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 reaction of the glutathione transferase from human placenta with a maleimide spin label derivative has been followed by EPR. Incubation of the enzyme at pH 7.0 with 50-fold molar excess of the spin label reagent gives rise to an immobilized nitroxyl EPR spectrum indicative of two reacting thiol groups per dimer of enzyme as evaluated by double integration of the EPR spectrum; the activity is lost in parallel. The same type of spectrum can be obtained simply by adding 2 eq of the spin label reagent to the enzyme. The binding is completed after less than 1 min at pH 8.0; it requires 2 min at pH 7.0 and more than 10 min at pH 6.0. These data indicate that the maleimide derivative reacts, in each subunit, with a thiol group which plays a crucial role for the maintenance of the catalytic activity and is characterized by a low pK. Inactivation of the enzyme at pH 7.0 in the presence of 2 eq of spin label reagent per mol of enzyme requires 15 min, suggesting the occurrence of a structural rearrangement after the binding of the thiol blocking agent. The same binding in the presence of S-methylglutathione or protoporphyrin IX shows a decreased reaction rate with respect to the reaction in the absence of inhibitors, indicating that the thiols are in proximity of both the glutathione and the porphyrin binding sites. For this latter case, this is unambiguously demonstrated by the titration of spin-labeled enzyme with hemin, which produces a decrease of the EPR signal amplitude from which an interspin distance of about 10 A can be evaluated.
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PMID:Electron paramagnetic resonance identification of a highly reactive thiol group in the proximity of the catalytic site of human placenta glutathione transferase. 184 10

Kinetic and binding studies with substrates, products, and a spin-labeled product analogue of glutathione (sl-glutathione) have been used to characterize the kinetic mechanism and properties of the catalytic site of the homodimer YaYa of glutathione S-transferase. Product inhibition studies and inhibition by sl-glutathione indicate the random addition of substrates. The kinetically determined dissociation constant for the product S-(2,4-dinitrophenyl)glutathione is approximately 7 microM. A newly described spin-labeled product analogue, S-[[(2,2,5,5,-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl] glutathione (sl-glutathione), acts as a competitive inhibitor with respect to both substrates (glutathione and 1-Cl-2,4-dinitrobenzene) with a kinetically determined dissociation constant of approximately 40 microM. Analysis of the glutathione S-transferase X sl-glutathione complex by EPR gives a rigid limit spectrum indicative of highly immobilized spin label. Kinetic and EPR results support the proposal that sl-glutathione binds as a bisubstrate or product analogue by occupying both the glutathione and hydrophobic substrate sites. Binding studies of sl-glutathione by EPR give a dissociation constant of 28 microM and a single binding site per homodimer. Displacement of sl-glutathione by substrates and product have been used to directly determine enzyme-ligand dissociation constants. Dissociation constants of 2.1 mM, 17 microM, and 25 microM were obtained for glutathione, 1-Cl-2,4-dinitrobenzene and S-(2,4-dinitrophenyl)glutathione when enzyme was added to a mixture of sl-glutathione and the competing ligand. The dissociation constants for glutathione and 1-Cl-2,4-dinitrobenzene but not for S-(2,4-dinitrophenyl) glutathione were dependent on the order of addition, consistent with the existence of several kinetically stable conformations for the enzyme. The sl-glutathione described here may provide a useful analogue for similar studies with other glutathione S-transferases or other enzymes which bind glutathione.
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PMID:Kinetic studies and active site-binding properties of glutathione S-transferase using spin-labeled glutathione, a product analogue. 631 84

