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At least 433 records · Page 24Linked to original sources
PubMed1988

Levels of polyamines, glutathione and glutathione-spermidine conjugates during growth of the insect trypanosomatid Crithidia fasciculata.

Levels of the polyamines spermidine and putrescine and the major intracellular thiols glutathione (GSH), glutathionylspermidine (GSH-SPD) and dihydrotrypanothione [bis-(glutathionyl)spermidine); T[SH]2] were measured by high performance liquid chromatography throughout the growth cycle of the insect trypanosomatid Crithidia fasciculata. The amount of total spermidine, putrescine and glutathione (free and conjugated to spermidine) was found to be elevated during growth. Of the total spermidine, 30 to 50% was found conjugated to glutathione during the exponential growth phase, increasing to 60 to 70% at stationary phase. T[SH]2 was the principal intracellular thiol during exponential growth (12.1 to 17.4 nmol per 10(8) cells), whereas GSH-SPD was the major thiol in stationary phase (26.2 nmol per 10(8) cells). GSH levels changed little during the growth cycle and represented a constant proportion (10 to 12%) of the total intracellular glutathione. On dilution of stationary phase cells into fresh medium, a rapid decrease in GSH-SPD levels was observed to be associated with synthesis of T[SH]2. This process reached 90% completion by 15 min, with steady state achieved by 120 min. As the total spermidine and glutathione pools did not increase during this interval, it could be calculated that this rapid redistribution of metabolites resulted in the release of 13 nmol per 10(8) cells unconjugated spermidine without de novo synthesis. This mechanism for rapidly elevating the intracellular concentration of free spermidine may be advantageous to this organism in rapidly adapting to favourable growth conditions.

Amino Acids↗
PubMedDate not supplied

Protective effect of glutathione (GSH) over glutathione monoethyl-ester (GSH-E) on cold preservation of isolated rat liver cells.

Hepatocyte suspensions provide a rapid method to determine how hypothermic storage affects liver cell metabolism and viability. We investigated whether reduced Glutathione (GSH) inclusion into a modified University of Wisconsin (UW) solution, has a protective effect over Glutathione derivatives, such as Glutathione-monoethylester (GSH-E), when suspensions of hepatocytes are cold stored for several days. Isolated rat liver cells were cold preserved 96 h in UW, UW plus 3 mM GSH and UW plus 3 mM GSH-E. During the cold storage, not significant changes in cell viability were observed, but the total Glutathione content was higher in systems with extracellular GSH over those with GSH-E or without. After cold storage, the liver cells were gently resuspended in Krebs-Henseleit-1% Albumin and used for 120 min of normothermic (37 degrees C) incubation. We evaluate the functional response of the cells measuring the exclusion of Trypan Blue (TBE). This response was clearly different in preserved cells in presence of GSH. These results indicate a protective role of extracellular Glutathione, due to an accumulation of it, rather than the derivative, for hepatic cell during the cold storage in UW solutions. And also, it is possible to extend experiments with hepatocytes from a single cell isolation over 4 or more consecutive days.

Animals↗
PubMed2002

Glutathione redox regulates lipopolysaccharide-induced IL-12 production through p38 mitogen-activated protein kinase activation in human monocytes: role of glutathione redox in IFN-gamma priming of IL-12 production.

We examined whether changes in intracellular reduced (GSH) or oxidized (GSSG) glutathione of human monocytes regulate lipopolysaccharide (LPS)-induced IL-12 production and defined the molecular mechanism that underlies glutathione redox regulation. Monocytes exposed to glutathione reduced form ethyl ester (GSH-OEt) or maleic acid diethyl ester (DEM) increased or decreased the intracellular GSH/GSSG ratio, respectively. LPS-induced IL-12 production and p38 mitogen-activated protein (MAP) kinase activation were enhanced by GSH-OEt but suppressed by DEM. Selective p38 inhibitors showed that p38 promoted GSH-OEt-enhanced IL-12 production. Furthermore, IFN-gamma priming increased the GSH/GSSG ratio and enhanced IL-12 production through p38, and DEM negated the priming effect of IFN-gamma on p38 activation and IL-12 production as well as on the GSH/GSSG ratio. These findings reveal that glutathione redox regulates LPS-induced IL-12 production from monocytes through p38 MAP kinase activation and that the priming effect of IFN-gamma on IL-12 production is partly a result of the glutathione redox balance.

