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PubMed1985

Inactivation of glutamine synthetase by a purified rabbit liver microsomal cytochrome P-450 system.

Several mixed-function oxidation systems catalyze inactivation of Escherichia coli glutamine synthetase and other key metabolic enzymes. In the presence of NADPH and molecular oxygen, highly purified preparations of cytochrome P-450 reductase and cytochrome P-450 (isozyme 2) from rabbit liver microsomes catalyze enzyme inactivation. The inactivation reaction is stimulated by Fe(III) or Cu(II) and is inhibited by catalase, Mn(II), Zn(II), histidine, and the metal chelators o-phenanthroline and EDTA. The inactivation of glutamine synthetase is highly specific and involves the oxidative modification of a histidine in each glutamine synthetase subunit and the generation of a carbonyl derivative of the protein which forms a stable hydrazone when treated with 2,4-dinitrophenylhydrazine. We have proposed that the mixed-function oxidation system (the cytochrome P-450 system) produces Fe(II) and H2O2 which react at the metal binding site on the glutamine synthetase to generate an activated oxygen species which oxidizes a nearby susceptible histidine. This thesis is supported by the fact that (a) Mn(II) and Zn(II) inhibit inactivation and also interfere with the reduction of Fe(III) to Fe(II) by the P-450 system; (b) Fe(II) and H2O2 (anaerobically), in the absence of a P-450 system, catalyze glutamine synthetase inactivation; (c) inactivation is inhibited by catalase; and (d) hexobarbital, which stimulates the rate of H2O2 production by the P-450 system, stimulates the rate of glutamine synthetase inactivation. Moreover, inactivation of glutamine synthetase by the P-450 system does not require complex formation because inactivation occurs when the P-450 components and the glutamine synthetase are separated by a semipermeable membrane. Also, if endogenous catalase is inhibited by azide, rabbit liver microsomes catalyze the inactivation of glutamine synthetase.

Animals↗
PubMed1995

Effect of an enterally administered glutamine-rich protein on the catabolic response to a zymosan challenge in rats.

Glutamine is considered to be a conditionally essential amino acid during critical illness and has, therefore, been advocated to be included in nutritional support supplied in these situations. We investigated whether a diet containing a protein source rich in glutamine can restore depleted glutamine pools (plasma and muscle) and counteract muscle wasting, in a rat model of critical illness (intraperitoneal injection of zymosan). A glutamine-rich protein source was obtained by mixing a wheat protein hydrolysate (25% glutamine) with a whey protein isolate (to prevent essential amino acid deficiency). Feeding healthy control rats for 2 weeks with an adequate diet containing this protein source increased the two main glutamine pools (plasma and muscle). However, no effect was observed on the following zymosan-induced changes: 1) decreased glutamine and arginine concentrations (plasma and muscle), 2) wasting of muscle protein, and 3) decreased mitochondrial content in skeletal muscle. We conclude that a diet containing a glutamine-rich protein source can be used to increase plasma and muscle glutamine concentrations in healthy rats, but is of limited value to counteract wasting of skeletal muscle in zymosan-treated rats.

Journal Article↗
PubMedDate not supplied

Starvation alters the activity and mRNA level of glutaminase and glutamine synthetase in the rat intestine.

