Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Alkene monooxygenase (AMO) from Rhodococcus rhodochrous (formerly Nocardia corallina) B-276 is a three-component enzyme system encoded by the four-gene operon amoABCD. AMO catalyses the stereoselective epoxygenation of aliphatic alkenes, yielding primarily R enantiomers. The presumed site of alkene oxygenation is a dinuclear iron centre similar to that in the soluble methane monooxygenases of methanotrophic bacteria, to which AMO exhibits a significant degree of amino acid sequence identity. The AMO complex was not expressed in Escherichia coli, at least partly because that host did not produce all of the AMO polypeptides. Expression of AMO was achieved in Streptomyces lividans by cloning the AMO genes into the thiostrepton-inducible expression plasmid pIJ6021. No background of AMO activity was detected in S. lividans cells without amoABCD and expression of AMO activity, at a level comparable to that from wild-type R. rhodochrous B-276, coincided with appearance of the AMO subunits. Recombinant AMO activity in cell-free extracts of S. lividans was stimulated by the addition of NADH and produced R-epoxypropane with comparable enantiomeric excess to AMO purified from the original organism. Although the whole AMO complex could not be expressed in E. coli, the functional coupling protein (AmoB) and reductase (AmoD) were expressed individually in E. coli as fusions with glutathione S-transferase. The expression systems described here now allow structure/function studies on AMO to be carried out by site-directed mutagenesis.
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PMID:Heterologous expression of alkene monooxygenase from Rhodococcus rhodochrous B-276. 1009 80

4-aminobenzoate hydroxylase (4ABH) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate to 4-hydroxyaniline. For use as a clinical reagent, the gene encoding 4ABH from Agaricus bisporus was cloned by the RACE method. Also, the cDNA encoding 4ABH was expressed in Escherichia coli cells as a fusion protein with glutathione S-transferase (GST). The expressed GST-4ABH fusion protein (recombinant 4ABH) in the soluble fraction exhibits decarboxylative hydroxylation and additional NADH oxidation activities.We investigated a new ultraviolet spectrometric method for determining serum gamma-glutamyltransferase (gamma-GT) using recombinant 4ABH as a coupling enzyme. The principle of the method is as follows. Using gamma-glutamyl-3-choloro-4-aminobenzoate (L-gamma-glu-PAClBA) and glycylglycine as the donor and acceptor substrates, 3-choloro-4-aminobenzoate (PAClBA) is formed by the catalysis of serum gamma-GT. PAClBA is stoichiometrically converted to 3-choloro-4-hydroxyaniline (PHClA) and NAD(+) by 4ABH and NADH. However, NADH oxidation results in a high reagent blank, which is considered as a drawback for use as a clinical reagent. Using recombinant 4ABH, we examined the effects of pH and detergents on these two activities, and found that several detergents suppress the additional NADH oxidation activity with little or no effect on hydroxylation activity. The results indicate a promising approach to establishing an ultraviolet spectrophotometric method for determining serum gamma-GT activity using L-gamma-glu-PAClBA as the donor substrate and recombinant 4ABH as a coupling enzyme.
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PMID:A new UV method for serum gamma-glutamyltransferase assay using recombinant 4-aminobenzoate hydroxylase as a coupling enzyme. 1042 28

