Specificity of the glutamine-binding site involved in the reguation of glutamine-synthetase activity in hepatoma tissue-culture cells.

Glutamine accelerates the degradation of glutamine synthetase in hepatoma tissue culture cells. Compounds structurally related to glutamine were tested for their ability to mimic or antagonize this effect of glutamine. 6-Diazo-5-oxo-L-norleucine, like glutamine depressed the activity of glutamine synthetase in hepatoma tissue culture cells. L-Methionine sulfone, albizzine, L-methionine sulfoxide, L-gamma-glutamyl hydrazide and gamma-N-methyl-L-glutamine (listed in order of decreasing potency) were antagonists which prevented the effect of glutamine on glutamine synthetase activity. These antagonists had little effect on glutamine transport or protein synthesis of hepatoma tissue culture cells and their effects were reversible. The effects of compounds on gluatmine synthetase activity in cell-free extracts of the cells were examined. Diazo-oxonorleucine and albizzine inhibited neither the transferase nor the synthetase activity of glutamine synthetase. This observation is interpreted to mean that the glutamine-binding site involved in the regulation of glutamine synthetase activity of hepatoma tissue culture cells is not the active site of the enzyme.     

On the synthesis of glutamine before and after fertilization of the sea urchin, Strongylocentrotus purpuratus

Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, have a much lower capacity for glutamine synthesis than do fertilized eggs. This difference is not caused by an alteration of glutamine synthetase activity attendant upon fertilization. Neither the specific activity of glutamine synthetase nor its pattern of activation by divalent metal ions is affected by fertilization. The enzyme from both fertilized and unfertilized eggs is activated by α-ketoglutarate and inhibited by ultimate end products of glutamine metabolism. This type of regulation is similar to that seen with many other eucaryotic glutamine synthetases.Unfertilized eggs take up less glutamic acid than do fertilized eggs when the amino acid is presented at high concentrations (12.5 mM), whereas there is no difference in glutamic acid uptake at low concentrations (5 μM). Under conditions where glutamate uptake is identical, unfertilized eggs are dependent upon exogenous ammonia for glutamine synthesis in vivo; fertilized eggs are able to synthesize glutamine in the absence of added ammonia. Thus, our data suggest that the increased capacity for glutamine synthesis after fertilization is related to an increased availability of the substrate, ammonia.     

The regulation of glutamine and ketone-body metabolism in the small intestine of the long-term (40-day) streptozotocin-diabetic rat.

The small intestine is the major site of glutamine utilization in the mammalian body. During prolonged (40-day) streptozotocin-diabetes in the rat there is a marked increase in both the size and the phosphate-activated glutaminase activity of the small intestine. Despite this increased capacity, intestinal glutamine utilization ceases in diabetic rats. Mean arterial glutamine concentration fell by more than 50% in diabetic rats, suggesting that substrate availability is responsible for the decrease in intestinal glutamine use. When arterial glutamine concentrations in diabetic rats were elevated by infusion of glutamine solutions, glutamine uptake across the portal-drained viscera was observed. The effect of other respiratory fuels on intestinal glutamine metabolism was examined. Infusions of ketone bodies did not affect glutamine use by the portal-drained viscera of non-diabetic rats. Prolonged diabetes had no effect on the activity of 3-oxoacid CoA-transferase in the small intestine or on the rate of ketone-body utilization in isolated enterocytes. Glutamine (2 mM) utilization was decreased in enterocytes isolated from diabetic rats as compared with those from control animals. However, glutaminase activity in homogenates of enterocytes was unchanged by diabetes. In enterocytes isolated from diabetic rats the addition of ketone bodies or octanoate decreased glutamine use. It is proposed that during prolonged diabetes ketone bodies, and possibly fatty acids, replace glutamine as the major respiratory fuel of the small intestine.     

Cascade control of E. coli glutamine synthetase. II. Metabolite regulation of the enzymes in the cascade.

