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PubMed1989

Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange.

The regulation of purified glutathione S-transferase from rat liver microsomes was studied by examining the effects of various sulfhydryl reagents on enzyme activity with 1-chloro-2,4-dinitrobenzene as the substrate. Diamide (4 mM), cystamine (5 mM), and N-ethylmaleimide (1 mM) increased the microsomal glutathione S-transferase activity by 3-, 2-, and 10-fold, respectively, in absence of glutathione; glutathione disulfide had no effect. In presence of glutathione, microsomal glutathione S-transferase activity was increased 10-fold by diamide (0.5 mM), but the activation of the transferase by N-ethylmaleimide or cystamine was only slightly affected by presence of glutathione. The activation of microsomal glutathione S-transferase by diamide or cystamine was reversed by the addition of dithiothreitol. Glutathione disulfide increased microsomal glutathione S-transferase activity only when membrane-bound enzyme was used. These results indicate that microsomal glutathione S-transferase activity may be regulated by reversible thiol/disulfide exchange and that mixed disulfide formation of the microsomal glutathione S-transferase with glutathione disulfide may be catalyzed enzymatically in vivo.

Animals↗
PubMed2006

Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH.

BACKGROUND: Glutathione, a ubiquitous tripeptide, is an important cellular constituent, and measurement of reduced and oxidized glutathione is a measure of the redox state of cells. Glutathione-S-transferase (GST) fusion proteins bind naturally to beads derivatized with glutathione, and elution of such bead-bound fusion proteins with buffer containing millimolar glutathione is a commonly used method of protein purification. Many protein-protein interactions have been established by using GST fusion proteins and measuring binding of fusion protein binding partners by GST pulldown assays, usually monitored by Western blot methodology. METHODS: Dextran beads suitable for flow cytometry were derivatized with glutathione. A fusion protein of GST and green fluorescent protein was used to define kinetic and equilibrium binding characteristics of GST fusion proteins to glutathione beads. Free glutathione competes with this binding, and this competition was used to measure free glutathione concentration. RESULTS: A 10 microl assay can measure 5 microl of 20 microM glutathione (100 pmol glutathione) in 2 h by flow cytometry. This concentration is two orders of magnitude lower than cellular glutathione concentrations, and three orders of magnitude lower than affinity chromatography eluates. One important result is that by generating high site density, the GST fusion proteins can be constrained to the surface of one bead without hopping to the next bead in multiplex assays. CONCLUSIONS: Glutathione in cellular lysates and GST-fusion protein affinity chromatography eluates can be measured by flow cytometry. Many interactions between GST fusion proteins and their fluorescent binding partners should be quantifiable by flow cytometry. Although a system may have the disadvantage that it has a low affinity and a correspondingly quick off-rate in solution, it may remain on beads if the site density can be increased to offer a slow apparent off rate.

Binding Sites↗
PubMed1985

The fall and rise of cellular glutathione levels: enzyme-based approaches.

Present knowledge of the pathways of metabolism and transport of glutathione and of its functions has been substantially advanced by study of the enzymes involved in glutathione metabolism, especially by investigations that have elucidated their mechanisms of action, active site topology, and other aspects of their behavior. Such information has made it possible to design selective enzyme inhibitors, which have served as valuable experimental tools and which may also be useful in therapy. Cellular glutathione levels may be lowered or raised by appropriate biochemical procedures. Glutathione synthesis is effectively blocked by administration of buthionine sulfoximine, which selectively binds to the active site of gamma-glutamylcysteine synthetase. Cellular levels of glutathione may be increased by administration of cysteine precursors such as L-2-oxothiazolidine-4-carboxylate, which is effectively transported into cells and converted by 5-oxoprolinase to cysteine, which is utilized for glutathione synthesis. Administration of gamma-glutamylcysteine, gamma-glutamylcysteine disulfide, or gamma-glutamylcysteine also leads to increased cellular levels of glutathione, especially in the kidney. These gamma-glutamyl amino acids are transported and provide substrate for glutathione synthesis. Another way of increasing cellular glutathione levels consists of administration of glutathione esters. For example, the ester of glutathione in which the glycine carboxyl group is esterified with ethanol is well transported, and is converted to glutathione intracellularly. These studies indicate that manipulation of the cellular levels of glutathione can be achieved, and may be of value in the selective destruction of cells, and in their protection.

