Regulation of Glutamine Synthetase X. Effect of Growth Conditions on the Susceptibility of Escherichia coli Glutamine Synthetase to Feedback Inhibition

The kinetic properties of Escherichia coli glutamine synthetase are markedly influenced by the manner in which the organism is grown. Enzyme obtained from stationary-phase cells grown on glycerol and glutamate is strongely inhibited by each of the eight feedback effectors known to influence this enzyme; however, the enzyme from log-phase cells grown on glucose and growth-limiting concentrations of NH(4)Cl is stimulated by some of these effectors. Of the growth variables examined, nitrogen source and time of harvest were the most important; carbon source and aeration seemed to have no effect. Two purified enzyme preparations have been obtained from cells grown under two different conditions, designated enzymes I and II for convenience. Enzyme I is stimulated by adenosine 5'-monophosphate, histidine, and tryptophan in the transfer assay, whereas enzyme II is strongly inhibited by all effectors tested. Enzyme I has a higher specific activity in the forward assay in the presence of Mg(++) or Co(++), whereas enzyme II is more active in the presence of Mn(++).     

斜茎黄芪根瘤菌谷氨酰胺合成酶基因多样性研究

研究了22株代表性斜茎黄芪根瘤菌的谷氨酰胺合成酶基因多样性。首先对供试菌株进行了谷氨酰胺合成酶glnA和glnII基因扩增,结果显示从来自Mesorhizobium和Rhizobium属的多数代表菌株都可以扩增到约1kp、大小一致的glnA基因产物,而从Agrobacterium sp.的4株代表菌株未能得到glnA PCR扩增产物。基因glnII的扩增结果显示几乎从所有测试菌株都能够得到基因产物,来自Mesorhizobium septentrionale、M.temperatum和Mesorhizobium spp.的代表菌株都得到了单一的、大小约400bp～500bp的glnII PCR扩增产物,而从Agrobacterium sp.的4株代表菌株扩增得到的glnII PCR扩增产物明显不同于其它斜茎黄芪根瘤菌代表菌,它们都有一条约1 kb的特征PCR扩增产物条带,SDW052和R084还出现了另外2～3个扩增产物条带。此外,基因glnA的RFLP分析结果与我们先前的16S rRNA基因分析结果具有很好的一致性,这些结果都进一步证实了这些根瘤菌的染色体基因多样性。     

大肠杆菌邻氨基苯甲酸合成酶编码基因trpED的克隆与表达

目的:通过基因重组的方法获得在体外具有邻氨基苯甲酸合成酶活性的基因trpED,为进一步解除反馈抑制的基因改造寻找靶标。方法:分别构建重组质粒pBV220-trpE和pBV220-trpD;由于trpE和trpD存在翻译偶联现象,将其自大肠杆菌基因组中经PCR扩增得到,构建新的表达质粒pBV220-trpED。结果:SDS-PAGE显示,包含质粒pBV220-trpED的重组菌在相对分子质量约53000(trpE基因表达)和56000(trpD基因表达)处的蛋白表达量明显增多,且邻氨基苯甲酸合成酶活性是对照的3倍。结论:翻译偶联的存在直接影响蛋白活性的发挥;重组质粒pBV220-trpED的获得为进一步的基因改造,以提高色氨酸产量奠定了研究基础。     

Inhibition of Glutamine Synthetase from Pea by Tabtoxinine-beta-lactam

Tabtoxinine-β-lactam, a hydrolytic product of tabtoxin produced by Pseudomonas syringae pv. tabaci, apparently inactivates pea seed glutamine synthetase. Inhibition of the enzyme's initial velocity is linear over a range of 0.5 to 5 millimolar tabtoxinine-β-lactam in the presence of 10 millimolar glutamate. A method for the purification of glutamine synthetase from dried peas is presented which gives a 30% yield with a 2,000-fold increase in specific activity. A method for obtaining highly purified tabtoxinine-β-lactam and tabtoxin in good yields is also presented. The authenticity and purity of tabtoxinine-β-lactam and tabtoxin were verified by chromatography, biological activity, and ¹H and ¹³C nuclear magnetic resonance spectroscopy.     

Glutamine Production by Coupling with Energy Transfer: Employing Yeast Cells and Gluconobacter Glutamine Synthetase

A glutamine production process was established by combining alcoholic fermentation of baker's yeast cells with glutamine synthetase from the bacterium Gluconobacter suboxydans. The maximum amount of glutamine formed under optimum conditions was about 20 mM in 3 hr with 80% yield based on glutamate, substrate. The fermentation proceeded in two steps: the accumulation of energy in a form of fructose 1,6-diphosphate (FDP) by yeast fermentation of sugar based on the Harden-Young effect and the fermentation of FDP coupled with glutamine synthetase reaction (an endergonic reaction) through an ATP-ADP system. The following factors were found to be important: (a) the ratio of the activities of yeast fermentation of sugar and glutamine synthetase, (b) effect of contaminating enzyme(s) in glutamine synthetase preparation, and (c) enzymatic properties of glutamine synthetase.     

Selection and Neutrality in Lactose Operons of Escherichia Coli

The kinetics of the permeases and beta-galactosidases of six lactose operons which had been transduced into a common genetic background from natural isolates of Escherichia coli were investigated. The fitnesses conferred by the operons were determined using chemostat competition experiments in which lactose was the sole growth-limiting factor. The cell wall is demonstrated to impose a resistance to the diffusion of galactosides at low substrate concentrations. A steady state model of the flux of lactose through the metabolic pathway (diffusion, uptake and hydrolysis) is shown to be proportional to fitness. This metabolic model is used to explain why an approximately twofold range in activity among the permease alleles confers a 13% range in fitness, whereas a similar range in activity among alleles of the beta-galactosidase confers a 0.5% range in fitness. This metabolic model implies that selection need not be maximized when a resource is scarce.     

