J. Biol. Chem. 253, 2615-2623 (1978)[PubMed:24639]
Human erythrocyte gamma-glutamylcysteine synthetase is inactivated by the disulfide cystamine (2,2'-dithiobis-(ethylamine)) at pH 8.2 with a rate constant of 1020 min-1 mM-1. Magnesium ion and various combinations of substrates and products confer differing degrees of protection against cystamine inactivation, thus allowing the detection and quantification of certain enzyme-ligand interactions. By measuring inactivation rates as a function of ligand concentrations in incomplete reaction mixtures, we have obtained evidence for the following complexes: enzyme . Mg2+; enzyme . Mg2+ . MgATP2-; enzyme . Mg2+ . L-glutamate; enzyme . Mg2+ . MgATP2- . L-glutamate; enzyme . Mg2+ . L-gamma-glutamyl-L-alpha-aminobutyrate. The data also imply the existence of enzyme . (Mg2+)2 . MgATP2- . L-glutamate and several enzyme forms resulting from the weak binding to L-alpha-aminobutyrate. The methods used permit the calculation of cystamine inactivation rates for most of these enzyme forms and also give values for the equilibrium constants describing their formation.
Interacting selectively and non-covalently with a coenzyme, any of various nonprotein organic cofactors that are required, in addition to an enzyme and a substrate, for an enzymatic reaction to proceed.
Evidence
1:
Inferred from Physical InteractionUniProtKB
Biochem. J. 336 ( Pt 3), 675-680 (1998)[PubMed:9841880]
Glutamate-cysteine ligase (GLCL) catalyses the rate-limiting step in glutathione biosynthesis. To identify cysteine residues in GLCL that are involved in its activity, eight conserved cysteine residues in human GLCL catalytic subunit (hGLCLC) were replaced with glycine residues by PCR-based site-directed mutagenesis. Both recombinant hGLCLC and hGLCL holoenzyme were expressed and purified with a baculovirus expression system. The activity of purified hGLCL holoenzyme with the mutant hGLCLC-C553G was 110+/-12 micromol/h per mg of protein compared with 370+/-20 micromol/h per mg of protein for the wild-type. Holoenzymes with hGLCLC-C52G, -C248G, -C249G, -C295G, -C491G, -C501G or -C605G showed activities similar to the wild type. The Km values of hGLCL containing hGLCLC-C553G were slightly lower than those of the wild type, indicating that the replacement of cysteine-553 with Gly in hGLCLC did not significantly affect substrate binding by the enzyme. hGLCLC-C553G was more easily dissociated from hGLCLR than the wild-type hGLCLC. GLCL activity increased by 11% after hGLCLC-C553G was incubated with an equimolar amount of purified hGLCL regulatory subunit (hGLCLR) at room temperature for 30 min, but increased by 110% after wild-type hGLCLC was incubated with hGLCLR for 10 min. These results indicate that cysteine-553 in hGLCLC is involved in heterodimer formation between hGLCLC and hGLCLR.
Evidence
2:
Inferred from Physical InteractionUniProtKB
gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the ATP-dependent ligation of L-glutamate and L-cysteine to form L-gamma-glutamyl-L-cysteine; this is the first and rate-limiting step in glutathione biosynthesis. Inhibitors of gamma-GCS such as buthionine sulfoximine are widely used as tools for elucidating glutathione metabolism in vivo and as pharmacological agents for reversing glutathione-based resistance to chemotherapy and radiation therapy in certain cancers. Although gamma-GCS is readily isolated from rat kidneys, future drug design efforts are better based on structure-activity relationships established with the human enzyme. We report here the coexpression in Escherichia coli BL21(DE3) of the human gamma-GCS catalytic (heavy) subunit and regulatory (light) subunit using pET-3d and pET-9d vectors, respectively. Intracellular assembly of the holoenzyme occurred without difficulty, and levels of expression were acceptable (approximately 32 mg holoenzyme/100 g cells). Recombinant human gamma-GCS was purified to homogeneity in an overall yield of 45% by ammonium sulfate fractionation followed by sequential chromatography on Q-Sepharose ion-exchange, Superdex 200 gel filtration and ATP-affinity resins. Trace amounts of E. coli gamma-GCS were removed by immunoaffinity chromatography. The specific activity of the isolated enzyme was >1500 units/mg, comparable to the best preparations from rat kidney. The Km values for L-glutamate, L-cysteine, L-gamma-aminobutyrate (an L-cysteine surrogate), and ATP are 1.8, 0.1, 1.3, and 0.4 mM, respectively. Recombinant human gamma-GCS, like native rat gamma-GCS, is feedback inhibited by glutathione and is potently inhibited by buthionine sulfoximine and cystamine.
