UMLS:C1260386 - FACTA Search

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Query: UMLS:C1260386 (GSH)
38,102 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutathione (GSH) is the major intracellular antioxidant and is essential to normal cell function and replication. Cysteine and other thiol compounds have been considered rate-limiting for GSH biosynthesis, but recent studies have demonstrated that glutamine (GLN) becomes essential during metabolic stress to replete tissue GSH levels which have become depleted. To determine the role of GLN supplementation in the resting, nonstressed state, we studied three groups of Wistar rats. The animals were catheterized and randomly assigned to one of three groups; (1) chow ad libitum group receiving iv saline (control), (2) standard total parenteral nutrition (STA-TPN) group, and (3) glutamine-enriched TPN (GLN-TPN) group. The intravenously fed animals received no rat chow. The infusions were administered at a rate of 2.2 ml/hr for 4 days and all animals were harvested on the fifth day of study. The GLN-TPN group had a significantly higher plasma GSH level than STA-TPN or control animals (P < 0.01). The hepatic concentration of GSH and the oxidized GSH/reduced GSH were similar in all groups. GLN-TPN had a significantly lower plasma ALT level than the control group (P < 0.05). The control group had a significantly higher ALP level than STA-TPN and GLN-TPN animals (P < 0.01). There were no significant differences in other measures of hepatic functions among the three groups. Our data demonstrate that in this model GLN-enriched TPN enhances plasma GSH concentrations, while maintaining hepatic GSH stores. This suggests that GSH turnover is altered during glutamine-enriched TPN, which may explain how dietary GLN supplementation enhances tissue antioxidant capacity.
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PMID:Glutamine-enriched total parenteral nutrition enhances plasma glutathione in the resting state. 876 39

Since the toxicity of diesel exhaust particles (DEP) after intratracheal injection, was suppressed by pretreatment with superoxide dismutase (SOD) modified with polyethylene glycol (Sagai et al. Free Rad. Biol. Med. 14: 37-47; 1993), the possibility that superoxide could be enzymatically and continuously generated from diesel exhaust particles (DEP), was examined. Nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) oxidation was stimulated during interaction of a methanol extract of DEP with the Triton N-101 treated microsomal preparation of mouse lung whereas the cytosolic fraction was less active, suggesting that DEP contains substrates for NADPH-cytochrome P450 reductase (EC 1.6.2.4, P450 reductase) rather than DT-diaphorase. When purified P450 reductase was used as the enzyme source, the turnover value was enhanced approximately 260-fold. Quinones appeared to be served as substrate for P450 reductase because reaction was inhibited by addition of glutathione (GSH) to form those GSH adduct or pretreatment with NaBH4 to reduce those to the hydroxy compounds although a possibility of nitroarenes as the alternative substrates cannot be excluded. A methanol extract of DEP (37.5 micrograms) caused a significant formation of superoxide (3240 nmol/min/mg protein) in the presence of P450 reductase. Electron spin resonance (ESR) experiments revealed that hydroxyl radical was formed as well. The reactive species generated by DEP in the presence of P450 reductase caused DNA scission which was reduced in the presence of superoxide dismutase (SOD), catalase, or hydroxyl radical scavenging agents. Taken together, these results indicate that DEP components, probably quinoid or nitroaromatic structures, that appear to promote DNA damage through the redox cycling based generation of superoxide.
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PMID:Generation of reactive oxygen species during interaction of diesel exhaust particle components with NADPH-cytochrome P450 reductase and involvement of the bioactivation in the DNA damage. 898 Oct 40

The inhibition of Saccharomyces cerevisiae aldehyde dehydrogenase (AlDH) by gaseous nitric oxide (NO) in solution and by NO generated from diethylamine nonoate was time and concentration dependent. The presence of oxygen significantly reduced the extent of inhibition by NO, indicating that NO itself rather than an oxidation product of NO such as N2O3 is the inhibitory species under physiological conditions. A cysteine residue at the active site of the enzyme was implicated in this inhibition based on the following observations: a) NAD+ and NADP+, but not reduced cofactors, significantly enhanced inhibition of AlDH by NO; b) the aldehyde substrate, benzaldehyde, blocked inhibition; and c) inhibition was accompanied by loss of free sulfhydryl groups on the enzyme. Activity of the NO-inactivated enzyme was readily restored by treatment with dithiothreitol (DTT), but not with GSH. This difference was attributed, in part, to a redox process leading to the formation of a cyclic DTT disulfide. Based on the chemistry deduced from model systems, the reaction of NO with AlDH sulfhydryls was shown to produce intramolecular disulfides and N2O. These disulfides were shown to be intrasubunit disulfides by nonreducing SDS-PAGE analysis of the NO- inhibited enzyme. Following complete inhibition of AlDH by NO, four of the eight titratable (Ellman's reagent) sulfhydryl groups of AlDH were found to be oxidized to disulfides. These results suggest that a) the sulfhydryl group of active site Cys-302 and a proximal cysteine are oxidized to form an intrasubunit disulfide by NO; b) only two of the four subunits of AlDH are catalytically active; and c) NO preferentially oxidizes sulfhydryl groups of the catalytically active subunits. A detailed mechanism for the inhibition of AlDH by NO is presented.
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PMID:Mechanism for the inhibition of aldehyde dehydrogenase by nitric oxide. 908 20

