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

The gene for the large subunit of glutathione synthetase (EC 6.3.2.3) of Schizosaccharomyces pombe was cloned from a S. pombe genomic DNA library by complementation of cadmium hypersensitivity of a glutathione synthetase deficient mutant of S. pombe. A long open reading frame was found in the cloned DNA sequence. Amino acid sequence predicted from the long open reading frame coincided with amino acid sequences of peptides obtained by V8 protease digestion of the large subunit of the purified glutathione synthetase. The glutathione synthetase deficient mutant which harbored plasmids containing the glutathione synthetase large subunit gene exhibited glutathione synthetase activity higher than the activity in the wild type strain, though the plasmid did not contain the gene for the small subunit of the enzyme.
 ...
PMID:Cloning and sequencing of the gene encoding the large subunit of glutathione synthetase of Schizosaccharomyces pombe. 195 12

A strain of Escherichia coli, enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase activities by recombinant DNA techniques, is more resistant to the lethal effects of gamma-irradiation than is the corresponding wild strain. Although the gene-enriched strain has higher glutathione levels than the wild strain, the observed radioresistance appears to be associated with the increased capacity of the gene-enriched strain to synthesize glutathione when irradiated rather than to the cellular levels of glutathione per se. Thus, resistance was abolished in the presence of buthionine sulfoximine, a selective inactivator of gamma-glutamylcysteine synthetase that decreases glutathione synthesis but that does not act directly to lower cellular glutathione levels. Conclusions drawn from studies on this E. coli model system may have relevance to protection of mammalian cells by glutathione.
 ...
PMID:Increased capacity for glutathione synthesis enhances resistance to radiation in Escherichia coli: a possible model for mammalian cell protection. 256 2

The Escherichia coli structural gene for glutathione synthetase, gshB, was cloned into pBR322. Plasmids containing gshB were able to complement the glutathione requirement of a trxA gshB double mutant, and cells containing the plasmids were found to have elevated levels of glutathione synthetase. A mutant gshB allele was constructed by inserting the kan gene from pUC4K into a unique HpaI site located within gshB. The resulting plasmid-encoded allele was used to replace a genomic gshB+ by homologous recombination. The resulting strain had no detectable glutathione synthetase activity. The gshB allele containing the kan insertion was used to map gshB on the E. coli chromosome by P1 transduction. The results indicated that gshB is located at 63.4 min, between metK and speC. The allele was further localized to a region of 3,100 to 3,120 kilobase pairs on the physical map (restriction map) of E. coli by DNA-DNA hybridization to a series of lambda bacteriophages (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987).
 ...
PMID:In vitro construction of gshB::kan in Escherichia coli and use of gshB::kan in mapping the gshB locus. 267 Sep 10

A procedure for synthesis of glutathione selectivity labeled with isotopes is described. A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques is immobilized in a carrageenan matrix and treated with toluene to render the cells more permeable to the substrates. The immobilized cell matrix is incubated with a mixture containing the appropriately labeled amino acid, the other amino acid constituents of glutathione, ATP, and acetylphosphate. The radiolabeled product is isolated by column chromatography.
 ...
PMID:Glutathione specifically labeled with isotopes. 286 14

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.
 ...
PMID:Glutathione metabolism in normal and cystinotic fibroblasts. 288 73