Biochem. J. 336 ( Pt 3), 675-680 (1998)[PubMed:9841880]
Glutamate-cysteine ligase (GLCL) catalyses the rate-limiting step in glutathione biosynthesis. To identify cysteine residues in GLCL that are involved in its activity, eight conserved cysteine residues in human GLCL catalytic subunit (hGLCLC) were replaced with glycine residues by PCR-based site-directed mutagenesis. Both recombinant hGLCLC and hGLCL holoenzyme were expressed and purified with a baculovirus expression system. The activity of purified hGLCL holoenzyme with the mutant hGLCLC-C553G was 110+/-12 micromol/h per mg of protein compared with 370+/-20 micromol/h per mg of protein for the wild-type. Holoenzymes with hGLCLC-C52G, -C248G, -C249G, -C295G, -C491G, -C501G or -C605G showed activities similar to the wild type. The Km values of hGLCL containing hGLCLC-C553G were slightly lower than those of the wild type, indicating that the replacement of cysteine-553 with Gly in hGLCLC did not significantly affect substrate binding by the enzyme. hGLCLC-C553G was more easily dissociated from hGLCLR than the wild-type hGLCLC. GLCL activity increased by 11% after hGLCLC-C553G was incubated with an equimolar amount of purified hGLCL regulatory subunit (hGLCLR) at room temperature for 30 min, but increased by 110% after wild-type hGLCLC was incubated with hGLCLR for 10 min. These results indicate that cysteine-553 in hGLCLC is involved in heterodimer formation between hGLCLC and hGLCLR.
BACKGROUND: The bronchial epithelium is exposed to reactive oxygen species (ROS) derived from cigarette smoke, air pollutants and activated leucocytes. Glutathione (GSH) prevents ROS-mediated loss of cell function, tissue injury and inflammation, and its synthesis is regulated by gamma-glutamylcysteine synthetase (gamma-GCS). However, the capacity of bronchial epithelial cells to adapt to oxidative stress and the mechanisms involved are not known. OBJECTIVE: To investigate the effects of oxidative stress on the regulation of GSH synthesis in human bronchial epithelial (NCI-H292) cells. METHODS: NCI-H292 cells were exposed to menadione and intracellular GSH concentrations were measured by spectrophotometry. gamma-GCS activity was measured by HPLC assay and changes in gamma-GCS mRNA by Northern blotting. RESULTS: Exposure to menadione (MQ, 10-200 microm, 30-120 min) decreased total cellular GSH content, measured immediately after exposure to MQ. However, GSH content measured 6-12 h after withdrawal of the oxidant stress (MQ, 50 microm, 30 min), increased c. two fold over baseline levels (P < 0.001). gamma-GCS activity measured 6 h (21.7 +/- 3.4 nmol/min/mg, SD, n = 5, P < 0.01) or 12 h (23.2 +/- 4.6, P < 0.001) after MQ treatment was also significantly increased compared with untreated cells (12.8 +/- 1.0). Similarly, gamma-GCS mRNA expression increased 1.3-1.6-fold relative to GAPDH mRNA, 3-6 h after MQ treatment. The MQ-induced increase in gamma-GCS mRNA expression was completely inhibited by actinomycin D. CONCLUSIONS: Bronchial epithelial (NCI-H292) cells respond rapidly and sensitively to oxidant stress, and this adaptive response is mediated by increased gamma-GCS mRNA transcription and enzyme activity.