Both sorbitol accumulation-linked osmotic stress and "pseudohypoxia" [increase in NADH/NAD+, similar to that in hypoxic tissues, and attributed to increased sorbitol dehydrogenase (1-iditol:NAD+ 5-oxidoreductase; EC 1.1.1.14; SDH) activity] have been invoked among the mechanisms underlying oxidative injury in target tissues for diabetic complications. We used the specific SDH inhibitor SDI-157 [2-methyl-4(4-N,N-dimethylaminosulfonyl-1-piperazino)pyrimid ine] to evaluate the role of osmotic stress versus "pseudohypoxia" in oxidative stress occurring in diabetic precataractous lens. Control and diabetic rats were treated with or without SDI-157 (100 mg/kg/day for 3 weeks). Lens malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HA), MDA, GSH, and ascorbate levels, as well as the GSSG/GSH ratios, were similar in SDI-treated and untreated control rats, thus indicating that SDI-157 was not a prooxidant. Intralenticular osmotic stress, manifested by sorbitol levels, was more severe in SDI-treated diabetic rats (38.2+/-6.8 vs 21.2+/-3.5 micromol/g in untreated diabetic and 0.758+/-0.222 micromol/g in control rats, P<0.01 for both), while the decrease in the free cytosolic NAD+/NADH ratio was partially prevented (120+/-16 vs 88+/-11 in untreated diabetic rats and 143+/-13 in controls, P<0.01 for both). GSH and ascorbate levels were decreased, while MDA plus 4-HA and MDA levels were increased in diabetic rats versus controls; both antioxidant depletion and lipid aldehyde accumulation were exacerbated by SDI treatment. Superoxide dismutase (superoxide:superoxide oxidoreductase; EC 1.15.1.1), GSSG reductase (NAD[P]H:oxidized-glutathione oxidoreductase; EC 1.6.4.2), GSH transferase (glutathione S-transferase; EC 2.5.1.18), GSH peroxidase (glutathione:hydrogen-peroxide oxidoreductase; EC 1.11.1.9), and cytoplasmic NADH oxidase activities were increased in diabetic rats versus controls, and all the enzymes but GSH peroxidase were up-regulated further by SDI. In conclusion, sorbitol accumulation and osmotic stress generated oxidative stress in diabetic lens, whereas the contribution of "pseudohypoxia" was minor. SDIs provide a valuable tool for exploring mechanisms of oxidative injury in sites of diabetic complications.
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PMID:Interaction between osmotic and oxidative stress in diabetic precataractous lens: studies with a sorbitol dehydrogenase inhibitor. 1059 Nov 49

The mitochondrial outer membrane enzyme kynurenine 3-hydroxylase (K3H) is an NADPH-dependent flavin mono-oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS-1 cells as a glutathione S-transferase (GST) fusion protein, and the pure recombinant protein (rec-K3H) was obtained with a specific activity of about 2000 nmol.min-1.mg-1. Rec-K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non-covalently bound FAD per molecule of enzyme. The mechanism of the rec-K3H-catalyzed reaction was investigated by overall initial-rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl-, and that NADPH-catalyzed oxidation occurred even in the absence of kynurenine if 3-hydroxykynurenine was present, suggesting an uncoupling effect of 3-hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3-hydroxykynurenine in vivo.
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PMID:Functional characterization and mechanism of action of recombinant human kynurenine 3-hydroxylase. 1067 18

Recessive congenital methemoglobinemia due to nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is classified into 2 clinical types: type 1 (erythrocyte type) and type 2 (generalized type). We found a Chinese family with type 1 recessive congenital methemoglobinemia, the patients from which were diagnosed according to clinical symptoms and b5R enzyme activity in the blood cells. To learn the molecular basis of type 1 recessive congenital methemoglobinemia in this Chinese family, we isolated total RNA from the peripheral leukocytes of the propositus and b5R complementary DNA (cDNA) by reverse transcription- polymerase chain reaction (RT-PCR). The coding region of the b5R cDNA was analyzed by sequencing the cloned PCR products. The results showed that the propositus was homozygous for a G-->A transition at codon 203 in exon 7, changing a cysteine to a tyrosine (Cys203Tyr). To characterize the mutant enzyme, both glutathione S-transferase (GST)-fused wild-type b5R and GST-fused mutant Cys203Tyr b5R were expressed in Escherichia coli and affinity purified. The results showed that the catalytic activity of the enzyme was not much affected by this amino acid substitution, but the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin. These properties of the mutant enzyme would account for the restricted b5R deficiency and mild clinical manifestations of these type 1 patients. The finding of this novel mutation makes codon 203 the only position within the b5R gene at which more than 1 mutation has been found.
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PMID:A novel mutation in the NADH-cytochrome b5 reductase gene of a Chinese patient with recessive congenital methemoglobinemia. 1080 96

