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:2.5.1.47 (cysteine synthase)
625 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutathione (GSH) is one of several intracellular hydrogen donating species thought to compete with O2, a damaging species, to repair radiation induced free radical damage. The O2 K factor was determined for normal Chinese hamster V79 cells, V79 cells made acutely thiol deficient (no detectable GSH or NPSH) using the gamma-glutamyl-cysteine synthetase enzyme inhibitor D,L-Buthionine-S,R-sulfoximine (D,L-BSO), or a human skin fibroblast cell line GM3877 which, because of the nature of its genetic defect, has chronically low levels of GSH (7% of normal skin fibroblasts). The K factors for normal V79 cells, treated with BSO or GSH deficient human fibroblasts, were 0.54, 0.15, and 0.1% O2, respectively. While thiol depletion affects the O2 K factor, V79 cells without any detectable GSH were still not as sensitive as the genetically deficient line GM3877 with 7% of normal GSH values. Other factors which may influence the results are whether the GSH levels remain low or regenerate following irradiation and the intracellular distribution of GSH.
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PMID:Oxygen concentration and the OER for acutely or chronically thiol deficient cells. 374 29

The effects of depletion of cellular glutathione (GSH) on the sensitivity of cultured EMT6/SF cells to chemotherapy agents or x rays under hypoxic and aerated conditions were investigated. Buthionine sulfoximine (BSO), a potent inhibitor of the enzyme gamma-glutamyl-cysteine synthetase, was used to deplete cellular GSH. Addition of BSO (50 microM) to EMT6/SF cultures depleted cellular GSH with a half-time of approximately 2 hr. Cellular GSH reached very low levels within hours of addition of BSO. After removal of BSO, cellular GSH recovered with approximately the same kinetics as was seen for depletion. Incubation of EMT6/SF cells with BSO concentrations of up to 1 mM did not reduce the viability or inhibit growth when exposure was limited to times less than 24 hr. However, for longer exposure times, toxicity and growth inhibition were demonstrated in a dose dependent fashion. EMT6/SF cells were treated with chemotherapy agents under either aerated or extremely hypoxic conditions. Cells were more sensitive to cis-dichlorodiammino Pt(II) (DDP), mitomycin C (MitC), L-phenylalanine mustard (L-PAM), and nitrogen mustard (HN2) when treatment was under hypoxic conditions. The magnitude of this sensitization under hypoxic conditions ranged from a dose modifying factor (DMF) of 1.4 (HN2) to 4.1 (MitC), measured at the 0.1 level of cell survival. Hypoxic EMT6/SF cells were more resistant to the cytotoxic effects of actinomycin D (ActD) under hypoxic conditions (DMF = 10 at SF = 0.3). When cellular GSH was depleted to less than 5% of control by treatment with 50 microM BSO for 12-14 hr, cells were sensitized to DDP, L-PAM and HN2 under both aerated and hypoxic conditions. DMF's ranged from 1.4-6.5, depending on the agent. Hypoxic cell sensitization was never significantly greater than that seen in aerated cells, as was the case for X radiation (DMF = 1.3 for hypoxic cells only). GSH depletion also sensitized to MitC, but only under aerated conditions (DMF = 2.1). Hypoxic EMT6/SF cells were not sensitized to MitC by depletion of GSH. GSH depletion afforded slight protection against ActD toxicity under both aerated and hypoxic conditions. These studies suggest that cellular GSH plays an important role in modifying cellular response to cytotoxic drugs. GSH depletion may sensitize tumor cells to some chemotherapy agents, but differential sensitization of tumors compared to normal tissues, based on hypoxic tumor cells as targets, would not be expected based on these in vitro experiments.
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PMID:Effects of glutathione depletion by buthionine sulfoximine on the sensitivity of EMT6/SF cells to chemotherapy agents or X radiation. 374 36