Evidence
2:
Inferred from Mutant PhenotypeUniProtKB
Glutathione deficiency is an early biochemical feature that occurs during apoptotic neuronal death associated with certain neurological disorders such as Parkinson disease. However, whether specific targeting of glutathione biosynthesis in neurons is sufficient to trigger neurodegeneration remains undetermined. To address this issue, we used a vector-based small hairpin RNA (shRNA) strategy to knock down each subunit of glutamate-cysteine ligase (GCL; gamma-glutamylcysteine synthetase), the heterodimeric enzyme that catalyzes the rate-limiting step of glutathione biosynthesis. Independent targeting of the catalytic and modulatory subunits by shRNA caused disruption of GCL as assessed by Northern and Western blotting, enzyme activity, and glutathione concentrations. Silencing each subunit in primary cortical neurons spontaneously elicited time-dependent apoptotic death, an effect that was synergistic with glutamate or nitric oxide treatment. Moreover, neuronal apoptosis by GCL knockdown was rescued by expressing the corresponding subunit full-length cDNA carrying silent mutations within the shRNA target cDNA sequence and by incubating neurons with gamma-glutamylcysteine or glutathione ethyl ester. In contrast, supplying glutathione precursors to neurons from co-cultured astrocytes did not prevent the apoptotic death triggered by GCL knockdown. Finally, overexpressing the catalytic (but not modulatory) GCL subunit full-length cDNA increased enzyme activity and glutathione concentrations, yielding neurons more resistant to glutamate- or nitric oxide-mediated apoptosis. Thus, specific and independent disruption of each subunit of GCL in neurons can be said to cause a primary decrease in glutathione that is sufficient to promote neurodegeneration.
J. Biol. Chem. 268, 20366-20372 (1993)[PubMed:8104187]
Effect of heat shock on a glutathione-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and ATP-dependent outward transport of glutathione S-conjugate was characterized using K562 erythroid cells. When K562 cells grown at 37 degrees C were shifted to 42 degrees C for 2 h, an approximate 1.7-fold increase in the activity of gamma-GCS was observed. Treatment of K562 cells with erythropoietin (EP) for 12 h resulted in a decrease in the activity of gamma-GCS to 64% of the control. However, responsiveness of this enzyme activity in the cells treated with EP to heat shock was similar to that in untreated cells. Changes in the immunological activity of gamma-GCS were also observed in parallel with those in the enzymatic activity. On Northern blot analysis of total RNAs isolated from the cells with human cDNA for gamma-GCS, a substantial induction of mRNA level was found by heat shock and a reduction of EP. These changes were modest but correlated to the mRNA expression of a heat shock protein, HSP 70. Heat shock also had an effect of 1.8-fold stimulation on glutathione S-conjugate transport in K562 cells previously incubated with 1-chloro-2,4-dinitrobenzene. Treatment of the cells with EP resulted in a decrease in this transport by 62%. Similarly, the levels of glutathione S-conjugate-stimulated Mg(2+)-ATPase, which enzyme is thought to be involved in the transport of glutathione S-conjugate, were responsive to heat shock and EP. These results suggest that glutathione synthesis and transport process of glutathione metabolites are responsive to heat shock and play a role in the defense system against stresses. It is also suggested that the regulatory site of the expression of these enzymes by heat shock is independent of that by EP.
J. Biol. Chem. 253, 2615-2623 (1978)[PubMed:24639]
Human erythrocyte gamma-glutamylcysteine synthetase is inactivated by the disulfide cystamine (2,2'-dithiobis-(ethylamine)) at pH 8.2 with a rate constant of 1020 min-1 mM-1. Magnesium ion and various combinations of substrates and products confer differing degrees of protection against cystamine inactivation, thus allowing the detection and quantification of certain enzyme-ligand interactions. By measuring inactivation rates as a function of ligand concentrations in incomplete reaction mixtures, we have obtained evidence for the following complexes: enzyme . Mg2+; enzyme . Mg2+ . MgATP2-; enzyme . Mg2+ . L-glutamate; enzyme . Mg2+ . MgATP2- . L-glutamate; enzyme . Mg2+ . L-gamma-glutamyl-L-alpha-aminobutyrate. The data also imply the existence of enzyme . (Mg2+)2 . MgATP2- . L-glutamate and several enzyme forms resulting from the weak binding to L-alpha-aminobutyrate. The methods used permit the calculation of cystamine inactivation rates for most of these enzyme forms and also give values for the equilibrium constants describing their formation.
