林肯链霉菌谷氨酰胺合成酶活力调节的研究

对不同氮源生长条件下林肯链霉菌无细胞粗提液中谷氨酰胺合成酶 (GS)的研究结果表明 ,高浓度NH+4阻遏了GS的生物合成。从不同氮源生长条件下林肯链霉菌中分离纯化了GS ,其性质没有差别。以受腺苷化调节的产气克雷伯氏菌GS作对照 ,林肯链霉菌GS没有明显的氨休克作用 ,经蛇毒磷酸二酯酶处理后 ,其活力没有变化。这些结果都说明林肯链霉菌GS不存在腺苷化共价修饰这一调节方式。反馈抑制作用是林肯链霉菌GS的一种重要的调节方式 ,这种抑制作用是以累积的方式进行的 ,这表明各种抑制剂对GS作用位点不同 ,各种抑制剂对GS的抑制作用是相互独立的。由此推测 ,林肯链霉菌GS是一种变构酶。     

Regulation of nitrogenase A and R concentrations in Rhodopseudomonas capsulata by glutamine synthetase.

Nitrogen-starved purple non-sulphur bacteria have an active unregulated form of nitrogenase (nitrogenase A); however, the nitrogenase of a glutamine synthetase-negative mutant of Rhodopseudomonas capsulata, when nitrogen-starved, was predominantly inactive and required activation by Mn2+ and activating-factor protein. This regulatory form of nitrogenase has been called nitrogenase R. Treatment of wild-type cells (containing nitrogenase A) with methionine sulphoximine, an inhibitor of glutamine synthetase, converted the enzyme into nitrogenase R. Glutamine synthetase thus appears to control the intracellular concentrations of nitrogenase A and R and in this way regulates nitrogenase activity in the photosynthetic bacterium.     

Effects of glutamine,methionine sulfone and dexamethasone on rates of synthesis of glutamine synthetase in cultured hepatoma cells

Glutamine synthetase (EC 6.3.1.2) activity of hepatoma tissue culture cells is elevated by cortocisteroids and depressed by glutamine (Kulka, R.G., Tomkins, G.M. and Crook, R.B. (1972) J. Cell Biol., 54, 175–179). The transfer of cells from high (1–5 mM) to low (0.2–0.4 mM) concentrations of glutamine causes a marked increase in glutamine synthetase activity. The addition of a glutamine antagonist, methionine sulfone (1 mM) to cells suspended in high (1 mM) concentrations of glutamine also causes an increase of glutamine synthetase activity which is greater than that elicited by the transfer of cells to low concentrations of glutamine. Rates of synthesis of glutamine synthetase have been measured by radioimunoprecipitation in hepatoma tissue culture cells incubated under various conditions. Incubation of cells with the synthetic corticosteroid hormone, dexamethasone, markedly stimulates the relative rate of glutamine synthetase biosynthesis. Glutamine, or its analogue, methionine sulfone, have no effect on the relative rate of synthesis of the enzyme. However, total protein and RNA synthesis increase markedly with increasing external glutamine concentration in the range 0–1 mM. Methionine sulfone (1 mM) inhibits the degradation of glutamine synthetase in the presence of 1 mM glutamine. The data are consistent with the conclusion that the corticosteroid, dexamethasone, elevates glutamine synthetase activity by stimulating its rate of synthesis, whereas methionine sulfone elevates glutamine synthetase activity by inhibiting the glutamine-stimulated degradation of preformed enzyme.     

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803.

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.     

Properties of the Bacillus licheniformis A5 glutamine synthetase purified from cells grown in the presence of ammonia or nitrate.

The glutamine synthetase from Bacillus licheniformis A5 was purified by using a combination of polyethylene glycol precipitation and chromatography on Bio-Gel A 1.5m. The resulting preparation was judged to be homogeneous by the criteria of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, equilibrium analytical ultracentrifugation, and electron microscopic analysis. The enzyme is a dodecamer with a molecular weight of approximately 616,000, and its subunit molecular weight is 51,000. Under optimal assay conditions (pH 6.6, 37 degrees C) apparent Km values for glutamate, ammonia, and manganese.adenosine 5'-triphosphate (1:1 ratio) were 3.6, 0.4, and 0.9 mM, respectively. Glutamine synthetase activity was inhibited approximately 50% by the addition of 5 mM glutamine, alanine, glycine, serine, alpha-ketoglutarate, carbamyl phosphate, adenosine 5'-diphosphate, or inosine 5'-triphosphate to the standard glutamine synthetase assay system, whereas 5 mM adenosine 5'-monophosphate or pyrophosphate caused approximately 90% inhibition of enzyme activity. Phosphorylribosyl pyrophosphate at 5 mM enhanced activity approximately 60%. We were unable to detect any physical or kinetic differences in the properties of the enzyme when it was purified from cells grown in the presence of ammonia or nitrate as sole nitrogen source. The data indicate that B. licheniformis A5 contains one species of glutamine synthetase whose catalytic activity is not regulated by a covalent modification system.     

