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)

Subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex was over-expressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-RSIV) using the expression vector, pGEX/RSIV. Maximum yield of soluble active recombinant fusion protein was obtained from cells harvested 3 h after induction of growth at 37 degrees C in LB medium. Subunit IV was released from the fusion protein by proteolytic cleavage with thrombin. When subjected to SDS-polyacrylamide gel electrophoresis, isolated recombinant subunit IV of R. sphaeroides cytochrome b-c1 complex. Although the isolated recombinant subunit IV is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1000 kDa. The addition of detergent deaggregates the isolated protein, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. When the three-subunit core cytochrome b-c1 complex, purified from RS delta IV-adapted chromatophores containing a fraction of the wild type cytochrome b-c1 complex activity, was reacted with varying amounts of recombinant subunit IV, the activity increased as the subunit IV concentration increased. Maximum activity restoration was reached when 1 mol of subunit IV/mol of three-subunit core complex was used. The reconstituted cytochrome b-c1 complex is similar to the wild-type complex in molecular size, apparent Km for Q2H2, and inhibitor sensitivity, indicating that recombinant subunit IV is properly assembled into the active cytochrome b-c1 complex. A tryptophan residue in subunit IV was found to be involved in the interaction with the three-subunit core complex.
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PMID:Functional expression of subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex and reconstitution of recombinant protein with three-subunit core complex. 856 59

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 present study was designed to elucidate the inhibitory potential of Momordica charantia (Bitter Gourd) peel, pulp, seed and whole fruit extract on mouse skin papillomagensis with the modulatory influence of biotransformation system enzymes. Topical application of Momordica whole fruit extract (100 microl/animal per day) during the peri-initiation stage (1 week before and 2 weeks after initiation) by 7,12-dimethylbenz[a]anthracene (DMBA) and/or during the tumor promotion stage reduced the (i) tumor burden to 4.26, 3.72 and 3.11 (positive control value: 5.42); (ii) cumulative number of papillomas to 81, 67 and 53 (positive control value: 103); and (iii) percent incidence of mice bearing papillomas to 100, 94 and 94, respectively (positive control value: 100). In a comparison of the anticarcinogenic efficacy of Momordica peel, pulp, seed and whole fruit extract (100 microl/animal per day), after topical treatment during the peri-initiation and during the tumor promotion stage, revealed the modulation of the (i) tumor burden (tumors/mouse) to 3.06, 3.61, 3.17 and 3.11; (ii) cumulative number of papillomas to 49, 65, 54 and 53; and (iii) percent incidence of mice bearing papillomas to 84, 100, 94 and 94, respectively. Significant elevation in the sulfhydryl (-SH) level was observed in the liver and skin tissues by the topical treatment of Momordica peel, pulp, seed and whole fruit extract. Elevation in the hepatic levels of cytosolic glutathione S-transferase (GST) and microsomal cytochrome b, was also observed by the topical treatment of Momordica peel, seed and whole fruit extract. The results suggest the maximum chemopreventive potential is in the Momordica peel. Equivocal efficacy is in the Momordica seed and whole fruit extract. Biotransformation system enzymes may be the cause of this reduced papillomagenesis.
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PMID:Momordica charantia (Bitter Gourd) peel, pulp, seed and whole fruit extract inhibits mouse skin papillomagenesis. 954 97

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. This subunit is involved in Q binding and the structural integrity of the complex. When the cytochrome bc1 complex is photoaffinity labeled with [3H]azido-Q derivative, radioactivity is found in subunits IV and I (cytochrome b), indicating that these two subunits are responsible for Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R. sphaeroides chromosome, the resulting strain (RSdeltaIV) requires a period of adaptation before the start of photosynthetic growth. The cytochrome bc1 complex in adapted RSdeltaIV chromatophores is labile to detergent treatment (60-75% inactivation), and shows a four-fold increase in the Km for Q2H2. The first two changes indicate a structural role of subunit IV; the third change supports its Q-binding function. Tryptophan-79 is important for structural and Q-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GST fusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunit IV is functionally active as it can restore the bc1 complex activity from the three-subunit core complex to the same level as that of wild-type or complement complex. Three regions in the subunit IV sequence, residues 86-109, 77-85, and 41-55, are essential for interaction with the core complex because deleting one of these regions yields a subunit completely or partially unable to restore cytochrome bc1 from the core complex.
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PMID:The role of the supernumerary subunit of Rhodobacter sphaeroides cytochrome bc1 complex. 1059 31

Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively.
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PMID:Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides. Localization of regions essential for interaction with the three-subunit core complex. 1074 84

We examined three primary variables in the preparation of human liver microsomes. In three experiments, each using three livers, we manipulated 1) the force of the first centrifugation (9,000, 10,500, or 12,000g); 2) the presence of sucrose in the homogenization buffer; and 3) the number of homogenizing strokes (6, 8, or 10). Sedimentation plots for the marker enzymes succinate dehydrogenase, NADPH cytochrome P450 reductase (reductase), and glutathione S-transferase in the resulting premicrosomal, microsomal, and cytosolic fractions suggest that enhanced purity of microsomes can be obtained by reducing force of centrifugation, including sucrose, and increasing the number of homogenization strokes. Each microsomal fraction was also assayed for protein content, cytochrome P450, NADH cytochrome b(5) reductase, cytochrome b(5), absorbance at 420, p-nitrophenol hydroxylation, tolbutamide hydroxylation, dextromethorphan N- and O-demethylation, glucuronidation of morphine and 1-naphthol, and ester cleavage of p-nitrophenolacetate. These microsomal indicators were ranked and tested for statistical differences. The use of 9000g statistically increased optimal recovery (per gram of liver) and specific activity (per milligram of protein). The inclusion of sucrose improved activity specific to reductase activity. Ten homogenization strokes improved activity specific to reductase activity. Substrate-dependent activities of dextromethorphan O-demethylation to dextrorphan and the N-demethylation of l-alpha-acetylmethadol (LAAM) to norLAAM and dinorLAAM were compared in microsomes prepared with or without sucrose and microsomes prepared using 9,000 or 12,000g force, respectively. No significant differences were found in the concentration-dependent activities. Variation of the methods used to prepare human liver microsomes can significantly affect the recovery and specific activity of microsomal components; however, they do not appear to affect enzyme kinetics.
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PMID:Variables in human liver microsome preparation: impact on the kinetics of l-alpha-acetylmethadol (LAAM) n-demethylation and dextromethorphan O-demethylation. 1118 2

The bioactivation of N-nitrosamines and polycyclic aromatic hydrocarbons (PAHs) is mediated primarily by the mixed-function oxidase system, which includes dimethylnitrosamine N-demethylase I, arylhydrocarbon [benzo(a)pyerne] hydroxylase, cytochrome P450, cytochrome b(5), and ethoxycoumarin deethylase. Most of carcinogens and xenobiotics are conjugated and detoxified by phase II drug-metabolizing enzymes such as glutathione S-transferase. The present study showed the influence of Schistosoma haematobium on the activity of the above-mentioned enzymes in 13 schistosome-infected human bladder tissues compared with those of 15 schistosome-free samples. The contents of cytochrome P450 and cytochrome b(5) were increased in the bladder tissues by 48 and 69%, respectively. Moreover, the activities of dimethylnitrosamine N-demethylase I and arylhydrocarbon hydroxylase, ethoxycoumarin O-deethylase, ethoxyresourfin O-deethylase, and pentoxyresorufin O-pentoxyresorufin were increased by 75, 159, 49, 63 and 44%, respectively. The signal intensity for cytochrome P450 2E1 was greatly increased over the control. Also, the activity of glutathione S-transferase was increased by 89%. On the other hand, the activity of glutathione reductase and the level of reduced glutathione were decreased by 40 and 57%, respectively. Interestingly, the level of free radical, thiobarbituric acid reactive substance, was increased in the schistosome-infected human bladder tissues by 125%. The present study clearly demonstrated that S. haematobium changes the activity of carcinogen-metabolizing enzymes. We conclude that S. haematobium could enhance the carcinogenicity of polycyclic aromatic hydrocarbons (e.g. benzo(a)pyrene) and N-nitrosamines (e.g. dimethylnitrosamine) through induction of their corresponding bioactivating enzymes in human bladder tissues.
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PMID:Changes in the expression of cytochrome P450 2E1 and the activity of carcinogen-metabolizing enzymes in Schistosoma haematobium-infected human bladder tissues. 1131 57