Cells, Cultured↗
PubMed2005

[Glutathione and glutathione assays].

Glutathione, the very important intracellular antioxidant, is present in intracelullar environment in milimolar concentrations. Glutathione is a tripeptide molecule, which plays an essential role in the antioxidant system, as well as in maintenance of the intracellular redox state. This thiol compound exists in two forms, the reduced (GSH) and the oxidized (GSSG), and the ratio of both forms is crucial for the characterization of the oxidative stress in cells. Number of analytical methods have been developed for the measurement of the glutathione. Especially, High Performance Liquid Chromatography methods (HPLC) are mostly used linked to different types of detection, including electrochemical, UV/VIS or fluorimetric detection. Another approach for glutathione assay is using the spectral methods, either fluorimetric or spectrophotometric assays. In enzymatic assay, glutathione reductase reduces GSSG with simultaneous oxidation of specific substrate, which is sequentially photometrically detected. The fluorimetric method is based on the detection of derivatized GSH molecule.

Chromatography, High Pressure Liquid↗
PubMed1975

[Optimized determination and properties of NADPH-dependent glutathione reductase in serum. Studies on serum glutathione reductase, I. (author's transl)].

Reaction conditions were optimized for the determination of serum glutathione reductase, which has not yet been investigated systematically. Imidazole was found to be the most suitable buffer material; the highest glutathione reductase activity in serum was always obtained with imidazole/HCl buffer, which, in contrast to all other tested buffers, also resulted in the maximal enzyme activity without preincubation. In imidazole buffer, the pH-activity curve of serum glutathione reductase shows a broad optimum between pH 6.5 and 6.9. A GSSG concentration of 2 mmol/l and a NADPH concentration of 0.43 mmol/l gave maximal enzyme activity and a linear reaction over 10 min up to 20 U/l test solution. An investigation of serum glutathione reductase activity from 100 clinically healthy probands gave values between 20 and 50 U/l. In the optimized assay system the glutathione reductase in the serum reacts specifically with GSSG and NADPH.

Autoanalysis↗
PubMed1986

alpha-Bromoisovalerylurea as model substrate for studies on pharmacokinetics of glutathione conjugation in the rat. I. (Bio-) synthesis, analysis and identification of diastereomeric glutathione conjugates and mercapturates.

In order to find a model substrate for kinetic characterization of glutathione conjugation in vivo alpha-bromoisovalerylurea (BIU) was studied. After administration of racemic [14C]urea BIU to rats, two radioactive metabolites were found in bile by high-performance liquid chromatography. The identity of these metabolites was established by various methods. Based on the hydrolytic activity of gamma-glutamyltranspeptidase (presence of the gamma-glutamyl moiety), high resolution nuclear magnetic resonance (isovaleryl and glutathionyl moieties) and fast atom bombardment mass spectrometry (molecular weight and fragmentation pattern), they were identified as glutathione conjugates of BIU. Because both conjugates in bile had these characteristics in common they must be diastereomers. Incubation of BIU with glutathione in the presence of rat liver cytosol resulted in formation of the same diastereomeric glutathione conjugates. Chemical synthesis of the diastereomers confirmed their identity. The major urinary excretion products of [14C]urea BIU in the rat were identified as diastereomeric mercapturates. A convenient chromatographic separation of the diastereomeric glutathione conjugates and the mercapturic acids is described. Electrochemical detection was used to determine the presence of the thioethers in both urine and bile. Pharmacokinetic results on BIU conjugation are described in the accompanying paper.

Acetylcysteine↗
PubMed1994

Transport of the glutathione-methylmercury complex across liver canalicular membranes on reduced glutathione carriers.

