Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.3.2.3 (glutathione synthetase)
 678 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A flow cytometric method to determine cellular GSH contents has been developed. This method is fast and simple and enables the determination of GSH contents in intact cells. Results obtained with the new method correlate well with the results obtained by a specific biochemical assay for GSH (r = 0.9984; n = 7). The method has been used to determine GSH recovery rates in cultured fibroblasts from healthy subjects and from patients with Werner's syndrome, Spielmeyer-Vogt syndrome and Fanconi's anemia. No obvious differences in GSH recovery rates were observed. GSH recovery rates were also not affected after in vitro ageing. Experiments with cells deficient in GSH synthetase revealed that the observed GSH recovery is exclusively due to de novo synthesis.
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PMID:De novo synthesis of glutathione in human fibroblasts during in vitro ageing and in some metabolic diseases as measured by a flow cytometric method. 375 23

Cumene hydroperoxide (Chp) and 4-hydroxynonenal (HNE) were used to investigate the effect of peroxidative challenge upon the glutathione (GSH) metabolism of human skin fibroblasts. Cellular GSH contents decreased during short-term incubations with Chp and oxidised glutathione (GSSG) was formed concomitantly. During longer incubations the GSH level was restored and the substrate flux through the pentose phosphate shunt increased. So in the presence of hydroperoxides the GSH level is maintained by reduction of GSSG. HNE caused a strong decrease in cellular GSH contents. Prolonged incubation with HNE lead to a rise in GSH contents above the basal level. The flux through the pentose phosphate shunt did not change during exposure to HNE. Hence, during incubation with HNE the cell maintains its GSH content by de novo synthesis of GSH. This conclusion is further substantiated by the findings with a cell strain deficient in GSH synthetase. These cells survived if incubated with Chp but not if exposed to HNE. GSH contents of normal cells from phase II (young) cultures and from phase III (aged) cultures responded similarly to Chp during short-term incubations and during a week of culture with the test compound. The flux through the pentose phosphate shunt rose much more in phase III than in phase II cells when incubated with the same concentration series of Chp. We conclude that during in vitro ageing the amount of NADPH needed to maintain cellular GSH levels in the presence of hydroperoxides increases, while the capacity to respond to such a challenge is not affected.
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PMID:Influence of cumene hydroperoxide and 4-hydroxynonenal on the glutathione metabolism during in vitro ageing of human skin fibroblasts. 380 87

Human fetal and adult liver were found to have similar concentrations of acid soluble sulfhydryl (SH) groups (7.4 mmol/kg) in the same range as is found in adult mouse and rat liver. The concentration was 4-fold higher than in human fetal adrenal gland tissue. Methods specific for glutathione (GSH) associated SH groups revealed that the postmortem levels of GSH is very low (0.4 mmol/kg) in relation to total SH groups. In contrast, the levels of cysteine were high (2.8 mmol/kg), indicating a rapid cleavage of GSH. Only negligible amounts of gamma-glutamylcysteine and cysteinylglycine were measured. Our findings may be explained by high fetal activity of gamma-glutamyl transpeptidase (which metabolizes GSH) that has been documented previously both in man and in experimental animals. High activities of the two GSH-synthesizing enzymes, gamma-glutamylcysteine synthetase and GSH synthetase were found in the human fetal liver (7.1 and 3.0 mukat/kg, respectively). The activities of these enzymes were in the same range as in human adult liver, whereas that of gamma-glutamyl transpeptidase was 3-fold higher in the fetal liver. Our results demonstrate the presence of high concentration of SH groups and capacity to synthesize GSH already in the first and second trimester of the human fetal gestation. This has more than theoretical interest, since we assume that the SH groups (GSH) have importance for the protection of the fetus against drugs and foreign compounds and their (toxic) metabolites, the formation of which is catalyzed by the fetus itself.
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PMID:Glutathione and gamma-glutamyl cycle enzymes in human fetal liver. 611 63