Enzymes and regulatory proteins involved in the cascade control of glutamine synthetase activity of Escherichia coli have been separated from one another and the effects of numerous metabolites on each step in the cascade have been determined. The adenylyl transferase (ATase) -catalyzed adenylylation of glutamine synthetase, which requires the presence of the unmodified form of the regulatory protein PII is enhanced by glutamine and is inhibited by either α-ketoglutarate (α-KG) or the uridylylated form (PII·UMP) of the regulatory protein. PII·UMP and α-KG act synergistically to inhibit this activity. In contrast, the PII·UMP-dependent, ATase-catalyzed deadenylylation of glutamine synthetase requires α-KG and ATP and is inhibited by glutamine or PII and synergistically by glutamine plus PII. The capacity of uridylyl transferase (UTase) to catalyze the uridylylation of PII is dependent on the presence of α-KG and ATP and is inhibited by glutamine. The deuridylylation of PII·UMP by the uridylyl removing enzyme (UR) is enhanced by glutamine but is unaffected by α-KG. However, CMP, UMP, and CoA all inhibit activity at 10^?6m. High concentrations of ATase inhibit both UR and UTase activities, presumably by binding the regulatory protein. Of more than 50 substances that alter the activity of at least one enzyme in the cascade, only α-KG and glutamine affect the activity at every step. This accounts for the observation that glutamine synthetase activity in vivo is very sensitive to the intracellular ratio of α-KG to glutamine.     

Derepression of the Glutamine Synthetase in Neuroblastoma Cells at Low Concentrations of Glutamine

Abstract: Regulation of the biosynthesis of glutamine synthetase was studied in neuroblastoma cells (Neuro-2A) by use of a recently developed, sensitive radioisotopic assay. The removal of glutamine from the culture medium of these cells for 24 h resulted in a 10-fold increase in glutamine synthetase specific activity (15-fold after 2 weeks) compared with the basal level found in cells grown in the presence of 2 m M glutamine. Following the growth of these cells for 2 weeks in the presence of various concentrations of glutamine, a negative linear correlation was observed between the specific activity of glutamine synthetase (from 1.7 to 0.14 unit/mg) and the concentration of glutamine in the growth medium (from 0.5 to 2 m M ). Cycloheximide or actinomycin D blocked the increase in glutamine synthetase activity observed in the absence of glutamine. These results suggest that the removal of glutamine led to the induction of glutamine synthetase by stimulating new enzyme synthesis. The enzyme was not degraded, but only diluted, by growth upon readdition of glutamine to the medium. The influence of glutamine depletion is also reported for C-6 glioma cells and glial cells in primary cultures.     

The effect of glutamine concentration on the activity of carbamoyl-phosphate synthase II and on the incorporation of [3H]thymidine into DNA in rat mesenteric lymphocytes stimulated by phytohaemagglutinin.

The maximum catalytic activities of carbamoyl-phosphate synthase II, a limiting enzyme for pyrimidine nucleotide synthesis, are very much less than those of glutaminase, a limiting enzyme for glutamine utilization, in lymphocytes and macrophages; and the flux through the pathway for pyrimidine formation de novo is only about 0.4% of the rate of glutamine utilization by lymphocytes. The Km of synthase II for glutamine is about 16 microM and the concentration of glutamine necessary to stimulate lymphocyte proliferation half-maximally is about 21 microM. This agreement suggests that the importance of glutamine for these cells is provision of nitrogen for biosynthesis of pyrimidine nucleotides (and probably purine nucleotides). However, the glutamine concentration necessary for half-maximal stimulation of glutamine utilization (glutaminolysis) by the lymphocytes is 2.5 mM. The fact that the rate of glutamine utilization by lymphocytes is markedly in excess of the rate of the pathway for pyrimidine nucleotide synthesis de novo and that the Km and 'half-maximal concentration' values are so different, suggests that the glutaminolytic pathway is independent of the use of glutamine nitrogen for pyrimidine synthesis.     

Role of the four conserved histidine residues in the amidotransferase domain of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from ATP, bicarbonate, and glutamine. The amidotransferase activity of this enzyme is catalyzed by the smaller of the two subunits of the heterodimeric protein. The roles of four conserved histidine residues within this subunit were probed by site-directed mutagenesis to asparagine. The catalytic activities of the H272N and H341N mutants are not significantly different than that of the wild-type enzyme. The H353N mutant is unable to utilize glutamine as a nitrogen source in the synthetase reaction or the partial glutaminase reaction. However, binding to the glutamine active site is not impaired in the H353N enzyme since glutamine is found to activate the partial ATPase reaction by 40% with a Kd of 54 microM. The H312N mutant has a Michaelis constant for glutamine that is 2 orders of magnitude larger than the wild-type value, but the maximal rate of glutamine hydrolysis is unchanged. These results are consistent with His-353 functioning as a general acid/base catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site.     

