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)

Blood samples from 722 unrelated patients with anemia and/or reticulocytosis were submitted to our laboratory for red cell enzyme assay during the past 7 years. Among these 722 cases, we found 82 cases of 7 different red cell enzyme deficiencies and 2 of unstable hemoglobin. Abnormalities of pyruvate kinase (PK) were found to cause hemolysis in 55 patients. Although their average PK activity was about 35% of the normal level, 5 showed normal and 2 demonstrated high PK activity. Among 17 patients in whom pyruvate kinase assays or screening tests had been carried out in routine laboratories, the correct diagnoses had been made in only 4. Glucose-6-phosphate dehydrogenase (G6PD) deficiency was found in 15 patients, pyrimidine 5'-nucleotidase deficiency in 5, glucose phosphate isomerase deficiency in 3, adenylate kinase deficiency in 2, phosphoglycerate kinase deficiency in 1, and glutathione synthetase deficiency in 1 patient. Even after we performed a panel of over 20 different red cell enzyme assays, 519 patients still remained undiagnosed.
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PMID:Enzymatic diagnosis in non-spherocytic hemolytic anemia. 335 12

Ethylene dichloride (EDC) is metabolized by two competing pathways both of which consume glutathione (GSH). EDC undergoes oxidation to form chloroacetaldehyde (CAA) which is detoxified by GSH and also reacts directly with GSH to form 2-(s-chloroethyl)-GSH. A physiological pharmacokinetic model developed for EDC was extended to describe tissue GSH turnover and its depletion after EDC exposures. This GSH model was necessary to keep track of GSH concentrations with time, as EDC metabolism is affected by GSH status. Reactions of GSH with EDC and GSH with CAA were defined as second-order. Steady-state GSH formation was modeled as zero-order and GSH loss as first-order. GSH rebound effects after its depletion were controlled by a GSH synthetase reaction, which allowed time- and GSH concentration-dependent feedback for increased GSH resynthesis. The model was developed for liver GSH in the rat and was extrapolated to include the lung. Allometric scaling was used to extrapolate the model to other animal species. Experimental observations in the rat and mouse were consistent with model predictions.
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PMID:Physiological model for tissue glutathione depletion and increased resynthesis after ethylene dichloride exposure. 336 6

Evaluation of idiosyncratic drug reactions in predisposed individuals is limited by ethical concerns arising from rechallenge with the suspected offending agent. A previously developed in vitro method using human lymphocytes and a murine microsomal drug metabolizing system has been used to examine toxicity due to acetaminophen (APAP), sulfonamide antibiotics and aromatic anticonvulsants. An improved method is described in which toxic APAP metabolites are generated by a purified and reconstituted cytochrome P-450 system, minimizing the amount of exogenous detoxification enzymes in the assay. Toxicity is assessed by an objective, automated method based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to an insoluble purple formazan by the mitochondria of viable cells and correlates with that based on trypan blue exclusion. Toxicity required cytochrome P-450 and NADPH, and was inhibited by SKF 525A. Exogenous glutathione also decreased toxicity in a concentration-dependent manner. Lymphocytes from a glutathione synthetase-deficient patient exhibited markedly enhanced toxicity to APAP exceeding the 95% CL of 10 control subjects over a concentration range of 10 to 1000 micrograms/ml. The data are consistent with the generation of cytochrome P-450-dependent reactive metabolites which subsequently can be detoxified by glutathione. This method allows one to address specifically individual differences in detoxification pathways. The use of an automated assessment of cell viability may prove useful in preclinical screening of new compounds for their propensity to cause "idiosyncratic" drug reactions in a predisposed population.
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PMID:Drug metabolite toxicity assessed in human lymphocytes with a purified, reconstituted cytochrome P-450 system. 338 48

Perchloric acid extracts of LLC-PK1/Cl4 cells, a renal epithelial cell line, incubated with either [2-13C]glycine L-[3-13C]alanine, or D,L-[3-13C]aspartic acid were investigated by 13C-NMR spectroscopy. All amino acids, except labelled glycine, gave rise to glycolytic products and tricarboxylic acid cycle (TCA) intermediates. For the first time we also observed activity of gamma-glutamyltransferase activity and glutathione synthetase activity in LLC-PK1 cells, as is evident from enrichment of reduced glutathione. Time courses showed that only 6% of the labelled glycine was utilized in 30 min, whereas 31% of L-alanine and 60% of L-aspartic acid was utilized during the same period. 13C-NMR was also shown to be a useful tool for the determination of amino acid uptake in LLC-PK1 cells. These uptake experiments indicated that glycine, alanine and aspartic acid are transported into Cl4 cells via a sodium-dependent process. From the relative enrichment of the glutamate carbons, we calculated the activity of pyruvate dehydrogenase to be about 61% when labelled L-alanine was the only carbon source for LLC-PK1/Cl4 cells. Experiments with labelled D,L-aspartic, however, showed that about 40% of C-3-enriched oxaloacetate (arising from a de-amination of aspartic acid) reached the pyruvate pool.
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PMID:A 13C-NMR study on the influxes into the tricarboxylic acid cycle of a renal epithelial cell line, LLC-PK1/Cl4: the metabolism of [2-13C]glycine, L-[3-13C]alanine and L-[3-13C]aspartic acid in renal epithelial cells. 340 8

