头状轮生链霉菌谷氨酰胺合成酶的研究 Ⅱ.酶的性质和调节

头状轮生链霉菌(Streptoverticillium caespitosus)谷氨酰胺合成酶(Glutamine synthetase,GS)生物活性的最适pH为7。测活系统中必须加入Mn~(2 )或Mg~(2 ),二者分别作激活离子时得到的酶的参数不同。Mn~(2 )对GS有稳定作用。一些金属离子如Ca~(2 )等强烈地抑制生物活性。GS的最适温度为50℃,对ATP、谷氨酸和NH_4Cl的Km值分别为1.75、2.78和5mmol/L。NH_4Cl的浓度小于10mmol/L时对酶有正协同效应,大于10mmol/L时对酶有负协同效应。GS可被氨阻遏,比活能被硝酸盐促进。一些代谢物对GS有累积反馈抑制作用。发酵过程中加入氨后无“氨休克”现象,高氨条件下的GS不受蛇毒磷酸二酯酶(SVPDE)的作用,所以此酶无腺苷酰化—去腺苷酰化调节。     

头状轮生链霉菌谷氨酰胺合成酶的研究 Ⅱ.酶的性质和调节

头状轮生链霉菌(Streptoverticillium caespitosus)谷氨酰胺合成酶(Glutamine synthetase,GS)生物活性的最适pH为7。测活系统中必须加入Mn~(2+)或Mg~(2+),二者分别作激活离子时得到的酶的参数不同。Mn~(2+)对GS有稳定作用。一些金属离子如Ca~(2+)等强烈地抑制生物活性。GS的最适温度为50℃,对ATP、谷氨酸和NH_4Cl的Km值分别为1.75、2.78和5mmol/L。NH_4Cl的浓度小于10mmol/L时对酶有正协同效应,大于10mmol/L时对酶有负协同效应。GS可被氨阻遏,比活能被硝酸盐促进。一些代谢物对GS有累积反馈抑制作用。发酵过程中加入氨后无“氨休克”现象,高氨条件下的GS不受蛇毒磷酸二酯酶(SVPDE)的作用,所以此酶无腺苷酰化—去腺苷酰化调节。     

氨和谷氨酰胺对浑球红假单胞菌（Rhodobacter sphaeroides）固氮酶...

浑球红假单胞菌菌株601具有迅速对外源氨作出“关闭”固氮酶活性的反应。氨对固氮酶的抑制作用,可被谷氨酰胺合成酶(GS)抑制剂MSX所解除。反之,加入Glu代谢抑制剂DON,可延长氨抑制的持续时间。Gln对固氮酶也有抑制作用。在脱腺苷化GS的透性细胞中,加入Gln可抑制固氮酶活性,同时,GS腺苷化状态提高。然而,氨则对透性细胞的固氮酶活性和GS腺苷化状态没有影响。     

光合细菌Rhodopseudomonas capsulata谷氨酰胺合成酶的某些特性

研究了荚膜红假单孢光合细菌菌株N-3谷氨酰胺合成酶(GS)的腺苷态/脱腺苷态间互相转换和调节方式,对十六烷基三甲基溴化铵添加后的稳定,以及不同氮源对酶量的变化和腺苷态的影响。在光暗的调节上,光强在500～50001x范围内,此酶的活性几乎处于同一水平,其腺苷态和脱腺苷态之比亦不发生显著变化,但一经黑暗处理,GS活性迅速下降,相似于固氮酶活性受光、暗的调节。借助于谷氨酰胺合成酶的抑制剂MSX加入反应系统,消除了氨对固氮酶活性的瞬间抑制,但消除不了暗对固氮酶活性的瞬间抑制,表明谷氨酰胺合成酶参与了氨对固氮酶活性的瞬间调节,但并不参与固氮酶活性的光,暗瞬间调节。     

