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 structural gene for glyoxalase I (GLO1) of Saccharomyces cerevisiae was identified. The GLO1 gene contained an open reading frame with 326 amino acids, and the molecular weight of the gene product (Glo1p) deduced from the DNA sequence was calculated to be 37,207.06. Glyoxalase I activity increased approximately 95-fold when the GLO1 gene was introduced into the yeast cell with a multicopy plasmid, and the resultant transformant showed the increased resistance against methylglyoxal. Since the knockout mutant of the GLO1 gene of haploid strain of S. cerevisiae was still viable, the GLO1 gene was thought to be unnecessary for growth of the yeast. The GLO1 gene was overexpressed in two kinds of glutathione-deficient mutants, gamma-glutamylcysteine synthetase-deficient (gsh1(-)) and glutathione synthetase-deficient (gsh2(-)), respectively, and the sensitivites to methylglyoxal were compared. The gsh1-deficient mutant, which could not produce glutathione at all, was hypersensitive to methylglyoxal, and overproduction of the Glo1p did not restore the growth arrest caused by exogenously added methylglyoxal. The gsh2-deficient mutant, which accumulates gamma-glutamylcysteine (an intermediate of glutathione biosynthesis), was also sensitive to methylglyoxal compared with the isogenic wild type strain, although the growth arrest caused by methylglyoxal was partially restored by overexpression of the GLO1 gene. Purified glyoxalase I from yeast could use gamma-glutamylcysteine as a substrate (kcat/Km = 1.89 x 10(7) M-1 s-1, glutathione; 3.47 x 10(4) M-1 s-1, gamma-glutamylcysteine).
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PMID:Identification of the structural gene for glyoxalase I from Saccharomyces cerevisiae. 882 31

Mutants in the adenine biosynthetic pathway of yeasts (ade1 and ade2 of Saccharomyces cerevisiae, ade6 and ade7 of Schizosaccharomyces pombe) accumulate an intense red pigment in their vacuoles when grown under adenine-limiting conditions. The precise events that determine the formation of the pigment are however, still unknown. We have begun a genetic investigation into the nature and cause of pigmentation of ade6 mutants of S. pombe and have discovered that one of these pigmentation defective mutants, apd1 (adenine pigmentation defective), is a strict glutathione auxotroph. The gene apd1+ was found to encode the first enzyme in glutathione biosynthesis, gamma-glutamylcysteine synthetase, gcs1+. This gene when expressed in the mutant could confer both glutathione prototrophy and the characteristic red pigmentation, and disruption of the gene led to a loss in both phenotypes. Supplementation of glutathione in the medium, however, could only restore growth but not the pigmentation because the cells were unable to achieve sufficient intracellular levels of glutathione. Disruption of the second enzyme in glutathione biosynthesis, glutathione synthetase gsh2+, also led to glutathione auxotrophy, but only a partial defect in pigment formation. A reevaluation of the major amino acids previously reported to be present in the pigment indicated that the pigment is probably a glutathione conjugate. The ability of vanadate to inhibit pigment formation indicated that the conjugate was transported into the vacuole through a glutathione-conjugate pump. This was further confirmed using strains of S. cerevisiae bearing disruptions in the recently identified glutathione-conjugate pump, YCF1, where a significant reduction in pigment formation was observed. The pump of S. pombe is distinct from the previously identified vacuolar pump, hmt1p, for transporting cadystin peptides into vacuoles of S. pombe.
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PMID:apd1+, a gene required for red pigment formation in ade6 mutants of Schizosaccharomyces pombe, encodes an enzyme required for glutathione biosynthesis: a role for glutathione and a glutathione-conjugate pump. 901 91

Tumor necrosis factor (TNF) is an inflammatory cytokine that causes cell injury by generation of oxidative stress. Since glutathione (GSH) is a key cellular antioxidant that detoxifies reactive oxygen species, the purpose of our work was to examine the regulation of cellular GSH, the expression of heavy subunit chain of gamma-glutamylcysteine synthetase (gamma-GCS-HS), and control of intracellular generation of reactive oxygen species in cultured rat hepatocytes treated with TNF. Exposure of cells to TNF (10,000 units/ml) resulted in depletion of cellular GSH levels (50-70%) and overproduction of hydrogen peroxide (2-3-fold) and lipid peroxidation. However, cells treated with lower doses of TNF (250-500 units/ml) exhibited increased levels of GSH (60-80% over control). TNF treatment increased (70-100%) the levels of gamma-GCS-HS mRNA, the catalytic subunit of the regulating enzyme in GSH biosynthesis. Furthermore, intact nuclei isolated from hepatocytes treated with TNF transcribed the gamma-GCS-HS gene to a greater extent than control cells, indicating that TNF regulates gamma-GCS-HS at the transcriptional level. The capacity to synthesize GSH de novo determined in cell-free extracts incubated with GSH precursors was greater (50-70%) in hepatocytes that were treated with TNF; however, the activity of GSH synthetase remained unaltered by TNF treatment indicating that TNF selectively increased the activity of gamma-GCS. Despite activation of nuclear factor-kappaB (NF-kappaB) by TNF, this transcription factor was not required for TNF-induced transcription of gamma-GCS-HS as revealed by deletion constructs of the gamma-GCS-HS promoter subcloned in a chloramphenicol acetyltransferase reporter vector and transfected into HepG2 cells. In contrast, a construct containing AP-1 like/metal response regulatory elements increased chloramphenicol acetyltransferase activity upon exposure to TNF. Thus, TNF increases hepatocellular GSH levels by transcriptional regulation of gamma-GCS-HS gene, probably through AP-1/metal response element-like binding site(s) in its promoter, which may constitute a protective mechanism in the control of oxidative stress induced by inflammatory cytokines.
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PMID:Tumor necrosis factor increases hepatocellular glutathione by transcriptional regulation of the heavy subunit chain of gamma-glutamylcysteine synthetase. 937 27