Animals↗
PubMed1997

Abnormal glutathione conjugation in patients with tyrosinaemia type I.

Previous studies have suggested that tyrosinaemia type I may be associated with reduced glutathione availability due to conjugation of tyrosinaemia-associated reactive intermediates with glutathione. In the present study, the glutathione/ glutathione S-transferase system of two tyrosinaemia patients and three healthy controls were characterized by administering the racemic sedative drug bromisoval, a probe drug for assessing glutathione conjugation activity in vivo. Furthermore, concentrations of glutathione and glutathione S-transferase class alpha (GSTA) isoenzymes as well as the glutathione S-transferase class mu phenotype were assessed in the blood of six tyrosinaemia patients. The excretion of bromisoval mercapturates in healthy children was comparable to that observed in healthy adults. Tyrosinaemia patients were found to have a very high urinary recovery of bromisoval mercapturates (> or = 60% of the dose compared to about 30% for healthy, age-matched children and adults), which could be attributed mainly to a higher urinary excretion of the mercapturate derived from S-bromisoval. Healthy children and adults predominantly excrete the (R)-bromisoval mercapturate. The differences in amount excreted as well as in stereoselectivity of the urinary excretion of bromisoval mercapturates in tyrosinaemia patients are possibly related to an increased activity of specific glutathione S-transferase isoenzymes. Plasma glutathione and blood cell glutathione disulphide concentrations in tyrosinaemia patients were normal. Low blood cell glutathione concentrations were in general found only in two patients with a poor clinical condition. These results indicate that, in contrast to previous suggestions, reduced glutathione availability is not a generalized problem in (stabilized) tyrosinaemia patients.

Adult↗
PubMed2000

Astrocytes provide cysteine to neurons by releasing glutathione.

Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine<-->cysteine + cysteine-glutathione disulfide. Conditioned medium from neuron-enriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS.

Animals↗
PubMed2004

Glutathione-S-transferase P1-1 protects aberrant crypt foci from apoptosis induced by deoxycholic acid.

BACKGROUND & AIMS: Aberrant crypt foci, precursors of colonic adenoma, are frequently positive for glutathione-S-transferase P1-1. Because deoxycholic acid is an apoptosis-inducing xenobiotic in the colon, we examined the possibility that aberrant crypt foci, through the cytoprotecting function of glutathione-S-transferase P1-1, resist deoxycholic acid-induced apoptosis, thereby surviving to become adenomas and subsequently cancer. METHODS: Glutathione-S-transferase P1-1 or cyclooxygenase-2 expression and the percentage of apoptotic cells in aberrant crypt foci were examined by immunohistochemistry and by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, respectively. Glutathione-S-transferase P1-1 was transfected into colon cancer cells (M7609) and human lung fibroblasts, and deoxycholic acid-induced apoptosis was evaluated by a dye-uptake assay and flow cytometry. Binding of deoxycholic acid to glutathione-S-transferase P1-1 was analyzed by circular dichroism and immunoprecipitation. Caspase activities were determined by colorimetric protease assay, and sulindac binding to glutathione-S-transferase P1-1 was determined by inhibition assay of glutathione-S-transferase P1-1 activity. RESULTS: Aberrant crypt foci showed positive immunostaining for glutathione-S-transferase P1-1 but negative staining for cyclooxygenase-2. The percentage of apoptotic cells in aberrant crypt foci was significantly lower than in healthy epithelium, and the difference became more apparent with deoxycholic acid treatment. The impaired sensitivity of aberrant crypt foci to deoxycholic acid was restored by the glutathione-S-transferase P1-1-specific inhibitor gamma-glutamyl-S-(benzyl)cysteinyl-R-phenylglycine diethylester. By transfection of glutathione-S-transferase P1-1, M7609 cells became more resistant to deoxycholic acid-induced apoptosis than mock transfectants. Direct binding of glutathione-S-transferase P1-1 to deoxycholic acid was proven by circular dichroism and by immunoprecipitation. The aberrant crypt foci in adenoma patients treated with sulindac, which was shown to bind to glutathione-S-transferase P1-1, underwent apoptosis in 4 days and mostly regressed in 2-3 months. CONCLUSIONS: Glutathione-S-transferase P1-1 protects aberrant crypt foci from deoxycholic acid-induced apoptosis and may play a pivotal role in early colon carcinogenesis.

