Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

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

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

The effect of protoporphyrin (PP) administration on the activities of enzymes related to and/or involved in lipid peroxidation and on the content of reduced glutathione (GSH) was investigated in rat liver. PP, at an intravenous dose of 20 mg/kg, increased GSH content, caused a weak suppression of NADPH-cytochrome c reductase activity and a slight increase of gamma-glutamyl transpeptidase activity 24 h after dosing, but had no effect on the activities of other enzymes such as xanthine oxidase, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, gamma-glutamylcysteine synthetase or glutathione synthetase. Treatment of rats with diethyl maleate following PP injection resulted in the disappearance of antioxidative action of PP. Furthermore, sinusoidal, but not canalicular, efflux of hepatic GSH was decreased by the PP treatment. The increase of liver GSH content by PP treatment due to the decrease of sinusoidal efflux of GSH from the liver, thus would be involved in the exertion of antioxidative action of PP.
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PMID:Antioxidative effect of protoporphyrin and increase of glutathione in protoporphyrin-administered rat liver. 810 76

Glutathione (GSH) synthetase (EC 6.3.2.3) was purified from the fission yeast Schizosaccharomyces pombe L972h- and from the GSH synthetase deficient mutant MN101/pYS41, which harbors a plasmid containing the GSH synthetase gene of the fission yeast. GSH synthetase is expressed at 10 times higher the amount in MN101/pYS41 than in wild-type L972h-. The purified enzyme gave a single band on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (native PAGE). The molecular weight of this enzyme was determined to be 1.2 x 10(5) by Sepharose CL-6B gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that this enzyme was composed of two kinds of subunits, A (M(r) = 33 x 10(3)) and B (M(r) = 26 x 10(3)), and existed as a heterotetramer (A2B2). The enzyme purified from the wild-type fission yeast, which did not harbor the plasmid, showed the same electrophoretic mobilities on both native PAGE and SDS-PAGE and similar catalytic properties under standard conditions. This enzyme is most active at 45 degrees C and pH 8.0-8.5 with 20 mM Mg2+ + 10 mM ATP and 50 mM K+. The strict requirement for the monovalent cation is rather specific for the enzymes from yeasts. The presence of sugar components in the enzyme is also observed, similar to that in the rat kidney enzyme.
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PMID:Glutathione synthetase from the fission yeast. Purification and its unique heteromeric subunit structure. 819 97

Glutathione (GSH) was shown to regulate the generation of IL-2-dependent activated killer cells. Generation of alpha CD3-activated killer cells CD3-AK was regulated by both IL-2 and IL-4. In the present study the role of GSH in the regulation of IL-4-dependent CD3-AK cells was examined. After initial activation of mouse splenocytes by alpha CD3, subculturing the CD3-AK cells in IL-4 resulted in the production of IL-4-dependent killer cells whose proliferative and cytolytic activities were abrogated by alpha IL-4 antibody 11B11. Adding graded doses of BSO, a GSH synthetase inhibitor, into CD3-AK cells culturing in IL-4 resulted in the reduction of their proliferative and cytotoxic responses. Adding exogenous GSH reversed the inhibitory effect of BSO and restored the proliferation and cytolytic activity of IL-4-dependent CD3-AK cells. The dose requirement for BSO to affect the IL-4-dependent CD3-AK cells was similar to that for the IL-2-dependent CD3-AK cells. These findings indicate that GSH also regulates the function of IL-4 in the activation and differentiation of CD3-AK cells. To further study the mechanism for the GSH regulation of the cytolytic activity of CD3-AK cells, we found that BSO did not reduce the production of BLT-esterase which contained mostly the cytolytic granules; in fact, BLT-esterase production was often increased by BSO. Furthermore, the exocytosis and effector function of cytolytic granules were also not affected by BSO. Thus it appears that reduction of cellular GSH may result in the accumulation of defective cytolytic granules which accounts for the reduction of killer cell cytolytic activity.
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PMID:Regulation by glutathione of the activation and differentiation of IL-4-dependent activated killer cells. 833 Mar 19