This study was designed to (1) evaluate retinal lipid peroxidation in early diabetes by the method specific for free malondialdehyde and 4-hydroxyalkenals, (2) identify impaired antioxidative defense mechanisms and (3) assess if enhanced retinal oxidative stress in diabetes is prevented by the potent antioxidant, DL-alpha-lipoic acid. The groups included control and streptozotocin-diabetic rats treated with or without DL-alpha-lipoic acid (100 mg kg(-1) day(-1), i.p., for 6 weeks). All parameters were measured in individual retinae. 4-Hydroxyalkenal concentration was increased in diabetic rats (2.63+/-0.60 vs. 1.44+/-0.30 nmol/mg soluble protein in controls, P<0.01), and this increase was prevented by DL-alpha-lipoic acid (1.20+/-0.88, P<0.01 vs. untreated diabetic group). Malondialdehyde, reduced glutathione (GSH) and oxidized glutathione (GSSG) concentrations were similar among the groups. Superoxide dismutase, glutathione peroxidase (GSHPx), glutathione reductase (GSSGRed) and glutathione transferase (GSHTrans) activities were decreased in diabetic rats vs. controls. Quinone reductase was upregulated in diabetic rats, whereas catalase and cytoplasmic NADH oxidase activities were unchanged. DL-alpha-Lipoic acid prevented changes in superoxide dismutase and quinone reductase activities induced by diabetes without affecting the enzymes of glutathione metabolism. In conclusion, accumulation of 4-hydroxyalkenals is an early marker of oxidative stress in the diabetic retina. Increased lipid peroxidation occurs in the absence of GSH depletion, and is prevented by DL-alpha-lipoic acid.
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PMID:Early changes in lipid peroxidation and antioxidative defense in diabetic rat retina: effect of DL-alpha-lipoic acid. 1085 58

Fullerene (C60, C70, etc.) is a third carbon allotrope discovered in 1985, and a great deal of attention has been focused on its physical and chemical properties in recent years. We are very interested in its biological properties for use fullerene as a pharmacophore. We first developed a method of solubilizing fullerene itself in water to perform in vitro biological screening. The concentrations of aqueous C60 and C70 solution with 5% poly(vinylpyrorridone) (PVP) are 400 and 200 micrograms/mL, respectively. By using aqueous fullerene solutions prepared in this manner, we have clarified a series of biological activities of fullerene, consisting of DNA-cleavage, hemolysis, cancer-initiation, and cell-toxicity under photoirradiation, and chondrogenesis and inhibition of glutathione S-transferase activity without photoirradiation. The biological activity of photo-excited fullerene was found to be promising, because fullerene is a highly efficient photo-sensitizer. We synthesized a C60 derivative with an acridine moiety as a DNA-chelating function and assessed its effective DNA-cleaving activity. What kind of active species is involved in the biological action of photo-excited fullerene is our next concerns. Two pathways have been reported for the photo-excitation of fullerene. The so-called Type II energy transfer pathway generates singlet oxygen (1O2), while the Type I electron transfer pathway gives a fullerene radical anion (C60.-, C70.-). In order to clarify the effective oxygen species actually responsible for the biological action of photo-excited fullerene, we performed DNA-cleaving tests and EPR spectroscopic analyses under several conditions. The results showed that the photo-induced biological activity of fullerene is not caused by 1O2, but by reduced oxygen species (O2.-, .OH) generated by the electron transfer reaction of C60.-, with molecular oxygen. Its specificity is thought to be mainly attributed to the high-reducible property of fullerene. Since the reductive activation of molecular oxygen by photo-excited fullerene was observed at physiological concentrations of NADH as the reductant, fullerene can be classified as an oxyl-radical-generating photosensitizer. Pharmaceutical application of fullerene to cancer photo-dynamic therapy appears promising.
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PMID:[Biological activity of photoexcited fullerene]. 1085 36

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was irreversibly and (S)-selectively inactivated by the enantiomers of racemic 4-hydroxy-2(E)-nonenal (HNE), a reactive product released from biomembranes by lipid peroxidation in cells. Rates of the enzyme inactivations were 1.7, 3.0, and 6.0 M(-1).s(-1) for (R)-, racemic and (S)-HNEs respectively. In rat liver cytosol the HNE was detoxified 2.5-fold more (S)-selectively by GSH conjugation and 2. 4-fold more (R)-selectively by NADH-dependent reduction mediated by alcohol dehydrogenase (ADH) than the opposite enantiomers. However, in the cytosol the GSH conjugation of (R)-HNE proceeded at a much higher rate than did its ADH-mediated reduction. The minor glutathione S-transferase (GST) isoform, A4-4, in the rat (r) liver had a major role in the cytosolic (S)-selective GSH conjugation. The catalytic efficiency, k(cat)/K(m), of purified rGSTA4-4 was 4-fold higher for (S)-HNE than for (R)-HNE; the K(m) was 3-fold higher for (R)-HNE than for (S)-HNE. (S)-HNE was preferentially detoxified to (R)-HNE by rGSTA4-4 when racemic HNE was used as a substrate.
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PMID:4-Hydroxy-2(E)-nonenal enantiomers: (S)-selective inactivation of glyceraldehyde-3-phosphate dehydrogenase and detoxification by rat glutathione S-transferase A4-4. 1090 33

