Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kidney and several other thyroid hormone-responsive tissues contain a NADP-regulated thyroid hormone (TH)-binding protein (THBP), with an apparent molecular mass of 36 kDa on SDS-PAGE, responsible for most of the intracellular high-affinity T3 and T4 binding. THBP was purified to homogeneity from human kidney cytosol and used to generate proteolytic peptides. Microsequencing of four peptides revealed identity to amino acid sequences deduced from a human cDNA homolog to a cDNA encoding kangaroo mu-crystallin. This protein is a major structural kangaroo lens protein with no known function in other species. A full-sized cDNA (TH5.9) was isolated by 5'- and 3'-rapid amplification of cDNA ends using a human brain cDNA library and gene-specific PCR primers, confirming identity to the previously cloned human cDNA. The TH5.9 cDNA encodes a 314-residue protein (theoretical mol wt = 33,775) with significant homologies (40 to 60%) with two bacterial enzymes: lysine cyclodeaminase and ornithine cyclodeaminase. The TH5.9 cDNA was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Purified GST fusion protein, but not GST, bound T3 specifically with high affinity [dissociation constant (Kd) = 0.5 nM] in the presence of NADPH, and was labeled by UV-driven cross-linking of underivatized [(125)I]T3. T3 binding and photoaffinity labeling of GST fusion protein were activated by NADPH [activation constant (K[act]) = 10(-8) M], but not by NADH. The expressed protein displays the appropriate binding properties, indicating that TH5.9 cDNA encodes the NADP-regulated THBP characterized in human tissues.
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PMID:Purification, molecular cloning, and functional expression of the human nicodinamide-adenine dinucleotide phosphate-regulated thyroid hormone-binding protein. 932 54

Native biliverdin IX alpha reductase (BVR-A) is a monomer of molecular mass 34 kDa. We have developed an expression vector that allows the isolation of 40 mg of a glutathione S-transferase (GST)-BVR-A fusion protein from 1 litre of culture. The fusion protein (60 kDa) behaves as a dimer on gel filtration (120 kDa), so that we have artificially created a BVR-A dimer. The recombinant rat kidney enzyme exhibits pre-steady-state 'burst' kinetics that show a pH dependence similar to that already described for ox kidney BVR-A. Similar behaviour was obtained in the presence and absence of the GST domain both for the burst kinetics and during initial-rate studies in the presence and absence of albumin. The stereospecificity of the BVR-A-catalysed oxidation of [4-3H]NADH, labelled at the A and B faces, was shown to occur exclusively via the B face.
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PMID:Cloning and overexpression of rat kidney biliverdin IX alpha reductase as a fusion protein with glutathione S-transferase: stereochemistry of NADH oxidation and evidence that the presence of the glutathione S-transferase domain does not effect BVR-A activity. 935 30

Monensin, a polyether ionophore antibiotic used worldwide for its anticoccidial and growth-promoting properties, is reported to act as anin vivo inducer or inhibitor of drug-metabolizing enzyme systems in various species according to dosage regimens and duration of exposure. When incubated at a concentration up to 0.25 mM with hepatic subfractions from either untreated- (UT) or phenobarbital- (PB) induced rats, monensin did not induce appreciable changes in cytochrome P450 content and functions as well as in NADPH cytochrome c reductase or glutathione S-transferase. On the other hand, monensin concentrations ranging from 0.05 to 0.25 mM proved to increase the initial rate of NADPH oxidation up to 63% in UT-microsomes, and the in vitro addition of the ionophore to microsomes resulted in the formation of a characteristic type I binding spectrum. The rate of monensin O-demethylation was 0.34+/-0. 01 and 0.99+/-0.07 nmol min-1 per mg of protein in UT- and PB-microsomes, respectively. In the latter, this reaction was consistently depressed when NADPH was omitted or replaced with NADH, or upon the addition of 1 mM metyrapone, a known P450 inhibitor. It is concluded that monensin does not behave as a direct in vitro inhibitor of drug metabolizing enzymes and appears to be a substrate of P450-dependent monooxygenases.
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PMID:'In vitro' interactions of monensin with hepatic xenobiotic metabolizing enzymes. 936 71

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


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