Role of manganese accumulation in increased brain glutamine of the cirrhotic rat

Cirrhosis promotes increases of both manganese and glutamine in brain. Manganese is a modulator and glutamine is the product of glutamine synthetase. This work studies the relationship between manganese and glutamine synthetase in a model of cirrhosis in the rat. We administered manganese (1 g/L) in the drinking water of sham-operated and bile-duct obstructed rats. We evaluated the manganese and glutamine accumulation and the glutamine synthetase activity in frontal cortex, striatum, and pallidum after 2, 4, and 6 weeks of biliary obstruction or sham surgery. Cirrhotic rats receiving manganese increased their brain content of metal about 400%–600% after 4 weeks of treatment (P < .05) and also remarkably accumulated glutamine through time in the three regions studied (P < .05 at week 6). Interestingly, bile-duct obstructed rats treated with manganese showed no effect on glutamine synthetase activity. Results from this study suggest that manganese induces increases of brain glutamine independently of its synthesis.     

Genetics of the glutamine transport system in Escherichia coli.

The active transport of glutamine by Escherichia coli occurs via a single osmotic shock-sensitive transport system which is known to be dependent upon a periplasmic binding protein specific for glutamine. We obtained a mutant that had elevated levels of glutamine transport and overproduced the glutamine binding protein. From this strain many point mutants and deletion-carrying strains defective in glutamine transport were isolated by a variety of techniques. The genetic locus coding for the glutamine transport system, glnP, and the regulatory mutation which causes overproduction of the transport system were both shown to map at 17.7 min on the E. coli chromosome, and it was demonstrated that the glnP locus contains the structural gene for the glutamine binding protein. Evidence was also obtained that the glutamine transport system, by an unknown mechanism, plays a direct role in the catabolism of glutamate and, hence, of glutamine and proline as well.     

Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium.

Glutamine synthetase activity from Synechocystis sp. strain PCC 6803 is regulated as a function of the nitrogen source available in the medium. Addition of 0.25 mM NH4Cl to nitrate-grown cells promotes a clear short-term inactivation of glutamine synthetase, whose enzyme activity decreases to 5 to 10% of the initial value in 25 min. The intracellular levels of glutamine, determined under various conditions, taken together with the results obtained with azaserine (an inhibitor of transamidases), rule out the possibility that glutamine per se is responsible for glutamine synthetase inactivation. Nitrogen starvation attenuates the ammonium-mediated glutamine synthetase inactivation, indicating that glutamine synthetase regulation is modulated through the internal balance between carbon-nitrogen compounds and carbon compounds. The parallelism observed between the glutamine synthetase activity and the internal concentration of alpha-ketoglutarate suggests that this metabolite could play a role as a positive effector of glutamine synthetase activity in Synechocystis sp. Despite the similarities of this physiological system to that described for enterobacteria, the lack of in vivo 32P labeling of glutamine synthetase during the inactivation process excludes the existence of an adenylylation-deadenylylation system in this cyanobacterium.     

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.     

Evidence of covalent modification of glutamine synthetase in the purple sulfur bacterium Thiocapsa roseopersicina

Abstract It was shown that glutamine synthetase of purple sulfur bacterium Thiocapsa roseopersicina is regulated by covalent modification. This conclusion is made on the basis of results showing that: (i) incubation of cells under conditions of nitrogen deprivation in the light lead to an increase of glutamine synthetase activity; (ii) addition of ammonium to nitrogen-starved cell suspensions caused a rapid decrease of glutamine synthetase activity; (iii) inhibition of glutamine synthetase by feedback modifiers was higher in ammonium-treated cells than in those starved for a nitrogen source; (iv) treatment of purified glutamine synthetase and cell-free extracts with phosphodiesterase was accompanied by an increase of glutamine synthetase activity, indicating the cleavage of modifying residues covalently bound to glutamine synthetase molecules.     