Preferential degradation of the oxidatively modified form of glutamine synthetase by intracellular mammalian proteases

Four intracellular proteases partially purified from liver preferentially degraded the oxidatively modified (catalytically inactive) form of glutamine synthetase. One of the proteases was cathepsin D which is of lysosomal origin; the other three proteases were present in the cytosol. Two of these were calcium-dependent proteases with different calcium requirements. The low-calcium-requiring type (calpain I) accounted for most of the calcium-dependent activity of both mouse and rat liver. The calcium-independent cytosolic protease, referred to as the alkaline protease, has a molecular weight of 300,000 determined by gel filtration. Native glutamine synthetase was not significantly degraded by the cytosolic proteases at physiological pH, but oxidative modification of the enzyme caused a dramatic increase in its susceptibility to attack by these proteases. In contrast, trypsin and papain did degrade the native enzyme and the degradation of modified glutamine synthetase was only 2- to 4-fold more rapid. Adenylylation of glutamine synthetase had little effect on its susceptibility to proteolysis. Although major structural modifications such as dissociation, relaxation, and denaturation also increased the rate of degradation, the oxidative modification is a specific type of covalent modification which could occur in vivo. Oxidative modification can be catalyzed by a variety of mixed function oxidase systems present within cells and causes inactivation of a number of enzymes. Moreover, the presence of cytosolic proteases which recognize the oxidized form of glutamine synthetase suggests that oxidative modification may be involved in intracellular protein turnover.     

Glutamine synthetase activity and glutamine content in brain: modulation by NMDA receptors and nitric oxide

Acute intoxication with large doses of ammonia leads to rapid death. The main mechanism for ammonia elimination in brain is its reaction with glutamate to form glutamine. This reaction is catalyzed by glutamine synthetase and consumes ATP. In the course of studies on the molecular mechanism of acute ammonia toxicity, we have found that glutamine synthetase activity and glutamine content in brain are modulated by NMDA receptors and nitric oxide. The main findings can be summarized as follows.Blocking NMDA receptors prevents ammonia-induced depletion of brain ATP and death of rats but not the increase in brain glutamine, indicating that ammonia toxicity is not due to increased activity of glutamine synthetase or formation of glutamine but to excessive activation of NMDA receptors.Blocking NMDA receptors in vivo increases glutamine synthetase activity and glutamine content in brain, indicating that tonic activation of NMDA receptors maintains a tonic inhibition of glutamine synthetase.Blocking NMDA receptors in vivo increases the activity of glutamine synthetase assayed in vitro, indicating that increased activity is due to a covalent modification of the enzyme. Nitric oxide inhibits glutamine synthetase, indicating that the covalent modification that inhibits glutamine synthetase is a nitrosylation or a nitration.Inhibition of nitric oxide synthase increases the activity of glutamine synthetase, indicating that the covalent modification is reversible and it must be an enzyme that denitrosylate or denitrate glutamine synthetase.NMDA mediated activation of nitric oxide synthase is responsible only for part of the tonic inhibition of glutamine synthetase. Other sources of nitric oxide are also contributing to this tonic inhibition.Glutamine synthetase is not working at maximum rate in brain and its activity may be increased pharmacologically by manipulating NMDA receptors or nitric oxide content. This may be useful, for example, to increase ammonia detoxification in brain in hyperammonemic situations.     

Purification and partial characterization of glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum

Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) from the photosynthetic bacterium Rhodospirillum rubrum grown under nitrogen fixing conditions has been purified to homogeneity. The purification procedure involves affinity chromatography on ADP-agarose type 2 as the major purification step. The recovery in the purification is 70%. The specific activity of the purified enzyme is about 10-times higher in the gamma-glutamyl transferase assay than in the coupled biosynthetic assay. The molecular weight was determined to 530,000 by native gradient polyacrylamide gel electrophoresis and to 500,000 by gel filtration. The subunits have an apparent molecular weight of 52,000. Glutamine synthetase isolated from Rsp. rubrum which had been exposed to ammonium ions ('switch-off') before harvest had about 20% of the transferase activity compared with the enzyme purified from nitrogen-starved cells. The low-activity form showed two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

What type of glutamine synthetase is important forStreptomyces coelicolor A3(2) under nitrogen-limited growth conditions?

Summary Glutamine synthetase I activity ofStreptomyces coelicolor was strongly repressed by ammonia and was induced 56.8 fold in a nitrogen-free medium. Glutamine synthetase II activity was not induced even by a long-term nitrogen starvation. Therefore, glutamine synthetase I is the only active enzyme ofStreptomyces coelicolor.     

