138 Molecular Evolution of Glutamine Synthetase In Protists

The binding of fluorescein isothiocyanate (FITC) conjugated lectins to gametes of Aglaothamnion byssoides Itono during the fertilization was studied by the use of confocal microscope. The physiological effects of lectins and carbohydrates on gamete binding were also examined. Three lectins, concanavalin A (ConA), Soybean agglutinin (SBA) and wheat germ agglutinin (WGA) bound to the surface of spermatia, but each lectin labeled different region of the spermatium. SBA bound only to the spermatial appendages but ConA bound to the whole spermatial surface except spermatial appendages. WGA labeled narrow region which connects spermatial body and appendages. During fertilization, ConA and WGA specific substances on the spermatial surface moved towards the area contacting with trichogyne and accumulated on the surface of fertilization canal. Spermatial binding to trichogynes was inhibited by pre-incubation of spermatia with SBA, while trichogyne receptors were blocked by the complementary carbohydrate, N-acetyl-D-galactosamine. WGA and its complementary carbohydrate had little effect on gamete binding. For searching the step of sexual isolation, crossing experiment was performed between Aglaothamnion byssoides and twelve other red algal species. Results showed that the gamete recognition was genus-specific: the gametes bound freely with their partners of the same genus. When two species from same genus were crossed, sexual isolation occurs gradually during the fertilization process. Therefore, sexual isolation in red algae appears to be determined by multi-step process and gamete binding is the initial step.     

Molecular Mechanisms of Lead Neurotoxicity

Epidemiological studies have shown a strong relationship between the level of lead in blood and bone as assessed by performance on IQ tests and other psychometric tests. Approximately 1 out of 10 children in the United States have blood lead levels above 10g/dl, which has been established as the level of concern. Studies on experimental animals exposed to lead after birth have shown learning deficits at similar blood lead levels. Since learning requires the remodeling of synapses in the brain, lead may specifically affect synaptic transmission. Although the molecular targets for lead are unknown, a vast amount of evidence accumulated over many years has shown that lead disrupts processes that are regulated by calcium. Our laboratory has been studying the effect of lead on protein kinase C, a family of isozymes some of which require calcium for activity. We and others have shown that picomolar concentrations of lead can replace micromolar concentrations of calcium in a protein kinase C enzyme assay. Furthermore, lead activates protein kinase C in intact cells and induces the expression of new genes by a mechanism dependent on protein kinase C. We propose that the learning deficits caused by lead are due to events regulated by protein kinase C that most likely occur at the synapse.     

Molecular Mechanisms of Disease-Causing Missense Mutations

Genetic variations resulting in a change of amino acid sequence can have a dramatic effect on stability, hydrogen bond network, conformational dynamics, activity and many other physiologically important properties of proteins. The substitutions of only one residue in a protein sequence, so-called missense mutations, can be related to many pathological conditions and may influence susceptibility to disease and drug treatment. The plausible effects of missense mutations range from affecting the macromolecular stability to perturbing macromolecular interactions and cellular localization. Here we review the individual cases and genome-wide studies that illustrate the association between missense mutations and diseases. In addition, we emphasize that the molecular mechanisms of effects of mutations should be revealed in order to understand the disease origin. Finally, we report the current state-of-the-art methodologies that predict the effects of mutations on protein stability, the hydrogen bond network, pH dependence, conformational dynamics and protein function.     

Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors

The majority of ovarian tumors eventually recur in a drug resistant form. Using cisplatin sensitive and resistant cell lines assembled into 3D spheroids we profiled gene expression and identified candidate mechanisms and biological pathways associated with cisplatin resistance. OVCAR-8 human ovarian carcinoma cells were exposed to sub-lethal concentrations of cisplatin to create a matched cisplatin-resistant cell line, OVCAR-8R. Genome-wide gene expression profiling of sensitive and resistant ovarian cancer spheroids identified 3,331 significantly differentially expressed probesets coding for 3,139 distinct protein-coding genes (Fc >2, FDR < 0.05) (S2 Table). Despite significant expression changes in some transporters including MDR1, cisplatin resistance was not associated with differences in intracellular cisplatin concentration. Cisplatin resistant cells were significantly enriched for a mesenchymal gene expression signature. OVCAR-8R resistance derived gene sets were significantly more biased to patients with shorter survival. From the most differentially expressed genes, we derived a 17-gene expression signature that identifies ovarian cancer patients with shorter overall survival in three independent datasets. We propose that the use of cisplatin resistant cell lines in 3D spheroid models is a viable approach to gain insight into resistance mechanisms relevant to ovarian tumors in patients. Our data support the emerging concept that ovarian cancers can acquire drug resistance through an epithelial-to-mesenchymal transition.     