The metabolism of glutamine, the main respiratory fuel of enterocytes, is governed by the activity of glutaminase and glutamine synthetase. Because starvation induces intestinal atrophy, it might alter the rate of intestinal glutamine utilization. This study examined the effect of starvation on the activity, level of mRNA, and distribution of mRNA of glutaminase and glutamine synthetase in the rat intestine. Rats were randomized into groups and were either: (1) fed for 2 days with rat food ad libitum or (2) starved for 2 days. Standardized segments of jejunum and ileum were removed for the estimation of enzyme activity, level of mRNA, and in situ hybridization analysis. The jejunum of the fed rats had a greater activity of both enzymes per centimeter of intestine (P < 0.01), a greater glutaminase specific activity (1.97 +/- 0.45 vs. 1.09 +/- 0.34 micromol/hr/mg protein, P < 0.01), and a lower level of glutaminase and glutamine synthetase mRNA. The ileum of the fed rats had a greater activity of glutamine synthetase per centimeter of intestine (162.9 +/- 50.6 vs. 91.0 +/- 23.1 nmol/hr/cm bowel, P < 0.01), a lower level of glutaminase mRNA, and a greater level of glutamine synthetase mRNA. In situ hybridization analysis showed that starvation does not alter the distribution of glutaminase and glutamine synthetase mRNA in the intestinal mucosa. This study confirms that starvation decreases the total intestinal activity per centimeter of both glutaminase and glutamine synthetase. More importantly, the results indicate that the intestine adapts to starvation by accumulating glutaminase mRNA. This process prepares the intestine for a restoration of intake.

Journal Article↗
PubMed2002

Standardized parenteral alanyl-glutamine dipeptide supplementation is not beneficial in autologous transplant patients: a randomized, double-blind, placebo controlled study.

We conducted a controlled, double-blind study of parenteral glutamine supplementation in an unselected group of consecutive autologous transplant patients. Patients received 30 g of alanyl-glutamine dipeptide (Dipeptiven; Fresenius-Kabi, Bad Homburg, Germany) or glutamine-free amino acid solution i.v. from day +1 to day +14 or to discharge. All patients were assessed for clinical status, mucositis, blood counts, oral intake and immune reconstitution. Parenteral nutrition was administered according to predefined guidelines. Forty patients were randomized; 21 into the glutamine and 19 into the placebo arm. Glutamine patients had less days with diarrhoea (3.3 +/- 4.0 vs 4.3 +/- 3.0, P = 0.03), but they had more severe oral mucositis (mean 4 +/- 4.7 vs 1.4 +/- 2.3 days of mucositis score >13, P = 0.04), spent more days on opioids (mean 3.5 +/- 4.2 vs 1.2 +/- 2.2 days, P = 0.03) and left hospital later than placebo patients (mean 13.5 +/- 3.1 vs 11.7 +/- 2.4 days after transplant, P = 0.06). There were more relapses (P = 0.02) and deaths (P = 0.05) in the glutamine group. The cost of supportive care (mean 2960 +/- 1694 vs 1534 +/- 513 Euro, P = 0.002) was also greater for glutamine patients, mainly due to the cost of glutamine dipeptide itself. The described mode and dosage of glutamine administration did not produce meaningful benefit in our autologous transplant patients and it was certainly not cost-effective.

Adult↗
PubMed1997

Expression and efficient export of enzymatically active Mycobacterium tuberculosis glutamine synthetase in Mycobacterium smegmatis and evidence that the information for export is contained within the protein.

We have investigated the expression and extracellular release of active, recombinant Mycobacterium tuberculosis glutamine synthetase (EC 6.3.1.2), an enzyme that is a potentially important determinant of M. tuberculosis infection and whose extracellular release is correlated with pathogenicity. The M. tuberculosis glutamine synthetase gene encodes a polypeptide of 478 amino acids; 12 such subunits comprise the active enzyme. Northern blot, nuclease S1, and primer extension analyses revealed glutamine synthetase specific transcripts of approximately 1,550 and 1,650 nucleotides produced under low and high nitrogen conditions, respectively. Expression of recombinant M. tuberculosis glutamine synthetase in Escherichia coli YMC21E, a glutamine synthetase deletion mutant, led to transcomplementation of the mutant but not to release of active enzyme. Expression in Mycobacterium smegmatis 1-2c, from the gene's own promoter, resulted in the release of >95% of all recombinant enzyme. No hybrid molecules containing M. tuberculosis and M. smegmatis glutamine synthetase subunits were detected. Native and recombinant exported and intracellular glutamine synthetase molecules were indistinguishable from one another by mass, N-terminal amino acid sequence, antibody reactivity, and enzymatic activity. Since M. tuberculosis glutamine synthetase is similar to other, strictly intracellular, bacterial glutamine synthetases and the DNA sequence upstream of the structural gene does not encode a leader peptide, the information to target the protein for export must be contained in its amino acid sequence and/or conformation.