Adenoma↗
PubMed1991

Glutathione deficiency leads to mitochondrial damage in brain.

Glutathione deficiency induced in newborn rats by giving buthionine sulfoximine, a selective inhibitor of gamma-glutamylcysteine synthetase, led to markedly decreased cerebral cortex glutathione levels and striking enlargement and degeneration of the mitochondria. These effects were prevented by giving glutathione monoethyl ester, which relieved the glutathione deficiency, but such effects were not prevented by giving glutathione, indicating that glutathione is not appreciably taken up by the cerebral cortex. Some of the oxygen used by mitochondria is known to be converted to hydrogen peroxide. We suggest that in glutathione deficiency, hydrogen peroxide accumulates and damages mitochondria. Glutathione, thus, has an essential function in mitochondria under normal physiological conditions. Observations on turnover and utilization of brain glutathione in newborn, preweaning, and adult rats show that (i) some glutathione turns over rapidly (t 1/2, approximately 30 min in adults, approximately 8 min in newborns), (ii) several pools of glutathione probably exist, and (iii) brain utilizes plasma glutathione, probably by gamma-glutamyl transpeptidase-initiated pathways that account for some, but not all, of the turnover; thus, there is recovery or transport of cysteine moieties. These studies provide an animal model for the human diseases involving glutathione deficiency and are relevant to oxidative phenomena that occur in the newborn.

Aging↗
PubMed1997

Dietary selenium increases cellular glutathione peroxidase activity and reduces the enhanced susceptibility to lipid peroxidation of plasma and low-density lipoprotein in kidney transplant recipients.

The glutathione system plays a major role in the protection of cells against oxidative stress in humans. The aim of the present study was to find out the relationship between the glutathione system and plasma lipid peroxidation in six renal transplant recipients (who are under oxidative stress and thus at high risk for atherosclerosis), by using dietary selenium to activate the glutathione system. 2,2'-Azobis-2-amidinopropane hydrochloride (AAPH)-induced plasma lipid peroxidation was increased (by 60%) in all six patients in comparison to normal subjects. A similar pattern of increased plasma lipid peroxidation was found even in the basal state (in the absence of added AAPH). CuSO4-induced low-density lipoprotein (LDL) oxidation measured by peroxide formation was also significantly increased by 2.3-fold in the patients' LDL in comparison to normal LDL. Even in the absence of CuSO4, the LDL oxidation state was also increased in the patients' LDL in comparison to normal LDL. We thus analyzed the effect of dietary selenium (0.2 mg/day for a period of 3 months, followed by an additional 3 months on placebo) on plasma and on LDL lipid peroxidation. Selenium treatment resulted in a 50% reduction in AAPH-induced plasma lipid peroxidation. The susceptibility of the patients' plasma to lipid peroxidation returned toward baseline values 3 months after termination of the selenium treatment. Similar results, although less pronounced (only 15% reduction), were obtained for CuSO4-induced LDL oxidation. Analyses of the patients' red blood cell (RBC) glutathione system revealed low levels of reduced glutathione and decreased activities of RBC glutathione peroxidase and glutathione reductase by 23%, 18%, and 20%, respectively, in comparison to normal RBC. Selenium treatment resulted in a significant elevation of RBC glutathione peroxidase and glutathione reductase activities and in reduced glutathione content by 64%, 57%, and 11%, respectively; this effect was also paralleled by a 39% reduction in the RBC oxidized glutathione content. On termination of the selenium treatment, and after 3 months on placebo, all of these values of the glutathione system elements returned toward baseline levels. We thus conclude that dietary selenium, which activates the glutathione system, is a potent antioxidant against plasma and LDL lipid peroxidation in renal transplant recipients, and may thus be considered antiatherogenic.