Muscle glutamine depletion in the intensive care unit

Glutamine is primarily synthesized in skeletal muscle and enables transfer of nitrogen to splanchnic tissues, kidneys and immune system. Discrepancy between increasing rates of glutamine utilization at whole body level and relative impairment of de novo synthesis in skeletal muscle leads to systemic glutamine deficiency and characterizes critical illness. Glutamine depletion at whole body level may contribute to gut, liver and immune system disfunctions, whereas its intramuscular deficiency may directly contribute to lean body mass loss. Severe intramuscular glutamine depletion also develops because of outward transport system upregulation, which is not counteracted by increased de novo synthesis. The negative impact of systemic glutamine depletion on critically ill patients is suggested both by the association between a lower plasma glutamine concentration and poor outcome and by a clear clinical benefit after glutamine supplementation. Enteral glutamine administration preferentially increases glutamine disposal in splanchnic tissues, whereas parenteral supplementation provides glutamine to the whole organism. Nonetheless, systemic administration was ineffective in preventing muscle depletion, due to a relative inability of skeletal muscle to seize glutamine from the bloodstream. Intramuscular glutamine depletion could be potentially counteracted by promoting de novo glutamine synthesis with pharmacological or nutritional interventions.     

Identification of the promoter elements involved in the stimulation of ASCT2 expression by glutamine availability in HepG2 cells and the probable involvement of FXR/RXR dimers

Expression of the glutamine transport protein ASCT2 in the human hepatoma cell line HepG2 is increased when cells are cultured in the presence of glutamine and this has been shown to be due to stimulation of the ASCT2 promoter. Analysis of a number of promoter constructs localised the activation site to be between bases -653 and -543. Gel shift assays identified an IR-1 repeat within a 24bp region of this sequence which bound at least two nuclear proteins. Protein binding to this site was significantly higher in cells grown in glutamine-containing medium than when glutamine was absent. The identity of the higher molecular weight species binding to this promoter element was likely to be FXR/RXR dimers. Simultaneous overexpression of FXR and RXR increased the promoter activity in cells grown without glutamine to the same extent as did glutamine addition; the effects of glutamine and FXR/RXR expression were not additive. Mutagenesis of the FXR/RXR binding site in the promoter construct abolished glutamine and FXR/RXR stimulation. Real-time PCR showed levels of FXR mRNA were significantly increased in response to glutamine. The activity of the FXR promoter was also increased in response to glutamine. These results show that the stimulation of ASCT2 expression in response to glutamine in part involves binding of FXR/RXR to the ASCT2 promoter.     

Cancer cell metabolism: the essential role of the nonessential amino acid,glutamine

Biochemistry textbooks and cell culture experiments seem to be telling us two different things about the significance of external glutamine supply for mammalian cell growth and proliferation. Despite the fact that glutamine is a nonessential amino acid that can be synthesized by cells from glucose‐derived carbons and amino acid‐derived ammonia, most mammalian cells in tissue culture cannot proliferate or even survive in an environment that does not contain millimolar levels of glutamine. Not only are the levels of glutamine in standard tissue culture media at least ten‐fold higher than other amino acids, but glutamine is also the most abundant amino acid in the human bloodstream, where it is assiduously maintained at approximately 0.5 mM through a combination of dietary uptake, de novo synthesis, and muscle protein catabolism. The complex metabolic logic of the proliferating cancer cells' appetite for glutamine—which goes far beyond satisfying their protein synthesis requirements—has only recently come into focus. In this review, we examine the diversity of biosynthetic and regulatory uses of glutamine and their role in proliferation, stress resistance, and cellular identity, as well as discuss the mechanisms that cells utilize in order to adapt to glutamine limitation.     