Amino Acid Sequence↗
PubMed1999

An inhibitor of exported Mycobacterium tuberculosis glutamine synthetase selectively blocks the growth of pathogenic mycobacteria in axenic culture and in human monocytes: extracellular proteins as potential novel drug targets.

Mycobacterium tuberculosis and other pathogenic mycobacteria export abundant quantities of proteins into their extracellular milieu when growing either axenically or within phagosomes of host cells. One major extracellular protein, the enzyme glutamine synthetase, is of particular interest because of its link to pathogenicity. Pathogenic mycobacteria, but not nonpathogenic mycobacteria, export large amounts of this protein. Interestingly, export of the enzyme is associated with the presence of a poly-L-glutamate/glutamine structure in the mycobacterial cell wall. In this study, we investigated the influence of glutamine synthetase inhibitors on the growth of pathogenic and nonpathogenic mycobacteria and on the poly-L-glutamate/glutamine cell wall structure. The inhibitor L-methionine-S-sulfoximine rapidly inactivated purified M. tuberculosis glutamine synthetase, which was 100-fold more sensitive to this inhibitor than a representative mammalian glutamine synthetase. Added to cultures of pathogenic mycobacteria, L-methionine- S-sulfoximine rapidly inhibited extracellular glutamine synthetase in a concentration-dependent manner but had only a minimal effect on cellular glutamine synthetase, a finding consistent with failure of the drug to cross the mycobacterial cell wall. Remarkably, the inhibitor selectively blocked the growth of pathogenic mycobacteria, all of which release glutamine synthetase extracellularly, but had no effect on nonpathogenic mycobacteria or nonmycobacterial microorganisms, none of which release glutamine synthetase extracellularly. The inhibitor was also bacteriostatic for M. tuberculosis in human mononuclear phagocytes (THP-1 cells), the pathogen's primary host cells. Paralleling and perhaps underlying its bacteriostatic effect, the inhibitor markedly reduced the amount of poly-L-glutamate/glutamine cell wall structure in M. tuberculosis. Although it is possible that glutamine synthetase inhibitors interact with additional extracellular proteins or structures, our findings support the concept that extracellular proteins of M. tuberculosis and other pathogenic mycobacteria are worthy targets for new antibiotics. Such proteins constitute readily accessible targets of these relatively impermeable organisms, which are rapidly developing resistance to conventional antibiotics.

Aminobutyrates↗
PubMed1982

Induction of glutamine synthetase by dibutyryl cyclic AMP in C-6 glioma cells.

Glutamine synthetase was found to be increased in C-6 glioma cells as a result of increasing culture passage and N-6,2'-O-dibutyryl cyclic AMP (dbcAMP) treatment. At low passage dbcAMP produced a 2.5-fold increase in glutamine synthetase activity per unit of cellular protein. At high passage control glutamine synthetase was approximately double that seen at low passage, but dbcAMP produced an additional 65% increase. Lactate dehydrogenase activity was also increased by dbcAMP treatment at both low and high passage, but culture passage produced no change in the lactate dehydrogenase. With increasing culture passage, the ratio of cellular protein to DNA doubled. Therefore, expression of data per unit of protein tended to minimize the apparent changes in activity. The maximum increase in glutamine synthetase activity produced by both dbcAMP and increasing culture passage and expressed on a DNA basis was 5.6-fold. The increase in glutamine synthetase activity was generally linear during the first 20 h of drug treatment, after which enzyme activity remained nearly constant up to 72 h. Ninety percent or more of the dbcAMP remained in the medium at the end of 48-h exposure of cells to dbcAMP. 8-br-Cyclic AMP also increased glutamine synthetase activity of C-6-cels, but n-butyrate did not. Isoproterenol, which increases cyclic AMP in C-6-cells, increased glutamine synthetase activity. The effect of isoproterenol on glutamine synthetase was inhibited by the beta-adrenergic blocking agent sotalol. Cycloheximide (10 micrograms/ml) inhibited the dbcAMP effect on glutamine synthetase activity and also decreased the control enzyme activity by 60%.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Neural control of glutamine synthetase activity in rat skeletal muscles.

