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 (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

To understand more about the role of glutathione (GSH) in metabolism, we have cloned both cDNA and genomic sequences for mouse glutathione synthetase (GSH syn), the enzyme that catalyzes the last step in the synthesis of glutathione. The mouse cDNA contains an open reading frame (ORF) of 474 aa and shares 64 and 95% deduced amino acid sequence identity with Xenopus cDNA and rat cDNA, respectively. The cDNA complements Schizosaccaromyces pombe strains deficient in GSH syn. The gene is a single-copy gene spanning approximately 30 kb and is composed of at least 15 exons. Steady-state RNA levels and enzyme activity levels are highest in kidney, about 3-fold lower in liver, and 8- to 10-fold lower in lung and brain. We have identified six different GSH syn RNAs: three, termed types A1, A2, and A3, have different 5' ends that localize to different sites in the gene, but appear to encode the same protein (474 aa). Types B, C1, and C2 all have unique 5' ends and type-specific ORFs, which are shorter than that for types A1, A2, and A3. In liver only type A1 GSH syn RNA is detectable, while in kidney 90% of GSH syn RNA is type A1 and types B and C account for about 10%.
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PMID:A single mouse glutathione synthetase gene encodes six mRNAs with different 5' ends. 866 Jul 1

The present study demonstrates the activities of antioxidant and glutathione-associated enzymes and the level of glutathione in Wilms' tumour (nephroblastoma) samples after chemotherapy (mainly actinomycin D and vincristine). We observed higher activity of superoxide dismutase in Wilms' tumour compared to adjacent morphologically unchanged kidney. On the other hand, in this tumour lower activities of catalase and the glutathione-associated enzymes glutathione synthetase, gamma-glutamyl transpeptidase, glutathione reductase and total glutathione S-transferases (GST) were found. Using isoelectric focusing we separated different forms of GST in tested tissues and revealed lower activities of the basic enzymes in Wilms' tumour, which may be responsible for the decrease of total GST activity. Moreover, we found the acidic isoenzymes to be the predominant class of GST in nephroblastoma. In Wilms' tumours with unfavourable histology a high activity of these isoenzymes together with a high level of GSH were observed. We suggest that these parameters may participate in the known phenomenon of anticancer drug resistance of tumours with unfavourable histology.
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PMID:Antioxidant and glutathione-associated enzymes in Wilms' tumour after chemotherapy. 869 48

Eisai hyperbilirubinemic rats (EHBR) are mutant Sprague-Dawley rats that exhibit impaired biliary organic anion and reduced glutathione (GSH) secretion. In addition, liver GSH levels are twice that of age-matched controls. The mechanisms for the defect in biliary GSH secretion and the increase in cell GSH are not fully understood. We previously showed that canalicular membrane-enriched vesicles isolated from EHBR livers exhibited normal GSH transport. In the present study, we examined the steady-state rat canalicular reduced glutathione transporter (RcGshT) messenger RNA (mRNA) and protein levels, as well as the mechanisms for the increase in cell GSH. Both Northern and Western blot analyses of EHBR livers showed nearly identical RcGshT mRNA and polypeptide levels, respectively, as compared with controls. Treatment with phenobarbital, which increased steady-state RcGshT mRNA by five- to sixfold, RcGshT polypeptide, and biliary GSH secretion by onefold in controls, had a smaller effect on steady-state RcGshT-mRNA level in EHBR (by 1.5-fold) and did not increase RcGshT polypeptide or biliary GSH secretion. In examining possible mechanisms for increased liver GSH, both cysteine level and gamma-glutamylcysteine synthetase (GCS) activity were significantly higher than controls, while the activity of GSH synthetase was unchanged. Northern and Western blot analyses also showed increased steady-state GCS heavy subunit (GCS-HS) mRNA and polypeptide levels, respectively. In addition to liver, GSH levels in kidney, duodenal, jejunal, and ileal mucosa of EHBR were 200% to 300% of age-matched control rats. GCS activity was also increased in kidney cytosol of EHBR. Thus, the defect in biliary GSH secretion in EHBR most likely is either at the posttranslational level of RcGshT or in the inhibition exerted by retained endogenous organic anions. In addition, there is a widespread up-regulation of GSH synthesis capacity in the tissues of EHBR.
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PMID:Alterations in glutathione homeostasis in mutant Eisai hyperbilirubinemic rats. 870 71