Tumor necrosis factor (TNF) is a highly pleiotropic cytokine whose activity is at least partially regulated by the redox status of the cell. The cellular redox status is controlled primarily by glutathione, a major cellular antioxidant, whose synthesis is regulated by the rate-limiting enzyme gamma-glutamylcysteine synthetase (gamma-GCS). In the present report we investigated the effect of gamma-GCS overexpression on the TNF-induced activation of nuclear transcription factors NF-kappa B and AP-1, stress-activated protein kinase/c-Jun amino-terminal kinase (JNK) and apoptosis. Transfection of cells with gamma-GCS cDNA blocked TNF-induced NF-kappa B activation, cytoplasmic I kappa B alpha degradation, nuclear translocation of p65, and NF-kappa B-dependent gene transcription. gamma-GCS overexpression also completely suppressed NF-kappa B activation induced by phorbol ester and okadaic acid, whereas that induced by H2O2, ceramide, and lipopolysaccharide was minimally affected. gamma-GCS also abolished the activation of AP-1 induced by TNF and inhibited TNF-induced activation of JNK and mitogen-activated protein kinase kinase. TNF-mediated cytotoxicity and activation of caspase-3 were both abrogated in gamma-GCS-overexpressing cells. Overall, our results indicate that most of the pleiotropic actions of TNF are regulated by the glutathione-controlled redox status of the cell.
gamma-Glutamylcysteine synthetase is one of the enzymes of glutathione (GSH) synthesis. A deficiency of this enzyme has been found only once previously in humans: it was associated with spinocerebellar degeneration and hemolytic anemia. We report the case of a woman, daughter of fifth cousins, who was gamma-glutamylcysteine-synthetase-deficient. Modest decreases in the amount of GSH in cultured lymphoblasts and fibroblasts could be documented. The amount of residual enzyme was insufficient to permit detailed studies of the characteristics of the mutant enzymes, but no major abnormality in its Km for cysteine and glutamic acid or in its heat stability were found. In contrast to the earlier report, the only manifestation of the enzyme deficiency was hemolytic anemia. This leads us to conclude that either the occurrence of neurologic symptoms in the other reported family was a chance association or that the clinical expression of this rare defect is pleomorphic.
gamma-Glutamylcysteine synthetase is one of the enzymes of glutathione (GSH) synthesis. A deficiency of this enzyme has been found only once previously in humans: it was associated with spinocerebellar degeneration and hemolytic anemia. We report the case of a woman, daughter of fifth cousins, who was gamma-glutamylcysteine-synthetase-deficient. Modest decreases in the amount of GSH in cultured lymphoblasts and fibroblasts could be documented. The amount of residual enzyme was insufficient to permit detailed studies of the characteristics of the mutant enzymes, but no major abnormality in its Km for cysteine and glutamic acid or in its heat stability were found. In contrast to the earlier report, the only manifestation of the enzyme deficiency was hemolytic anemia. This leads us to conclude that either the occurrence of neurologic symptoms in the other reported family was a chance association or that the clinical expression of this rare defect is pleomorphic.
Gamma-glutamylcysteine synthetase (gamma-GCS) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis: the adenosine triphosphate (ATP)-dependent ligation of glutamate and cysteine. gamma-GCS consists of a catalytic (gamma-GCSH) and modifier (gamma-GCSL) subunit. Hereditary deficiency of gamma-GCS has been reported in a small number of patients and is associated with low erythrocyte levels of gamma-GCS and GSH leading to hemolytic anemia. Here we report a novel gamma-GCSH mutation, isolated from the cDNA of 2 related patients diagnosed with gamma-GCS deficiency. Each was found to be homozygous for a C>T missense mutation at nucleotide 379, encoding for a predicted Arg127Cys amino acid change. Computerized structure modeling identified that the mutated amino acid lies within a cleft on the protein surface of gamma-GCSH, and the border of this cleft was shown to contain Cys249, an evolutionarily conserved residue that has been proven to lie near the binding site of gamma-GCSH. Transfection studies showed that the mutation is associated with decreased GSH production, and binding studies using purified recombinant protein showed that the mutant protein has markedly decreased enzymatic activity compared to wild type.