固氮粪产碱菌谷氨酰胺合成酶的分离纯化及其特性

联合固氮细菌粪产碱菌（Ａｌｃａｌｉｇｅｎｅｓｆａｅｃａｌｉｓ）Ａ１５０１菌体经超声破碎后,无细胞粗提液以ＰＥＧ－６０００分级沉淀,丙酮沉淀,再经蓝琼脂糖（ＢｌｕｅＳｅｐｈａｒｏｓｅＣＬ－６８）亲和层析分离、纯化。获得的纯谷氨酰胺合成酶（ＧＳ）在ＳＤＳ－ＰＡＧＥ和４－３０％梯度ＰＡＧＥ上均呈均一的一条带。ＧＳ亚基及整酶分子量分别为５５ｋＤ和６４５ｋＤ,亚基由４５６个氨基酸残基组成。ＧＳ的Ｋｍ值,在以Ｇｌｕ为氮源的介质中培养时分别为２０ｍｍｏｌ／Ｌ（Ｇｌｕ）,５０ｍｍｏｌ／Ｌ（ＡＴＰ）和４５ｍｍｏｌ／Ｌ（ＮＨ~＋_４）;在以ＮＨ~＋_４为氮源的介质中培养时则分别为７０ｍｍｏｌ／Ｌ（Ｇｌｕ）,４９ｍｍｏｌ／Ｌ（ＡＴＰ）和８０ｍｍｏｌ／Ｌ（ＮＨ~＋_４）,表明ＮＨ~＋_４培养下形成高度腺苷化的ＧＳ对Ｇｌｕ及ＮＨ~＋_４的亲和力有所下降。     

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

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

Glutamine synthetase activity in Huntington's disease

Glutamine synthetase activity was measured in seven brain areas post-mortem from control patients, and those with Huntington's disease. The activity of the enzyme was reduced in the frontal and temporal cortex, putamen and cerebellum, but not in the hippocampus, thalamus or olivary nucleus. The results do not suggest a generalised deficiency of glutamine synthetase in Huntington's disease. However, as this enzyme is localised to astrocytic cells, the reduction in activity in areas of neuronal devastation, where the ration of astrocytes to neurones is increased, may reflect a greater functional deficit. The enzyme plays a crucial role in cerebral ammonia assimilation and its inhibition in laboratory animals is known to produce neuronal toxicity. A reduction in its activity in Huntington's disease may well contribute to the neuronal pathology in certain areas.     

The purification and properties of the glutamine synthetase from the cytosol of Soya-bean root nodules.

The major portion of glutamine synthetase activity in root nodules of soya-bean plants is associated with the cytosol rather than with Rhizobium japonicum bacteroids. Glutamine synthetase accounts for about 2% of the total soluble protein in nodule cytosol. Glutamine synthetase from nodule cytosol has been purified by a procedure involving fractionation with protamine sulphate, ammonium sulphate and polypropylene glycol, chromatography on DEAE-Bio-Gel A and Bio-Gel A-5m and affinity chromatography on glutamate-agarose columns. The purified preparation appeared to be homogeneous in the analytical ultracentrifuge. From sedimentation-equilibrium experiments a mol. wt. of about 376000 was determined for the native enzyme and 47300 for the enzyme in guanidinium chloride. From these data and measurements of electron micrographs, we have concluded that glutamine synthetase from nodule cytosol consists of eight subunits arranged in two sets of planar tetramers which form a cubical configuration with dimensions of about 10 nm (100 A) across each side. Glutamine synthetase from nodule cytosol has a higher glycine and proline content and a lower content of phenylalanine than the glutamine synthetase that has been prepared from pea seed. The cytosol enzyme contains four half-cystine molecules per subunit, which is in contrast with two reported for the enzyme from pea seed. Enzyme activity is striking influenced by the relative proportion of Mg2+ and Mn2+ in the assay medium. Activity is inhibited by feedback inhibitors and is influenced by energy charge.     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase     

Physiological roles of glutamine synthetases I and II in ammonium assimilation in Rhizobium sp. 32H1

The two glutamine synthetases of Rhizobium sp. 32H1 appear to be structurally and functionally distinct. Glutamine synthetase I was reversibly adenylylated, and its synthesis was repressed only twofold by ammonium. When in the unadenylylated configuration, it was the enzyme which allowed the organism to grow, albeit marginally, on ammonium as a nitrogen source. There is no evidence to suggest that the second enzyme, glutamine synthetase II, is regulated by adenylylation. However, this enzyme was repressed at least 50-fold by even low amounts of ammonium. Glutamine synthetase II does not seem to function in ammonium assimilation but rather in purine biosynthesis.     