Human glutathione reductase (GR; which catalyzes the reaction NADPH + GSSG + H+ --> 2 GSH + NADP+) is an obligatory FAD-containing homodimer of known geometry. Native human GR, a potential target of antimalarial and cytostatic agents, cannot be dissociated by dilution or by means of subunit-interface mimetics, similarly to well-studied viral dimeric proteins. However, ab initio folding and/or dimerization of human GR can be inhibited by point mutations or by peptides corresponding to subunit-interface areas, for example synthetic peptide P11, which represents the intersubunit-contact helix H11. The structure of this peptide, which might assist inhibitor design, was solved by high-resolution NMR spectroscopy. Residues 440-453, were found to be alpha helical in the isolated peptide. To quantitate the efficacy of inhibitors such as P11, we developed the following unfolding/reactivation assay. The effects of various guanidine hydrochloride (Gdn/HCl) concentrations were studied by analytical ultracentrifugation. It was shown that human GR denatured by greater than 3 M Gdn/HCl is monomeric and free of FAD. Circular-dichroism experiments at 223 nm indicated a half-life of approximately 20 s at 20 degrees C for the unfolding process. To optimize the reactivation yield, four parameters [protein concentration (x) in the range 0.3-10 microg/ml, cofactor supplementation, temperature (y: 0-32 degrees C), and time (0-72 h)] were varied systematically, and a reactivation score z was given to each constellation of parameters. This type of analysis might be useful to optimize refolding and activation yields for other proteins. For human GR, the highest recovery was found not to occur at one of the corners of the x,y plane, but close to its center. Consequently, the optimal assay conditions for folding and dimerization inhibitors are as follows. The enzyme (at 300 microg/ml) is denatured by 5 M guanidine hydrochloride/5 mM dithiothreitol, then reactivated by dilution to 1 microg/ml at pH 6.9 and 20 degrees C. In the absence of inhibitors, this procedure leads to 70% of the control activity within 8 h. Peptides representing the upper subunit interface (for instance residues 436-478) of human GR were found to inhibit refolding with EC50% values in the micromolar range, whereas fragments from other regions of the protein had no influence on this process. For peptide P11, the EC50% value was 20 microM. In conclusion, hGR, enzyme with a tight intersubunit contact area of 21 nm2, appears to be suitable for studying protein folding, dimerization, and prosthetic-group complexation in the absence and presence of compounds that inhibit these processes. There is a shortage, at least for oligomeric enzymes of eukaryotes, of published systematic studies on protein (re)activation.
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PMID:Denaturation and reactivation of dimeric human glutathione reductase--an assay for folding inhibitors. 915 53

1. Morphinone, a toxic metabolite, and its glutathione adduct (MO-GSH) were identified in the bile of rat after subcutaneous injection of morphine (25 mg/kg) by hplc procedures. The amounts of morphinone and MO-GSH excreted in the 12-h bile were 0.8 +/- 0.3 and 8.4 +/- 4.3% respectively. 2. The 9000 g supernatants of rat, guinea pig, rabbit, mouse, hamster and bovine livers produced morphinone from morphine in the presence of either NAD+ or NADP+, NAD+ was a more efficient cofactor than NADP+ except in the guinea pig which equally utilized both cofactors. With NAD+ as cofactor, the amounts of morphinone formed in rat and guinea pig were 5.70 and 5.82 mumol/g liver/30 min respectively and were three-to-four times those in other species. 3. The enzyme activity responsible for formation of morphinone from morphine in the rat was almost exclusively distributed in the microsomal fraction, whereas guinea pig, hamster and bovine expressed the enzyme activity mainly in the cytosolic fraction. Rabbit and mouse gave higher activity in the cytosolic and microsomal fractions respectively, but other fractions of both species contained considerable activity. 4. The enzyme activities in male and female rat microsomes were characterized with respect to developmental pattern, kinetic parameters, pH dependency and susceptibility to inhibitors. 5. In conclusion the metabolism of morphine to morphinone in rat was confirmed by in vivo and in vitro experiments. It is also suggested that this pathway is a common route in morphine metabolism in several mammalian species.
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PMID:In vivo and in vitro formation of morphinone from morphine in rat. 925 42