The chemical reactions and pathways resulting in the formation of glutathione, the tripeptide glutamylcysteinylglycine, which acts as a coenzyme for some enzymes and as an antioxidant in the protection of sulfhydryl groups in enzymes and other proteins.
J. Biol. Chem. 268, 20366-20372 (1993)[PubMed:8104187]
Effect of heat shock on a glutathione-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and ATP-dependent outward transport of glutathione S-conjugate was characterized using K562 erythroid cells. When K562 cells grown at 37 degrees C were shifted to 42 degrees C for 2 h, an approximate 1.7-fold increase in the activity of gamma-GCS was observed. Treatment of K562 cells with erythropoietin (EP) for 12 h resulted in a decrease in the activity of gamma-GCS to 64% of the control. However, responsiveness of this enzyme activity in the cells treated with EP to heat shock was similar to that in untreated cells. Changes in the immunological activity of gamma-GCS were also observed in parallel with those in the enzymatic activity. On Northern blot analysis of total RNAs isolated from the cells with human cDNA for gamma-GCS, a substantial induction of mRNA level was found by heat shock and a reduction of EP. These changes were modest but correlated to the mRNA expression of a heat shock protein, HSP 70. Heat shock also had an effect of 1.8-fold stimulation on glutathione S-conjugate transport in K562 cells previously incubated with 1-chloro-2,4-dinitrobenzene. Treatment of the cells with EP resulted in a decrease in this transport by 62%. Similarly, the levels of glutathione S-conjugate-stimulated Mg(2+)-ATPase, which enzyme is thought to be involved in the transport of glutathione S-conjugate, were responsive to heat shock and EP. These results suggest that glutathione synthesis and transport process of glutathione metabolites are responsive to heat shock and play a role in the defense system against stresses. It is also suggested that the regulatory site of the expression of these enzymes by heat shock is independent of that by EP.
gamma-Glutamylcysteine synthetase (GCS) is of major importance in glutathione homeostasis. The GCS heterodimer is composed of catalytic (heavy subunit, GCSh) and regulatory (light subunit, GCSl) subunits. Regulation of the human GCSl subunit gene (GLCLR) expression was studied as GCSl has a critical role in glutathione synthesis. The minimal basal expression of GLCLR was found to be mediated by a region between nt -205 and -318. The major transcriptional start site in HT29 cells was located within this region at nt -283. A region between nt -411 and -447 was identified as having a potential involvement in the negative regulation of GLCLR expression. In order to study the transcriptional regulation of GCSl by oxidant stress, HepG2 cells were treated with sodium nitroprusside (SNP). SNP (1.5 mM) was found to increase glutathione levels by 2-fold, as well as GCS activity by 6-fold. This is accompanied by a co-ordinate increase in the levels of the both the GCSl and GCSh subunits, each by approximately 2-fold. The transcriptional activity of the GLCLR gene was increased by approximately 2.5-fold in SNP-treated cells.
Evidence
4:
Inferred from Mutant PhenotypeUniProtKB
Gamma-glutamylcysteine synthetase (gamma-GCS) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis: the adenosine triphosphate (ATP)-dependent ligation of glutamate and cysteine. gamma-GCS consists of a catalytic (gamma-GCSH) and modifier (gamma-GCSL) subunit. Hereditary deficiency of gamma-GCS has been reported in a small number of patients and is associated with low erythrocyte levels of gamma-GCS and GSH leading to hemolytic anemia. Here we report a novel gamma-GCSH mutation, isolated from the cDNA of 2 related patients diagnosed with gamma-GCS deficiency. Each was found to be homozygous for a C>T missense mutation at nucleotide 379, encoding for a predicted Arg127Cys amino acid change. Computerized structure modeling identified that the mutated amino acid lies within a cleft on the protein surface of gamma-GCSH, and the border of this cleft was shown to contain Cys249, an evolutionarily conserved residue that has been proven to lie near the binding site of gamma-GCSH. Transfection studies showed that the mutation is associated with decreased GSH production, and binding studies using purified recombinant protein showed that the mutant protein has markedly decreased enzymatic activity compared to wild type.