Regulation and properties of the glutamine synthetase purified from Photobacterium phosphoreum

Glutamine synthetase from a marine enterobacterium, Photobacterium phosphoreum, was purified to homogeneity from cells grown in glycerol-yeast extract medium. The purified enzyme had a molecular weight of approximately 670,000 and a subunit size of 56,000, i.e. larger than that of the enzyme from E. coli. Regulation of the glutamine synthetase activity by adenylylation/deadenylylation was demonstrated on snake venom phosphodiesterase treatment. The state of adenylylation appeared to influence both the biosynthetic and gamma-glutamyltransferase activities of P. phosphoreum glutamine synthetase similar to in the case of the E. coli enzyme. The enzyme activity was controlled by adenylylation and possibly in combination with feedback inhibition by alanine, serine, and glycine, metabolites which are especially effective in inhibiting P. phosphoreum glutamine synthetase. When either Mn2+ or Mg2+ was added to the relaxed (divalent cation-free) enzyme, similar UV-difference spectra were obtained for the enzyme, indicating that the conformational states induced by these cations were also similar. The profile of these spectra varied from those published for E. coli, and three peaks were four 1 at 282.5, 288.5, and 298 nm.     

Glutamine synthetase from pig brain: binding of adenosine triphosphate.

Glutamine synthetase was purified to homogeneity from fresh brain homogenates by a procedure that makes use of the affinity of this ATP requiring ligase to Cibacron 3G-A Blue-Sephadex G-75. It is shown that the interaction of pig brain enzyme with the dye does not concern the active centre but a non-catalytic although specific ATP binding site. Both sides contain a cysteine residue which may react with N-ethylmaleimide. The unprotected succinimidated enzyme is inactive, and aggregates. ATP alone protects only against aggregation, not against inactivation; the ATP/Mg2 complex also hinders inactivation. A tryptic heptadecapeptide isolated from the derivatized enzyme representing the carboxy terminal seems to belong to the active centre; its amino acid sequence was partially determined.     

Factors affecting the production of glutamine in cultured mouse cells

Glutamine synthetase activity of NCTC clone 929 mouse cells (strain L) was studied as a function of the prior nutritional experience of the cells. Small enzyme increases were recorded in response to either glutamine depletion or chronic serum supplementation of the growth medium. Somewhat greater increases resulted from the administration of cortisol or certain other steroids, particularly if the hormone treatment was combined with glutamine withdrawal. High concentrations of glutamate in the medium did not augment the glutamine synthetase content of the cells and even caused an apparent decrease in it. The presence of glutamine in the culture medium resulted in a fairly rapid rate of disappearance of the glutamine synthetase of previously induced cells. The data suggest that glutamine and cortisol act independently on the cells in regulating the level of the enzyme.     

Regulation of glutamine synthetase and glutaminase activities in cultured skeletal muscle cells

Glutamine is synthesized in skeletal muscle, released to the circulation, and transported to other tissues, where it may provide important substrate for gluconeogenesis, ammoniagenesis, and energy-yielding pathways. With the ultimate goal of delineating the factors that control glutamine production and release by skeletal muscle, we have studied the regulation of two key enzymes, glutamine synthetase and glutaminase, in the L6 line of rat skeletal muscle cells grown in monolayer culture. The cultured myotubes were found to have glutamine synthetase and phosphate-dependent glutaminase activities. Glutamine synthetase activity was increased following incubation (1) in glutamine-free medium (threefold); (2) in medium containing high glutamic acid concentrations (fourfold); and (3) in medium supplemented with dexamethasone (threefold). In each case the increase in glutamine synthetase activity required several hours to reach a maximum and was prevented by cycloheximide, suggesting that the change occurred through increased enzyme biosynthesis. No substances tested were found to affect glutaminase activity. We conclude that glutamine synthetase in cultured skeletal muscle is responsive to substrate, product, and hormonal regulation.     

New enzymic assays for glutamine synthetase adenylytransferase and its regulatory protein PIIA.

Glutamine synthetase from some gram-negative bacteria is regulated mainly by a covalent modification cascade. The activity of the enzyme is dependent on its state of adenylylation, which is catalyzed by adenylyltransferase (ATase), whose activity is modulated by a regulatory protein, P_II, which exists in two interconvertible forms, P_IIA and P_IID. In this report, two simple enzymic assays for ATase and P_IIA are described. These methods are based on the knowledge that unadenylylated and adenylylated glutamine synthetase exhibit different pH optima in the γ-glutamyl transferase reaction and that l-alanine, a noncompetitive inhibitor, inhibits them to different extents. These assays have been used in the purification of ATase and P_IIA and in mechanistic studies on adenylylation. The scope and limitations of these assays are discussed.