Glutamine Synthetase Activity, Relative Water Content and Water Potential in Maize Submitted to Drought

Primer screening and optimization for random amplified polymorphic DNA (RAPD) analysis of cashew (Anacardium occidentale L.) was investigated. Among four series (A, B, D and N) of 10-mer primers, A-series performed better amplification of fragments than other series. The maximum amplification fragments was obtained using OPA-02, OPA-03, OPA-09, OPB-06, OPB-10, OPD-03, OPD-05 and OPN-03 primers. The primers OPA-02 and OPN-03 produced maximum number of DNA fragments in Anacardium occidentale cv. H-320. Primers (OPB-08 and OPN-05 performed a least number of amplification fragments. RAPD profile also indicate that some primer did not produce good amplification. The primer OPA-02 amplified 12 number of polymorphic bands in 20 cultivars of cashew. Only one DNA fragment was produced in A. occidentale cv. Vridhachalam - 2 (M-44/3) by using the primer OPA-02. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immunolocalization of a Unique Form of Maize Kernel Glutamine Synthetase Using a Monoclonal Antibody

The pedicel (basal maternal tissue) of maize (Zea mays L.) kernels contains a physically and kinetically unique form of glutamine synthetase (GSp1) that is involved in the conversion of transport forms of nitrogen into glutamine for uptake by the developing endosperm (M.J. Muhitch [1989] Plant Physiol 91: 868-875). A monoclonal antibody has been raised against this kernel-specific GS that does not cross-react either with a second GS isozyme found in the pedicel or with the GS isozymes from the embryo, roots, or leaves. When used as a probe for tissue printing, the antibody labeled the pedicel tissue uniformly and also labeled some of the pericarp surrounding the lower endosperm. Silver-enhanced immunogold staining of whole-kernel paraffin sections revealed the presence of GSp1 in both the vascular tissue that terminates in the pedicel and the pedicel parenchyma cells, which are located between the vascular tissue and the basal endosperm transfer cells. Light staining of the subaleurone was also noted. The tissue-specific localization of GSp1 within the pedicel is consistent with its role in the metabolism of nitrogenous transport compounds as they are unloaded from the phloem.     

Regulation of Neurotransmitter Aspartate Metabolism by Glial Glutamine Synthetase

Aspartate levels and release from rat striatal slices following the inhibition of glutamine synthetase (GS) by methionine sulfoximine (MSO) were studied. Striatal levels of aspartate and glutamine were decreased over time in a manner that correlated with GS inhibition. Ca2+-dependent, K+-stimulated aspartate release was diminished in striatal tissue slices from animals pretreated with MSO. The decreased release of aspartate correlated over time with the inhibition of GS. The addition of glutamine to the perfusion medium completely reversed the effects of MSO on calcium-dependent aspartate release. It is suggested that glutamine is a major precursor for transmitter aspartate.     

Oxidative Modification of Glutamine Synthetase by Amyloid Beta Peptide

β-Amyloid peptide (Aβ), the main constituent of senile plaques and diffuse amyloid deposits in Alzheimer's diseased brain, was shown to initiate the development of oxidative stress in neuronal cell cultures. Toxic lots of Aβ form free radical species in aqueous solution. It was proposed that Aβ-derived free radicals can directly damage cell proteins via oxidative modification. Recently we reported that synthetic Aβ can interact with glutamine synthetase (GS) and induce inactivation of this enzyme. In the present study we present the evidence that toxic Aβ(25-35) induces the oxidation of pure GS in vitro. It was found that inactivation of GS by Aβ, as well as the oxidation of GS by metal-catalyzed oxidation system, is accompanied by an increase of protein carbonyl content. As it was reported previously by our laboratory, radicalization of Aβ is not iron or peroxide-dependent. Our present observations consistently show that toxic Aβ does not need iron or peroxide to oxidize GS. However, treatment of GS with the peptide, iron and peroxide together significantly stimulates the protein carbonyl formation. Here we report also that Aβ(25-35) induces carbonyl formation in BSA. Our results demonstrate that P-peptide, as well as other free radical generators, induces carbonyl formation when brought into contact with different proteins.     

Roles of Glutamine Synthetase Inhibition in Epilepsy

Glutamine synthetase (GS, E.C. 6.3.1.2) is a ubiquitous and highly compartmentalized enzyme that is critically involved in several metabolic pathways in the brain, including the glutamine-glutamate-GABA cycle and detoxification of ammonia. GS is normally localized to the cytoplasm of most astrocytes, with elevated concentrations of the enzyme being present in perivascular endfeet and in processes close to excitatory synapses. Interestingly, an increasing number of studies have indicated that the expression, distribution, or activity of brain GS is altered in several brain disorders, including Alzheimer’s disease, schizophrenia, depression, suicidality, and mesial temporal lobe epilepsy (MTLE). Although the metabolic and functional sequelae of brain GS perturbations are not fully understood, it is likely that a deficiency in brain GS will have a significant biological impact due to the critical metabolic role of the enzyme. Furthermore, it is possible that restoration of GS in astrocytes lacking the enzyme could constitute a novel and highly specific therapy for these disorders. The goals of this review are to summarize key features of mammalian GS under normal conditions, and discuss the consequences of GS deficiency in brain disorders, specifically MTLE.