谷氨酰胺合成酶产生菌的固定化研究

谷氨酰胺合成酶产生菌的固定化在酶法合成谷氨酰胺中的应用具有重要意义。实验首先从味精废水中筛选出谷氨酰胺合成酶高产菌株LNU018,然后分别用海藻酸钠、聚乙烯醇(PVA)为载体对谷氨酰胺合成酶高产菌棒杆菌进行固定化。探讨了固定化条件对固定化小球结构、机械强度、弹性、稳定性和培养后菌体的谷氨酰胺合成酶的活性情况的影响,分析确定最佳的固定化条件。研究结果表明,5%的海藻酸钠、11%的聚乙烯醇形成的固定化菌球大小合适,有弹性,但5%的海藻酸钠能更好的保持酶活性,比11%聚乙烯醇高16%,其为最佳的固定化条件。     

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(++).     

Crystallization of Glutamine Synthetase from Micrococcus glutamicus

For the defense mechanism against pathogenic bacteria in the digestive tracts of silkworm larvae reared on mulberry leaves, in vitro evidence of the formation of a true antibacterial substance was obtained. Caffeic acid (CA) derived from chlorogenic acid (ChA) was converted into caffeoquinone (CQ) by base-catalyzed oxidation in a buffer solution (pH 10.0). CQ was trapped as 6′- phenylsulfonylcaffeic acid (6′-sulfone) by the addition of benzenesulfinic acid (BSA).

The synergistc effects of amino compounds on the antibacterial activity of CQ are discussed in detail, and the probable reactions of CA with amino and thiol compounds in the alkaline solution are proposed.     

Molecular Basis of Differential Sensitivity of Myeloma Cells to Clinically Relevant Bolus Treatment with Bortezomib

The proteasome inhibitor bortezomib (Velcade) is prescribed for the treatment of multiple myeloma. Clinically achievable concentrations of bortezomib cause less than 85% inhibition of the chymotrypsin-like activity of the proteasome, but little attention has been paid as to whether in vitro studies are representative of this level of inhibition. Patients receive bortezomib as an intravenous or subcutaneous bolus injection, resulting in maximum proteasome inhibition within one hour followed by a gradual recovery of activity. In contrast, most in vitro studies use continuous treatment so that activity never recovers. Replacing continuous treatment with 1 h-pulse treatment increases differences in sensitivity in a panel of 7 multiple myeloma cell lines from 5.3-fold to 18-fold, and reveals that the more sensitive cell lines undergo apoptosis at faster rates. Clinically achievable inhibition of active sites was sufficient to induce cytotoxicity only in one cell line. At concentrations of bortezomib that produced similar inhibition of peptidase activities a different extent of inhibition of protein degradation was observed, providing an explanation for the differential sensitivity. The amount of protein degraded per number of active proteasomes correlated with sensitivity to bortezomib. Thus, (i) in vitro studies of proteasome inhibitors should be conducted at pharmacologically achievable concentrations and duration of treatment; (ii) a similar level of inhibition of active sites results in a different extent of inhibition of protein breakdown in different cell lines, and hence a difference in sensitivity.     

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.     

Phenotypic Changes Resulting from Distinct Point Mutations in the Azospirillum brasilense glnA Gene, Encoding Glutamine Synthetase

Sequencing the glnA genes of two chemically induced Azospirillum brasilense glutamine synthetase mutants revealed an Arg→Cys mutation, corresponding to the glutamate binding site, in one mutant and an Asp→Asn mutation, corresponding to the ammonium binding site, in the second mutant. The phenotypic changes in these mutants are discussed in relation to their genotypes.     

Tissue-Specific Distribution of Glutamine Synthetase in Potato Tubers

Cytosolic isoforms of the enzyme glutamine synthetase (GS) locatedin the phloem have been implicated in the mobilization of nitrogenfor intracellular transport in higher plants. The potato tuberrepresents an important reservoir of nitrogen and an approachwas made to the characterization of GS in this organ, particularlyat the stages of sprouting and of new tuber formation. By immunoblottingafter SDS-PAGE, and by immunological tissue printing, it waspossible to conclude that a cytosolic GS is present in tubersand sprouts, and that it is mainly expressed in the internalphloem, in a very precise tissue-specific pattern of distribution.These data provide additional clues to the interpretation ofthe functional role of GS in the mobilization of nitrogen andits utilization in growing parts of the plant. The importanceof morphological data and localization studies in complementingmolecular and biochemical work is emphasized. The proposed functionalimportance of the internal phloem inSolanum tuberosum organsis also reinforced. Solanum tuberosum L. cv. Desirée; potato; tubers; plant glutamine synthetase; tissue-specific distribution; phloem; nitrogen mobilization; in situlocalization; tissue printing     

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.     

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.     

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 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.     

Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L

Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined.

The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N₂-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides.

     

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.     

菠菜叶中存在两种谷氨酰胺合成酶同工酶

运用非变性聚丙稀酰胺凝胶电泳结合活性染色的方法,在菠菜(Spinacia oleracea L．)生长发育过程中,观察到叶片中至少存在2种谷氨酰胺合成酶(GS),其中一种GS的活性随发育进程而逐渐升高,而另一种GS的活性逐渐降低。在不同来源的成熟的菠菜叶片中同样观察到2种GS的存在。