trans-4,5-Dihydroxy-1,2-dithiane, the intramolecular disulfide form of dithiothreitol (DTTox) transcriptionally activates the stress-responsive genes gadd153(chop) and grp78. Herein, we used a renal epithelial cell line, LLC-PK1, to investigate the mechanism(s) whereby DTTox activates a molecular stress response. DTTox activated both grp78 and gadd153 transcriptionally, but gadd153 mRNA stability also increased suggesting that both transcriptional and posttranscriptional mechanisms are involved. DTTox did not activate hsp70 transcription indicating that a heat shock response was not induced. Structure-activity studies showed that DTTox analogues lacking the intramolecular disulfide were inactive. Furthermore, the ring-open intermolecular disulfide form of DTTox, 2-hydroxyethyl disulfide, was only a weak inducer of grp78 and gadd153 but was a strong inducer of hsp70 mRNA and a potent oxidant that lowered the NADPH/NADP+ ratio and depleted reduced glutathione (GSH). DTTox had little effect on the overall GSH and NADPH levels; thus cells were not undergoing oxidative stress; however, the NADPH/NADP+ ratio decreased slightly indicating that reducing equivalents were consumed. LLC-PK1 cells reduced DTTox to DTT, and the kinetics as well as the concentration dependence for reduction correlated with induction of both grp78 and gadd153 mRNA. Prior treatment with DTTox rendered cells tolerant to the potent nephrotoxicant S-(1,1,2, 2-tetrafluoroethyl)-L-cysteine. Bacitracin, an inhibitor of plasma membrane oxidoreductases, blocked DTTox reduction and gene activation as well as DTTox-induced tolerance. Thus, activation of stress genes and induction of cellular tolerance by DTTox is mediated by a novel mechanism involving cellular oxidoreductases.
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PMID:Reduction of trans-4,5-dihydroxy-1,2-dithiane by cellular oxidoreductases activates gadd153/chop and grp78 transcription and induces cellular tolerance in kidney epithelial cells. 926 5

The current study aimed to evaluate whether nicotinamide adenine dinucleotide phosphate (NADPH) alteration in erythrocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM) is responsible for the impaired glutathione (GSH) redox status, and to assess if short-term inhibition of the polyol pathway normalizes NADPH levels and GSH redox status via an amelioration of the NADPH/total NADP (tNADP) ratio. For this purpose, erythrocyte NADPH and GSH levels were measured in 18 NIDDM patients at baseline and then after 1 week of random double-blind assignment to treatment with either tolrestat (an aldose reductase inhibitor, 200 mg daily) (n = 12) or placebo (n = 6). A group of 16 healthy volunteers served as the control. In the basal condition, mean GSH (P < .0001) and NADPH (P < .0001) levels and NADPH/tNADP (P < .0001) and GSH/ glutathione disulfide (GSSG) (P < .005) ratios were lower in NIDDM patients than in control subjects. Tolrestat treatment increased GSH levels (P < .05 v placebo and baseline) and the NADPH/tNADP ratio (P < .05 v placebo and baseline). Interestingly, tolrestat-induced changes in GSH and NADPH levels and in GSH/GSSG and NADPH/tNADP ratios were significant only in patients who showed a decreased NADPH/tNADP ratio at baseline (n = 8). In these latter patients, we also found a direct correlation between percentage increments in GSH levels and NADPH/tNADP ratios after tolrestat treatment (r = .71, P < .05). In conclusion, our findings support the hypothesis that polyol pathway activation decreases NADPH and GSH levels. Accordingly, short-term inhibition of this enzymatic route increased both the GSH level and the NADPH/tNADP ratio. These changes were observable only in the subgroup of patients with an abnormal NADPH/tNADP ratio at baseline. Polyol pathway inhibition could be useful for decreasing oxidative stress in NIDDM.
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PMID:Polyol pathway activation and glutathione redox status in non-insulin-dependent diabetic patients. 932 6