The formic acid extracts of several glutathione-deficient strains of Escherichia coli have been assayed for the presence of the mixed disulfide of CoA and glutathione, CoASSG. Strains deficient in gamma-glutamyl-cysteine synthase (EC 6.3.2.2) produced only CoA dimer. Strains deficient in glutathione synthase (EC 6.3.2.3) produced the mixed disulfide of CoA and the gamma-glutamylcysteine dipeptide. The pool size of total CoA in the cell did not change significantly even in the absence of glutathione.
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PMID:Effect of glutathione deficiency on the pool of CoA-glutathione mixed disulfide in Escherichia coli. 611 90

The yield and rejoining of single-strand DNA breaks (ssb) was investigated after irradiation of cells which were deficient in glutathione (GSH) either due to a genetic defect of their GSH synthetase activity, or inhibition of gamma-glutamylcysteine synthetase activity by DL-buthionine-SR-sulfoximine (BSO). The results were concordant in indicating that decreased cellular GSH content is associated with an increased yield of ssb after anoxic, but not after aerobic radiation exposures. Rejoining of ssb was delayed and incomplete during a one hour's incubation period after oxic, but not after anoxic exposure of GSH-deficient cells. The defective rejoining capacity of these cells was restituted to nearly normal by the admixture of GSH-proficient cells in the incubation medium.
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PMID:Glutathione-dependent yield and repair of single-strand DNA breaks in irradiated cells. 642 3

Many studies have established the role of the glutathione S-transferases (GSTs) and glutathione (GSH) in the neoplastic process and the drug resistance of tumor. Using isoelectric focusing we separated different forms of GSTs in 28 renal cell carcinomas (RCCs) and in morphologically unchanged adjacent kidney. In addition we determined in RCCs and adjacent kidney the level of GSH and the activities of enzymes participating in synthesis and uptake of this thiol compound. We found higher activity of acidic GSTs and higher level of GSH in RCCs versus kidney. Therefore we suggest that both parameters may play the significant role in the well known phenomenon of intrinsic cytostatic drug resistance of RCC. We also observed the elevation of GSH synthetase activity in tumor tissues in comparison to the kidneys. It may indicate that GSH synthetase, catalysing the final step in GSH synthesis, may participate in the elevation of GSH concentration in RCCs. In this work we also compared the tested parameters in RCCs in relation to the size and local extent of primary tumor (T). We found significantly lower activity of gamma-glutamyl transpeptidase (GGT) as well as GSH synthetase in the group of T3 and T4 tumors than in T2 tumors. However, no substantial differences in GSH concentrations were observed between these distinguished groups.
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PMID:Glutathione S-transferase isoenzymes and glutathione in renal cell carcinoma and kidney tissue. 765 81

Glutathione (GSH) synthetase [gamma-L-glutamyl-L-cysteine:glycine ligase (ADP-forming), EC 6.3.2.3], an enzyme present in almost all cells, catalyzes the ATP-dependent synthesis of GSH from gamma-L-glutamyl-L-cysteine and glycine. Highly purified preparations of the enzyme have been obtained from rat kidney and several lower forms. The rat kidney enzyme (M(r), 118,000), which contains approximately 2% carbohydrate, is composed of two apparently identical subunits. The cDNA encoding rat kidney GSH synthetase was isolated from a rat kidney lambda gt11 cDNA library by immunoscreening with an antibody prepared against the isolated enzyme. The cDNA contains 1905 nucleotides and an open reading frame of 1422 nucleotides coding for 474 amino acids. The cDNA has a 3' untranslated region of 439 nucleotides, which includes a poly(A) tail. The deduced amino acid sequence (M(r), 52,344) contains all five of the peptide sequences that were independently determined by Edman degradation. The cDNA was expressed in Escherichia coli. The amino acid sequence of the rat kidney enzyme has no significant similarity to that of the enzyme from E. coli and shows some similarity to those deduced for the yeast and frog enzymes. Knowledge of this amino acid sequence is expected to facilitate elucidation of the sequence of the corresponding human enzyme and to lead to studies on the biochemical mechanisms involved in human GSH synthetase deficiency as well as to development of improved methods for prenatal diagnosis of these inborn diseases.
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PMID:Amino acid sequence of rat kidney glutathione synthetase. 786 66