Two modes of killing of Escherichia coli by hydrogen peroxide can be distinguished. Mode-one killing is maximal at 1-2 mM; at higher concentrations the killing rate is approximately half-maximal and is independent of H2O2 concentration but first order with respect to exposure time. Mutagenesis and induction of a phage lambda lysogen are similarly affected by H2O2 concentration, with reduced levels of response above 1-2 mM-H2O2. Mutagenesis is not affected by inactivation of umuC. Mode-one killing requires active metabolism during the H2O2 challenge and it results in sfiA-independent filamentation of both cells that survive and those that are killed by the challenge. This mode of killing is enhanced in xth, polA, recA and recB strains; however, it is unaffected by mutations in the nth, uvrA, uvrB, uvrC, uvrD, rep, gyrA, htpR and rel loci. Mode-one killing is normal in strains totally lacking catalase activity (katE, katG), glutathione reductase (gor) or glutathione synthetase (gshB), but enhanced in a strain lacking NADH dehydrogenase (ndh). Mode-one killing is accelerated by the presence of CN- or by an unidentified function that is induced by anoxic growth and is under the control of the fnr locus. A strain carrying both xth and recA mutations and certain polA mutants appear to undergo spontaneous mode-one killing only under aerobic conditions. Taken together, these observations imply that mode-one killing results from DNA damage that normally occurs at a low, non-lethal level during aerobic growth. Models for the resistance to mode-one killing at dose above 1-2 mM-H2O2 will be discussed. Mode-two killing occurs at high concentrations of H2O2 and longer times. It does not require active metabolism, and cells that are killed do not filament, although survivors demonstrate a dose-dependent growth lag followed by a period of filamentation. Mode-two killing is accompanied by enhanced mutagenesis, but strains with DNA repair defects were not observed to be especially sensitive to this mode of killing.
 ...
PMID:Toxicity, mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide. 330 21

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.
 ...
PMID:Enzymatic synthesis of novel glutathione analogs. 355 55

The enzymatic production of glutathione (GSH) has been studied in a bioreactor system using toluene-treated cells of Escherichia coli B transformed with recombinant plasmids for gamma-glutamylcysteine synthetase (GSH-I) and glutathione synthetase (GSH-II). As reported previously the genes for both enzymes were separately cloned onto vector plasmid pBR322. The plasmid for GSH-I was designated pGS100-2 and that for GSH-II as pGS200. The effect on GSH production in the bioreactor system, containing an ATP regenerating system, of using cells containing various hybrid plasmids has now been explored. Three kinds of hybrid plasmids, designated pGS300, pGS400, and pGS500, were constructed by subcloning the genes in pGS100-2 and pGS200 onto vector plasmid pBR325. pGS300 contained the E. coli B chromosomal DNA fragment with a gene for GSH-I in the PstI site of pBR325. pGS400 also contained E. coli B chromosomal DNA fragment with a gene for GSH-II in the HindIII site of pBR325. In contrast, pGS500 contained two kinds of DNA fragments with the genes for GSH-I and GSH-II in the PstI and HindIII sites of pBR325, respectively. All the hybrid plasmids thus prepared were stably maintained in E. coli cells when chloramphenicol was included at 10 micrograms/ml in the medium. The activity of the cells containing pGS300 was higher than that of the cells containing pGS400, although the former activity did not come up to that of cells having both pGS300 and pGS400. The highest glutathione-producing activity was found in the case of the cells transformed with pGS500 carrying both genes for GSH-I and GSH-II on the vector plasmid pBR325. About 5 mg/ml of glutathione was produced by E. coli cells with pGS500 from 80 mM L-glutamate, 20 mM L-cysteine, and 20 mM glycine within 3 h at 37 degrees C.
 ...
PMID:Construction of glutathione-producing strains of Escherichia coli B by recombinant DNA techniques. 614 39

The nucleotide sequence of the cloned DNA, 1,478 bp in length coding for glutathione synthetase (GSH-II) of E. coli B has been determined. Amino acid and nucleotide sequence analyses have assigned the open reading frame for GSH-II, starting with the ATG near its 5' terminus. The molecular weight calculated from the predicted amino acid sequence is 35,559 daltons, being in good agreement with that of a GSH-II subunit estimated by the SDS-PAGE method. Several signal sequences conserved in the promoter regions of E. coli were found in the non-coding regions of the gsh-II gene. They include the Shine-Dalgarno sequence, the Pribnow box and the sequence conserved in the "-35 region" with a preferable spacing from each other for an efficient transcription. Downstream from the termination codon, the inverted repeat sequences were present, followed by 6 successive T's. These structural features found in the non-coding regions have suggested to be involved in regulatory functions for the gsh-II gene expression.
 ...
PMID:Complete nucleotide sequence of the E. coli glutathione synthetase gsh-II. 639 55

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.
 ...
PMID:Glutathione-dependent yield and repair of single-strand DNA breaks in irradiated cells. 642 3


1 2 3 4 5 6 Next >>