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 roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis. Replacement of tyrosine 9 with phenylalanine (Y9F), threonine (Y9T), histidine (Y9H), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes. Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat. The estimated pK alpha values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK alpha value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase. These results indicate that tyrosine 9 is directly responsible for the lowering of the pKa of the sulfhydryl group of glutathione, presumably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine. To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine. All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive. Kinetic studies with four mutant enzymes (D93E, D93N, D101E, and D101N) indicate that only D101N exhibited a 5-fold increase in Km toward glutathione. Also, the binding of this mutant to the affinity column was greatly reduced. These results demonstrate that aspartic acid 101 plays an important role in glutathione interaction to YaYa. The role of aspartic acid 157 in catalysis remains to be determined.
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PMID:Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis. 140 Mar 2

Three mouse monoclonal antibodies (Moabs) have been obtained with specificity for the 7B2 protein, a proposed member of the granin family of neuroendocrine proteins. Bacterially produced hybrid proteins of 7B2 were used as immunogens. The Moabs were designated MON-100, MON-101, and MON-102. Furthermore, we report the construction of 35 deletion mutants of the glutathione S-transferase-7B2 (GST-7B2) fusion-gene using recombinant DNA technology. The hybrid proteins encoded by eleven of these mutants were used in epitope mapping experiments and the results of these studies strongly suggested that recognition of 7B2 by all three Moabs involved the same 16 amino acid region of 7B2 (from amino acid residue 128-135). This was further substantiated by the observation that MON-101 and MON-102 specifically recognized a conjugate between bovine serum albumin and the synthetic peptide Phe-Glu-Pro-Glu-His-Asp-Tyr-Pro-Gly-Leu-Gly-Lys based upon the deduced amino acid sequence of the predicted epitope region in 7B2. In an approach to generate a series of 7B2-specific Moabs targeted against another epitope region in the 7B2 protein, the hybrid protein encoded by deletion mutant pPV32 was used as the immunogen. This protein lacked the epitope region recognized by the first series of Moabs. A second series of three Moabs, designated MON-142, MON-143, and MON-144, was obtained and, in all three cases, the region of 7B2 from amino acid residue 64-94 appeared to be involved in specific recognition by the Moabs. The whole panel of six anti-7B2 antibodies appeared to be useful in immunoprecipitation and Western blot analysis of the 7B2 protein and specifically stained neuroendocrine cells in immunohistochemical experiments. Using a double determinant sandwich enzyme immunoassay, 7B2 protein levels in rat pituitary were determined as 20 ng/mg tissue.
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PMID:Application of recombinant DNA technology in epitope mapping and targeting. Development and characterization of a panel of monoclonal antibodies against the 7B2 neuroendocrine protein. 171 98

S-(4-Bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, has been synthesized and characterized by UV spectroscopy and thin-layer chromatography, as well as by bromide and primary amine analysis. Incubation of S-BDB-G (200 microM) with the 4-4 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. The kobs exhibits a nonlinear dependence on S-BDB-G concentration from 50 to 1000 microM, with a kmax of 0.078 min-1 and K1 = 66 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, completely protects against inactivation by S-BDB-G. About 1.3 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated concomitant with 100% inactivation, whereas only 0.48 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when activity is fully retained. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with NaBH4, carboxymethylated, and digested with trypsin. The tryptic digest was fractionated by reverse-phase high-performance liquid chromatography. Two radioactive peptides were identified: Lys82-His-Asn-Leu-X-Gly-Glu-Thr-Glu-Glu-Glu-Arg93, in which X is modified Cys86, and Leu109-Gln-Leu-Ala-Met-CmCys-Y-Ser-Pro-Asp-Phe-Glu-Arg121 , in which Y is modified Tyr115. Only the Lys82-Arg93 peptide was modified in the presence of S-hexylglutathione when the enzyme retained full activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:S-(4-Bromo-2,3-dioxobutyl)glutathione: a new affinity label for the 4-4 isoenzyme of rat liver glutathione S-transferase. 195 60

A plasmid vector was constructed that encodes the expression in Escherichia coli of a truncated form of GST2, a human Alpha-class glutathione transferase. The truncated enzyme, GST2del210, has 12 residues deleted from the C-terminus and has the last two residues of the new C-terminal mutated from aspartic acid and glutamic acid to histidine and glycine respectively. GST2del210 has substantially diminished specific activity with either 1-chloro-2,4-dinitrobenzene or cumene hydroperoxide as substrate. The affinity of the truncated enzyme for a GSH-agarose matrix was also diminished, but sufficient interaction remained to enable affinity purification. Inhibition of GST2del210 by bromosulphophthalein was not altered. In contrast, this truncated form was not inhibited by S-pentylglutathione, a competitive inhibitor of the wild-type GST2 isoenzyme. The results show that the C-terminal segment of the Alpha-class glutathione transferases may form a component of the hydrophobic substrate-binding site. In contrast, this region appears not to be directly involved in GSH binding and is not absolutely essential for catalytic activity.
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PMID:The contribution of the C-terminal sequence to the catalytic activity of GST2, a human alpha-class glutathione transferase. 201 73

