FTIR) was used to analyze the structure and thermal stability of the protein in detail. The data indicate that glutamine binding induces small changes in the secondary structure of the protein and that it renders the structure more thermostable and less flexible. Detailed analyses of IR spectra show a lower thermal sensitivity of alpha-helices than beta-sheets in the protein both in the absence and in the presence of glutamine. Generalized two-dimensional (2D) analyses of IR spectra reveal the same sequence of unfolding events in the protein in the absence and in the presence of glutamine, indicating that the amino acid does not affect the unfolding pathway of the protein. The data give new insight into the structural characteristics of GlnBP that are useful for both basic knowledge and biotechnological applications.

Carrier Proteins↗
PubMed1991

Respective effects of glucose and glutamine on the glutamine synthetase activity of human skin fibroblasts.

The activity of Glutamine Synthetase (GS) was measured during the growth of human diploid skin fibroblasts cultured for three weeks in the presence or absence of either glucose or glutamine or both. In medium free of both glucose and glutamine, a single late peak in GS activity was observed concomitantly with delayed small cell protein increment. In all media containing either glucose or glutamine or both. GS activity rose sharply during rapid cell growth, displayed a plateau, and then decreased once the cells had reached confluency. The variations in extracellular amino acid levels were also determined and were found to depend on the composition of the medium but not on the cell culture duration. These results demonstrate, for the first time as far as we know, that strong GS activity is present in rapidly growing skin fibroblasts. In contrast to many other mammalian cell types, GS activity in human skin fibroblasts appears not to be subject to regulation by extracellular glutamine. This difference may well be connected with cell differentiation.

Amino Acids↗
PubMed1976

Glutamine-stimulated modification and degradation of glutamine synthetase in hepatoma tissue culture cells.

Effects of glutamine on glutamine synthetase (GS) activity of hepatoma tissue culture (HTC) cells were studied with the aid of a specific goat anti-rat GS serum. Immunodiffusion and immunoelectrophoretic tests show that rat liver GS and HTC cell GS are immunologically similar but not identical. Immunotitrations of HTC cell extracts demonstrate that in cells incubated in high concentrations (5 mM) of glutamine, a cross-reacting form of GS with a decreased enzyme-specific activity accumulates. On prolonged incubation of cells in high glutamine, there is net degradation of GS to form immunologically inactive products. Radio-immunoprecipitation experiments show that glutamine acts by accelerating the degradation of preformed GS.

Animals↗
PubMed1978

Effect of glutamine on the degradation of glutamine synthetase in hepatoma tissue-culture cells.

In certain lines of hepatoma tissue-culture cells, the extracellular glutamine concentration regulates the specific activity of glutamine synthetase. By quantifying the radioactivity in immunoprecipitated glutamine synthetase on polyacrylamide gels, we found that the rate of degradation, but not of synthesis, of glutamine synthetase is a sensitive function of extracellular glutamine. The activiy that degrades this enzyme appears to be labile.

Animals↗
PubMed1985

Glutamine and glucose metabolism during thymocyte proliferation. Pathways of glutamine and glutamate metabolism.