The hybrid poplar (Populus tremula x P. alba) was transformed to express the Escherichia coli gene for gamma-glutamylcysteine synthetase (EC 6.3.2.2: gamma-ECS) in the cytosol. Four transformed lines of poplar were obtained. These were phenotypically indistinguishable from untransformed poplars. Three lines, ggs28 (Noctor et al. 1996, Plant Physiol 112: 1071-1078), ggs11 and ggs5 possessed high levels of bacterial gene transcripts. Line ggs17 had lower transcript levels. Antisera were prepared against bacterial gamma-ECS and bacterial glutathione synthetase (EC 6.3.2.3: GS). Using the antiserum prepared against the purified His-tagged E. coli gamma-ECS, lines ggs28, ggs11 and ggs5 were shown to possess abundant quantities of the bacterial protein, whereas ggs17 contained lower amounts. The antiserum prepared against the purified His-tagged E. coli GS was also effective in screening poplars transformed with the E. coli gene coding for this enzyme. Immunoblots of leaf extracts from poplars overexpressing GS using this antibody revealed two bands. The extractable foliar gamma-ECS activities of the gamma-ECS transformants were in quantitative agreement with the protein levels. Lines ggs28, ggs11 and ggs5 had approximately 30-fold higher gamma-ECS activity than untransformed poplars, whereas in ggs17 this activity was only augmented about 3-fold. The lines strongly overexpressing gamma-ECS, ggs28, ggs11 and ggs5, contained enhanced foliar levels of cysteine (up to 2-fold), gamma-glutamylcysteine (5- to 20-fold) and glutathione (2- to 4-fold). Foliar thiol contents in ggs17 were no different to those of untransformed plants.
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PMID:Modification of thiol contents in poplars (Populus tremula x P. alba) overexpressing enzymes involved in glutathione synthesis. 943 83

In roots of Brassica juncea L. cadmium (Cd) exposure (25 microM) induces a massive formation of phytochelatins (PCs), which is accompanied by an only moderate decrease (-20%) of the putative PC precursor glutathione (GSH). As PC formation in roots could be the result of local GSH de novo synthesis and/or depend on GSH import from the shoot, we have analyzed the expression of the enzymes involved in GSH synthesis in the root, namely OAS(thiol)lyase (OAS-TL; catalysing the last step in Cys biosynthesis), gamma-glutamylcysteine synthetase (gamma-ECS), and glutathione synthetase (GSHS). cDNA clones were isolated from a cDNA library prepared from heavy metal exposed roots. Protein sequences from cDNA clones encoding OAS-TL, gamma-ECS, and GSHS, all exhibited putative mitochondrial targeting sequences, however, for OAS-TL also two putative cytosolic isoforms were isolated. Furthermore, we have cloned several metallothionein cDNAs of the MT2 group. Northern blot analysis with coding region probes revealed that in roots of Cd-exposed plants transcript amounts for OAS-TL and GSHS were only moderately increased, whereas gamma-ECS mRNA showed a stronger increase. Expression analysis with 3'-UTR probes indicated that among the putative mitochondrial OAS-TL, gamma-ECS and GSHS isoforms only gamma-ECS was up-regulated in response to Cd exposure. Conversely, transcripts for MT2 appeared to be slightly reduced. The results indicate that in roots Cd-induced PC synthesis correlates with a moderate increase of expression of genes involved in GSH synthesis, the change for gamma-ECS being most pronounced.
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PMID:cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy-metal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial gamma-glutamylcysteine synthetase isoform. 962 Feb 67

Glutathione (GSH; gamma-glutamylcysteinylglycine) is ubiquitous in mammalian and other living cells. It has several important functions, including protection against oxidative stress. It is synthesized from its constituent amino acids by the consecutive actions of gamma-glutamylcysteine synthetase and GSH synthetase. gamma-Glutamylcysteine synthetase activity is modulated by its light subunit and by feedback inhibition of the end product, GSH. Treatment with an inhibitor, buthionine sulfoximine (BSO), of gamma-glutamylcysteine synthetase leads to decreased cellular GSH levels, and its application can provide a useful experimental model of GSH deficiency. Cellular levels of GSH may be increased by supplying substrates and GSH delivery compounds. Increasing cellular GSH may be therapeutically useful.
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PMID:Glutathione: an overview of biosynthesis and modulation. 967 38