The impact of intracellular glutathione depletion on chromosome damage induced by X irradiation under aerobic conditions was investigated in two different cell lines, Ehrlich ascites tumor cells (EATC) and Chinese hamster ovary cells (CHO-K1). Thiol-depleted cell cultures in plateau phase were obtained by prolonged incubation in growth medium containing DL-buthionine-SR-sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase. Cells were then assayed using the procedures of G. L. Ellmann (Arch. Biochem. Biophys. 82, 70-77 (1959)), F. Tietze (Anal. Biochem. 27, 502-522 (1969)), and J. Sedlack and R.H. Lindsay (Anal. Biochem. 25, 192-205 (1968)) for non-protein bound SH (NPSH), glutathione (GSH), and total SH (TSH). In both cell lines GSH was reduced to less than 10% of controls at higher BSO concentrations around 1 mM, whereas TSH and NPSH were affected to only 40-60%. In EATC pretreated with up to 1 mM BSO for 72 h, increased levels of spontaneously occurring micronuclei were found. At BSO concentrations above 200 microM, both cell lines showed a potentiation of chromosome lesions scored as micronuclei and induced under aerobic X irradiation when liquid holding recovery in the original nutrient-depleted medium was performed; the extent of chromosome damage eventually reached that which could be obtained by application of beta-arabinofuranosyladenine (beta-araA), known to inhibit DNA repair processes by blocking DNA polymerases. It is therefore suggested that GSH depletion causes impairment of repair of lesions leading to chromosome deletions and subsequently to micronuclei. In contrast to CHO cell cultures, EATC showed a reversion of the potentiation effect as indicated by a decrease in the micronucleus content during prolonged incubation in the presence of BSO in the millimolar range. This effect could not be correlated to the remaining GSH content of less than 10% but may be due to some accumulation of unknown NPSH components. Since addition of L-cysteine to EATC cultures pretreated with BSO decreased the micronucleus content, cysteine/cystine or a related thiol within the NPSH fraction may be involved in the reestablishment of repair. Thus at least in one cell line, a rather complex response to BSO administration indicated that not only GSH but also other thiols may determine the level of chromosome damage after liquid holding recovery.
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PMID:Glutathione depletion by DL-buthionine-SR-sulfoximine (BSO) potentiates X-ray-induced chromosome lesions after liquid holding recovery. 375 39

Salmonella typhimurium strains (OASS-positive) synthesize a toxic but non-mutagenic metabolite from cyanide and O-acetylserine. Salmonella typhimurium mutant DW379 (OASS-deficient) is neither able to carry out this reaction in vitro nor produce the toxic metabolite in vivo. L-Cysteine reverses the cyanide metabolite mediated inhibition and thus allows OASS-positive strains to grow in medium containing cyanide and O-acetylserine. The results suggest that the enzyme O-acetylserine sulfhydrylase catalyzes the reaction of cyanide and O-acetylserine to form the toxic metabolite. This metabolite is ninhydrin-positive, adheres strongly to the cation-exchange column, and migrates in TLC to an Rf value similar to that of beta-cyanoalanine.
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PMID:Activation of sodium cyanide to a toxic but non-mutagenic metabolite in Salmonella typhimurium. 393 44

Glutathione-depleted hepatocytes, by incubation with diethylmaleate (DEM) or phorone (2,6-dimethyl-2,5-heptadiene-4-one), i.e., substrates of the GSH S-transferases (EC 2.5.1.18), showed rates of gluconeogenesis from various precursors significantly lower than controls; however the rate of glucose synthesis from fructose was similar to that of controls. Isolated hepatocytes from rats pretreated with those substrates 1 h before isolation to deplete hepatic glutathione (GSH) also showed a decrease of the rate of gluconeogenesis from lactate plus pyruvate. Incubation of hepatocytes with L-buthionine sulfoximine, a specific inhibitor of gamma-glutamyl-cysteine synthetase (EC 6.3.2.2), resulted in a decreased rate of gluconeogenesis from lactate plus pyruvate only when GSH values were lower than 1 mumol/g cells. Freeze-clamped livers from GSH-depleted rats showed a higher concentration of malate and glycerol 3-phosphate, indicating that GSH depletion probably affects phosphoenolpyruvate carboxykinase and glycerol-3-phosphate dehydrogenase activities. Several indicators of cell viability, such as lactate dehydrogenase leakage, malondialdehyde accumulation, ATP concentration, or urea synthesis from different precursors, were not affected by GSH depletion under the experimental conditions used here. Besides, the GSH/GSSG ratio remained unchanged in all cases.
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PMID:Effects of glutathione depletion on gluconeogenesis in isolated hepatocytes. 402 24