Energy metabolism in proliferating cultured rat thymocytes was compared with that of freshly prepared non-proliferating resting cells. Cultured rat thymocytes enter a proliferative cycle after stimulation by concanavalin A and Lymphocult T (interleukin-2), with maximal rates of DNA synthesis at 60 h. Compared with incubated resting thymocytes, glucose metabolism by incubated proliferating thymocytes was 53-fold increased; 90% of the amount of glucose utilized was converted into lactate, whereas resting cells metabolized only 56% to lactate. However, the latter oxidized 27% of glucose to CO2, as opposed to 1.1% by the proliferating cells. Activities of hexokinase, 6-phosphofructokinase, pyruvate kinase and aldolase in proliferating thymocytes were increased 12-, 17-, 30- and 24-fold respectively, whereas the rate of pyruvate oxidation was enhanced only 3-fold. The relatively low capacity of pyruvate degradation in proliferating thymocytes might be the reason for almost complete conversion of glucose into lactate by these cells. Glutamine utilization by rat thymocytes was 8-fold increased during proliferation. The major end products of glutamine metabolism are glutamate, aspartate, CO2 and ammonia. A complete recovery of glutamine carbon and nitrogen in the products was obtained. The amount of glutamate formed by phosphate-dependent glutaminase which entered the citric acid cycle was enhanced 5-fold in the proliferating cells: 76% was converted into 2-oxoglutarate by aspartate aminotransferase, present in high activity, and the remaining 24% by glutamate dehydrogenase. With resting cells the same percentages were obtained (75 and 25). Maximal activities of glutaminase, glutamate dehydrogenase and aspartate aminotransferase were increased 3-, 12- and 6-fold respectively in proliferating cells; 32% of the glutamate metabolized in the citric acid cycle was recovered in CO2 and 61% in aspartate. In resting cells this proportion was 41% and 59% and in mitogen-stimulated cells 39% and 65% respectively. Addition of glucose (4 mM) or malate (2 mM) strongly decreased the rates of glutamine utilization and glutamate conversion into 2-oxoglutarate by proliferating thymocytes and also affected the pathways of further glutamate metabolism. Addition of 2 mM-pyruvate did not alter the rate of glutamine utilization by proliferating thymocytes, but decreased the rate of metabolism beyond the stage of glutamate significantly. Formation of acetyl-CoA in the presence of pyruvate might explain the relatively enhanced oxidation of glutamate to CO2 (56%) by proliferating thymocytes.

Animals↗
PubMed1989

Glutamine assimilation pathways in Neurospora crassa growing on glutamine as sole nitrogen and carbon source.

Neurospora crassa wild-type is almost unable to grow on glutamine as sole nitrogen and carbon source but a GDH-; GS +/- double mutant strain, lacking NADP-dependent glutamate dehydrogenase and partially lacking glutamine synthetase did grow. Under these conditions, the double mutant had a higher chemical energy content than the wild-type. Enzyme assays and labelling experiments with glutamine indicated that in the double mutant glutamine was degraded to ammonium and to carbon skeletons by glutamate synthase, the catabolic (NADH-dependent) glutamate dehydrogenase and the glutamine transaminase-omega-amidase pathway.

Amino Acids↗
PubMed1992

[Effect of glutamine and glutamine-dipeptide enriched amino acid solution on rat small bowel mucosa regeneration].

To mine the effects of glutamine and it's dipeptide enriched nutrition solution on the gastro-intestinal structure and protein metabolism, 40 male Wistar rats were studied for 8 days. The rats were randomly divided into four groups: sham operation group, standard amino acid solution group, glutamine enriched group and glutamine dipeptide enriched group. Observation parameters were: weight change; serum AA profile analysis; computerized morphometric analysis of mucosa thickness and villus height; total nitrogen balance; and liver, kidney functional results. The data were managed by multiple sample comparison methods (ANOVA of Statview 512 software) on Macintosh PC. Significant level was put on P < 0.05. The results showed that significant differences of body weight, serum glutamine level, nitrogen balance, intestinal mucosa thickness and villus height were found between the control and study groups. No significant difference in liver and kidney function was found. Gln-Ala dipeptide had same effects as free glutamine did.

Amino Acids↗
PubMed1983

The glutamine-utilizing site of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase.

Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with 6-diazo-5-oxo-L-norleucine resulted in complete loss of its ability to catalyze glutamine-dependent phosphoribosylamine formation and its glutaminase activity, whereas its ability to catalyze ammonia-dependent phosphoribosylamine formation and to hydrolyze phosphoribosylpyrophosphate was increased. The site of reaction with 6-diazo-5-oxo-L-norleucine was the NH2-terminal cysteine residue. The NH2-terminal sequence of the B. subtilis enzyme was homologous with that of the corresponding amidotransferase from Escherichia coli, for which the NH2-terminal cysteine is also essential for glutamine utilization (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536). The fact that the metal-free E. coli amidotransferase contains a glutamine-utilizing structure that is very similar to that found in B. subtilis amidotransferase, which contains an essential [4Fe-4S] center, indicates that the iron-sulfur center probably plays no role in glutamine utilization.

Amidophosphoribosyltransferase↗