A fluorimetric technique previously described for other tissues has been applied to determine levels of glutathione and its synthetic rates in ocular tissues of Hartley guinea-pigs. Monochlorobimane forms a stable, fluorescent adduct with glutathione in a reaction catalyzed by glutathione-S-transferase. The fluorescent signal recorded over time is directly proportional to the synthetic rate of glutathione. Lens, cornea and retina were homogenized and cytosolic fractions dialyzed overnight to deplete endogeneous glutathione. Glutathione synthetic rates were determined from a mixture of glutathione precursors and co-factors, viz. cysteine+dithiothreitol, glutamate+glycine, ATP and Mg++ in the presence of monochlorobimane. The mixture was supplemented with glutathione-S-transferase to catalyze the formation of the fluorescent adduct. Glutathione synthetic rates were determined in the absence and presence of buthionine sulfoximine, an inhibitor of gamma-glutamyl cysteine synthetase. The difference in fluorescence change over time in the presence and absence of buthionine sulfoximine was used to estimate glutathione synthesis. Basal levels of glutathione in pre-dialyzed cytosolic fractions of the lens, cornea, and retina were 21.8 +/- 2.2, 36.5 +/- 4.1 and 38.6 +/- 2.8 nmol mg-1 protein, respectively. The maximal glutathione synthetic rates in these tissues were 0.52 +/- 0.04, 2.25 +/- 0.67 and 3.35 +/- 0.65 nmol min-1 mg-1 protein, respectively. When gamma-glutamyl cysteine is used as a precursor instead of cysteine, the glutathione synthetase activities from lenses and retinas were 0.19 +/- 0.08 and 1.54 +/- 0.76 nmol-1 min mg-1 protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A simple technique to determine glutathione (GSH) levels and synthesis in ocular tissues as GSH-bimane adduct: application to normal and galactosemic guinea-pigs. 843 34

Monocrotaline (MONO), a pyrrolizidine alkaloid, causes veno-occlusive disease of the liver, pulmonary arterial hypertension, and right ventricular hypertrophy. Toxicity is due to the hepatic formation of a pyrolic metabolite that can be detoxified by conjugation with glutathione (GSH). We have shown that the GSH content of the liver affects the quantity of the pyrrolic metabolite that is released from the liver. We have now examined whether MONO, in turn, affects GSH metabolism. Twenty-four hours after administration of MONO to rats (65 mg/kg, i.p.), the highest concentration of bound pyrrolic metabolites was found in the liver, followed by the lung and kidney. Heart and brain contained lower concentrations of these metabolites. Significantly higher levels of GSH were found in liver and lungs of MONO-treated rats than in saline-injected control animals. In the liver, activities of the following enzymes were elevated: gamma-glutamylcysteine synthetase, GSH synthetase, gamma-glutamyl transpeptidase, dipeptidase, and microsomal GSH transferase. The same changes were seen in the lung. In the heart, gamma-glutamyl transpeptidase activity was decreased markedly, and cytosolic GSH transferase activity was elevated. In the kidney, the activities of GSH synthetase, gamma-glutamyl transpeptidase, and cytosolic GSH transferase were increased. Our results establish a mutual interaction of MONO and sulfur metabolism. It appears that an early metabolic action of MONO is to modify sulfur amino acid metabolism, diverting cysteine metabolism from oxidation to taurine towards synthesis of GSH.
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PMID:Effects of monocrotaline, a pyrrolizidine alkaloid, on glutathione metabolism in the rat. 857 5

Glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH) plays an important role in the protection of plants against various types of stress caused by reactive oxygen species, gazeous pollutants, heavy metals and xenobiotics. A cDNA fragment containing the entire coding unit for glutathione synthetase (GSH2) of Arabidopsis thaliana was cloned by complementation of the methylglyoxal sensitivity of a gsh2 mutant of the yeast Saccharomyces cerevisiae. The cDNA encodes a protein of 478 amino acids (deduced Mr: 53783), bearing clear sequence similarities to GSH2 products from frog embryos (Xenopus laevis), rat kidney (Rattus norvegicus) and from the fission yeast (Schizosaccharomyces pombe). A highly conserved glycine-rich domain close to the carboxy-terminus was found in the GSH2 product and appears to be typical for eukaryotic glutathione synthetases. The Mr is similar to those of soluble animal enzymes, suggesting that the Arabidopsis gene also codes for a cytosolic protein. Genomic DNA-blot analysis indicates the presence of a single GSH2 gene. The yeast gsh2 mutant becomes resistant to methylglyoxal and cadmium after transformation with the plasmid bearing the Arabidopsis GSH2 cDNA. Moreover, this increased resistance is correlated to the restoration of GSH content from below detectability in mutants to about 50% of the wild-type levels in transformed cells.
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PMID:Cloning of Arabidopsis thaliana glutathione synthetase (GSH2) by functional complementation of a yeast gsh2 mutant. 861 43

Three unrelated Japanese patients with chronic nonspherocytic hemolytic anemia wer found to have marked deficiency of red blood cell (RBC) reduced glutathoine (GSH) (4.4%, 13.1%, and 6.9% of normal, respectively). A panel of RBC enzyme assays showed that one patient had decreased glutathione synthetase activity and the other two were moderately deficient in gamma-glutamylcystine synthetase. Some family members of each patient showed mild deficiency of the respective enzymes. RBCs of these patients also showed a decreased level of glutathione-S-transferase as in previously described GSH-deficient cases. Hemolytic anemia was their only manifestation, and neither 5-oxoprolinemia nor 5-oxoprolinuria, which are usually associated with to generalized type of glutathione synthetase deficiency, was noted in our patients.
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PMID:Three cases of hereditary nonspherocytic hemolytic anemia associated with red blood cell glutathione deficiency. 863 59


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