The chemical reactions and pathways involving L-ascorbic acid, (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2,5-dihydrofuran-3-olate; L-ascorbic acid is vitamin C and has co-factor and anti-oxidant activities in many species.
Tumor necrosis factor (TNF) is a highly pleiotropic cytokine whose activity is at least partially regulated by the redox status of the cell. The cellular redox status is controlled primarily by glutathione, a major cellular antioxidant, whose synthesis is regulated by the rate-limiting enzyme gamma-glutamylcysteine synthetase (gamma-GCS). In the present report we investigated the effect of gamma-GCS overexpression on the TNF-induced activation of nuclear transcription factors NF-kappa B and AP-1, stress-activated protein kinase/c-Jun amino-terminal kinase (JNK) and apoptosis. Transfection of cells with gamma-GCS cDNA blocked TNF-induced NF-kappa B activation, cytoplasmic I kappa B alpha degradation, nuclear translocation of p65, and NF-kappa B-dependent gene transcription. gamma-GCS overexpression also completely suppressed NF-kappa B activation induced by phorbol ester and okadaic acid, whereas that induced by H2O2, ceramide, and lipopolysaccharide was minimally affected. gamma-GCS also abolished the activation of AP-1 induced by TNF and inhibited TNF-induced activation of JNK and mitogen-activated protein kinase kinase. TNF-mediated cytotoxicity and activation of caspase-3 were both abrogated in gamma-GCS-overexpressing cells. Overall, our results indicate that most of the pleiotropic actions of TNF are regulated by the glutathione-controlled redox status of the cell.
Tumor necrosis factor (TNF) is a highly pleiotropic cytokine whose activity is at least partially regulated by the redox status of the cell. The cellular redox status is controlled primarily by glutathione, a major cellular antioxidant, whose synthesis is regulated by the rate-limiting enzyme gamma-glutamylcysteine synthetase (gamma-GCS). In the present report we investigated the effect of gamma-GCS overexpression on the TNF-induced activation of nuclear transcription factors NF-kappa B and AP-1, stress-activated protein kinase/c-Jun amino-terminal kinase (JNK) and apoptosis. Transfection of cells with gamma-GCS cDNA blocked TNF-induced NF-kappa B activation, cytoplasmic I kappa B alpha degradation, nuclear translocation of p65, and NF-kappa B-dependent gene transcription. gamma-GCS overexpression also completely suppressed NF-kappa B activation induced by phorbol ester and okadaic acid, whereas that induced by H2O2, ceramide, and lipopolysaccharide was minimally affected. gamma-GCS also abolished the activation of AP-1 induced by TNF and inhibited TNF-induced activation of JNK and mitogen-activated protein kinase kinase. TNF-mediated cytotoxicity and activation of caspase-3 were both abrogated in gamma-GCS-overexpressing cells. Overall, our results indicate that most of the pleiotropic actions of TNF are regulated by the glutathione-controlled redox status of the cell.
Positive regulation of proteasomal ubiquitin-dependent protein catabolic processdefinition[GO:0032436]‹silver
Any process that activates or increases the frequency, rate or extent of the breakdown of a protein or peptide by hydrolysis of its peptide bonds, initiated by the covalent attachment of ubiquitin, and mediated by the proteasome.