The effect of hydroalcoholic (80% ethanol, 20% water) extract of leaves of Aegle marmelos was examined on carcinogen-metabolizing phase-I and phase-II enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase and lipid peroxidation, using two doses of dried extract (50 and 100 mg kg(-1) daily for 14 days), in the liver of mice. The modulatory effect of the extract was also examined on extrahepatic organs (lung, kidney and fore-stomach) for effects on the activity of glutathione S-transferase, DT-diaphorase, superoxide dismutase and catalase. Extract treatment significantly increased the basal levels of acid-soluble sulphydryl (-SH) content, cytochrome P450, NADPH-cytochrome P450 reductase, cytochrome b5, NADH-cytochrome b5 reductase, glutathione S-transferase, DT-diaphorase, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase in the liver. Aegle acted as a bifunctional inducer since it induced both phase-I and phase-II enzyme systems. Both doses significantly decreased the activity of lactate dehydrogenase and formation of malondialdehyde in liver, suggesting a role in cytoprotection as well as protection against pro-oxidant-induced membrane damage. Butylated hydroxyanisole (positive control) induced almost all the antioxidative parameters measured in this study. The extract was effective in inducing glutathione S-transferase, DT-diaphorase, superoxide dismutase and catalase in lung, glutathione S-transferase, DT-diaphorase and superoxide dismutase in fore-stomach, and DT-diaphorase and superoxide dismutase in lung. These significant changes in the levels of drug-metabolizing enzymes and antioxidative profiles are strongly indicative of the chemopreventive potential of this plant, especially against chemical carcinogenesis.
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PMID:Effect of Aegle marmelos on biotransformation enzyme systems and protection against free-radical-mediated damage in mice. 1100 71

The effect of two different doses (50 and 100 mg/kg body wt/day for 14 days) of 80% ethanolic extract of the leaves of Adhatoda vesica were examined on drug metabolizing phase I and phase II enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase and lipid peroxidation in the liver of 8 weeks old Swiss albino mice. The modulatory effect of the extract was also examined on extra-hepatic organs viz. lung, kidney and forestomach for the activities of glutathione S-transferase, DT-diaphorase, superoxide dismutase and catalase. Significant increase in the activities of acid soluble sulfhydryl (-SH) content, cytochrome P450, NADPH-cytochrome P450 reductase, cytochrome b5, NADH-cytochrome b5 reductase, glutathione S-transferase (GST), DT-diaphorase (DTD), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were observed in the liver at both dose levels of treatments. Adhatoda vesica acted as bifunctional inducer since it induced both phase I and phase II enzyme systems. Both the treated groups showed significant decrease in malondialdehyde (MDA) formation in liver, suggesting its role in protection against prooxidant induced membrane damage. The cytosolic protein was significantly inhibited at both the dose levels of treatment indicating the possibility of its involvement in the inhibition of protein synthesis. BHA has significantly induced the activities of GR and GSH in the present study. The extract was effective in inducing GST and DTD in lung and forestomach, and SOD and CAT in kidney. Thus, besides liver, other organs viz., lung, kidney and forestomach were also stimulated by Adhatoda, to increase the potential of the machinery associated with the detoxification of xenobiotic compounds. But, liver and lung showed a more consistent induction. Since the study of induction of the phase I and phase II enzymes is considered to be a reliable marker for evaluating the chemopreventive efficacy of a particular compound, these findings are suggestive of the possible chemopreventive role played by Adhatoda leaf extract.
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PMID:Modulatory influence of Adhatoda vesica (Justicia adhatoda) leaf extract on the enzymes of xenobiotic metabolism, antioxidant status and lipid peroxidation in mice. 1112 64


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