Adult↗
PubMed1990

Effects of hypoxia and reoxygenation on lung glutathione system.

Reexpansion pulmonary edema (RPE) parallels reperfusion (reoxygenation) injuries in other organs in that hypoxic and hypoperfused lung tissue develops increased vascular permeability and neutrophil infiltration after reexpansion. This study investigated the lung cellular glutathione system during hypoxia (produced by lung collapse) and after reoxygenation (produced by reexpansion). Two separate groups of rabbits were studied to determine effects of lung hypoxia-reoxygenation on 1) lung glutathione peroxidase and reductase enzyme activities and 2) lung tissue, plasma, and alveolar lavage fluid total (reduced glutathione plus glutathione disulfide) and oxidized glutathione. Neither lung collapse for 3-7 days nor reexpansion for 2 h after 7 days of collapse affected glutathione peroxidase [controls, 0.36 +/- 0.04 (left), 0.38 +/- 0.03 U/mg DNA (right)] or reductase [controls, 0.12 +/- 0.01 (left), 0.14 +/- 0.01 U/mg DNA (right)] activities. The concentration of glutathione disulfide increased markedly in right alveolar lavage fluid, but not in plasma, after right lung reexpansion. Right lung total glutathione decreased significantly (-19%) after 7 days of collapse. After right lung reexpansion, both left (-65%) and right (-68%) lung total glutathione decreased significantly. The percent of total glutathione present in the oxidized form increased significantly in both left (to 15.5 +/- 4.0% of total) and right (to 18.7 +/- 6.3% of total) lungs after reexpansion of the right lung. These data indicate that lung tissue hypoxia, produced by unilateral lung collapse, was associated with a unilateral decrease in lung total glutathione content. Right lung reoxygenation, due to rapid reexpansion, caused a bilateral decrease in lung total glutathione content and an increase in right lung and alveolar lavage fluid glutathione disulfide concentration.

Animals↗
PubMed1987

Protection by verapamil of mitochondrial glutathione equilibrium and phospholipid changes during reperfusion of ischemic canine myocardium.

Pretreatment of the ischemic myocardium with verapamil protects against mitochondrial respiratory depression observed during ischemic arrest as well as during reperfusion. Since ischemic mitochondrial function appears not to be altered further by reperfusion, the purpose of this study is to identify a biochemical event affecting mitochondria that is specifically associated with reperfusion injury. It has been proposed that increased cellular Ca2+ influx and oxygen toxicity may result from reintroduction of coronary flow. Increased cytosolic Ca2+ is transmitted to the mitochondria with subsequent activation of Ca2+-dependent events, including phospholipase A2. Net production of lysophospholipids (and loss of total diacylphospholipids from the mitochondria) will proceed when reacylation mechanisms are inhibited. Since acyl-CoA:lysophospholipid acyltransferase is a sulfhydryl-sensitive enzyme and since increased activity of glutathione peroxidase shifts the levels of the mitochondrial sulfhydryl buffer, glutathione, towards oxidation, levels of glutathione and its oxidation state were measured during reperfusion in the absence or presence of verapamil pretreatment. Ischemia lowers total glutathione and reduces the redox ratio (reduced glutathione: oxidized glutathione) by 85%. Reperfusion partially returns the redox ratio to control by causing oxidized glutathione to disappear from the matrix. Verapamil maintains both the concentration and the redox potential of glutathione at control levels. Concomitant with alterations in reduced glutathione:oxidized glutathione is a decrease in ischemic mitochondrial phospholipid content. During reperfusion, phosphatidylethanolamine and its major constituent fatty acids (C 18:0 and C 20:4) are specifically lost from the mitochondrial membrane. Accompanying the significant loss of arachidonic acid during reperfusion is the decreased content of 11-OH, 12-OH, and 15-OH arachidonate. These lipid peroxidation products are not increased in ischemia. It is proposed that oxidation of matrix glutathione to glutathione disulfide during ischemia results in formation of glutathione-protein mixed disulfides and inhibition of sulfhydryl-sensitive proteins, including acyl-CoA lysophosphatide acyltransferase. Thus, metabolic events occurring within the ischemic period set the stage for prolonged dysfunction during reperfusion.