GSH, GSSG, vitamin E, and ascorbate were measured in 14-day cultures of chick astrocytes and neurons and compared with levels in the forebrains of chick embryos of comparable age. Activities of enzymes involved in GSH metabolism were also measured. These included gamma-glutamylcysteine synthetase, GSH synthetase, gamma-glutamyl cyclotransferase, gamma-glutamyltranspeptidase, glutathione transferase (GST), GSH peroxidase, and GSSG reductase. The concentration of lipid-soluble vitamin E in the cultured neurons was found to be comparable with that in the forebrain. On the other hand, the concentration of vitamin E in the astrocytes was significantly greater in the cultured astrocytes than in the neurons, suggesting that the astrocytes are able to accumulate exogenous vitamin E more extensively than neurons. The concentrations of major fatty acids were higher in the cell membranes of cultured neurons than those in the astrocytes. Ascorbate was not detected in cultured cells although the chick forebrains contained appreciable levels of this antioxidant. GSH, total glutathione (i.e., GSH and GSSG), and GST activity were much higher in cultured astrocytes than in neurons. gamma-Glutamylcysteine synthetase activity was higher in the cultured astrocytes than in the cultured neurons. GSH reductase and GSH peroxidase activities were roughly comparable in cultured astrocytes and neurons. The high levels of GSH and GST in cultured astrocytes appears to reflect the situation in vivo. The data suggest that astrocytes are resistant to reactive oxygen species (and potentially toxic xenobiotics) and may play a protective role in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Vitamin E, ascorbate, glutathione, glutathione disulfide, and enzymes of glutathione metabolism in cultures of chick astrocytes and neurons: evidence that astrocytes play an important role in antioxidative processes in the brain. 790 54

Prenatal diagnosis for glutathione synthase (EC 6.3.2.3) deficiency in two pregnancies of an at-risk couple was performed on amniotic fluid taken at 16 weeks' gestation. 5-Oxoproline (pyroglutamic acid) levels were 970 and 790 mumol/l compared with the normal mean value of 29 mumol/l (range 13-51 mumol/l). The pregnancies were terminated and the diagnosis in one case was subsequently confirmed by assay of glutathione synthase in cultured fetal fibroblasts. In the other, post-mortem tissue samples failed to grow.
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PMID:Prenatal diagnosis of glutathione synthase deficiency. 793 85

Glutathione (GSH), an important physiological antioxidant, is synthesized de novo by the sequential reactions of gamma-glutamylcysteine synthetase (gamma GCS) and GSH synthetase. In the present studies, incubation with the quinones 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) and menadione (MQ), which generate superoxide and hydrogen peroxide, was used to investigate GSH synthesis in bovine pulmonary artery endothelial cells under oxidative stress. MQ can also cause initial depletion of GSH through conjugation, whereas DMNQ cannot. during continuous exposure to DMNQ (5 or 10 microM), elevation of GSH by DMNQ started after 6 h, almost doubled after 24 h, and remained at this level to 48 h. The elevation of GSH by DMNQ was mostly in the reduced form, and the ratio of reduced to oxidized glutathione remained unchanged for the first 24 h. Treatment with MQ (25 or 50 microM) for 30 min caused a significant decrease in GSH and total glutathione. After changing the medium to remove any residual MQ, GSH content doubled during the next 12 h. The enzymatic activity of gamma GCS, the rate-limiting enzyme of GSH biosynthesis, increased twofold after 12 h of exposure of cells to either 5 microM DMNQ or 50 microM MQ. Both DMNQ and MQ treatment caused concentration- and time-dependent increases in gamma GCS-mRNA expression. The elevation of gamma GCS-mRNA content by DMNQ for 12 h was completely blocked by coincubation with 0.05 microgram/ml actinomycin D but not 0.5 microgram/ml cycloheximide, suggesting the elevation of gamma GCS-mRNA content occurred through increased transcription. Our results suggest that increased de novo GSH synthesis, mediated by an elevation in gamma GCS, constitutes an adaptive response to oxidative stress.
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PMID:gamma-Glutamylcysteine synthetase and GSH increase in quinone-induced oxidative stress in BPAEC. 794 45


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