The role of glutathione (GSH) in lectin-induced lymphocyte activation can be studied by quantitating lectin-induced nuclear size transformation in the presence of variable degrees of GSH depletion. Buthionine sulfoximine (BSO) inhibits intracellular GSH synthesis by inhibition of the enzyme gamma-glutamyl-cysteine synthetase. By combining endogenous GSH depletion in cell cultures with BSO-induced inhibition of GSH synthesis, lectin-induced lymphocyte activation can be studied at various concentrations of soluble intracellular GSH. With this approach, the percentage of lymphocytes undergoing a nuclear size transformation is minimally affected despite depletion of soluble intracellular GSH to 0.27 nmol/10(7) cells (PBL), which represents approximately 95% depletion of intracellular GSH. When soluble intracellular GSH is depleted to undetectable levels (less than 0.10 nmol/10(7) cells) there is a 10 to 12% reduction in the number of cell nuclei transformed. However, in all BSO-pretreated cultures the lectin-induced nuclear size transformation is intermediate between resting and blast-transformed lymphocytes, suggesting only partial (or aborted) activation. The partial activation response observed in BSO-pretreated cultures may be due to mobilization of the protein-bound pool of GSH, which is relatively resistant to depletion by BSO. That the inhibition of full blast transformation is truly due to GSH depletion was proven by experiments in which GSH was repleted exogenously and a full blast transformation was restored. The results of previous work in our laboratory had shown that the sulfhydryl-reactive agent 2-cyclohexene-1-one (2-CHX) was a potent inhibitor of activation at soluble intracellular GSH concentrations well above 0.27 nmol/10(7) PBL. In the present study, the dose-dependent inhibition of activation by 2-CHX was confirmed, but it was shown that the degree of inhibition caused by 2-CHX could be at least partially dissociated from the level of intracellular GSH present at the time of lectin addition and that the inhibitory potential of 2-CHX exceeded that of BSO at comparable levels of soluble intracellular GSH. Thus, the inhibitory properties of 2-CHX cannot be accounted for solely on the basis of GSH depletion.
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PMID:The role of glutathione in lymphocyte activation. I. Comparison of inhibitory effects of buthionine sulfoximine and 2-cyclohexene-1-one by nuclear size transformation. 403 98

The two enzymes required for de novo glutathione synthesis, glutamyl cysteine synthetase and glutathione synthetase, have been demonstrated in hemolysates of human erythrocytes. Glutamyl cysteine synthetase requires glutamic acid, cysteine, adenosine triphosphate (ATP), and magnesium ions to form gamma-glutamyl cysteine. The activity of this enzyme in hemolysates from 25 normal subjects was 0.43+/-0.04 mumole glutamyl cysteine formed per g hemoglobin per min. Glutathione synthetase requires gamma-glutamyl cysteine, glycine, ATP, and magnesium ions to form glutathione. The activity of this enzyme in hemolysates from 25 normal subjects was 0.19+/-0.03 mumole glutathione formed per g hemoglobin per min. Glutathione synthetase also catalyzes an exchange reaction between glycine and glutathione, but this reaction is not significant under the conditions used for assay of hemolysates. The capacity for erythrocytes to synthesize glutathione exceeds the rate of glutathione turnover by 150-fold, indicating that there is considerable reserve capacity for glutathione synthesis. A patient with erythrocyte glutathione synthetase deficiency has been described. The inability of patients' extracts to synthesize glutathione is corrected by the addition of pure glutathione synthetase, indicating that there is no inhibitor in the patients' erythrocytes.
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PMID:Glutathione biosynthesis in human erythrocytes. I. Identification of the enzymes of glutathione synthesis in hemolysates. 554 17