Effect of applied benzyladenine on endogenous cytokinin content during the early stages of bud development of kiwifruit

The filamentous non-N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) was able to utilize glutamine as the sole nitrogen source. The addition to ammonium-grown cultures of the irreversible inhibitor of glutamine synthetase activity L-methionine-D, L-sulfoximine (MSX) inhibited cell growth. Supplying glutamine to the culture restored cell growth. This re-established growth was not due to interference by glutamine of MSX uptake by the cells, since glutamine synthetase (GS, EC 6.3.1.2) activity remained completely inhibited by MSX even when glutamine was simultaneously present. Both glutamine and ammonium exerted a negative effect on nitrate reductase (NR. EC 1.7.7.2) and nitrite reductase (NiR, EC 1.7.7.1) in vivo. This negative effect was reversed by MSX. When glutamine was added to MSX-treated cells, intracellular glutamine level was high, but the activity of both reductases remained at a high level. These results suggest that the presence of the active form of glutamine synthetase is required for the in vivo prevention of nitrate assimilation caused by ammonium and glutamine.     

Interaction Between the Glutamine Cycle and the Uptake of Large Neutral Amino Acids in Astrocytes

Abstract: When astrocyte cultures are incubated with glutamate and ammonium, the clearance of these substrates followed by the formation and export of glutamine simulates the action of the "glutamine cycle" that is believed to function in the CNS. In the present study this process was found to increase the uptake of large neutral amino acids (LNAAs), namely, histidine, kynurenine, leucine, phenylalanine, and tryptophan, by two-to threefold in mouse cerebral astrocytes. The enhancement of kynurenine uptake was shown to depend on the formation of glutamine and to saturate at low levels of glutamine. LNAAs transiently lowered the glutamine content of astrocytes that were incubated with glutamate and ammonium, but they did not affect net export of glutamine to the solution at normal physiological pH. However, on adjustment of the pH of the solution to 7.8, which causes a large increase in glutamine content without affecting export, kynurenine now significantly increased net glutamine export. These findings relate to proposed mechanisms of cerebral dysfunction in hyperammonemia.     

Regulation of glutamine uptake in the cyanobiont Nostoc ANTH

Abstract Glutamine uptake in the cyanobiont Nostoc ANTH was energy-dependent and repressed in ammonia-grown cells. l -Methionine- dl -sulphoximine (MSX), a glutamate analogue and an inhibitor of glutamine synthetase (GS), did not affect glutamine uptake whereas azaserine, an inhibitor of glutamate synthase (GOGAT) did, suggesting that GS activity is not necessarily involved in the glutamine uptake system and that increased intracellular glutamine level regulates its own uptake. Repression of glutamine uptake by ammonia did not require de novo protein synthesis but required GS activity, suggesting that ammonia itself was not the repressor signal. The derepression of the glutamine uptake system did not require GS activity but required de novo protein synthesis.     

Glutamine as a feedback inhibitor of the Rhodopseudomonas sphaeroides nitrogenase system.

In whole cells of Rhodopseudomonas sphaeroides, nitrogen fixation, as measured by hydrogen production and acetylene reduction, was totally inhibited by micromolar concentrations of ammonia. This inhibition could not be duplicated by glutamate or glutamine alone. The inhibition by ammonia was abolished by methionine sulfoximine, a glutamine synthetase inhibitor. Inhibition by glutamine was complete in the presence of methionine sulfone, a preferential inhibitor of glutamate synthase, presumably by permitting a rise in the glutamine pool. The results indicated that the level of the glutamine pool controlled the activity of nitrogenase. None of these effects could be duplicated with cell-free nitrogenase, indicating there is probably a mediator which responds to the glutamine pool and inhibits nitrogenase, rather than glutamine itself being a direct inhibitor.     

Glutamine Synthetases of Higher Plants : Evidence for a Specific Isoform Content Related to Their Possible Physiological Role and Their Compartmentation within the Leaf

The chromatographic properties of glutamine synthetase isoforms have been investigated in a wide range of higher plant leaves and shoots using ion exchange chromatography. Different patterns of glutamine synthetase isoform content were observed. Among higher plants, four patterns or groups could be recognized. The first group is characterized by having only cytosolic glutamine synthetase, whereas the second group is distinguished by having only chloroplastic glutamine synthetase. The third group is characterized by cytosolic glutamine synthetase being a minor component of the total leaf glutamine synthetase activity. The fourth group is distinct from the other groups in having high cytosolic and chloroplast glutamine synthetase activity. Immunological studies have been undertaken on a few species from each group to identify unambiguously both cytosolic and chloroplastic glutamine synthetases.