林肯链霉菌谷氨酰胺合成酶的酶学性质

在分离纯化的基础上,报道了pH、温度和金属离子对林肯链霉菌(Streptomyceslincolnensis)Z-512谷氨酸胺合成酶(GS)活力的影响及GS底物专一性的研究结果.在动力学性质的研究中,发现林肯链霉菌GS在生物合成反应系统中,对底物NH_4CI的饱和曲线不遵守米氏方程.Hill作图呈两相曲线.在NH_4CI浓度低的情况下,Hill系数大于1,具有正协同效应;当NH_4CI浓度增加到一定程度时,Hill系数小于1,具有负协同效应.这说明NH_4CI不仅作为林肯链霉菌GS的底物,而且作为一种效应物调节GS的活性.林肯链霉菌GS对底物Glu及ATP的饱和曲线遵守米氏方程.在不同的激活离子存在下,GS对Glu、ATP的Km值也不同.     

硝酸盐、镁盐对林可霉素生物合成的促进作用

在林肯链霉菌生物合成林可霉素代谢调节的研究中,发现硝酸盐可明显促进林可霉素的生物合成．加入硝酸钾０．８％,林肯链霉菌合成林可霉素的产量可增加３７％．在发酵９６ｈ之前加入硝酸盐均能促进林可霉毒的合成,但产量的增加随加入时间的延迟而降低．硝酸钾在促进产量的同时,使菌体生长减少,看来硝酸盐对林可霉素的合成与菌体生长之间起着调节作用．洗涤菌体试验指出,硝酸盐的加入诱导了林可霉素合成所需要的酶系,这可能是加入硝酸盐后,产生进一步氮代谢的结果;蛋白胨不能代替硝酸盐,进一步说明硝酸钾的作用并不是作为氮源利用．在蛋白质合成抑制剂氯霉素存在下,硝酸盐不再能促进林可霉素的合成,说明氯霉素抑制了硝酸盐或其代谢中间物所诱导的酶系的合成．同时还报导了镁盐促进林可霉素生物合成现象的初步观察结果．硫酸镁在促进林可霉素产量提高的同时,使菌体生长延迟．硫酸镁的这种作用机制可能是通过磷酸镁铵沉淀,降低了培养基中游离氨和可溶性磷酸盐浓度,解除了铵盐和磷酸盐对林可霉素合成的抑制．     

谷氨酰胺合成酶在固氮鱼腥藻固氮调节中的作用

在MSX(methlonine sulfoximine,谷氨酰胺合成酶的不可逆抑制剂)存在下,固氮鱼腥藻(Anabaena azotica)所分泌的氨量和谷氨酰胺合成酶(GS)活力有较好的负相关性,证明谷氨酰胺合成酶-谷氨酸合成酶(GS-GOGAT)是固氮鱼腥藻氨同化的重要途径。在蛋白质合成受到氯霉素拟制时,NH_4~ 对固氮酶的失活是迅速的,同时GS活力有较大下降,表明NH_4~ 的调控酶的失活或降解。在氮固定条件下,固氮酶活力半衰期小于4小时,GS活力半衰期大于10小时,则GS并不是固氮酶的正调节因子。NH_4~ 和谷氨酰胺(gln)对固氮酶的失活作用随它的浓度增加而提高,但GS并没有这种相关性,低浓度NH_4~ (0.1—0.5mmol/L·NH_4Cl)对GS活力没有抑制作用,高浓度gln(1.0—2.0mmol/L)也没有抑制GS活力,说明GS并不直接调控固氮酶。MSX能消除NH_4~ 和gln对固氮酶的抑制作用,并与藻龄有关。     

头状轮生链霉菌谷氨酰胺合成酶的研究 Ⅰ.酶的生成和纯化

以氨为氮源培养头状轮生链霉菌(Streptoverticillium caespitosus)时粗抽提液中谷氨酰胺合成酶(glutamine synthetase, GS)对热稳定,以硝酸盐为氮源时GS对热不稳定。以硫酸链霉素沉淀、热处理、聚乙烯亚胺(PEI)沉淀和Affini-gel Blue柱纯化了前者,以DE-52柱和Affini-gel Blue柱纯化了后者,纯化后两个酶分子量同为550000,亚基分子量同为56000,热稳定性相同,转谷氨酰基酶活力的最适pH均在6.4～6.7之间,对谷氨酰胺的K_m值同为11.1mmol/L,寸羟胺的K_m值同为1.6mmol/L,所以认为此菌中总是同一GS表现出活力。     

