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)

We used mice deficient in gamma-glutamyl transpeptidase (GGT) to analyze the effects of GGT deficiency and altered thiol levels on gene expression in liver. GGT-deficient mice have markedly reduced levels of glutathione (GSH), cysteine, methionine, and cysteinylglycine in liver. Steady-state RNA levels of the catalytic subunit of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis, are elevated 4-fold in these mice, while those for glutathione synthetase (GSH syn) are elevated 2-fold. RNA levels of cystathionase (cystathionine gamma-lyase), a key enzyme in the synthesis of cysteine from methionine, are elevated approximately 3.5-fold. In contrast, levels of RNA coding for multidrug resistance protein 2 (MRP2), which transports GSH into bile, are half wild-type values. We found no change in RNA levels of enzymes related to oxidative injury (CuZn and Mn superoxide dismutases [SOD], catalase, and glutathione peroxidase). Similarly, RNA levels of glutathione reductase and ribonucleotide reductase were unchanged. Furthermore, in contrast to previous in vitro results, methyl methanesulfonate did not induce stress-activated signal transduction as measured by c-jun phosphorylation in livers of GGT-deficient mice, despite further depletion of GSH by buthionine sulfoximine. Our findings indicate that GGT deficiency itself and/or altered thiol levels regulate expression of genes involved in GSH metabolism, but have no effect on the expression of other antioxidant genes.
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PMID:Altered gene expression in the liver of gamma-glutamyl transpeptidase-deficient mice. 1096 Apr 49

Synthesis of GSH occurs via two enzymatic steps, the first is catalyzed by gamma-glutamylcysteine synthetase (GCS) and the second is catalyzed by GSH synthetase (GS). A heavy (HS) and light subunit (LS) make up GCS; regulation of both subunits have been well characterized, whereas regulation of GS is largely unknown. In this study, we examined the effects of treatments known to influence the gene expression of GCS subunits on GS expression. Insulin and hydrocortisone treatment of rat hepatocytes or ethanol-feeding of rats for 9 weeks, which increased the expression of GCS-HS only, had no influence on GS expression. However, two-thirds partial hepatectomy in rats which increased the expression of GCS-HS only, also increased GS expression. Treatment of hepatocytes or rats with diethyl maleate, buthionine sulfoximine, tert-butylhydroquinone, or thioacetamide, which increased the expression of both GCS subunits, increased the expression of GS. The GSH synthesis capacity increased 50-100% by treatments that increased only the GCS-HS expression, whereas it increased 161-200% by treatments that increased both GCS-HS and GS expression. Thioacetamide treatment of Chang cells increased cell GSH and GS expression by 50%, but had minimal influence on GCS subunits. Thus, GS induction can further increase the cell's GSH synthetic capacity and in some cells may be as important as GCS in determining the rate of GSH synthesis.
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PMID:Inducers of gamma-glutamylcysteine synthetase and their effects on glutathione synthetase expression. 1097 6

In Arabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed by in situ hybridization the tissue-specific expression of the genes Atcys-3A and sat5, which encode O-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well as gsh1 and gsh2, which encode gamma-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes of Arabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238+/-25, 80+/-2, and 144+/-19 microM were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCl treatment, GSH biosynthesis is markedly decreased in trichomes. Atcys-3A, sat5, gsh1, and gsh2 mRNA levels show a decrease in transcript abundance, and [GSH](cyt) is reduced to 47+/-5 microM. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes.
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PMID:Glutathione biosynthesis in Arabidopsis trichome cells. 1099 73

Certain dietary constituents can protect against chemically induced carcinogenesis in rodents. A principal mechanism by which these chemopreventive compounds exert their protective effects is likely to be via induction of carcinogen detoxification. This can be mediated by conjugation with glutathione, which is synthesized by the sequential actions of glutamate-cysteine ligase (GLCL) and glutathione synthetase. We have demonstrated that dietary administration of the naturally occurring chemopreventive agents, ellagic acid, coumarin or alpha-angelicalactone caused an increase in GLCL activity of between approximately 3- and 5-fold in rat liver. Treatment with the synthetic antioxidant ethoxyquin or the classic inducer phenobarbital caused < 2-fold induction of GLCL activity in rat liver, which was not found to be significant. The increases in GLCL activity were accompanied by increases (between 2- and 4-fold) in levels of both the catalytic heavy subunit (GLCLC) and regulatory light subunit (GLCLR). No substantial induction of GLCL was observed in rat kidney. The glutathione S-transferase (GST) subunits A1, A3, A4, A5, P1 and M1 were all found to be inducible in rat liver by most of the agents. The greatest levels of induction were observed for GST P1, following treatment with coumarin (20-fold), alpha-angelicalactone (10-fold) or ellagic acid (6-fold), and GST A5, following treatment with coumarin (7-fold), alpha-angelicalactone (6-fold) and ethoxyquin (6-fold). Glutathione synthetase was induced approximately 1.5-fold by coumarin, alpha-angelicalactone, ellagic acid and ethoxyquin. The expression of glutathione-related enzymes was also examined in preneoplastic lesions induced in rat liver by aflatoxin B(1). The majority of gamma-glutamyltranspeptidase (GGT)-positive preneoplastic foci contained increased levels of GLCLC relative to the surrounding tissue. This was usually found to be accompanied by an increase in GLCLR. Cells in the inner cortex of rat kidney were found to contain the highest levels of both GLCLC and GLCLR. The same cells showed the strongest staining for GGT activity.
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PMID:Regulation of rat glutamate-cysteine ligase (gamma-glutamylcysteine synthetase) subunits by chemopreventive agents and in aflatoxin B(1)-induced preneoplasia. 1102 40