The conversion of gamma-glutamylcysteinylethyl ester (gamma-GCE) to glutathione in a reduced form (GSH) was examined using isolated rat hepatocytes pretreated with diethylmaleate, a GSH-depletor. Incubation of hepatocytes with 0.1 and 5.0 mM gamma-GCE (gamma-GCE-hepatocytes) over a 30-min period resulted in time-dependent increases in intracellular GSH and nonprotein-SH (NP-SH) concentrations. Hepatocytes incubated with 5.0 mM but not 0.1 mM GSH over a period of 30 min showed a time-dependent increase in intracellular GSH concentration. In the gamma-GCE-hepatocytes pretreated with bis-(p-nitrophenyl)phosphate (BNPP), a non-specific esterase inhibitor, an enhancement of intracellular GSH concentration was markedly reduced. gamma-GCE concentration in the gamma-GCE-hepatocytes with BNPP pretreatment was significantly higher than that in the cells without BNPP pretreatment, although there was no difference in the total amount of intracellular NP-SH, i.e., gamma-GCE, GSH, gamma-glutamylcysteine, cysteine ethyl ester, and cysteine between both gamma-GCE-hepatocytes. The present results indicate that gamma-GCE is transported into liver cells more easily than GSH itself, resulting in its conversion to GSH via esterase and glutathione synthetase within the cells.
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PMID:Conversion of gamma-glutamylcysteinylethyl ester to glutathione in rat hepatocytes. 878 49

The crystal structure of glutathione synthetase from Escherichia coli B complexed with ADP, glutathione, and sulfate has been determined at 2.0 A resolution. Concerning the chemical similarity of sulfate and phosphate, this quaternary complex structure represents a pseudo enzyme-substrate complex in the reverse reaction and consequently allows us to understand the active site architecture of the E. coli glutathione synthetase. Two Mg2+ ions are coordinated with oxygen atoms from the alpha- and beta-phosphate groups of ADP and from the sulfate ion. The flexible loops, invisible in the unliganded or the binary and ternary complex structures, are fixed in the quaternary complex. The larger flexible loop (Ile226-Arg241) includes one turn of a 310-helix that comprises the binding site of the glycine moiety of GSH. The small loop (Gly164-Gly167) is involved in nucleotide binding and acts as a phosphate gripper. The side chains of Arg210 and Arg225 interact with the sulfate ion and the beta-phosphate moiety of ADP. Arg 210 is likely to interact with the carboxylate of the C-terminal gamma-glutamylcysteine in the substrate-binding form of the forward reaction. Other positively charged residues in the active site (Lys125 and Lys160) are involved in nucleotide binding, directing the phosphate groups to the right position for catalysis. Functional aspects of the active site architecture in the substrate-binding form are discussed.
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PMID:A pseudo-michaelis quaternary complex in the reverse reaction of a ligase: structure of Escherichia coli B glutathione synthetase complexed with ADP, glutathione, and sulfate at 2.0 A resolution. 881 Sep 1