The activities and properties of the enzymes involved in the formation and degradation of pyroglutamic acid (2-pyrrolidone-5-carboxylic acid, 5-oxoproline) in guinea pig epidermis have been studied. The enzyme pattern was characterized by an extremely high activity of gamma-glutamyl cyclotransferase. The epidermal extracts possessed a measurable, but rather low activity of pyroglutamate hydrolase. It is suggested that the only major pathway by which pyroglutamate may be formed in epidermal tissue is from L-glutamate by a 2-step reaction, the first involving the formation of a gamma-glutamyl peptide by the action of gamma-glutamyl-cysteine synthetase, and the second cyclization of the gamma-glutamyl moiety by the action of gamma-glutamyl cyclotransferase. Abundant substrate supply, the extremely high cyclotransferase activity and the rather low capacity to degrade pyroglutamate may be the factors responsible for the accumulation of this compound in epidermal tissue. A relatively low content of reduced glutathione may also be a contributing factor.
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PMID:Studies on the accumulation of L-pyroglutamic acid in guinea pig epidermis. 610 5

Two brothers, aged 16 and 11 years, had recurrent episodes of vomiting, diarrhoea and abdominal pain, starting in infancy. In spite of extensive investigations no cause of their enterocolitis could be established. After several years symptomatic treatment was discontinued without any recurrence of symptoms. Their father and several paternal relatives have had kidney stones. Both boys developed urolithiasis and an oxalate-containing stone was removed from the elder brother's kidney. He had no hypercalciuria. His glomerular and tubular function tests were normal. Gas chromatography of urine from both brothers revealed massive excretion of L-5-oxoproline (pyroglutamic acid). Glutathione levels in erythrocytes of both patients were normal. The activities of enzymes of the gamma-glutamyl cycle were analysed in erythrocytes, leukocytes and cultured skin fibroblasts. The level of glutathione synthetase was normal, as was the affinity of this enzyme for its substrate gamma-glutamyl-cysteine. Feedback inhibition of gamma-glutamyl-cysteine synthetase by glutathione was also normal. Both patients had a specific deficiency of 5-oxoprolinase, the activity of which was 2-4% of that of control subjects. Their parents had intermediate 5-oxoprolinase activities in fibroblasts, indicating a recessive mode of inheritance. Thus, 5-oxoprolinuria in these two patients was due to a lack of 5-oxoprolinase, i.e., a new inborn error in the gamma-glutamyl cycle.
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PMID:5-oxoprolinuria due to hereditary 5-oxoprolinase deficiency in two brothers--a new inborn error of the gamma-glutamyl cycle. 611 26

The biochemical properties of red cells from normal sheep and sheep with three types of red cell glutathione (GSH)-deficiency were compared. One deficiency was due to an impaired transport system for amino acids (lesion 1), one was the result of a diminished activity of gamma-glutamyl cysteine synthetase (GC-S) (lesion 2) and the third was a combined deficiency produced by selective breeding to give animals with both lesions 1 and 2. Under normal husbandry conditions no clinical symptoms were apparent in sheep with lesion 2, but red cells from sheep with lesion 1 and lesions 1 + 2 showed an increased osmotic fragility, a greater tendency to form Heinz bodies and a shorter potential life span than normal. These deficiencies were not found in tissues other than blood. Normal and GSH-deficient red cells had the expected low concentrations of 5-oxoproline. The effects of the toxic agents phenylhydrazine, s-methylcysteine sulphoxide and nitrite in vivo were measured in sheep of the different types. GSH-deficient sheep responded earlier and more dramatically than normal sheep, showing greater methaemoglobin formation, and for phenylhydrazine and s-methylcysteine sulphoxide, more severe anaemia. Sheep with the combined lesions were in general the most susceptible, but even they had the ability to recover from moderately severe oxidative challenge.
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PMID:Red cell glutathione deficiency: clinical and biochemical investigations using sheep as an experimental model system. 611 41


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