The functionality of glutathione (GSH), which is present in separate mitochondrial and cytosolic pools, hinges on a steady supply of reducing equivalents, provided by NADPH, to convert glutathione disulfide (GSSG) to GSH. It is believed traditionally that glucose 6-phosphate (G6-P) via the pentose phosphate pathway is the main cellular source of NADPH. The current study examined the ability of NADH- and NADPH-linked cosubstrates to support cardiac cytosolic GSSG reduction. Exogenous NADP+ was added to the incubation mixtures because of the loss of this nucleotide during homogenization. Exogenous GSSG was added to all samples to levels that were approximately 60% of total glutathione. In both the 500 x g (with mitochondria) and 10,000 x g (without mitochondria) rat heart supernatants, isocitrate supported reduction of approximately 90% of available GSSG within 10 min. Malate, pyruvate and palmitoyl carnitine did not support GSSG reduction in either supernatant. G6-P yielded GSH levels within 10 min equal to 77% of total glutathione in the 1,0000 x g supernatant and 47% in the 500 x g supernatant. The current data indicate: (1) The pentose phosphate pathway, alone, is less efficient than isocitrate at supplying reducing equivalents for cytosolic GSSG reduction; and (2) some confounding factor(s) occur in the 500 x g and reconstituted 500 x g supernatants whereby G6-P-supported GSSG reduction is attenuated.
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PMID:Cosubstrates involved in the reduction of cytosolic glutathione disulfide in rat heart. 939 51

Bovine lens aldose reductase (ALR2), which catalyzes the NADPH-dependent reduction of 4-hydroxy-2-nonenal (HNE), is readily inactivated by its own substrate in a time- and concentration-dependent manner. Both DTT and NADP+ can prevent enzyme inactivation but neither extensive dialysis nor thiol-reducing treatment were able to restore enzyme activity once inactivation had occurred. Unlike the native enzyme, S-glutathionyl-modified ALR2 is unaffected by HNE, and can be easily reverted to the native form under thiol-reducing conditions. Evidence is presented of the involvement of Cys298 in the inactivation process. Zofenoprilat, an antioxidant thiol compound, mimics the effect of GSH. The possibility is raised that enzyme thiolation may function as a protection mechanism against the irreversible modification of ALR2.
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PMID:Site-specific inactivation of aldose reductase by 4-hydroxynonenal. 947 98

In order to characterize further the antilipoperoxidative enzyme system of human sperm, that part of the system designed to provide reducing equivalents for the reduction of highly reactive and potentially damaging lipid hydroperoxides to relatively inert hydroxylipids was examined. The substrate that provides the reducing equivalents directly to glutathione peroxidase (GPX) is reduced glutathione (GSH), which is in turn oxidized to glutathione disulfide (GSSG). The reducing equivalents needed for regeneration of GSH through the action of glutathione reductase (GRD) are provided by NADPH, produced by the action of glucose-6-phosphate dehydrogenase (G6P-DH) on substrates glucose-6-phosphate and NADP+. The kinetic properties of the enzymes GRD and G6P-DH were determined by standard enzyme activity assay at 24 and 37 degrees C. At 37 degrees C, the Vmax for GRD was found to be 36 nmol/min x 10(8) cells, with Km values for GSSG and NAPH of 150 microM and 16 microM, respectively; the Vmax for G6P-DH was 3.3 nmol/min x 10(8) cells with Km for NADP+ of 8 microM. This suggested that G6P-DH activity was limiting in this reductive pathway. The activity of GRD in situ in intact cells was estimated using the thiol-reactive fluorogenic probe ThioGlo-1, which is cell permeant and reacts rapidly with GSH to give a highly fluorescent adduct. Mixing a suspension of human sperm with the fluorogenic reagent at 37 degrees C gave an initial rapid increase in fluorescence, followed by a slower one. The rapid phase is due to reaction with intracellular GSH already present; the slow phase is due to reaction with GSH generated by the GRD-catalyzed reduction of GSSG. Both rates showed first-order kinetics. Calculation of the maximal rate as NADPH oxidation, attributable to in situ GRD activity, gave the value of 1.0 nmol/min x 10(8) cells, less than the maximum for NADPH production by the dehydrogenase. These results support the suggestion that NADPH production limits the capacity of the pathway leading to hydroperoxide reduction in human sperm. We propose that the antilipoperoxidative defense system of human sperm has just sufficient capacity to allow these cells to fulfill their function but is limited to allow their timely disposal from the female reproductive tract.
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PMID:Human sperm glutathione reductase activity in situ reveals limitation in the glutathione antioxidant defense system due to supply of NADPH. 950 91

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