Induction of glutathione S-transferase (GST) Ya gene expression by a variety of chemical agents is mediated by a regulatory element composed of two adjacent AP-1-like binding sites and activated by the Fos/Jun heterodimeric complex (AP-1). We have previously shown that the induction of GST Ya gene expression and of AP-1 binding activity is regulated by intracellular glutathione (GSH) levels. To study the role of reactive oxygen species in the induction of AP-1 activity and GST Ya gene expression and their effect on intracellular GSH levels, we have exposed hepatoma cells to adriamycin and two synthetic quinones, Qcb and Qn, with different capacities to generate oxygen radicals. The kinetics of quinone-mediated generation of hydroxyl radicals were monitored in intact cells by a spin trapping technique and EPR spectral measurements. We find that quinones which can chelate Fe(III) ions, adriamycin and Qcb, are more effective in hydroxyl radical production than the nonchelating quinone Qn. Furthermore, we show that the induction of AP-1 binding activity and GST Ya gene expression by these quinones correlates with their oxygen radical production, adriamycin and Qcb being stronger inducers that Qn. The present study indicates that the AP-1-mediated induction of GST Ya gene expression is part of the response to oxidative stress. A transient increase by 2.5-fold in the intracellular GSH level was observed 30 min after exposure of cells to quinone and was followed by a rapid depletion of GSH. This increase in the GSH level represents an induction of GSH synthesis since it was blocked by buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of quinone-mediated generation of hydroxyl radicals in the induction of glutathione S-transferase gene expression. 781 27

Transcription factors AP-1 and NF-kappaB have been implicated in the inducible expression of a variety of genes in response to oxidative stress. Recently, based on the observation that butylated hydroxyanisole (BHA) and pyrrolidine dithiocarbamate (PDTC) induce AP-1 binding activity and AP-1-dependent gene expression and assuming that these compounds exert an antioxidant effect, it was claimed that AP-1 is an antioxidant-responsive factor. To determine whether AP-1 can be responsive to both oxidant and antioxidant, we examined the nature of BHA and PDTC inducing activity. Using EPR spectroscopy to detect semiquinone radicals, we demonstrate the autoxidation of BHA metabolite tert-butylhydroquinone (TBHQ) to tert-butylquinone. The kinetics of TBHQ-mediated generation of .OH radicals were monitored in intact hepatoma HepG2 cells by EPR spin trapping technique. Exogenous catalase inhibited the rate and amount of .OH radical formation and the induction of AP-1-mediated glutathione S-transferase (GST) Ya gene expression by BHA and TBHQ, thus indicating the intermediate formation of H2O2 in the metabolism of these chemicals. Furthermore, we show that the induction of AP-1 and NF-kappaB activities and GST Ya gene expression by BHA and TBHQ is due to a pro-oxidant activity, since this induction was inhibited by thiol compounds N-acetyl cysteine and GSH. Similarly, induction of AP-1 and GST Ya gene expression by PDTC was inhibited by N-acetyl cysteine and GSH. The present findings do not support the notion that the induction of AP-1 by BHA, TBHQ, or PDTC is an antioxidant response and demonstrate that both AP-1 and NF-kappaB activities are induced by oxygen radicals.
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PMID:Role of oxidants and antioxidants in the induction of AP-1, NF-kappaB, and glutathione S-transferase gene expression. 866 87

The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overproduction in Escherichia coli of fusion, full-length, and truncated forms of the Rieske (PetC) protein from the cyanobacterium Nostoc sp. PCC 7906. A glutathione S-transferase/Rieske fusion protein was used to prepare specific chicken egg-yolk antibodies against the Rieske protein. Expression of the nonfusion petC gene in a T7 RNA polymerase promoter vector produced copious quantities of the full-length Rieske protein predominantly as inclusion bodies. The highly enriched, Rieske protein from inclusion bodies has been denatured in guanidine hydrochloride and refolded and the characteristic 2Fe-2S cluster reconstituted in vitro by incubation with iron and sulfide under reducing conditions. Purification by chromatography on Whatman DE52 cellulose and ultrafiltration through a 30000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein. Reconstituted Rieske preparations showed intense and highly characteristic gx = 1.74, gy = 1.89, and gz = 2.03 "Rieske-type" electron paramagnetic resonance signals at 15 K. Two methods of reconstitution yielded Rieske preparations in which 20-60% of the protein contained 2Fe-2S clusters as determined by EPR spin quantitation. The reconstituted Rieske protein was soluble and stable at 4 degrees C in buffers containing nonionic detergents and showed a redox midpoint potential of +321 mV at pH 7.0 as determined by optical circular dichroism (CD) spectroscopy. These data demonstrate the in vitro restoration of a Cys and His liganded 2Fe-2S cluster and provide the basis for mutational and structural analysis of a PetC Rieske protein of oxygenic photosynthesis.
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PMID:Reconstitution of the 2Fe-2S center and g = 1.89 electron paramagnetic resonance signal into overproduced Nostoc sp. PCC 7906 Rieske protein. 895 2