J. Am. Coll. Cardiol. 41, 539-545 (2003)[PubMed:12598062]
OBJECTIVES: The purpose of this study was to test the hypothesis that polymorphisms in the promoter region of the glutamate-cysteine ligase catalytic subunit (GCLC) gene may be associated with coronary endothelial vasomotor dysfunction and myocardial infarction (MI). BACKGROUND: Glutamate-cysteine ligase is a rate-limiting enzyme for synthesis of glutathione (GSH) that plays a crucial role in the intracellular antioxidant defense systems. Oxidants transcriptionally upregulate the GCLC gene for GSH synthesis, providing a protective mechanism against oxidant-induced endothelial dysfunction or activation, which plays a pathogenetic role in cardiovascular diseases. METHODS: The association of the possible polymorphisms with coronary arterial diameter responses to acetylcholine was determined in 62 male subjects. The frequency of polymorphisms was compared between 255 male patients with MI and 179 male control subjects. RESULTS: We found a polymorphism (-129C/T) in which the T allele showed lower promoter activity (50% to 60% of the activity of the C allele) in response to H(2)O(2) in human endothelial cells. Endothelium-dependent dilation of coronary arteries was impaired in subjects with the -129T allele (n = 31), as compared with the age-matched subjects without the -129T allele (n = 31). The T allele was highly frequent in patients with MI as compared with control subjects, and it was a significant risk factor for MI, independent of traditional coronary risk factors (odds ratio [OR] 1.81, 95% confidence interval [CI] 1.08 to 3.03; p = 0.03). CONCLUSIONS: The -129T polymorphism of the GCLC gene may suppress the GCLC gene induction response to an oxidant, and it is implicated in coronary endothelial vasomotor dysfunction and MI.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an arsenic stimulus from compounds containing arsenic, including arsenates, arsenites, and arsenides.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a heat stimulus, a temperature stimulus above the optimal temperature for that organism.
J. Biol. Chem. 268, 20366-20372 (1993)[PubMed:8104187]
Effect of heat shock on a glutathione-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and ATP-dependent outward transport of glutathione S-conjugate was characterized using K562 erythroid cells. When K562 cells grown at 37 degrees C were shifted to 42 degrees C for 2 h, an approximate 1.7-fold increase in the activity of gamma-GCS was observed. Treatment of K562 cells with erythropoietin (EP) for 12 h resulted in a decrease in the activity of gamma-GCS to 64% of the control. However, responsiveness of this enzyme activity in the cells treated with EP to heat shock was similar to that in untreated cells. Changes in the immunological activity of gamma-GCS were also observed in parallel with those in the enzymatic activity. On Northern blot analysis of total RNAs isolated from the cells with human cDNA for gamma-GCS, a substantial induction of mRNA level was found by heat shock and a reduction of EP. These changes were modest but correlated to the mRNA expression of a heat shock protein, HSP 70. Heat shock also had an effect of 1.8-fold stimulation on glutathione S-conjugate transport in K562 cells previously incubated with 1-chloro-2,4-dinitrobenzene. Treatment of the cells with EP resulted in a decrease in this transport by 62%. Similarly, the levels of glutathione S-conjugate-stimulated Mg(2+)-ATPase, which enzyme is thought to be involved in the transport of glutathione S-conjugate, were responsive to heat shock and EP. These results suggest that glutathione synthesis and transport process of glutathione metabolites are responsive to heat shock and play a role in the defense system against stresses. It is also suggested that the regulatory site of the expression of these enzymes by heat shock is independent of that by EP.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a hormone stimulus.
J. Biol. Chem. 268, 20366-20372 (1993)[PubMed:8104187]
Effect of heat shock on a glutathione-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and ATP-dependent outward transport of glutathione S-conjugate was characterized using K562 erythroid cells. When K562 cells grown at 37 degrees C were shifted to 42 degrees C for 2 h, an approximate 1.7-fold increase in the activity of gamma-GCS was observed. Treatment of K562 cells with erythropoietin (EP) for 12 h resulted in a decrease in the activity of gamma-GCS to 64% of the control. However, responsiveness of this enzyme activity in the cells treated with EP to heat shock was similar to that in untreated cells. Changes in the immunological activity of gamma-GCS were also observed in parallel with those in the enzymatic activity. On Northern blot analysis of total RNAs isolated from the cells with human cDNA for gamma-GCS, a substantial induction of mRNA level was found by heat shock and a reduction of EP. These changes were modest but correlated to the mRNA expression of a heat shock protein, HSP 70. Heat shock also had an effect of 1.8-fold stimulation on glutathione S-conjugate transport in K562 cells previously incubated with 1-chloro-2,4-dinitrobenzene. Treatment of the cells with EP resulted in a decrease in this transport by 62%. Similarly, the levels of glutathione S-conjugate-stimulated Mg(2+)-ATPase, which enzyme is thought to be involved in the transport of glutathione S-conjugate, were responsive to heat shock and EP. These results suggest that glutathione synthesis and transport process of glutathione metabolites are responsive to heat shock and play a role in the defense system against stresses. It is also suggested that the regulatory site of the expression of these enzymes by heat shock is independent of that by EP.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a nitrosative stress stimulus. Nitrosative stress is a state often resulting from exposure to high levels of nitric oxide (NO) or the highly reactive oxidant peroxynitrite, which is produced following interaction of NO with superoxide anions.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of oxidative stress, a state often resulting from exposure to high levels of reactive oxygen species, e.g. superoxide anions, hydrogen peroxide (H2O2), and hydroxyl radicals.