Methylmercury transport across liver canalicular membranes into bile, a major route of excretion of this toxic compound, is dependent upon intracellular GSH, and a glutathione-methylmercury complex (CH3Hg.SG) has been detected in liver tissue and bile. To examine whether the CH3Hg.SG complex is itself transported across the canalicular membrane and to identify the transport system involved, studies were performed in isolated rat liver canalicular plasma membrane vesicles. Uptake of CH3(203)Hg.SG (10 microM) into an osmotically active space was temperature-sensitive and unaffected by either ATP (5 mM) or an inwardly directed Na+ gradient (100 mM); however, CH3Hg.SG uptake was enhanced by a valinomycin-induced K+ diffusion potential (inside-positive) indicating that its transport was electrogenic. Transport of CH3Hg.SG exhibited saturation kinetics with both high affinity (Km = 12 +/- 2 microM, Vmax = 0.23 +/- 0.02 nmol.mg-1.20 s-1) and low affinity (Km = 1.47 +/- 0.22 mM, Vmax = 1.23 +/- 0.14 nmol.mg-1.20 s-1) components. Uptake of this complex was inhibited by GSH, the GSH analog ophthalmic acid, S-methyl, S-ethyl, S-butyl, S-hexyl, S-octyl, and S-dinitrophenyl glutathione, but not by GSSG, bile acids, amino acids, and P-glycoprotein inhibitors. Furthermore, GSH competitively inhibited (Ki = 83 microM) and trans-stimulated CH3Hg.SG uptake into the canalicular vesicles. These studies provide the first kinetic characterization of a transport system for glutathione-mercaptides and indicate that CH3Hg.SG is not a substrate for the ATP-dependent, canalicular GSSG or glutathione S-conjugate carriers, but appears to be a substrate for canalicular carriers that also transport GSH. Because efflux systems for GSH are found in all mammalian cells, transport of glutathione-metal complexes by such carriers may be a common mechanism for the removal of methylmercury and possibly other metals from cells.

Animals↗
PubMedDate not supplied

Hepatic glutathione and glutathione S-conjugate transport mechanisms.

Glutathione (GSH) plays a critical role in many cellular processes, including the metabolism and detoxification of oxidants, metals, and other reactive electrophilic compounds of both endogenous and exogenous origin. Because the liver is a major site of GSH and glutathione S-conjugate biosynthesis and export, significant effort has been devoted to characterizing liver cell sinusoidal and canalicular membrane transporters for these compounds. Glutathione S-conjugates synthesized in the liver are secreted preferentially into bile, and recent studies in isolated canalicular membrane vesicles indicate that there are multiple transport mechanisms for these conjugates, including those that are energized by ATP hydrolysis and those that may be driven by the electrochemical gradient. Glutathione S-conjugates that are relatively hydrophobic or have a bulky S-substituent are good substrates for the canalicular ATP-dependent transporter mrp2 (multidrug resistance-associated protein 2, also called cMOAT, the canalicular multispecific organic anion transporter, or cMrp, the canalicular isoform of mrp). In contrast with the glutathione S-conjugates, hepatic GSH is released into both blood and bile. GSH transport across both of these membrane domains is of low affinity and is energized by the electrochemical potential. Recent reports describe two candidate GSH transport proteins for the canalicular and sinusoidal membranes (RcGshT and RsGshT, respectively); however, some concerns have been raised regarding these studies. Additional work is needed to characterize GSH transporters at the functional and molecular level.

Adenosine Triphosphate↗
PubMed1997

Glutathione consumption and inactivation of glutathione-related enzymes in liver, erythrocytes and serum of rats after methanol intoxication.

The primary metabolic fate of methanol is oxidation to formaldehyde and then to formate. These processes are accompanied by formation of superoxide anion and further hydrogen peroxide. Glutathione plays a unique role in the cellular defense system against xenobiotics. The glutathione (GSH) content and glutathione peroxidase (GSH-Px) and glutathione reductase (GSSG-R) activities were measured in liver, erythrocytes and serum of rats. Rats were intoxicated with 3.0 and 6.0 g methanol/kg body wt. and measurements taken after 6, 12 and 24 h and 2, 5 and 7 days of intoxication. The decrease in GSH content and in GSH-related enzyme activity was observed during the whole time-course of the intoxication. The most significant changes were observed in the erythrocytes. The results obtained show that the protection against oxidative damage due to methanol intoxication in rats seems to be less efficient than in control rats.

Animals↗
PubMed1984

CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in glutathione-deficient human fibroblasts.

The effect of genetically determined glutathione deficiency on the fibroblast content of CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase was investigated. No significant differences between glutathione-deficient and -proficient human fibroblasts were revealed. There was a large variation in the content of the investigated enzymes in fibroblasts grown and analysed on different occasions. Whereas the contents of CuZn superoxide dismutase, catalase and glutathione peroxidase did not deviate much from what has been found in other human cell-lines and tissues, the fibroblasts were found to contain exceptional amounts of Mn superoxide dismutase.

Adult↗
PubMed2005

Kinetic studies on the glutathione peroxidase activity of selenium-containing glutathione transferase.