Acetyl-CoA C-Acyltransferase↗
PubMedDate not supplied

Effects of ethanol feeding and withdrawal on plasma glutathione elimination in the rat.

Chronic ethanol feeding increases hepatic turnover and sinusoidal efflux of glutathione in rats. The present study was performed to determine whether the observed increase in glutathione efflux was due to increased extrahepatic requirements for glutathione. The concentration and disposition of plasma glutathione were determined in rats fed liquid diets containing 36% of calories as ethanol or pair-fed an isocaloric mixture with carbohydrate replacing ethanol calories for 5 to 8 weeks. The half-life and plasma clearance of [35S]glutathione were found to be similar in ethanol-fed and control rats and in rats withdrawn 24 hr from ethanol. Uptakes of the sulfur moiety of [35S]glutathione by kidney, jejunal mucosa, liver, lung, spleen, muscle and heart were also unchanged by ethanol feeding. The plasma glutathione concentration was significantly higher in ethanol-withdrawn rats 22.30 +/- 3.06 nmoles per ml (p less than 0.05) compared to pair-fed controls (13.51 +/- 2.04), while rats continuing to drink ethanol had intermediate levels (16.96 +/- 2.22). Plasma cysteine levels were slightly, but not significantly, higher in ethanol-fed rats. These findings suggest that increased sinusoidal efflux of glutathione in ethanol-fed rats is due to a direct effect of ethanol on hepatic glutathione transport and not due to an alteration in extrahepatic disposition of glutathione. In order to characterize further the effects of ethanol feeding on glutathione-dependent detoxification, activities of glutathione S-transferase, glutathione reductase and gamma-glutamyltransferase were determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Feed↗
PubMed1990

Role of hepatic gamma-glutamyltransferase in the degradation of circulating glutathione: studies in the intact guinea pig perfused liver.

The role of hepatic gamma-glutamyltransferase in the breakdown of circulating glutathione was studied in the perfused guinea pig liver. Hepatic gamma-glutamyltransferase activity in the guinea pig is sevenfold higher than in the rat and is comparable to its activity in man. Guinea pig livers were found to remove, in a single pass, 50% to 90% of glutathione (10 to 50 mumol/L) added to the portal perfusate. Removal of portal glutathione was totally dependent on the activity of gamma-glutamyltransferase and led to the near quantitative appearance of cysteinyl-glycine and cysteine in the caval perfusate. Glutathione removal by the intact liver followed saturation with a Michaelis constant (Km) of 59 mumol/L for glutathione and a maximum velocity of 235 nmol glutathione/min/gm of liver weight. The capacity of the guinea pig liver to remove circulating glutathione was estimated to be sevenfold to 10-fold higher than its net rate of output of glutathione into the circulation. Inhibition of gamma-glutamyltransferase activity in the perfused liver led to threefold to sixfold increases in the hepatic output of glutathione into the circulation, indicating that more than two thirds of glutathione transported extracellularly is broken down. Data obtained demonstrate a major role of hepatic gamma-glutamyltransferase, both in the removal of portally carried glutathione and in the degradation of glutathione molecules released by the liver itself into the sinusoids. These findings suggest the existence of an intraorgan transport of glutathione in the liver, whereby periportal cells could provide glutathione precursors to pericentral cells.