The mechanism of glutamine synthetase induction in rat skeletal muscle after denervation or limb immobilization was investigated. Adult male rats were subjected to midthigh section of the sciatic nerve. At 1, 2, and 5 h and 1, 2, and 7 days after denervation, rats were killed and denervated, and contralateral control soleus and plantaris muscles were excised, weighted, homogenized, and assayed for glutamine synthetase. Glutamine synthetase activity increased approximately twofold 1 h after denervation in both muscles. By 7 days postdenervation enzyme activity had increased to three times the control level in plantaris muscle and to four times the control level in soleus muscle. Increased enzyme activity after nerve section was associated with increased maximum velocity with no change in apparent Michaelis constant. Immunotitration with an antiglutamine synthetase antibody suggested that denervation caused an increase in the number of glutamine synthetase molecules in muscle. However, Northern-blot analysis revealed no increase in the steady-state level of glutamine synthetase mRNA after denervation. A mixing experiment failed to yield evidence for the presence of a soluble factor involved in regulating the activity of glutamine synthetase in denervated muscle. A combination of denervation and dexamethasone injections resulted in additive increases in glutamine synthetase. Thus the mechanism underlying increased glutamine synthetase after denervation appears to be posttranscriptional and is distinct from that of the glucocorticoid-mediated glutamine synthetase induction previously described by us.

Animals↗
PubMed2000

Expression of glutamine synthetase in macrophages.

We studied the expression of glutamine synthetase in liver macrophages (Kupffer cells, KCs) in situ and in culture. Glutamine synthetase was detectable at the mRNA and protein level in freshly isolated and short-term-cultured rat liver macrophages. Enzyme activity and protein content were about 9% of that in liver parenchymal cells. In contrast, glutamine synthetase mRNA levels in liver macrophages apparently exceeded those in parenchymal liver cells (PCs). By use of confocal laser scanning microscopy and specific macrophage markers, immunoreactive glutamine synthetase was localized to macrophages in normal rat liver and normal human liver in situ. All liver macrophages stained positive for glutamine synthetase. In addition, macrophages in rat pancreas contained immunoreactive glutamine synthetase, whereas glutamine synthetase was not detectable at the mRNA and protein level in blood monocytes and RAW 264.7 mouse macrophages. No significant amounts of glutamine synthetase were found in isolated rat liver sinusoidal endothelial cells (SECs). The data suggest a constitutive expression of glutamine synthetase not only, as previously believed, in perivenous liver parenchymal cells but also in resident liver macrophages.

Animals↗
PubMedDate not supplied

Effects of parenteral nutrition supplemented with alanyl-glutamine on nutrition status in rats.