To gain insight into cellular metabolism underlying the glutathione (GSH) alterations induced by surgical trauma, we assessed postoperative skeletal muscle GSH metabolism and its redox status in 10 patients undergoing elective abdominal surgery. Muscle biopsy specimens were taken from the quadriceps femoris muscle before and at 24 and 72 h after surgery. GSH concentrations decreased by 40% at 24 h postoperatively compared with the paired preoperative values (P < 0.001) and remained low at 72 h (P < 0.01). The concentration of GSH disulfide (GSSG) did not significantly change throughout the study period, whereas the total GSH (as GSH equivalent) concentration decreased after surgery. Of the GSH constituent amino acids, the concentration of cysteine remained unchanged throughout the study period (from 28.2 +/- 10.1 preoperatively to 29.4 +/- 13.9 at 24 h postoperatively and to 28.3 +/- 15.6 micromol/kg wet wt at 72 h postoperatively). Despite a reduction in glutamate concentration by 40% 24 h after surgery, no correlation was established between GSH and glutamate concentrations postoperatively. Activity of gamma-glutamylcysteine synthetase did not change significantly after surgery, whereas GSH synthetase activity decreased postoperatively (from 66.4 +/- 19.1 preoperatively to 41.0 +/- 10.5 24 h postoperatively, P < 0.01, and to 46.0 +/- 11.7 microU/mg protein 72 h postoperatively, P < 0.05). The decrease of GSH was correlated to the reduced GSH synthetase activity seen at 24 h postoperatively. These results indicate that the skeletal muscle GSH pool is diminished in patients after surgical trauma. The depletion of the GSH pool is associated with a decreased activity of GSH synthetase, indicating a decreased GSH synthetic capacity in skeletal muscle tissue.
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PMID:Surgical trauma decreases glutathione synthetic capacity in human skeletal muscle tissue. 968 40

Elevation of activity and mRNA level of a cytosolic aldehyde dehydrogenase-1 (ALDH1), which oxidizes aldophosphamide, was previously observed in a cyclophosphamide-resistant murine leukemia cell line. However, changes in other enzyme(s) which may detoxify the drug or produce anti-alkylating agent(s), have not been examined. The human leukemia cell line, K562, was made 30-fold resistant against 4-hydroperoxycyclophosphamide (4HC) by exposing the cells to increasing concentrations of the drug. Resistance against cisplatin was also increased by about 3-fold. Activities of glucose-6-phosphate dehydrogenase (G6PD) and ALDH1 were elevated more than 7-fold in the resistant cells. The mRNA level of the two enzymes was also proportionally elevated. The concentration of reduced glutathione (GSH) was higher in the resistant cells (i.e., 21.1 versus 4.68 nmole per 10(6) cells), while activities of gamma-glutamylcysteine synthetase and glutathione synthetase, and the expressions of other human ALDH genes were not increased in the resistant cells. These findings suggest that the acquired resistance against 4HC is a consequence of transcriptional activation of two genes, i.e., one encoding the G6PD, a major enzyme regenerating anti-alkylating GSH, and the other encoding ALDH1, which has a high activity for oxidation of aldophosphamide derived from 4HC.
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PMID:Enhanced expressions of glucose-6-phosphate dehydrogenase and cytosolic aldehyde dehydrogenase and elevation of reduced glutathione level in cyclophosphamide-resistant human leukemia cells. 971

Glutathione plays a pivotal role in protecting plants from environmental stresses, oxidative stress, xenobiotics, and some heavy metals. Arabidopsis plants treated with cadmium or copper responded by increasing transcription of the genes for glutathione synthesis, gamma-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione reductase. The response was specific for those metals whose toxicity is thought to be mitigated through phytochelatins, and other toxic and nontoxic metals did not alter mRNA levels. Feeding experiments suggested that neither oxidative stress, as results from exposure to H2O2, nor oxidized or reduced glutathione levels were responsible for activating transcription of these genes. Jasmonic acid also activated the same suite of genes, which suggests that it might be involved in the signal transduction pathway for copper and cadmium. Jasmonic acid treatment increased mRNA levels and the capacity for glutathione synthesis but did not alter the glutathione content in unstressed plants, which supports the idea that the glutathione concentration is controlled at multiple levels.
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PMID:Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. 972 99

Poplars (Populus tremula x Populus alba) were transformed to overexpress Escherichia coli gamma-glutamylcysteine synthetase (gamma-ECS) or glutathione synthetase in the chloroplast. Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product. Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity. Enhanced chloroplastic gamma-ECS activity markedly increased gamma-glutamylcysteine and GSH levels. These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol. Similar to cytosolic gamma-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes. Light was required for maximal accumulation of GSH in poplars overexpressing gamma-ECS in the chloroplast. High chloroplastic, but not cytosolic, gamma-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast. We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased gamma-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast.
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PMID:Manipulation of glutathione and amino acid biosynthesis in the chloroplast 976 32


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