Analogues of GSH in which either the gamma-glutamyl or the glycyl moiety is modified were synthesized and tested as both substrates for and inhibitors of glutathione S-transferases (GSTs) 7-7 and 8-8. Acceptor substrates for GST 7-7 were 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) and for GST 8-8 CDNB, ETA and 4-hydroxynon-trans-2-enal (HNE). The relative ability of each combination of enzyme and GSH analogue to catalyse the conjugation of all acceptor substrates was similar with the exception of the combination of GST 7-7 and gamma-L-Glu-L-Cys-L-Asp, which used CDNB but not ETA as acceptor substrate. In general, GST 7-7 was better than GST 8-8 in utilizing these analogues as substrates, and glycyl analogues were better than gamma-glutamyl analogues as both substrates and inhibitors. These results are compared with those obtained earlier with GSH analogues and GST isoenzymes 1-1, 2-2, 3-3 and 4-4 [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] and the implications with respect to the nature of their active sites are discussed.
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PMID:Interaction of rat glutathione S-transferases 7-7 and 8-8 with gamma-glutamyl- or glycyl-modified glutathione analogues. 261 14

The aim of this study was to optimize the pH in the liver microsomal assay (LMA) in processing short-term mutagenicity tests. pH optimization would increase the sensitivity (i.e. decrease the presence of false negatives) and increase the specificity (decrease false positives). Such optimization is a function of the relative activities and stabilities of the liver microsomal cytochrome P-450- and FAD-containing monooxygenase-dependent biotransformation enzymes present in the incubation mixtures used. The enzyme activities ethoxyresorufin O-deethylase, dinemorphan N-demethylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase and thiobenzamide S-oxidase (as phase-I markers), were examined in terms of their exact incubation conditions for the LMA during a period of pre-incubation (1 h) over the pH range 6-9. As a comparison, the behaviours of glutathione S-transferase and epoxide hydrase activities (as phase-II markers) were also studied. Lipid peroxidation was also determined. Experiments were carried out on S9 fractions derived from Na-phenobarbital and beta-naphthoflavone induced mouse liver. The maximal value of the mean specific activity (Asp) was found at pH 7.8 for the phase-I drug metabolizing enzymes considered (30-45% increase). On the contrary, a lower increase of Asp for epoxide hydrase and glutathione S-transferase (approximately 14%), was observed between pH 7.4 and 7.8. Lipid peroxidation was not changed appreciably by varying pH. In vitro DNA binding of the well-known pre-mutagenic agent [14C]dimethylnitrosamine ([14C]DMNA), mediated by mouse hepatic microsomal enzymes, showed a significant increase of specific activity at pH 7.8 (2.8-fold) compared to the usual pH (7.4) employed. Additional support for the above results has come from mutagenesis experiments using DMNA on the diploid D7 strain of Saccharomyces cerevisiae as a biological test system. In fact, a significant enhancement of mitotic gene conversion (1.7-fold), mitotic cross-over (2.6-fold) and reverse point mutation (2.3-fold) frequencies were observed at pH 7.8 compared to pH 7.4. These data indicate that pH 7.8 provides a more favourable condition for in vitro mutagenesis tests resulting in greater rates of biotransformation (as measured by an increased Asp phase-I/Asp phase-II ratio), DNA binding and genotoxic response.
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PMID:Improvement of short-term tests for mutagenicity: on the optimal pH for the liver microsomal assay. 319 71

The procedure developed for purification of the N-ethylmaleimide-activated microsomal glutathione transferase was applied successfully to isolation of this same enzyme in unactivated form. The microsomal glutathione transferases, the unactivated and activated forms, were shown to be identical in terms of molecular weight, immunochemical properties, and amino acid composition. In addition the microsomal glutathione transferase purified in unactivated form could be activated 15-fold with N-ethylmaleimide to give the same specific activity with 1-chloro-2,4-dinitrobenzene as that observed for the enzyme isolated in activated form. This activation involved the binding of one molecule N-ethylmaleimide to the single cysteine residue present in each polypeptide chain of the enzyme, as shown by amino acid analysis, determination of sulfhydryl groups by 2,2'-dithiopyridyl and binding of radioactive N-ethylmaleimide. Except for the presence of only a single cysteine residue and the total absence of tryptophan, the amino acid composition of the microsomal glutathione transferase is not remarkable. The contents of aspartic acid/asparagine + glutamic acid/glutamine, of basic amino acids, and of hydrophobic amino acids are 15%, 12% and 54% respectively. The isoelectric point of the enzyme is 10.1. Microsomal glutathione transferase conjugates a wide range of substrates with glutathione and also demonstrates glutathione peroxidase activity with cumene hydroperoxide, suggesting that it may be involved in preventing lipid peroxidation. Of the nine substrates identified here, the enzymatic activity towards only two, 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide, could be increased by treatment with N-ethylmaleimide. This treatment results in increases in both the apparent Km values and V values for 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Thus, although clearly distinct from the cytosolic glutathione transferases, the microsomal enzyme shares certain properties with these soluble enzymes, including a relative abundance, a high isoelectric point and a broad substrate specificity. The exact role of the microsomal glutathione transferase in drug metabolism, as well as other possible functions, remains to be established.
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PMID:Microsomal glutathione transferase. Purification in unactivated form and further characterization of the activation process, substrate specificity and amino acid composition. 688 49