gamma-Glutamylcysteine synthetase (GCS) is of major importance in glutathione homeostasis. The GCS heterodimer is composed of catalytic (heavy subunit, GCSh) and regulatory (light subunit, GCSl) subunits. Regulation of the human GCSl subunit gene (GLCLR) expression was studied as GCSl has a critical role in glutathione synthesis. The minimal basal expression of GLCLR was found to be mediated by a region between nt -205 and -318. The major transcriptional start site in HT29 cells was located within this region at nt -283. A region between nt -411 and -447 was identified as having a potential involvement in the negative regulation of GLCLR expression. In order to study the transcriptional regulation of GCSl by oxidant stress, HepG2 cells were treated with sodium nitroprusside (SNP). SNP (1.5 mM) was found to increase glutathione levels by 2-fold, as well as GCS activity by 6-fold. This is accompanied by a co-ordinate increase in the levels of the both the GCSl and GCSh subunits, each by approximately 2-fold. The transcriptional activity of the GLCLR gene was increased by approximately 2.5-fold in SNP-treated cells.
BACKGROUND: The bronchial epithelium is exposed to reactive oxygen species (ROS) derived from cigarette smoke, air pollutants and activated leucocytes. Glutathione (GSH) prevents ROS-mediated loss of cell function, tissue injury and inflammation, and its synthesis is regulated by gamma-glutamylcysteine synthetase (gamma-GCS). However, the capacity of bronchial epithelial cells to adapt to oxidative stress and the mechanisms involved are not known. OBJECTIVE: To investigate the effects of oxidative stress on the regulation of GSH synthesis in human bronchial epithelial (NCI-H292) cells. METHODS: NCI-H292 cells were exposed to menadione and intracellular GSH concentrations were measured by spectrophotometry. gamma-GCS activity was measured by HPLC assay and changes in gamma-GCS mRNA by Northern blotting. RESULTS: Exposure to menadione (MQ, 10-200 microm, 30-120 min) decreased total cellular GSH content, measured immediately after exposure to MQ. However, GSH content measured 6-12 h after withdrawal of the oxidant stress (MQ, 50 microm, 30 min), increased c. two fold over baseline levels (P < 0.001). gamma-GCS activity measured 6 h (21.7 +/- 3.4 nmol/min/mg, SD, n = 5, P < 0.01) or 12 h (23.2 +/- 4.6, P < 0.001) after MQ treatment was also significantly increased compared with untreated cells (12.8 +/- 1.0). Similarly, gamma-GCS mRNA expression increased 1.3-1.6-fold relative to GAPDH mRNA, 3-6 h after MQ treatment. The MQ-induced increase in gamma-GCS mRNA expression was completely inhibited by actinomycin D. CONCLUSIONS: Bronchial epithelial (NCI-H292) cells respond rapidly and sensitively to oxidant stress, and this adaptive response is mediated by increased gamma-GCS mRNA transcription and enzyme activity.
Protein involved in the synthesis of the tripeptide glutathione (Gamma-Glu-Cys-Gly). Glutathione sulphydryl group is kept largely in the reduced state; this allows it to act as a sulphydryl buffer, reducing any disulphide bonds formed within cytoplasmic proteins to cysteines. Glutathione is also important as a cofactor for the enzyme glutathione peroxidase, in the uptake of amino acids and participates in leucotriene synthesis. Glutathione contains an unusual peptide linkage between the carboxyl group of the glutamate side chain and the amine group of cysteine.
Enzyme that catalyzes the joining of two molecules coupled with the breakdown of a pyrophosphate bond in ATP or a similar triphosphate. Sometimes the terms "synthase", "synthetase" or "carboxylase" are also used for this class of enzymes.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
My favorites 
My labels 
Login
