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)

The biosynthesis of reduced glutathione (GSH) is carried out by the enzymes gamma-glutamylcysteine synthetase (GCL) and GSH synthetase. GCL is the rate-limiting step and represents a heterodimeric enzyme comprised of a catalytic subunit (GCLC) and a ("regulatory"), or modifier, subunit (GCLM). The nonhomologous Gclc and Gclm genes are located on mouse chromosomes 9 and 3, respectively. GCLC owns the catalytic activity, whereas GCLM enhances the enzyme activity by lowering the K(m) for glutamate and increasing the K(i) to GSH inhibition. Humans have been identified with one or two defective GCLC alleles and show low GSH levels. As an initial first step toward understanding the role of GSH in cellular redox homeostasis, we have targeted a disruption of the mouse Gclc gene. The Gclc(-/-) homozygous knockout animal dies before gestational day 13, whereas the Gclc(+/-) heterozygote is viable and fertile. The Gclc(+/-) mouse exhibits a gene-dose decrease in the GCLC protein and GCL activity, but only about a 20% diminution in GSH levels and a compensatory increase of approximately 30% in ascorbate-as compared with that in Gclc(+/+) wild-type littermates. These data show a reciprocal action between falling GSH concentrations and rising ascorbate levels. Therefore, the Gclc(+/-) mouse may be a useful genetic model for mild endogenous oxidative stress.
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PMID:Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous. 1111 86

The genes(gsh-I,gsh-II) for gamma-glutamyl-cysteine synthetase(GSH-I) and glutathione synthetase(GSH-II) from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively. The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc-gsh. E. coli BL21 was transformed by pTrc-gsh for expression of the related enzymes. Analysis of SDS-PAGE showed that the expected products were expressed. E. coli BL21(pTrc-gsh) were incubated at 37 degrees C and pH 7.2 to OD550 = 0.5. The conditions were then switched to 34 degrees C and pH6.7 after the addition of 0.1 mmol/L IPTG. The expressed products were up to 25% of the total protein of the bacteria. Acetone-treated cells of the engineered strain could synthesize GSH efficiently.
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PMID:[Cloning and expression of the genes of glutathione synthetases]. 1133 Jan 98

The present study examined how the multidrug resistance protein (MRP1), which is an ATP-dependent anionic conjugate transporter, also mediates the transport of reduced glutathione (GSH) and the co-transport of the cationic drug, daunorubicin, with GSH in living GLC4/Adr cells. To obtain information on the affinity of GSH for the multidrug resistance protein in GLC4/Adr cells, we investigated the GSH concentration dependence of the ATP-dependent GSH efflux. The intracellular GSH concentration was modulated by preincubation of the cells with 25 microM buthionine sulfoximine, an inhibitor of GSH synthetase, for 0-24 h. The transport of GSH was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued. Fitting of the obtained data according to the Michaelis-Menten equation revealed a Km of 3.4+/-1.4 mM and a Vmax of 1.5+/-0.2x10(-18) mol/cell/s. The ATP-dependent transport of GSH was inhibited by 3-([[3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl]-[(3-dimethylamino-3-oxopropyl)-thio]-methyl]thio)propanoic acid (MK571), with 50% inhibition being obtained with 1.4 microM MK571. We investigated the GSH concentration dependence of the MRP1-mediated ATP-dependent transport of daunorubicin under conditions where the transport of daunorubicin became saturated. The daunorubicin transport was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued. We were therefore in the situation where GSH acted as an activator: its presence was necessary for the binding and transport of daunorubicin by MRP1. However, GSH was also transported by the multidrug resistance protein. The concentration of GSH that gave half the maximal rate of daunorubicin efflux was 2.1+/-0.8 mM, very similar to the Km value obtained for GSH. In conclusion, the rate of daunorubicin efflux, under conditions where the transport of daunorubicin became saturated, and the rate of GSH efflux determined at any intracellular concentration of GSH were very similar, yielding a 1:1 stoichiometry with respect to GSH and daunorubicin transport. These results support a model in which daunorubicin is co-transported with GSH.
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PMID:Kinetics of glutathione and daunorubicin efflux from multidrug resistance protein overexpressing small-cell lung cancer cells. 1140 43

Glutathione (GSH) and homo-GSH (hGSH) are the major low-molecular weight thiols synthesized in Medicago truncatula. Two M. truncatula cDNAs (gshs1 and gshs2) corresponding to a putative GSH synthetase (GSHS) and a putative hGSH synthetase (hGSHS) were characterized. Heterologous expression of gshs1 and gshs2 cDNAs in an Escherichia coli strain deficient in GSHS activity showed that GSHS1 and GSHS2 are a GSHS and an hGSHS, respectively. Leucine-534 and proline-535 present in hGSHS were substituted by alanines that are conserved in plant GSHS. These substitutions resulted in a strongly stimulated GSH accumulation in the transformed E. coli strain showing that these residues play a crucial role in the differential recognition of beta-alanine and glycine by hGSHS. Phylogenetic analysis of GSHS2 and GSHS1 with other eukaryotic GSHS sequences indicated that gshs2 and gshs1 are the result of a gene duplication that occurred after the divergence between Fabales, Solanales, and Brassicales. Analysis of the structure of gshs1 and gshs2 genes shows they are both present in a cluster and in the same orientation in the M. truncatula genome, suggesting that the duplication of gshs1 and gshs2 occurred via a tandem duplication.
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PMID:A Medicago truncatula homoglutathione synthetase is derived from glutathione synthetase by gene duplication. 1150 May 68