BACKGROUND: Glutamine, a nonessential amino acid, has received increasing attention because it becomes essential during stress and catabolic conditions. Many investigations have shown that during severe stress, the consumption of glutamine exceeds glutamine synthesis, resulting in depletion of glutamine stores. The aim of this study was to evaluate the effects of supplementing parenteral diets with a glutamine-containing dipeptide, L-alanyl-L-glutamine, on rat nutrition status. METHODS: Male Wistar rats were used. Animals (n = 36) were centrally catheterized and randomly assigned to 1 of the following groups based on method of parenteral nutrition (PN): control group with oral nutrition and IV infusion of a saline solution; standard parenteral nutrition (SPN) group; or alanyl-glutamine-supplemented PN (ALA-GLN PN) group (20 g/L). Parenteral nutrition was isocaloric and isonitrogenous. Infusions were administered at a rate of 2.0 mL/h over 5 days. Nutrition status was assessed by body weight change, plasma proteins, accumulated urinary creatinine, and nitrogen balance. RESULTS: Accumulated urinary creatinine increased significantly after day 4 in the ALA-GLN PN group, compared with the SPN group and the controls. Body weight change significantly differed on day 5 between the ALA-GLN PN and SPN groups. After 3 days, nitrogen balance was significantly lower and nitrogen retention higher in the ALA-GLN PN group when compared with the SPN group. Albumin and transferrin concentrations decreased significantly in the SPN group, but did not differ from the controls in the ALA-GLN PN group. CONCLUSIONS: Weight, plasma proteins, urinary accumulated creatinine, and nitrogen retention showed a better evolution in the group supplemented with the glutamine dipeptide when compared with the SPN group. Our results suggest a more suitable nutrition support in animals receiving L-alanyl-L-glutamine.

Animals↗
PubMed1987

Inactivation of pea seed glutamine synthetase by the toxin, tabtoxinine-beta-lactam.

Glutamine synthetase of plants is the physiological target of tabtoxinine-beta-lactam, a toxin produced by several disease-causing pathovars of Pseudomonas syringae. This toxin, a unique amino acid, is an active site-directed, irreversible inhibitor of glutamine synthetase from pea. ATP is required for inactivation. Neither ADP, AMP, nor adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) supports inactivation. Adenyl-5'-yl imidophosphate (AMP-PNP) is slowly hydrolyzed by glutamine synthetase to produce adenyl-5'-yl phosphoramidate (AMP-PN) and inorganic phosphate as identified by 31P NMR spectroscopic analysis. AMP-PNP also supports a slow inactivation of glutamine synthetase by tabtoxinine-beta-lactam. These data are consistent with gamma-phosphate transfer being involved in the inactivation. Completely inactivated glutamine synthetase has 0.9 mumol of toxin bound/mumol of subunit. One mumol of ATP is bound per mumol of subunit of glutamine synthetase in the absence of either the toxin or another active site-directed inhibitor, methionine sulfoximine; whereas, a 2nd mumol of either [alpha- or gamma-32P]ATP is bound per mumol of subunit when glutamine synthetase is incubated in the presence of either toxin or methionine sulfoximine until all enzyme activity is lost. These data suggest that the gamma-phosphate hydrolyzed from ATP during inactivation remains with the enzyme-inhibitor complex, as well as the ADP. The open chain form, tabtoxinine, was neither a reversible nor an irreversible inhibitor of glutamine synthetase, suggesting that the beta-lactam ring is necessary for inhibition. The inactivation of glutamine synthetase with tabtoxinine-beta-lactam is pseudo-first-order when done in buffer containing 15% (v/v) ethylene glycol. The rate constant for this reaction is 3 X 10(-2) S-1, and the Ki for the toxin is 1 mM. Removal of the ethylene glycol from the buffer allows the reaction to proceed in a non-first-order manner with the apparent rate constant decreasing with time. As the enzyme is inactivated in these conditions, the binding affinity for the toxin appears to decrease, while the Km observed for glutamate does not change.

Adenosine Diphosphate↗
PubMed1995

Heterogeneous hepatocellular expression of glutamine synthetase in developing mouse liver and in testicular transplants of fetal liver.