Selenium-containing glutathione transferase (seleno-GST) was generated by biologically incorporating selenocysteine into the active site of glutathione transferase (GST) from a blowfly Lucilia cuprina (Diptera: Calliphoridae). Seleno-GST mimicked the antioxidant enzyme glutathione peroxidase (GPx) and catalyzed the reduction of structurally different hydroperoxides by glutathione. Kinetic investigations reveal a ping-pong kinetic mechanism in analogy with that of the natural GPx cycle as opposed to the sequential one of the wild type GST. This difference of the mechanisms might result from the intrinsic chemical properties of the incorporated residue selenocysteine, and the selenium-dependent mechanism is suggested to contribute to enhancement of the enzymatic efficiency.

Animals↗
PubMed1999

Glutathione and glutathione-related enzymes in reproduction. A review.

Glutathione and glutathione-related enzymes are pivotal for the normal functioning of several important biological processes in humans. Glutathione and glutathione-related enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as reactive oxygen species. The role of reactive oxygen species in reproduction was the subject of many investigations, and there is compelling evidence for the involvement of these species in the physiology and pathology of both male and female reproductive systems. The glutathione/glutathione-related enzyme system was extensively studied in gynaecological oncology, but to a lesser extent in other topics related to reproduction. In this paper a review is provided on the glutathione/glutathione-related enzyme system in reproduction. Attention is given to its role as a detoxicating system, and as an early marker for disease.

Female↗
PubMed1999

Protective effect of N-acetylcysteine on reduced glutathione, reduced glutathione-related enzymes and lipid peroxidation in methanol intoxication.

The primary metabolic appropriation of methanol is oxidation to formaldehyde and then to formate. These processes are accompanied by formation of superoxide anion and hydrogen peroxide. This paper reports data on the effect of N-acetylcysteine (NAC) on reduced glutathione (GSH) and on activity of some GSH-metabolising enzymes in the liver, erythrocytes and serum of rats intoxicated with methanol (3 g/kg b.w.) during 7 days after intoxication. Methanol administration, increasing concentration of the lipid peroxidation products, decreased the liver glutathione-peroxidase and glutathione reductase (GSSG-R) activities, GSH concentration and total antioxidant status (TAS). The use of NAC after methanol ingestion apparently diminished lipid peroxidation, elevated the GSH level in the liver and erythrocytes, and increased activity of GSH-related enzymes in the serum, erythrocytes and in the liver. These results suggest that NAC exerts its protective effect by acting as a precursor for glutathione, the main low molecular antioxidant and as a free radical scavenger.

Acetylcysteine↗
PubMed2000

Intracellular reduction of selenite into glutathione peroxidase. Evidence for involvement of NADPH and not glutathione as the reductant.

Selenium (Se) in selenite is present in an oxidized state, and must be reduced for it to be incorporated as selenocysteine into selenoenzymes such as glutathione peroxidase (GPx). In vitro, Se, as in selenite, can be reduced utilizing glutathione (GSH) and glutathione reductase (GRed). We determined the effects of decreasing GSH levels, inhibiting GRed activity, and decreasing cellular NADPH on the selenite-dependent rate of GPx synthesis in cultured cells: PC3, CHO, and the E89 glucose-6-phosphate dehydrogenase (G-6-PD)-deficient cell line. A novel statistical analysis method was developed (using Box Cox transformed regression and a bootstrap method) in order to assess the effects of these manipulations singly and in combinations. Buthionine sulfoximine (BSO) was used to decrease GSH levels, 1,3 bis-(2 chloroethyl)-1 -nitrosourea (BCNU) was used to inhibit GRed activity and methylene blue (MB) was used to decrease cellular NADPH levels. This statistical method evaluates the effects of BSO, BCNU, MB and selenite alone and in combinations on GPx activity. Decreasing the GSH level (< 5% of control) did not have an effect on the selenite-dependent rate of GPx synthesis in PC3 or CHO cells, but did have a small inhibitory effect on the rate of GPx synthesis in E89 cells. Inhibiting GRed activity was also associated with either no effect (CHO, E89) or a small effect (PC3) on GPx activity. In contrast, decreasing NADPH levels in cells treated with MB was associated with a large decrease in the selenite-dependent rate of GPx synthesis to 36, 34 and 25% of control in PC3, CHO, and E89 cells, respectively. The effects of BSO plus BCNU were not synergistic in any of the cell lines. The effects of BSO plus MB were synergistic in G-6-PD-deficient E89 cells, but not in PC3 or CHO cells. We therefore conclude that under normal culture conditions, NADPH, and not glutathione, is the primary reductant of Se in selenite to forms that are eventually incorporated into GPx. For cells with abnormal ability to generate NADPH, lowering the GSH levels had a small effect on selenite-dependent GPx synthesis. GRed activity is not required for the selenite-dependent synthesis of GPx.