GSH is the major low-molecular-mass thiol in most organisms. The tripeptide maintains a reduced intracellular environment and protects cellular components from damaging oxidation. GSH is synthesized by the action of two ATP-dependent enzymic steps, in which gamma-glutamylcysteine synthetase (gamma-GCS) catalyses the ligation of glutamate and cysteine and subsequently glutathione synthetase (GS) adds glycine to the dipeptide. Recently it was shown that the synthesis of gamma-glutamylcysteine is crucial for the survival of the erythrocytic stages of the malaria parasite Plasmodium falciparum by using the specific gamma-GCS inhibitor buthionine sulphoximine. In order to investigate further the synthetic pathway of the tripeptide in the parasite, GS was cloned and expressed recombinantly. The deduced amino acid sequence of P. falciparum GS shares only a moderate degree of identity with other known GSs, but the residues responsible for substrate and co-factor binding are almost all conserved, with the exception of the ones involved in gamma-glutamylcysteine binding. The protein is active as a dimer, with a subunit molecular mass of 77 kDa, and the addition of reducing reagents such as dithiothreitol is essential in maintaining enzymic activity, indicating that thiol groups are important for stability and enzymic activity. The K(app)(m) values for gamma-glutamyl-alpha-aminobutyrate, ATP and glycine were determined to be 107.1 microM, 59.1 microM and 5.04 mM, respectively, and the V(max) of 5.24 +/- 0.7 micromol.min(-1).mg(-1) was in the same range as that of the mammalian enzymes. However, the negative co-operativity observed for gamma-glutamylcysteine binding to the rat enzyme was not found for the parasite protein. This may be due to the alteration of several amino acids in the gamma-glutamylcysteine-binding site.
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PMID:Glutathione synthetase from Plasmodium falciparum. 1196 86

We used mice with a targeted disruption in g-glutamyl transpeptidase (GGT-deficient mice) to study the role of glutathione (GSH) in protection against oxygen-induced lung injury. These mice had reduced levels of lung GSH and restricted ability to synthesize GSH because of low levels of cysteine. When GGT-deficient mice were exposed to 80% oxygen, they developed diffuse pulmonary injury and died within eight days. Ten of 12 wild-type mice were alive after 18 days. Administration of N-acetylcysteine (NAC) to GGT-deficient mice corrected GSH values and prevented the development of severe pulmonary injury and death. Oxygen exposure induced an increase in lung GSH levels in both wild-type and GGT-deficient mice, but induced levels in the mutant mice were <50% of those in wild-type mice. Cysteine levels were approximately 50-fold lower than GSH levels the lungs of both wild-type and GGT-deficient mice. Levels of lung RNA coding for the heavy subunit of g-glutamyl cysteine synthetase rose three- to fourfold after oxygen exposure in both wild-type and GGT-deficient mice. In contrast, oxygen exposure failed to provoke increases in glutathione synthetase, glutathione peroxidase, glutaredoxin, or thioredoxin.
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PMID:Oxygen-induced pulmonary injury in gamma-glutamyl transpeptidase-deficient mice. 1197 99

Mice that lack the Nrf2 basic-region leucine-zipper transcription factor are more sensitive than wild-type (WT) animals to the cytotoxic and genotoxic effects of foreign chemicals and oxidants. To determine the basis for the decrease in tolerance of the Nrf2 homozygous null mice to xenobiotics, enzyme assay, Western blotting and gene-specific real-time PCR (TaqMan) have been used to examine the extent to which hepatic expression of GSH-dependent enzymes is influenced by the transcription factor. The amounts of protein and mRNA for class Alpha, Mu and Pi glutathione S-transferases were compared between WT and Nrf2 knockout (KO) mice of both sexes under both constitutive and inducible conditions. Among the class Alpha and class Mu transferases, constitutive expression of Gsta1, Gsta2, Gstm1, Gstm2, Gstm3, Gstm4 and Gstm6 subunits was reduced in the livers of Nrf2 mutant mice to between 3% and 60% of that observed in WT mice. Induction of these subunits by butylated hydroxyanisole (BHA) was more marked in WT female mice than in WT male mice. TaqMan analyses showed the increase in transferase mRNA caused by BHA was attenuated in Nrf2(-/-) mice, with the effect being most apparent in the case of Gsta1, Gstm1 and Gstm3. Amongst class Pi transferase subunits, the constitutive hepatic level of mRNA for Gstp1 and Gstp2 was not substantially affected in the KO mice, but their induction by BHA was dependent on Nrf2; this was more obvious in female mutant mice than in male mice. Nrf2 KO mice exhibited reduced constitutive expression of the glutamate cysteine ligase catalytic subunit, and, to a lesser extent, the expression of glutamate cysteine ligase modifier subunit. Little variation was observed in the levels of glutathione synthase in the different mouse lines. Thus the increased sensitivity of Nrf2(-/-) mice to xenobiotics can be partly attributed to a loss in constitutive expression of multiple GSH-dependent enzymes, which causes a reduction in intrinsic detoxification capacity in the KO animal. These data also indicate that attenuated induction of GSH-dependent enzymes in Nrf2(-/-) mice probably accounts for their failure to adapt to chronic exposure to chemical and oxidative stress.
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PMID:Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. 1199 5