Animals↗
PubMed1993

Selenium regulation of glutathione peroxidase in human hepatoma cell line Hep3B.

Glutathione peroxidase is an important enzyme in cellular antioxidant defense systems, detoxifying peroxides and hydroperoxides. As a component of the glutathione cycle, it protects the liver from reactive oxygen metabolites. Selenocysteine is present at the catalytic site of glutathione peroxidase, and selenium availability regulates glutathione peroxidase enzyme activity. Hep3B cells, a well-differentiated human hepatoma-derived cell line, exhibited time-dependent decrease in glutathione peroxidase activity (nmol NADPH oxidized/min/mg protein, mean +/- SE) when incubated in selenium-free medium for 10 days (Day 0, 21.8 +/- 7.3; Day 2, 10.9 +/- 1.2; Day 4, 7.9 +/- 0.8; Day 6, 4.0 +/- 0.7; Day 8, 4.5 +/- 0.6; Day 10, 1.6 +/- 0.4). With the reintroduction of selenium, glutathione peroxidase activity returned. A second human hepatoma cell line, HepG2, demonstrated a similar pattern when depleted of and then repleted with selenium. To assess protein synthesis, glutathione peroxidase activity was measured in deficient and replete Hep3B cells incubated with and without selenium and with and without cycloheximide. Deficient cells (mean +/- SE) (4.9 +/- 0.2) showed an increase in glutathione peroxidase activity after 24 h in selenium-containing medium (11.6 +/- 0.2), but not when cycloheximide was included in the medium (6.9 +/- 0.5) or when cycloheximide and no selenium was included (5.3 +/- 0.8). Replete Hep3B cells (40.1 +/- 1.1) demonstrated decreased glutathione peroxidase after 24 h in medium without selenium (34.0 +/- 1.4), medium with both cycloheximide and selenium (34.0 +/- 2.6), and medium without selenium and containing cycloheximide (37.6 +/- 1.3). These data suggest that protein synthesis is needed for selenium repletion to exert control on glutathione peroxidase activity. Using a cDNA for human glutathione peroxidase (GPx1), selenium-deficient and replete Hep3B cell RNA was analyzed by Northern blot. mRNA for GPx was quantified by densitometry. The steady-state mRNA level for glutathione peroxidase in deficient cells was 40% of that in replete cells. Nuclear run-on studies to determine the rate of GPx-specific mRNA synthesis showed no difference between nuclei from selenium-replete and selenium-deficient cells. This finding eliminated the possibility of differential transcription rates as an explanation for the observed reduction in mRNA brought about by selenium deficiency and suggested instead a stabilization of mRNA in selenium-replete cells. While selenium deficiency decreased mRNA levels by 60%, glutathione peroxidase enzyme activity decreased by 93%, suggesting a co- and/or post-translational control mechanism in addition to the effect on mRNA stability.

Carcinoma, Hepatocellular↗
PubMed1997

Molecular organization of the glutathione reductase gene in Drosophila melanogaster.