Previously we reported that expression of GSH1 (gamma-glutamylcysteine synthetase) and GSH2 (glutathione synthetase) of the yeast Saccharomyces cerevisiae was increased by heat-shock stress in a Yap1p-dependent fashion and consequently intracellular glutathione content was increased [Sugiyama, Izawa and Inoue (2000) J. Biol. Chem. 275, 15535-15540]. In the present study, we discuss the physiological role of glutathione in the heat-shock stress response in this yeast. Both gsh1 and gsh2 mutants could acquire thermotolerance by mild heat-shock stress and induction of Hsp104p in both mutants was normal; however, mutant cells died faster by heat shock than their parental wild-type strain. After pretreatment at a sublethal temperature, the number of respiration-deficient mutants increased in a gsh1 mutant strain in the early stages of exposure to a lethal temperature, although this increase was partially suppressed by the addition of glutathione. These results lead us to suspect that an increase of glutathione synthesis during heat-shock stress is to protect mitochondrial DNA from oxidative damage. To investigate the correlation between mitochondrial DNA damage and glutathione, mitochondrial Mn-superoxide dismutase (the SOD2 gene product) was disrupted. As a result, the rate of generation of respiration-deficient mutants of a sod2 delta strain was higher than that of the isogenic wild-type strain and treatment of the sod2 delta mutant with buthionine sulphoximine, an inhibitor of glutathione synthesis, inhibited cell growth. These results suggest that glutathione synthesis is induced by heat shock to protect the mitochondrial DNA from oxidative damage that may lead to cell death.
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PMID:Role of glutathione in heat-shock-induced cell death of Saccharomyces cerevisiae. 1106 59

The thiol tripeptides glutathione (GSH) and homoglutathione (hGSH) are very abundant in legume root nodules and their synthesis is catalyzed by the enzymes gamma-glutamylcysteine synthetase (gammaECS), GSH synthetase (GSHS), and hGSH synthetase (hGSHS). As an essential step to elucidate the role of thiols in N(2) fixation we have isolated cDNAs encoding the three enzymes and have quantified the transcripts in nodules. Assay of enzyme activities in highly purified nodule organelles revealed that gammaECS is localized in the plastids, hGSHS in the cytosol, and GSHS in the cytosol and mitochondria. These results are consistent with sequence analyses. Subcellular fractionation of nodules also showed that bacteroids contain high thiol concentrations and high specific gammaECS and GSHS activities. Results emphasize the role of nodule plastids in antioxidant protection and in control of thiol synthesis, and suggest that plastids may be important in the stress response of nodules. Overall, our results provide further evidence that thiol synthesis is critical for nodule functioning.
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PMID:Glutathione and homoglutathione synthetases of legume nodules. Cloning, expression, and subcellular localization. 1108 Mar 13

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

This article provides an overview of the mechanisms by which cancer chemopreventive blocking agents increase the expression of detoxication and antioxidant genes. These agents all appear capable of transcriptionally activating a gene battery that includes NAD(P)H:quinone oxidoreductase, aldo-keto reductases, glutathione S-transferases, gamma-glutamylcysteine synthetase, glutathione synthetase and heme oxygenase. Gene induction occurs through the antioxidant responsive element (ARE), a process that is dependent on the Nuclear Factor-Erythroid 2p45-related factors, Nrf1 and Nrf2. Under basal conditions, these basic region leucine zipper (bZIP) transcription factors are located in the cytoplasm of the cell bound to Keap1, and upon challenge with inducing agents, they are released from Keap1 and translocate to the nucleus. Within the nucleus, Nrf1 and Nrf2 are recruited to the ARE as heterodimers with either small Maf proteins, FosB, c-Jun, JunD, activating transcription factor 2 (ATF2) or ATF4. The role of protein kinases in transducing chemical stress signals to the bZIP factors that affect gene induction through the ARE is discussed.
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PMID:Molecular basis for the contribution of the antioxidant responsive element to cancer chemoprevention. 1168 85

In the present study we investigated the effects of L-pyroglutamic acid (PGA), which predominantly accumulates in the inherited metabolic diseases glutathione synthetase deficiency (GSD) and gamma-glutamylcysteine synthetase deficiency (GCSD), on some in vitro parameters of energy metabolism and lipid biosynthesis. We evaluated the rates of CO2 production and lipid synthesis from [U-14C]acetate, as well as ATP levels and the activities of creatine kinase and of the respiratory chain complexes I-IV in cerebral cortex of young rats in the presence of PGA at final concentrations ranging from 0.5 to 3 mM. PGA significantly reduced brain CO2 production by 50% at the concentrations of 0.5 to 3 mM, lipid biosynthesis by 20% at concentrations of 0.5 to 3 mM and ATP levels by 52% at the concentration of 3 mM. Regarding the enzyme activities, PGA significantly decreased NADH:cytochrome c oxireductase (complex I plus CoQ plus complex III) by 40% at concentrations of 0.5-3.0 mM and cytochrome c oxidase activity by 22-30% at the concentration of 3.0 mM, without affecting the activities of succinate dehydrogenase, succinate:DCPIP oxireductase (complex II), succinate:cytochrome c oxireductase (complex II plus CoQ plus complex III) or creatine kinase. The results strongly indicate that PGA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by these diseases.
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PMID:L-pyroglutamic acid inhibits energy production and lipid synthesis in cerebral cortex of young rats in vitro. 1188 78

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


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