The thiol tripeptide glutathione (GSH; gammaGlu-Cys-Gly) is very abundant in legume nodules where it performs multiple functions that are critical for optimal nitrogen fixation. Some legume nodules contain another tripeptide, homoglutathione (hGSH; gammaGlu-Cys-betaAla), in addition to or instead of GSH. We have isolated from a pea (Pisum sativum L.) nodule library a cDNA, GSHS2, that is expressed in nodules but not in leaves. This cDNA was overexpressed in insect cells and its protein product was identified as a highly active and specific hGSH synthetase. The enzyme, the first of this type to be completely purified, is predicted to be a homodimeric cytosolic protein. It shows a specific activity of 3400 nmol hGSH min-1 mg-1 protein with a standard substrate concentration (5 mM beta-alanine) and Km values of 1.9 mM for beta-alanine and 104 mM for glycine. The specificity constant (Vmax/Km) shows that the pure enzyme is 57.3-fold more specific for beta-alanine than for glycine. Southern blot analysis revealed that the gene is present as a single copy in the pea genome and that there are homologous genes in other legumes. We conclude that the synthesis of hGSH in pea nodules is catalysed by a specific hGSH synthetase and not by a GSH synthetase with broad substrate specificity.
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PMID:Cloning and functional characterization of a homoglutathione synthetase from pea nodules. 1201 Apr 68

Inorganic arsenicals are important environmental toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenicals often undergo methylation, forming compounds such as dimethyarsinic acid (DMA). Recent evidence indicates DMA is a complete carcinogen in rodents while evidence for inorganic arsenicals as carcinogens in rodents remains equivocal. Thus, we studied the molecular mechanisms of in vitro cytolethality of DMA compared to that of the trivalent inorganic arsenical, sodium arsenite, using a rat liver epithelial cell line (TRL 1215). Arsenite was very cytotoxic in these cells (LC(50) = 35 microM after 48 h of exposure). With arsenite exposure, most dead cells showed histological and biochemical evidence of necrosis. Arsenite cytotoxicity increased markedly when cellular GSH was depleted with the glutathione synthase inhibitor, L-buthionine-[S,R]-sulfoximine (BSO). In contrast, DMA was nearly 3 orders of magnitude less cytotoxic (LC(50) = 1.5 mM) although evidence showed the predominating form of death was apoptosis. Surprisingly, GSH depletion actually decreased DMA-induced apoptosis. A glutathione scavenger, diethyl maleate (DEM), and a glutathione reductase inhibitor, carmustine, also prevented DMA-induced apoptosis. These data indicate that DMA requires intracellular GSH to induce apoptosis. Ethacrynic acid (EA), an inhibitor of glutathione S-transferase (GST) that catalyzes GSH-substrate conjugation, acivicin, an inhibitor of gamma-glutamyltranspeptidase (GGT) which catalyzes the initial breakdown of GSH-substrate conjugates, and aminooxyacetic acid (AOAA), an inhibitor of beta-lyase which catalyzes the final breakdown of GSH-substrate conjugates, all were effective in suppressing DMA-induced apoptosis. These findings indicate that DMA likely is conjugated in some form with GSH, and that it is this conjugate that induces apoptosis during subsequent metabolic reactions.
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PMID:A major human arsenic metabolite, dimethylarsinic acid, requires reduced glutathione to induce apoptosis. 1201 83

An assay of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in crude extracts of cultured cells and tissues is described. It represents a novel combination of known methods, and is based on the formation of glutathione (GSH) from cysteine, glutamate and glycine in the presence of rat kidney GS for the assay of gamma-GCS, or from gamma-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the gamma-glutamyl transpeptidase (GGT) inhibitor acivicin in order to prevent the degradation of gamma-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and gamma-glutamylcysteine. gamma-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of gamma-GCS GS in GGT-containing tissues and for the studies of induction of gamma-GCS and GS in tissue cultures.
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PMID:A spectrophotometric assay of gamma-glutamylcysteine synthetase and glutathione synthetase in crude extracts from tissues and cultured mammalian cells. 1204 60


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