5-Oxoprolinuria (pyroglutamic aciduria) resulting from glutathione synthetase (GSS) deficiency is an inherited autosomal recessive disorder characterized, in its severe form, by massive urinary excretion of 5-oxoproline, metabolic acidosis, haemolytic anaemia and central nervous system damage. The metabolic defect results in low GSH levels presumably with feedback over-stimulation of gamma-glutamylcysteine synthesis and its subsequent conversion to 5-oxoproline. In this study, we cloned and characterized the human GSS gene and examined three families with four cases of well-documented 5-oxoprolinuria. We identified seven mutations at the GSS locus on six alleles: one splice site mutation, two deletions and four missense mutations. Bacterial expression and yeast complementation assays of the cDNAs encoded by these alleles demonstrated their functional defects. We also characterized a fifth case, an homozygous missense mutation in the gene in an individual affected by a milder-form of the GSS deficiency, which is apparently restricted to erythrocytes and only associated with haemolytic anaemia. Our data provide the first molecular genetic analysis of 5-oxoprolinuria and demonstrate that GSS deficiency with oxoprolinuria and GSS deficiency without 5-oxoprolinuria are caused by mutations in the same gene.
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PMID:Mutations in the glutathione synthetase gene cause 5-oxoprolinuria. 889 73

Under pathological conditions, the induction of nitric oxide synthase (NOS) in macrophages is responsible for NO production to a cytotoxic concentration. We have investigated changes to, and the role of, intracellular glutathione in NO production by the activated murine macrophage cell line J774. Total glutathione concentrations (reduced, GSH, plus the disulphide, GSSG) were decreased to 45% of the control 48 h after cells were activated with bacterial lipopolysaccharide plus interferon gamma. This was accompanied by a decrease in the GSH/GSSG ratio from 12:1 to 2:1. The intracellular decrease was not accounted for by either GSH or GSSG efflux; on the contrary, rapid export of glutathione in control cells was abrogated during activation. The loss of intra- and extracellular glutathione indicates either a decrease in synthesis de novo, or an increase in utilization, rather than competition for available NADPH. All changes in activated cells were prevented by pretreatment with the NOS inhibitor L-N-(1-iminoethyl)ornithine. Basal glutathione levels in J774 cells were manipulated by pretreatment with (1) buthionine sulphoximine (glutathione synthase inhibitor), (2) acivicin (gamma-glutamyltranspeptidase inhibitor), (3) bromo-octane (glutathione S-transferase substrate) and (4) diamide/zinc (thiol oxidant and glutathione reductase inhibitor). All treatments significantly decreased the output of NO following activation. The degree of inhibition was dependent on (i) duration of treatment prior to activation, (ii) rate of depletion or subsequent recovery and (iii) thiol end product. The level of GSH did not significantly affect the production of NO, after induction of NOS. Thus, glutathione redox status appears to plays an important role in NOS induction during macrophage activation.
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PMID:Induction of nitric oxide synthesis in J774 cells lowers intracellular glutathione: effect of modulated glutathione redox status on nitric oxide synthase induction. 906 66

Astrocyte death from glucose deprivation appears to be mediated by free radicals. Reduced glutathione (GSH) was used as a measure of antioxidant defenses in primary cultures of cortical astrocytes. Glucose deprivation caused progressive, near complete loss of reduced glutathione (GSH). Astrocytes were protected by increasing endogenous GSH levels. Depletion of GSH to 21.4 +/- 3.3% of controls by the glutathione synthetase inhibitor buthionine sulfoximine resulted in more rapid injury by glucose deprivation, yet depletion of glutathione alone did not kill astrocytes. Both enhanced lipid peroxidation and membrane rigidification were caused by glucose deprivation, both indicators of oxidative damage. Membrane peroxidation was detected as a 24 +/- 2% decrease in cis-parinaric acid fluorescence, membrane rgidification as a 6.3 +/- 0.8% increase in fluorescence anisotropy using diphenylhexatriene. Glucose deprivation under normoxic conditions may occur clinically in patients such as diabetics. In addition, oxidative damage in the setting of energy depletion occurs with other insults, including ischemic brain injury. Glucose deprivation may thus be a clinically relevant model of hypoglycemic astrocyte injury, and may be useful to investigate the effects of glutathione and redox modulation on second messenger systems and gene regulation.
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PMID:Vulnerability to glucose deprivation injury correlates with glutathione levels in astrocytes. 906 56


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