Animals↗
PubMed2000

Of humans and hamsters: comparative analysis of lipid peroxidation, glutathione, and glutathione-dependent enzymes during oral carcinogenesis.

We examined the extent of lipid peroxidation and the amount of reduced glutathione (GSH), and activities of the GSH-dependent enzymes, glutathione peroxidase (GPx) and glutathione S-transferase (GST), in human oral tumour tissue and 7, 12-dimethylbenz[a]anthracene-(DMBA)-induced hamster buccal pouch tumour tissues. Diminished lipid peroxidation in the oral and hamster tumours was accompanied by a significant reduction in the concentration of phospholipids and an increase in the cholesterol:phospholipid ratio. The concentration of glutathione and the activities of GPx and GST were raised in both human oral and hamster tumour tissues. Our results show that the patterns of changes in human oral squamous cell carcinomas and chemically-induced carcinomas of the hamster buccal pouch are similar.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

Glutathione S-transferase Pi has at least three distinguishable xenobiotic substrate sites close to its glutathione-binding site.

Benzyl isothiocyanate (BITC), present in cruciferous vegetables, is an efficient substrate of human glutathione S-transferase P1-1 (hGST P1-1). BITC also acts as an affinity label of hGST P1-1 in the absence of glutathione, yielding an enzyme inactive toward BITC as substrate. As monitored by using BITC as substrate, the dependence of k of inactivation (K(I)) of hGST P1-1 on [BITC] is hyperbolic, with K(I) = 66 +/- 7 microM. The enzyme incorporates 2 mol of BITC/mol of enzyme subunit upon complete inactivation. S-Methylglutathione and 8-anilino-1-naphthalene sulfonate (ANS) each yield partial protection against inactivation and decrease reagent incorporation, whereas S-(N-benzylthiocarbamoyl)glutathione or S-methylglutathione + ANS protects completely. Mapping of proteolytic digests of modified enzyme by using mass spectrometry reveals that Tyr(103) and Cys(47) are modified equally. S-Methylglutathione reduces modification of Cys(47), indicating this residue is at/near the glutathione binding region, whereas ANS decreases modification of Tyr(103), suggesting this residue is at/near the BITC substrate site, which is also near the binding site of ANS. The Y103F and Y103S mutant enzymes were generated, expressed, and purified. Both mutants handle substrate 1-chloro-2,4-dinitrobenzene normally; however, Y103S exhibits a 30-fold increase in K(m) for BITC and binds ANS poorly, whereas Y103F has a normal K(m) for BITC and K(d) for ANS. These results indicate that an aromatic residue at position 103 is essential for the binding of BITC and ANS. This study provides evidence for the existence of a novel xenobiotic substrate site in hGST P1-1, which can be occupied by benzyl isothiocyanate and is distinct from that of monobromobimane and 1-chloro-2,4 dinitrobenzene.

Binding Sites↗
PubMed1984

A simple assay for monoamine oxidase using glutathione peroxidase and glutathione reductase.

A new fluorometric assay for the determination of monoamine oxidase activity that is applicable to any substrates including dopamine and serotonin is described. Hydrogen peroxide formed during the monoamine oxidase reaction was reduced in the presence of glutathione and glutathione peroxidase, and the oxidized glutathione was measured fluorometrically as NADP+ via oxidation of NADPH by glutathione reductase. This method was applied for inhibitor studies using clorgyline and deprenyl.

Animals↗
PubMed1987

Effects of co-administration of monomethylaminoantipyrine and cobaltous chloride on hepatic glutathione level and glutathione-related enzyme activities in rats.

Concurrent administration of monomethylaminoantipyrine (MAA) and CoCl2 caused a significant decrease of hepatic reduced glutathione and oxidized glutathione levels. Furthermore, the increase of glutathione S-transferase activity by combined treatment resulted in the decrease of Se-dependent glutathione peroxidase activity.

Aminopyrine↗