BACKGROUND: Glutamine synthetase is exclusively expressed in pericentral hepatocytes in mammalian liver, but its regulation mechanism is still largely unknown. EXPERIMENTAL DESIGN: Heterogeneous expression of glutamine synthetase was examined in detail during mouse liver development by immunohistochemistry and by in situ hybridization. Heterogeneous expression of this enzyme was also analyzed in immature liver fragments transplanted to an ectopic site where no portal blood flow exists. RESULTS: At 18.5 days of gestation, a random, spotty distribution of low levels of glutamine synthetase mRNA was observed all over the liver parenchyma, but the enzyme protein was not detectable immunohistochemically in the liver at any fetal stage. Glutamine synthetase and its mRNA began to be heterogeneously expressed in pericentral hepatocytes 2 to 3 days after birth, when glycogen accumulation in the liver parenchyma was rather homogeneous. In the early postnatal development, a mosaic distribution of positive and negative hepatocytes with respect to glutamine synthetase protein and mRNA was noted around the central veins. Subsequently, mRNA distribution gradually became continuous, although some hepatocytes still lacked protein, indicating partial regulation at the translational level. When fetal liver fragments that had not yet heterogeneously expressed glutamine synthetase were cultured under the testis capsule of male mice, only pericentral hepatocytes expressed this enzyme after 2 months. However, the distribution of glutamine synthetase protein- and mRNA-positive hepatocytes around the central veins was patchy rather than continuous, as in perinatal livers. CONCLUSIONS: These results support the importance of local interactions of hepatocytes with intrahepatic cell populations and/or structural elements. Furthermore, they demonstrate that the capacity for the positional expression of glutamine synthetase is already established at a fetal age before expression of glutamine synthetase can be detected.

Animals↗
PubMed1975

Reversible reaction of cyanate with a reactive sulfhydryl group at the glutamine binding site of carbamyl phosphate synthetase.

Carbamyl phosphate synthetase from Escherichia coli reacts stoichiometrically (one to one) with [14C]cyanate to give a 14C-labeled complex which can be isolated by gel filtration. The formation of the complex is prevented if L-glutamine is present or if the enzyme is first reacted with 2-amino-4-oxo-5-chloropentanoic acid, a chloro ketone analog of glutamine which has been shown to react with a specific SH group in the glutamine binding site. The rate of complex formation is increased by ADP and decreased by ATP and HCO3-. The isolated complex is inactive with respect to glutamine-dependent synthetase activity. However, the reaction of cyanate with the enzyme is reversible. The rate of dissociation of the isolated complex is not affected by pH (over the pH range 6-10), is greatly increased by ATP and HCO3-, and is decreased by ADP. The allosteric effectors ornithine and UMP have no effect on either the rate of formation or the rate of dissociation of the complex; however, the apparent affinity of the enzyme for ATP is decreased by UMP and increased by ornithine. The site of reaction of cyanate with carbamyl phosphate synthetase, which is composed of a light and a heavy subunit, is with an SH group in the light subunit to give an S-carbamylcysteine residue. The binding of L-[14C]glutamine to the enzyme and the inhibition of glutamine-dependent synthetase activity by the chloroketone analog are both prevented by the presence of cyanate. The reaction with cyanate is considered to be with the same essential SH group which is located in the glutamine binding site and is alkylated by 2-amino-4-oxo-5-chloropentanoic acid. The bicarbonate-dependent effects of ATP suggest that formation of the activated carbon dioxide intermediate is accompanied by changes in the heavy subunit which functionally alter the properties of the glutamine binding site on the light subunit. The allosteric effects of ornithine and UMP are probably not related to this intersubunit interaction.

Adenosine Triphosphate↗
PubMed1994

Parenteral glutamine dipeptide supplementation does not ameliorate chemotherapy-induced toxicity.

