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)

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

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

This paper reports that the glutathione (GSH)-deficient mutant, cad2-1, of Arabidopsis is deficient in the first enzyme in the pathway of GSH biosynthesis, gamma-glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, gamma-glutamylcysteine. In vitro enzyme assays showed that the cad2-1 mutant has 40% of wild-type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. The CAD2 locus maps to chromosome 4 and is tightly linked to a gene, GSHA, identified by a previously isolated cDNA. A genomic clone of GSHA complements both the phenotypic and biochemical deficiencies of the cad2-1 mutant. The nucleotide sequence of the gene has been determined and, in the mutant, this gene contains a 6 bp deletion within an exon. These data demonstrate that the CAD2 gene encodes GCS. The cad2-1 mutation is close to the conserved cysteine which is believed to bind the substrate glutamate and the specific inhibitor L-buthionine-[S,R] sulfoximine (BSO). Both root growth and GCS activity of the cad2-1 mutant was less sensitive than the wild-type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.
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PMID:The glutathione-deficient, cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in gamma-glutamylcysteine synthetase. 980 29

By complementation screening of a cadmium-sensitive Schizosaccharomyces pombe mutant deficient in phytochelatin synthesis, but with 44% of the wild-type glutathione content, we cloned a DNA fragment involved in phytochelatin synthesis. Sequence analysis revealed that it encodes the second enzyme involved in glutathione (GSH) biosynthesis, glutathione synthetase (GSH2) (E.C.6.3.2.3, Wang and Oliver, 1997). The mutant allele shows a single base-pair exchange at the 3' end of the reading frame leading to a single amino acid change from glycine to aspartate. This mutation leads to a significant reduction of phytochelatin synthesis, whereas glutathione synthesis is impaired to a far lesser extent. Complementation with the Arabidopsis thaliana GSH2 cDNA led to a partial restoration of phytochelatin synthesis. These data strongly suggest that the GSH2 gene encodes a bifunctional enzyme that is able to catalyse both the synthesis of GSH by adding glycine to the dipeptide (gammaGlu-Cys) and the synthesis of phytochelatins. The sequence has been submitted to EMBL, Accession No. Y08414.
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PMID:Biosynthesis of phytochelatins in the fission yeast. Phytochelatin synthesis: a second role for the glutathione synthetase gene of Schizosaccharomyces pombe. 1021 97

Glutathione (GSH), a major antioxidant in most aerobic organisms, is perceived to be particularly important in plant chloroplasts because it helps to protect the photosynthetic apparatus from oxidative damage. In transgenic tobacco plants overexpressing a chloroplast-targeted gamma-glutamylcysteine synthetase (gamma-ECS), foliar levels of GSH were raised threefold. Paradoxically, increased GSH biosynthetic capacity in the chloroplast resulted in greatly enhanced oxidative stress, which was manifested as light intensity-dependent chlorosis or necrosis. This phenotype was associated with foliar pools of both GSH and gamma-glutamylcysteine (the immediate precursor to GSH) being in a more oxidized state. Further manipulations of both the content and redox state of the foliar thiol pools were achieved using hybrid transgenic plants with enhanced glutathione synthetase or glutathione reductase activity in addition to elevated levels of gamma-ECS. Given the results of these experiments, we suggest that gamma-ECS-transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast.
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PMID:Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress 1040 29

High-performance liquid chromatography (HPLC) with fluorescence detection was used to study thiol metabolism in legume nodules. Glutathione (GSH) was the major non-protein thiol in all indeterminate nodules examined, as well as in the determinate nodules of cowpea (Vigna unguiculata), whereas homoglutathione (hGSH) predominated in soybean (Glycine max), bean (Phaseolus vulgaris), and mungbean (Vigna radiata) nodules. All nodules had greater thiol concentrations than the leaves and roots of the same plants because of active thiol synthesis in nodule tissue. The correlation between thiol tripeptides and the activities of glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) in the nodules of eight legumes, and the contrasting thiol contents and activities in alfalfa (Medicago sativa) leaves (98% hGSH, 100% hGSHS) and nodules (72% GSH, 80% GSHS) indicated that the distribution of GSH and hGSH is determined by specific synthetases. Thiol contents and synthesis decreased with both natural and induced nodule senescence, and were also reduced in the senescent zone of indeterminate nodules. Thiols and GSHS were especially abundant in the meristematic and infected zones of pea (Pisum sativum) nodules. Thiols and gamma-glutamylcysteinyl synthetase were also more abundant in the infected zone of bean nodules, but hGSHS was predominant in the cortex. Isolation of full-length cDNA sequences coding for gamma-glutamylcysteinyl synthetase from legume nodules revealed that they are highly homologous to those from other higher plants.
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PMID:Glutathione and homoglutathione synthesis in legume root nodules. 1055 36

Glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH) is synthesized from its constituent amino acids by the sequential action of gamma-glutamylcysteine synthetase (gamma-GCS) and GSH synthetase. The intracellular GSH concentration, typically 1-8 mM, reflects a dynamic balance between the rate of GSH synthesis and the combined rate of GSH consumption within the cell and loss through efflux. The gamma-GCS reaction is rate limiting for GSH synthesis, and regulation of gamma-GCS expression and activity is critical for GSH homeostasis. Transcription of the gamma-GCS subunit genes is controlled by a variety of factors through mechanisms that are not yet fully elucidated. Glutathione synthesis is also modulated by the availability of gamma-GCS substrates, primarily L-cysteine, by feedback inhibition of gamma-GCS by GSH, and by covalent inhibition of gamma-GCS by phosphorylation or nitrosation. Because GSH plays a critical role in cellular defenses against electrophiles, oxidative stress and nitrosating species, pharmacologic manipulation of GSH synthesis has received much attention. Administration of L-cysteine precursors and other strategies allow GSH levels to be maintained under conditions that would otherwise result in GSH depletion and cytotoxicity. Conversely, inhibitors of gamma-GCS have been used to deplete GSH as a strategy for increasing the sensitivity of tumors and parasites to certain therapeutic interventions.
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PMID:Biologic and pharmacologic regulation of mammalian glutathione synthesis. 1056 25

Human immunodeficiency virus (HIV) progressively depletes GSH content in humans. Although the accumulated evidence suggests a role of decreased GSH in the pathogenesis of HIV, significant controversy remains concerning the mechanism of GSH depletion, especially in regard to envisioning appropriate therapeutic strategies to help compensate for such decreased antioxidant capacity. Tat, a transactivator encoded by HIV, is sufficient to cause GSH depletion in vitro and is implicated in AIDS-associated Kaposi's sarcoma and B cell lymphoma. In this study, we report a decrease in GSH biosynthesis with Tat, using HIV-1 Tat transgenic (Tat+) mice. A significant decline in the total intracellular GSH content in liver and erythrocytes of Tat+ mice was accompanied by decreased gamma-glutamylcysteine synthetase regulatory subunit mRNA and protein content, which resulted in an increased sensitivity of gamma-glutamylcysteine synthetase to feedback inhibition by GSH. Further study revealed a significant reduction in the activity of GSH synthetase in liver of Tat+ mice, which was linearly associated with their GSH content. Therefore, Tat appears to decrease GSH in vivo, at least partially, through modulation of GSH biosynthetic enzymes.
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PMID:Molecular mechanism of decreased glutathione content in human immunodeficiency virus type 1 Tat-transgenic mice. 1065 68

To search for compounds that reverse the drug resistance induced by glutathione (GSH), an original screening system to detect intracellular GSH depleters was established. Among 8843 microbes derived from the soil samples tested, the extracts of two Streptomyces species named KS6701 and KS8846, lowered the intracellular GSH level of Saccharomyces cerevisiae 5 x 47. From both the microbes, 5-hydroxy-4-oxo-L-norvaline (HON) was isolated as the active compound. At a concentration of 50-100 micrograms/ml, HON also decreased the GSH/protein level of the human ovarian tumor cell line, 2008/C13*5.25 and reversed its resistance to cisplatin. We also investigated the mechanism of the depletion. HON had little effect on gamma-glutamylcysteine synthetase (gamma-GCS) or glutathione synthetase, but HON decreased the quantity of thiol substances when it was spontaneously reacted with them. This suggested that the GSH depletion by HON occurred through a mechanism different from that of buthionine sulfoximine, a selective gamma-GCS inhibitor.
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PMID:5-Hydroxy-4-oxo-L-norvaline depletes intracellular glutathione: a new modulator of drug resistance. 1068 Nov 31

Increasing evidence suggests that glutathione (GSH) synthesis is a regulated process. Documented increases in gamma-glutamylcysteine synthetase (GCS) occur in response to oxidants, in tumors, on plating cells at a low cell density, and with nerve growth factor stimulation, suggesting that GSH synthesis may be related to the cell growth and transformation. Previously, extracellular acidic fibroblast growth factor (FGF-1) has been demonstrated to cause transformation and aggressive cell growth in murine embryonic fibroblasts. In the present investigation, we sought to determine whether FGF-1, with its growth inducing properties, resulted in the modulation of GSH biosynthetic enzymes, GCS and GSH synthetase. Murine fibroblasts transduced with (hst/KS)FGF-1, a chimeric human FGF-1 gene containing a signal peptide sequence for secretion, displayed elevated gene expression of both heavy and light subunits of GCS. Activity of GSH synthetase was also elevated in these cells compared with control cells. Nonetheless, GSH was decreased in the FGF-1-transduced cells along with high energy phosphates, adenine nucleotides, NADH, and the redox poise. However, GSSG was not elevated in these cells. Fibroblasts stably expressing human immunodeficiency virus type 1 Tat, which induces intrinsic FGF-1 secretion, resulted in similar changes in GCS, GS, and GSH. The results suggest that although increases in the enzymes of GSH synthesis are a common response to growth factors, an increase in GSH content per se is not required for altered cell growth.
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PMID:Modulation of glutathione synthetic enzymes by acidic fibroblast growth factor. 1068 68


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