In order to examine the role of cellular glutathione (GSH) in the in vitro aging of human diploid fibroblasts, we studied the effects of manipulated cellular GSH levels on their in vitro life span. An increase in cellular GSH level was produced by the addition of N-acetylcysteine (NAC), a carrier of cysteine across cell membranes, into the culture medium, while a decrease in GSH level was produced by the addition of L-buthionine-(R,S)-sulfoximine (BSO), a specific inhibitor of GSH synthetase. When the cells were serially subcultivated in a medium containing NAC or BSO, their life spans were markedly extended or shortened, respectively, in comparison to the life span of cells grown in a control medium. These results suggest that the cellular GSH level is a determinant of the in vitro life span of human diploid cells.
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PMID:Relationships between the cellular glutathione level and in vitro life span of human diploid fibroblasts. 340 54

The activity and the kinetic properties of glutathione synthetase and the concentrations of non-protein bound thiols of the gamma-glutamyl cycle were measured in 11 human fibroblast cell strains. Six of these strains were derived from patients suffering from 5-oxoprolinuria, a recessive genetic disease characterized by a deficiency in glutathione synthetase; the other cell strains were derived from healthy heterozygous or homozygous relatives of the patients. The glutathione synthetase activities of homozygous deficient strains were 1/3 of control values while those of heterozygous strains were 2/3 of control values. The total thiol concentration was lower in only 3 of the 6 deficient homozygotes and that of glutathione (GSH) was lower in only 4 of the 6 deficient homozygotes. This lower GSH level was at least partly offset by an accumulation of gamma-glutamylcysteine, a precursor of GSH, which is almost completely absent from control cells. The total quantities of thiols and GSH in plateau phase cells were about 50% and 30% respectively of the levels in growth phase cells. Approximately 80% of the GSH was in the reduced form in both quiescent and growing cells.
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PMID:Low molecular weight thiol content in glutathione synthetase-deficient human fibroblasts. 343 51

The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect.
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PMID:Radioprotective effect of cysteamine in glutathione synthetase-deficient cells. 348 71

Using a human fibroblast strain deficient in glutathione synthetase and a related proficient control strain, the role of glutathione (GSH) in repair of potentially lethal damage (PLD) has been investigated in determining survival by plating cells immediately or 24 h after irradiation. After oxic or hypoxic irradiation, both cell strains repair radiation-induced damage. However, under hypoxic conditions, the proficient cells repair PLD as well as under oxic conditions while the deficient cells repair less PLD after irradiation under hypoxic than under oxic conditions. Therefore, the oxygen enhancement ratio (o.e.r.) for proficient cells is similar whether the cells are plated immediately or 24 h later (2.0 and 2.13, respectively). In contrast, the o.e.r. for deficient cells is lower when the cells are plated 24 h after irradiation than when they are plated immediately thereafter (1.16 as compared to 1.55). The results indicate that GSH is involved in PLD repair and, in particular, in the repair of damage induced by radiation delivered under hypoxic conditions.
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PMID:Reduced repair of potentially lethal radiation damage in glutathione synthetase-deficient human fibroblasts after X-irradiation. 348 89

Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements. The effects of ligands of dihydrofolate reductase on the reaction of E. coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase. The E. coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim. Methotrexate was studied in detail and proved to bind to an ATP binding site of the E. coli B enzyme with K1 value of 0.1 mM. The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase. These analyses would indicate that the homologous portion of the amino acid sequence of the E. coli B enzyme provides the ATP binding site. This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.
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PMID:Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site. 355 73

A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques has been immobilized in a carrageenan matrix and used for the synthesis of various types of isotopically labeled glutathione (L-gamma-glutamyl-L-cysteinyl-glycine) (K. Murata, W. A. Abbott, R. J. Bridges, and A. Meister (1985) Anal. Biochem. 150, 235-237). In the present work, this E. coli matrix was used as the basis of a method for the synthesis of glutathione analogs. Thus, amino acid analogs were used in place of the corresponding amino acid constituents of glutathione (e.g., 4-fluoroglutamate was substituted for glutamate) in the reaction mixtures. Using this method we have synthesized several analogs of glutathione including L-gamma-glutamyl-(beta-chloro)-L-alanyl-glycine, (R,S)-4-fluoro-DL-gamma-glutamyl-L-cysteinyl-glycine, D-gamma-glutamyl-L-cysteinyl-glycine, and L-gamma-glutamyl-L-homocysteinyl-glycine. This method may also be used for the synthesis of a number of L- and D-gamma-glutamyl amino acids. The analogs are purified by gel-filtration and ion-exchange chromatography. The analogs are used to examine the substrate specificity and mechanisms of action of glutathione-utilizing enzymes and for studies on glutathione metabolism and function. Fluorine-containing analogs may be used for NMR studies. The enzymatically prepared compounds may also be used as intermediates in the chemical synthesis of other analogs of glutathione and glutathione disulfide.
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PMID:Enzymatic synthesis of novel glutathione analogs. 355 55


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