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.
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PMID:Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV. 753 46

The ligand-binding domain of the low-density lipoprotein receptor comprises seven cysteine-rich repeats, which have been highly conserved through evolution. This domain mediates interactions of the receptor with two lipoprotein apoproteins, apo E and apo B-100, putatively through a calcium-dependent association of the ligands with a cluster of acidic residues on the receptor. The second repeat (rLB2) of the receptor binding domain has been expressed as a thrombin-cleavable GST fusion protein, cleaved, and purified. On oxidation the protein refolded to give a single peak on reverse-phase HPLC. The aqueous solution structure of rLB2 has been determined using two-dimensional 1H NMR spectroscopy. In contrast to the amino-terminal repeat, rLB1, rLB2 has a very flexible structure in water. However, the conformation of rLB2 is markedly more ordered in the presence of a 4-fold molar excess of calcium chloride; the proton resonance dispersion and the number of NOESY cross-peaks are greatly enhanced. The three-dimensional structure of rLB2, obtained from the NMR data by molecular geometry and restrained molecular dynamics methods, parallels that of rLB1, with an amino-terminal hairpin structure followed by a succession of turns. However, there are clear differences in the backbone topology and structural flexibility. As for rLB1, the acidic residues are clustered on one face of the module. The side chain of Asp 37, which is part of a completely conserved SDE sequence thought to be involved in ligand binding, is buried, as is its counterpart (Asp 36) in rLB1. These results provide the first experimental support for the hypothesis that each of the repeats in the ligand-binding domain has a similar global fold but also highlight significant differences in structure and internal dynamics.
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PMID:Three-dimensional structure of the second cysteine-rich repeat from the human low-density lipoprotein receptor. 757 52

The SH2 domains of cytoplasmic signaling proteins bind to autophosphorylated growth factor receptors by direct recognition of specific phosphotyrosine-containing sites. To identify the phosphotyrosine involved in association of phospholipase C (PLC)-gamma 1 with the beta platelet-derived growth factor receptor (PDGFR), and to investigate which contiguous residues confer specificity for PLC-gamma 1, phosphotyrosine-containing glutathione S-transferase (GST) fusion proteins possessing different regions of the beta-PDGFR were incubated with lysates of Rat-2 cells that overexpress PLC-gamma 1. The phosphorylated C-terminal tail of the PDGFR bound PLC-gamma 1, but did not associate with phosphatidylinositol (PI) 3'-kinase or GTPase-activating protein (GAP). High-affinity binding of PLC-gamma 1 was dependent on phosphorylation of Tyr-1021. Creation of a new phosphorylation site by replacing Asp-1018 with tyrosine did not restore binding of PLC-gamma 1 in the absence of Tyr-1021, indicating that the location of the phosphorylated tyrosine is important for PLC-gamma 1 binding. Substitution of the proline at the +3 position relative to Tyr-1021 with methionine (Y1021IIP-->Y1021IIM) in the phosphorylated PDGFR tail did not alter PLC-gamma 1 association, but conferred binding activity towards PI 3'-kinase, indicating that this residue is critical in discriminating between PLC-gamma 1 and PI 3'-kinase. Progressive conversion of the three residues C-terminal to Tyr-1021 to the consensus for PI 3'-kinase binding (YMDM) allowed PI 3'-kinase association, but did not block PLC-gamma 1 binding, suggesting that additional residues other than the three residues immediately following the phosphotyrosine may contribute to the association of PLC-gamma 1 with the PDGFR. These results indicate that phosphorylation at Tyr-1021 in the tail of the PDGFR creates a specific binding site for PLC-gamma 1. Proline at the +3 position relative to Tyr-1021 is crucial in conferring specificity for binding to PLC-gamma 1.
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PMID:Identification of residues in the beta platelet-derived growth factor receptor that confer specificity for binding to phospholipase C-gamma 1. 768 24


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