Glutathione reductase catalyzes the conversion of the oxidized form of glutathione to regenerate reduced glutathione, which acts as a versatile intracellular reductant. The present study provides initial characterization of the glutathione reductase gene in Drosophila melanogaster and its response to experimentally induced oxidative stress. Drosophila cDNA clones were isolated, based on cross-hybridization to the Musca domestica glutathione reductase cDNA. Genomic clones were isolated by cross-hybridization with the Drosophila cDNA as hybridization probe. Northern analysis of adult Drosophila poly(A)+ RNA, utilizing the Drosophila cDNA probe, revealed a hybridization signal in the 2-kb range. The entire sequence of one cDNA was determined. In addition to a coding domain of 1431 bases, the sequence included 206 bases upstream of a putative start codon and 355 bases downstream of a putative stop codon. Based on the cDNA sequence, the 476 amino acid sequence of the Drosophila glutathione reductase gene was deduced and was found to have extensive similarities with the glutathione reductase gene from other species. Gene mapping of a 13-kb genomic fragment revealed that the glutathione reductase gene consists of at least two exons spanning approximately 5 kb. A first exon contains sequence for only the first 5 amino acids and the first base of the sixth and appears to be separated by a ca. 2.5-kb intron from the remainder of the coding region, which is confined to <2 kb. The Drosophila glutathione reductase is single copy and its cytogenetic position, as determined by in situ hybridization, is 7D-E on the X chromosome. mRNA levels of glutathione reductase, measured by RT-PCR, increased in response to exposure to 100% ambient oxygen by almost twofold and administration of paraquat by greater than threefold. Exposure of flies to hyperoxia also induced a 60% increase in the activity of glutathione reductase and augmented the concentration of total glutathione by ca. 40% following an initial drop. The present study, besides providing an initial molecular characterization of the glutathione reductase gene in Drosophila, demonstrates its dynamic involvement in response to experimentally induced oxidative stress.

Amino Acid Sequence↗
PubMed1993

A simple technique to determine glutathione (GSH) levels and synthesis in ocular tissues as GSH-bimane adduct: application to normal and galactosemic guinea-pigs.

A fluorimetric technique previously described for other tissues has been applied to determine levels of glutathione and its synthetic rates in ocular tissues of Hartley guinea-pigs. Monochlorobimane forms a stable, fluorescent adduct with glutathione in a reaction catalyzed by glutathione-S-transferase. The fluorescent signal recorded over time is directly proportional to the synthetic rate of glutathione. Lens, cornea and retina were homogenized and cytosolic fractions dialyzed overnight to deplete endogeneous glutathione. Glutathione synthetic rates were determined from a mixture of glutathione precursors and co-factors, viz. cysteine+dithiothreitol, glutamate+glycine, ATP and Mg++ in the presence of monochlorobimane. The mixture was supplemented with glutathione-S-transferase to catalyze the formation of the fluorescent adduct. Glutathione synthetic rates were determined in the absence and presence of buthionine sulfoximine, an inhibitor of gamma-glutamyl cysteine synthetase. The difference in fluorescence change over time in the presence and absence of buthionine sulfoximine was used to estimate glutathione synthesis. Basal levels of glutathione in pre-dialyzed cytosolic fractions of the lens, cornea, and retina were 21.8 +/- 2.2, 36.5 +/- 4.1 and 38.6 +/- 2.8 nmol mg-1 protein, respectively. The maximal glutathione synthetic rates in these tissues were 0.52 +/- 0.04, 2.25 +/- 0.67 and 3.35 +/- 0.65 nmol min-1 mg-1 protein, respectively. When gamma-glutamyl cysteine is used as a precursor instead of cysteine, the glutathione synthetase activities from lenses and retinas were 0.19 +/- 0.08 and 1.54 +/- 0.76 nmol-1 min mg-1 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Relationship between 1-chloro-2,4-dinitrobenzene-induced cytoskeletal perturbations and cellular glutathione.