浑球红假单胞菌氨同化途径的研究

本文测定了浑球红假单胞菌(Rhodobacter sphaeroides)菌株601谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、谷氨酸脱氢酶(GDH)和丙氨酸脱氢酶(ADH)的活性。低氨时,GS/GOGAT活力高,GDH活力低,高氨时,GS/GOGAT活力低,GDH活力高。在以分子氮或低浓度氨为氮源的培养条件下,加入GS抑制刑MSX(L—methionine—DL—sulphoximine),细菌生长受到抑制。但是,生长在以谷氨酸为氮源的细菌则不受影响。上述结果表明,浑球红假单胞菌菌株601氨同化是通过GS/GOGAT途径和GDH途径。     

头状轮生链霉菌谷氨酰胺合成酶的研究 Ⅰ.酶的生成和纯化

以氨为氮源培养头状轮生链霉菌(Streptoverticillium caespitosus)时粗抽提液中谷氨酰胺合成酶(glutamine synthetase, GS)对热稳定,以硝酸盐为氮源时GS对热不稳定。以硫酸链霉素沉淀、热处理、聚乙烯亚胺(PEI)沉淀和Affini-gel Blue柱纯化了前者,以DE-52柱和Affini-gel Blue柱纯化了后者,纯化后两个酶分子量同为550000,亚基分子量同为56000,热稳定性相同,转谷氨酰基酶活力的最适pH均在6.4～6.7之间,对谷氨酰胺的K_m值同为11.1mmol/L,寸羟胺的K_m值同为1.6mmol/L,所以认为此菌中总是同一GS表现出活力。     

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.     

Evidence for a role of glutamine synthetase in assimilation of amino acids as nitrogen source in the cyanobacterium Nostoc muscorum.

Methylammonium/ammonium ion, glutamine, glutamate, arginine and proline uptake, and their assimilation as nitrogen sources, was studied in Nostoc muscorum and its glutamine synthetase-deficient mutant. Glutamine served as nitrogen source independent of glutamine synthetase activity. Glutamate was not metabolised as a nitrogen source but still inhibited nitrogenase activity and diazotrophic growth. Glutamine synthetase activity was essential for the assimilation of N2, ammonia, arginine and proline as nitrogen sources but not for the control of their transport, heterocyst formation, and production of ammonia or aminoacid dependent repressor signal for N2-fixing heterocysts. These results also suggest that glutamine synthetase serves as the sole route of ammonia assimilation and glutamine synthesis, and ammonia per se as the repressor signal for N2-fixing heterocysts and methylammonium (ammonium) transport.     

Regulation of nitrogen catabolic enzymes in Streptomyces clavuligerus

The levels of several enzymes involved in assimilation of different nitrogen compounds were investigated in Streptomyces clavuligerus in relation to the nitrogen source supplied to the cultures. Threonine dehydratase, serine dehydratase, proline dehydrogenase, histidase and urocanase were not decreased in the presence of ammonium. The latter two enzymes were induced by histidine in the culture medium, while proline dehydrogenase was induced by proline. Glutamine synthetase, urease and ornithine aminotransferase levels were higher with poor nitrogen sources and were repressed by ammonium. Arginase was induced by arginine and repressed by ammonium. Glutamine synthetase was rapidly inactivated upon addition of ammonium to the culture, and could be reactivated in vitro by treatment with snake venom phosphodiesterase, which suggested that adenylylation is involved in the inactivation. Three previously isolated mutants with abnormal glutamine synthetase activities showed pleiotropic effects on urease formation. All these data point to a mechanism controlling preferential utilization of some nitrogen sources in this species.     