The QPs1 subunit of bovine heart mitochondrial succinate-ubiquinone reductase was overexpressed in Escherichia coli DH5 alpha cells as a glutathione S-transferase fusion protein (GST-QPs1) using the expression vector, pGEX/QPs1. The yield of soluble active recombinant GST-QPs1 fusion protein depends on the IPTG concentration, induction growth time, temperature, and medium. Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth with 0.5 mM IPTG at 27 degrees C in an enriched medium containing betaine and sorbitol. QPs1 is released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPs1 shows one protein band in SDS-polyacrylamide gel electrophoresis corresponding to subunit III of mitochondrial succinate-ubiquinone reductase. However, partial N-terminal amino acid sequence analysis of recombinant QPs1 shows two extra amino acid residues, glycine and serine, at the N-terminus of mature QPs1, resulting from the recombinant manipulation. When isolated recombinant QPs1 is dispersed in 0.01% dodecyl maltoside, it is in a highly aggregated form with an apparent molecular mass of over 1 million. Recombinant GST-QPs1 contains little cytochrome b-560 heme. However, addition of hemin chloride restores the spectral characteristics of cytochrome b-560. Cytochrome b-560 restoration varies with the amount of hemin used. Maximum reconstitution is obtained when the molar ratio of heme to fusion protein used in the system is 0.6. Reconstituted cytochrome b-560 shows a EPR signal at g = 2.91 which corresponds to one of the EPR signals of cytochrome b-560 in a QPs preparation. When GST-QPs1 with reconstituted cytochrome b-560 is treated with thrombin to cleave GST from QPs1, no change in the absorption and EPR characteristics of cytochrome b-560 is observed, indicating that the bis-histidine ligands of reconstituted cytochrome b-560 are provided by QPs1.
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PMID:Reconstitution of cytochrome b-560 (QPs1) of bovine heart mitochondrial succinate-ubiquinone reductase. 951 6

The smallest membrane-anchoring subunit (QPs3) of bovine heart succinate:ubiquinone reductase was overexpressed in Escherichia coli JM109 as a glutathione S-transferase fusion protein using the expression vector pGEX2T/QPs3. The yield of soluble active recombinant glutathione S-transferase-QPs3 fusion protein was isopropyl-1-thio-beta-D-galactopyranoside concentration-, induction growth time-, temperature-, and medium-dependent. Maximum yield of soluble recombinant fusion protein was obtained from cells harvested 3.5 h post-isopropyl-1-thio-beta-D-galactopyranoside (0.4 mM)-induction growth at 25 degrees C in 2.0% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose (SOC medium) containing 440 mM sorbitol and 2.5 mM betaine. QPs3 was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPs3 shows one protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that corresponds to subunit V of mitochondrial succinate:ubiquinone reductase. Although purified recombinant QPs3 is dispersed in 0.01% dodecylmaltoside, it is in a highly aggregated form, with an apparent molecular mass of more than 1 million. The recombinant QPs3 binds ubiquinone, causing a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that recombinant QPs3 may be in the functionally active state. Two amino acid residues, serine 33 and tyrosine 37, in the putative ubiquinone binding domain of QPs3 are involved in ubiquinone binding because the S33A- or Y37A-substituted recombinant QPs3s do not cause the spectral blue shift of ubiquinone. Although recombinant QPs3 contains little cytochrome b560 heme, the spectral characteristics of cytochrome b560 are reconstituted upon addition of hemin chloride. Reconstituted cytochrome b560 in recombinant QPs3 shows a EPR signal at g = 2.92. Histidine residues at positions 46 and 60 are responsible for heme ligation because the H46N- or H60N-substituted QPs3 fail to restore cytochrome b560 upon addition of hemin chloride.
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PMID:Identification of quinone-binding and heme-ligating residues of the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate:ubiquinone reductase. 1008 11