Exposure of 3T3 cells to micromolar doses of 1-chloro-2,4-dinitrobenzene, a substrate for glutathione-S-transferase, resulted in a rapid depletion of total cellular glutathione accompanied by disassembly of microtubules as visualized by fluorescence microscopy. However, prolonged incubation resulted in cellular recovery from 1-chloro-2,4-dinitrobenzene insult as evidenced by a steady rise in total cellular glutathione accompanied by microtubule reassembly to their normal organization 5 hours after treatment. To evaluate the role of total cellular glutathione in modulating the 1-chloro-2,4-dinitrobenzene-induced cytoskeletal perturbation, we used 1-chloro-2,4-dinitrobenzene and/or buthionine sulfoximine, an effective irreversible inhibitor of glutathione synthesis, to manipulate cellular glutathione levels. Incubation of 3T3 cells with 2.5 microM 1-chloro-2,4-dinitrobenzene and 250 microM buthionine sulfoximine for 5 hours resulted in a complete depletion of total cellular glutathione accompanied by essentially complete loss of microtubules and marked alterations in the density and distribution pattern of microfilaments. Buthionine sulfoximine enhanced markedly the extent and duration of cellular glutathione depletion and the severity of microtubule disruption of 3T3 cells over the level achieved by 1-chloro-2,4-dinitrobenzene treatment alone. Furthermore, buthionine sulfoximine also prevented the restoration of cellular glutathione content and microtubule reassembly that normally were evident 5 hours after 1-chloro-2,4-dinitrobenzene treatment. Exposure of 3T3 cells to 50 microM 2-cyclohexene-1-one, which depletes free glutathione by conjugation, resulted in a complete depletion of total cellular glutathione content without altering the microtubule organization. These results suggest that the total glutathione content may be important for cellular recovery from 1-chloro-2,4-dinitrobenzene-mediated cytoskeletal injuries, and that microtubule disassembly observed in 1-chloro-2,4-dinitrobenzene-treated cells probably results from depletion of cellular glutathione coupled with binding to tubulin and/or other microtubule components.

Animals↗
PubMed1992

The systemic availability of oral glutathione.

When the plasma glutathione concentration is low, such as in patients with HIV infection, alcoholics, and patients with cirrhosis, increasing the availability of circulating glutathione by oral administration might be of therapeutic benefit. To assess the feasibility of supplementing oral glutathione we have determined the systemic availability of glutathione in 7 healthy volunteers. The basal concentrations of glutathione, cysteine, and glutamate in plasma were 6.2, 8.3, and 54 mumol.l-1 respectively. During the 270 min after the administration of glutathione in a dose of 0.15 mmol.kg-1 the concentrations of glutathione, cysteine, and glutamate in plasma did not increase significantly, suggesting that the systemic availability of glutathione is negligible in man. Because of hydrolysis of glutathione by intestinal and hepatic gamma-glutamyltransferase, dietary glutathione is not a major determinant of circulating glutathione, and it is not possible to increase circulating glutathione to a clinically beneficial extent by the oral administration of a single dose of 3 g of glutathione.

Administration, Oral↗
PubMed1994

Reduction and binding of arsenate and dimethylarsinate by glutathione: a magnetic resonance study.

By observing the chemical shifts of the proton and carbon-13 nuclei of reduced glutathione, the interactions of arsenate, arsenite and dimethylarsinate with this tripeptide have been characterized. These spectral studies show the reduction and complexation of arsenic to be a two-step process. Initially, the oxidation of 2 mol of glutathione reduces arsenate to arsenite. Then, 3 mol of glutathione are consumed in the formation of a glutathione-arsenite complex. Similar experiments with arsenite identified a (glutathione)3-arsenite complex; however, no oxidized glutathione was detected. The arsenite binding site in the glutathione-arsenite complex is the cysteinyl sulfhydryl. The glutathione-arsenite complex is stable over the pH range from 1.5 to 7.0-7.5. At higher pH, dissociation occurs releasing reduced glutathione. For a glutathione to dimethylarsinate ratio of 3, oxidized glutathione is also coupled with a reduction to trivalent dimethylarsinous acid, prior to the formation of a 1:1 glutathione-dimethylarsinite complex. The role of reduced glutathione in the metabolism of arsenic is consistent with the previously described effects of this agent on the organismic toxicity of arsenic.

Arsenates↗