Glutamine synthetase of Streptomyces cattleya: purification and regulation of synthesis

Glutamine synthetase (GS; EC 6.3.1.2) from Streptomyces cattleya was purified using a single affinity-gel chromatography step, and some of its properties were determined. Levels of GS in S. cattleya cells varied by a factor of 8 depending upon the source of nitrogen in the growth medium. Of 24 nitrogen sources examined only glutamine or NH4Cl utilization resulted in very low GS activity. Addition of NH4Cl to a culture with high GS levels appeared to stop further synthesis and resulted in a progressive decrease in the specific activity of the enzyme. The GS inhibitor methionine sulphoximine (MSX) inhibited GS activity but had no effect on exponentially growing cells. The presence of MSX either lengthened or shortened the period between spore inoculation and initiation of exponential growth, depending on the source of nitrogen. In glutamine minimal medium MSX produced earlier and more efficient spore germination while in glutamate or nitrate minimal medium germination was delayed by its presence.     

Regulation of glutamine synthetase in the blue-green alga Anabaena L-31

In N2-grown cultures of Anabaena L-31, in which protein synthesis was prevented by chloramphenicol, presence of NH+4 caused a drastic decrease of glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) activity indicating NH+4-mediated inactivation or degradation of the enzyme. The half-life of glutamine synthetase was more than 24 h, whereas that of nitrogenase (reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolysing), EC 1.18.2.1) was less than 4 h, suggesting that glutamine synthetase may not act as positive regulator of nitrogenase synthesis in Anabaena. Glutamine synthetase purified to homogeneity was subject to cumulative inhibition by alanine, serine and glycine. The amino acids, however, exhibited partial antagonism in this behaviour. Glyoxylate, an intermediate in photorespiration, virtually prevented the amino acid inhibition. Kinetic studies revealed inhibition of the enzyme activity by high Mg2+ concentration under limiting glutamate level and by high glutamate in limiting Mg2+. Maximum enzyme activity occurred when the ratio of glutamate to free Mg2+ was 0.5 to 1.0. The results demonstrate that the enzyme is subject to multiple regulation by various metabolites involved in nitrogen assimilation.     

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     

Regulation of glutamine synthetase from Saccharomyces cerevisiae by repression, inactivation and proteolysis

Glutamine synthetase activity is modulated by nitrogen repression and by two distinct inactivation processes. Addition of glutamine to exponentially grown yeast leads to enzyme inactivation. 50% of glutamine synthetase activity is lost after 30 min (a quarter of the generation time). Removing glutamine from the growth medium results in a rapid recovery of enzyme activity. A regulatory mutation (gdhCR mutation) suppresses this inactivation by glutamine in addition to its derepressing effect on enzymes involved in nitrogen catabolism. The gdhCR mutation also increases the level of proteinase B in exponentially grown yeast. Inactivation of glutamine synthetase is also observed during nitrogen starvation. This inactivation is irreversible and consists very probably of a proteolytic degradation. Indeed, strains bearing proteinase A, B and C mutations are no longer inactivated under nitrogen starvation.     

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.     

In vitro reactivation of in vivo ammonium-inactivated glutamine synthetase from Synechocystis sp. PCC 6803.

Glutamine synthetase from Synechocystis sp. strain PCC 6803 is inactivated by ammonium addition to cells growing with nitrate as the nitrogen source. The enzyme can be reactivated in vitro by different methods such as alkaline phosphatase treatment, but not phosphodiesterase, by raising the pH of the crude extract to values higher than 8, by increasing the ionic strength of the cell-free extract, or by preincubation with organic solvents, such as 2-propanol and ethanol. These results suggest that the loss of glutamine synthetase activity promoted by ammonium involves the non-covalent binding of a phosphorylated compound to the enzyme and support previous results that rule out the existence of an adenylylation/deadenylylation system functioning in the regulation of cyanobacterial glutamine synthetase.