BACKGROUND: Glutamine-supplemented total parenteral nutrition (TPN) improved the nitrogen balance in catabolic situations. In animal studies, parenteral glutamine supplementation appeared to maintain gut integrity. This study was performed to evaluate the possible positive effects of glutamine supplementation in catabolic hematologic patients. METHODS: This was a prospective double-blind placebo-controlled pilot study, in which 20 treatment cycles in unselected hematologic patients with intensive chemotherapy were studied. Glutamine was given as a dipeptide. Patients were randomized per treatment cycle to receive isonitrogenous TPN (0.272 g nitrogen/kg of body weight) and isoenergetic TPN (2200 kcal NPE/day) without or with 40 g L-alanyl-L-glutamine (26 g glutamine) until the neutrophil count was greater than 0.5 x 10(9)/L. The daily oral food intake was recorded and analyzed carefully. Toxicity grades for performance status, mucositis, and diarrhea were scored according to the World Health Organization classification. RESULTS: No differences in neutropenic period, fever, extra antibiotics, and toxicity scores were observed, except for a gain in body weight per treatment cycle in favor of the glutamine-supplemented TPN. No side effects or allergic reactions were noted after the dipeptide administration. CONCLUSION: Supplementation of glutamine dipeptide was safe but had no significant positive clinical effect.

Adult↗
PubMed1994

Determination of amino- and amide-15N glutamine enrichment with tertiary butyldimethylsilyl derivatives.

We have developed a simple and rapid method for the selective synthesis of tetra-tertiarybutyldimethylsilyl (TBDMS) glutamine, which allows the simultaneous quantitation of glutamine (2-15N) and (5-15N) isotopic enrichment by selected ion monitoring (SIM) gas chromatographic/mass spectrometric analysis. The tetra-TBDMS glutamine (4S-gln) has an electron impact mass fragment at m/z 258 that contains only the amino-N and larger fragments (e.g., at m/z 545) that have both nitrogens. Derivatization with acetonitrile (ACN) and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide yields primarily tri-TBDMS glutamine (3S-gln) and quantities of 4S-gln too small to allow accurate SIM and tracer/tracee ratio determinations. However, when N,N-dimethylformamide, a more polar aprotic solvent, was substituted for ACN and the sample was heated for 30 min at 125 degrees C, greater than 80% of derivatized glutamine appeared as 4S-gln. Derivatized plasma samples that had been mixed with amide- and/or amino-15N glutamine and analyzed by SIM demonstrated strong agreement (r > or = 0.998, p = 0.0001) between theoretical and observed enrichment values for the 4S-gln fragments at m/z 258 and 545. Deamidation of glutamine to glutamate is negligible during sample processing and analysis. This procedure will facilitate the investigation of the specific sources and fates of glutamine amide and amino nitrogen as well as stable isotope studies involving amino acid transamination, ammonia clearance, urea production and other areas of nitrogen metabolism.

Acetamides↗
PubMedDate not supplied

Glucose and glutamine utilization by rat lymphocytes, monocytes and neutrophils in culture: a comparative study.

Glucose and glutamine utilization and production of glutamate and lactate were determined for up to 48 h in lymphocytes, monocytes and neutrophils cultured in medium rich in metabolites and vitamins. Glucose was utilized by the three cell types in culture in the following order: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the order: monocytes > neutrophils > lymphocytes. The consumption of glucose followed the activity of glucose-6-phosphate dehydrogenase but it was not related to hexokinase activity. Glutamine was consumed by the three leukocyte types in culture as follows: neutrophils > lymphocytes > or = monocytes. The consumption of glutamine was not fully related to the activity of phosphate-dependent glutaminase. The production of glutamate was not remarkably different among the three cell types. For comparison, glutamine and glucose utilization and glutamate and lactate production were also evaluated using 1-h incubated leukocytes. Under this condition, only glucose or glutamine was added to the medium. Glucose was utilized as follows: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the following order: monocytes > or = neutrophils > lymphocytes. Glutamine was consumed as follows: neutrophils > lymphocytes > monocytes, whereas glutamate was produced as follows: neutrophils > or = monocytes = lymphocytes. The ratio of the amount of glucose/glutamine consumed by 1-h incubated cells was 0.5 for neutrophils, 1.5 for monocytes, and 0.3 for lymphocytes. However, the three cell types cultured for 48 h utilized glucose to a much higher degree than glutamine. The ratio of the amount of glucose/glutamine utilized by the cultured cells was 8.9 for neutrophils, 16.4 for monocytes, and 6.7 for lymphocytes. These observations support the proposition that glutamine is required in much higher amounts than glucose to accomplish the total metabolic requirement of leukocytes. Under conditions closer to physiological when the availability of a variety of metabolites and vitamins is not restricted, glucose is the preferred substrate for lymphocytes, monocytes and neutrophils.