The effects of two doses (50 and 100 mg/kg body wt/day for 14 days) of an 80% hydroalcohol extract of Andrographis paniculata and butylated hydroxyanisole (BHA) were examined on drug metabolizing enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase (LDH) and lipid peroxidation in the liver of Swiss albino mice (6-8 weeks old). The effect of the extract and BHA were also examined on lung, kidney and forestomach for the activities of glutathione S-transferase (GST), DT-diaphorase (DTD), superoxide dismutase (SOD) and catalase. A significant increase in the levels of acid soluble sulphydryl (-SH) content, cytochrome P450, cytochrome P450 reductase, cytochrome b5 reductase, GST, DTD and SOD were observed at both dose levels of extract treatment while catalase, glutathione peroxidase and glutathione reductase (GR) showed significant increases only at the higher dose in the liver. Both Andrographis treated groups showed a significant decrease in activity of LDH and malondialdehyde (MDA) formation. BHA treated mice showed a significant increase in the levels of cytochrome b(5), GST, DTD, -SH content, GR and catalase in liver; while LDH and MDA levels were reduced significantly compared with their control values. In the lung, SOD, catalase and DTD, in the kidney catalase, DTD and GST, and in the forestomach SOD and DTD showed a significant increase at both dose levels of treatment. In BHA treated mice GST, DTD and catalase were significantly induced in the lung and along with these enzymes SOD was also induced in the kidney. In the case of the forestomach of BHA treated mice GST, DTD and SOD were enhanced significantly. These findings indicate the chemopreventive potential of Andrographis paniculata against chemotoxicity including carcinogenicity.
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PMID:Modulatory influence of Andrographis paniculata on mouse hepatic and extrahepatic carcinogen metabolizing enzymes and antioxidant status. 1150 28

The intake of heat-damaged proteins from food causes various effects, like the loss of essential amino acids and a reduced protein digestibility. There is also an influence on gastrointestinal microorganisms and different digestion enzymes. Until now, very little is known about the influence of heat-treated proteins on the enzymes of the biotransformation system. In the present study, the influence of protein-bound L-lysino-D,L-alanine, N(epsilon)-fructoselysine, and N(epsilon)-carboxymethyllysine (CML) on selected enzymes of the biotransformation in liver, kidney, and intestinal mucosa of male Wistar rats was examined. The contents of cytochrome P-450 and cytochrome b(5) and the activity of NADPH-cytochrome c reductase served as indicators of phase I biotransformation. The influence on phase II biotransformation was shown by the content of glutathione and the glutathione S-transferase activity. The results showed that treatment with heat-damaged proteins mainly affected phase II biotransformation enzymes with CML, yielding the strongest effect. The activity of glutathione S-transferase in the kidney was 86% higher in animals treated with diets containing 4,930 mg.kg(-1) protein-bound CML than in animals of the control group which received a diet without any detectable CML. In addition, a higher level of glutathione was found in the kidneys of animals fed on diets containing CML. The glutathione S-transferase activity was 64% higher in the intestinal mucosa of animals fed on protein-bound N(epsilon)-fructoselysine (2,700 mg.kg(-1)). The glutathione S-transferase activity was higher (p >0.05) in the intestinal mucosa of animals fed on protein-bound L-lysino-D,L-alanine (2,582 and 12,474 mg.kg(-1)). In conclusion, ingestion of heat-treated proteins led to an activation of the enzymes of phase II biotransformation. Whether or not the released pure compounds or the degradation products of the test proteins are responsible for the altered enzyme activities remains to be evaluated.
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PMID:Effect of heat-treated proteins on selected parameters of the biotransformation system in the rat. 1191 10

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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