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 proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8 is composed of 14 subunits (designated Nqo1-14). This NDH-1 houses nine putative iron-sulfur binding sites, eight of which are generally found in bacterial NDH-1 and its mitochondrial counterpart (complex I). The extra site contains a CXXCXXXCX(27)C motif and is located in the Nqo3 subunit. This motif was originally found in Escherichia coli NDH-1 and was assigned to a binuclear cluster (g(z, y, x) = 2.00, 1.95, 1.92) and named N1c. In this report, the Thermus Nqo3 fragment containing this motif was heterologously overexpressed, using a glutathione S-transferase fusion system. This fragment contained a small amount of iron-sulfur cluster, whose content was significantly increased by in vitro reconstitution. The UV-visible and EPR spectroscopic properties of this fragment indicate that the ligated iron-sulfur cluster is tetranuclear with nearly axial symmetry (g( parallel, perpendicular) = 2.045, approximately 1.94). Site-directed mutants show that all four cysteines participate in the ligation of a [4Fe-4S] cluster. Considering the fact that the same motif coordinates only tetranuclear clusters in other enzymes so far known, we propose that the CXXCXXXCX(27)C motif in the Nqo3 subunit most likely ligates the [4Fe-4S] cluster.
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PMID:Characterization of the iron-sulfur cluster coordinated by a cysteine cluster motif (CXXCXXXCX27C) in the Nqo3 subunit in the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8. 1170 68

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex molybdenum-, cytochrome b(557)- and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. To facilitate structure/function studies of the individual molybdenum center, we have developed bacterial expression systems for the heterologous production of the 541 residue amino-terminal, molybdenum center-containing domain of spinach nitrate reductase either as a six-histidine-tagged variant or as a glutathione-S-transferase-tagged fusion protein. Expression of the his-tagged molybdenum domain in Escherichia coli BL21(DE3) cells under anaerobic conditions yielded a 55-kDa domain with a specific activity of 1.5 micromol NO(3)(-) consumed/min/nmol enzyme and with a K(mapp)(NO(3)(-)) of 8 mciroM. In contrast, expression of the molybdenum domain as a GST-tagged fusion protein in E. coli TP1000(MobA(-) strain) cells under aerobic conditions yielded an 85-kDa fusion protein with a specific activity of 10.8 micromol NO(3)(-) consumed/min/nmol enzyme and with a K(mapp)(NO(3)(-)) of 12 microM. Fluorescence analysis indicated that both forms of the molybdenum domain contained the cofactor, MPT, although the MPT content was higher in the GST-fusion domain. Inductively coupled plasma mass spectrometric analysis of both the his-tagged and GST-fusion protein domain samples indicated Mo/protein ratios of 0.44 and 0.93, respectively, confirming a very high level of Mo incorporation in the GST-fusion protein. Expression of the GST-fusion protein in TP1000 cells in the presence of elevated tungsten concentrations resulted in an 85-kDa fusion protein that contained MPT but which was devoid of nitrate-reducing activity. Partial reduction of the molybdenum domain resulted in the generation of an axial Mo(V) EPR species with g values of 1.9952, 1.9693, and 1.9665, respectively, and exhibiting superhyperfine coupling to a single exchangeable proton, analogous to that previously observed for the native enzyme. In contrast, the tungsten-substituted MPT-containing domain yielded a W(V) EPR species with g values of 1.9560, 1.9474, and 1.9271, respectively, with unresolved superhyperfine interaction. NADH:nitrate reductase activity could be reconstituted using the GST-molybdenum domain fusion protein in the presence of the recombinant forms of the spinach nitrate reductase' flavin- and heme-containing domains.
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PMID:Bacterial expression of the molybdenum domain of assimilatory nitrate reductase: production of both the functional molybdenum-containing domain and the nonfunctional tungsten analog. 1213 73


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