Animals↗
PubMed1984

Stimulation by glutamine of the formation of N6-hydroxylysine in a cell-free extract from Aerobacter aerogenes 62-1.

Glutamine may serve as an activator and/or regulator of the N6-hydroxylase (E.C. 1.14.99) of Aerobacter aerogenes 62-1. Activation and stabilization of N6-hydroxylase activity was observed both in vivo and in vitro. Growth in a glutamine-supplemented medium resulted in (1) maximum N6-hydroxylase activity at an earlier stage of growth and (2) higher N6-hydroxylase activity and continued aerobactin synthesis into stationary phase. Storage of P2 in the presence of L-glutamine (1 mM) significantly increased the lifetime of the labile N6-hydroxylase activity. Inclusion of L-glutamine in the incubation mixture typically resulted in a 2-3-fold activation of the hydroxylase activity. The stimulatory effect of glutamine was independent of and additive to the enhancement of N6-hydroxylation by the active component(s) in the supernatant, S2 fraction. Glutamic acid-gamma-semihydrazide activated slightly in the absence of glutamine but activation of the system by glutamine was decreased by this compound. Azaserine was shown to be an uncompetitive inhibitor with respect to lysine and this inhibition was not reversed by glutamine.

Azaserine↗
PubMed1981

Regulation of glutamine and pyruvate oxidation in cultured adrenocortical cells by cortisol, antioxidants, and oxygen: effects on cell proliferation.

The regulation of CO2 production from [U-14C]glutamine and C2 of [2-14C]pyruvate was investigated in cultured bovine adrenocortical cells, and the effect of alterations in the relative rates of oxidation of these substrates on cell proliferation, particularly in the presence of an inhibitor of transamination reactions, was examined. 14CO2 production from 2 mM [U-14C]glutamine and 2 mM [2-14C]pyruvate was measured in the presence of 100 microM 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. Treatment of primary cultures of 24 h with 50 microM cortisol increased the oxidation of [14C]glutamine relative to that of [14C]pyruvate, an effect dependent on prior low cell density. Cortisol treatment also resulted in a prolonged delay in the onset of proliferation from low density, and completely inhibited growth in the presence of 2 mM aminooxyacetate, which reduces mitochondrial utilization of glutamine. The effects on glutamine and pyruvate metabolism and on cell growth, with or without aminooxyacetate, were prevented by simultaneous treatment with the antioxidants dimethyl sulfoxide (10 mM) and butylated hydroxyanisole (100 microM), suggesting the involvement of lipid peroxidation in the action of cortisol, as previously demonstrated for its action on 11 beta-hydroxylase. During continued proliferation of adrenocortical cells in the absence of cortisol there was also a slower increase in the oxidation of [14C]glutamine relative to that of [14C]pyruvate as a function of population doubling level. The rate of this increase was slowed by growth of cells in 2% O2 rather than the standard 19% O2, and accelerated by continued growth of cells in the presence of cortisol. The rate of increase in the oxidation of [14C]glutamine relative to that of [14C]pyruvate under these three conditions correlated with inhibition of cell growth by aminooxyacetate. In contrast to the complete inhibition of growth in aminooxyacetate demonstrated by cortisol-treated cells, control cells (19% O2) did proliferate, although growth was limited, whereas cells at 2% O2 proliferated to a much greater extent. In the absence of aminooxyacetate the rate of growth in primary adrenocortical cell cultures under these three conditions was similar. Lipid peroxidation appears to make cultured adrenocortical cells dependent on glutamine for mitochondrial function and proliferation by inhibiting the utilization of the normal substrate, pyruvate.

Adrenal Cortex↗