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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)

Purification and characterization of microsomal glutathione S-transferase produced by Aspergillus ochraceus TS are reported. The isozymes are located in microsomes and were active against 1-chloro-2,4-dinitrobenzene, ethacrynic acid, 1,2-dichloro-4-nitrobenzene, trans-4-phenyl-3-buten-2-one,p-nitrobenzyl chloride and bromosulphophthalein. They were inhibited by N-ethylmaleimide and bromosulphophthalein. The GST isozymes produced by Aspergillus ochraceus TS are indistinguishable in respect of their molecular mass both in native and denatured state. The subunit of the purified protein had an apparent M(r) of 11 kDa while molecular mass of the native protein is around 56 kDa. The substrate specificity and pI values of the isozymes were different. The GSTs produced by Aspergillus ochraceus TS fairly share functional properties with mammalian cytosolic isozymes.
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PMID:Characterization of a novel microsomal glutathione S-transferase produced by Aspergillus ochraceus TS. 148 53

The role of the hydroxyl group of tyrosine 6 in the catalytic mechanism of isoenzyme 3-3 of rat glutathione S-transferase has been examined by x-ray crystallography and site-specific replacement of the residue with phenylalanine and evaluation of the catalytic properties of the mutant enzyme. This particuar tyrosine residue is conserved in the sequences of all of the cytosolic enzymes and is found, in crystal structures of both isoenzyme 3-3 from the mu-gene class and an isoenzyme from the pi-gene class, to be proximal to the sulfur of glutathione (GSH) or glutathione sulfonate bound at the active site. The 2.2-A structure of the binary complex of isoenzyme 3-3 and GSH indicates that the hydroxyl group of Tyr6 is located 3.2-3.5 A from the sulfur of GSH, well within hydrogen bonding distance. Removal of the hydroxyl group of Tyr6 has essentially no effect on the dissociation constant (22 +/- 3 microM) for GSH. Nevertheless the Y6F mutant exhibits a turnover number which is only about 1% that of the native enzyme when assayed at pH 6.5 with either 1-chloro-2,4-dinitrobenzene (CDNB) or 4-phenyl-3-buten-2-one. UV difference spectra of the binary enzyme-GSH complexes suggest that the predominant ionization state of GSH in the active site of the Y6F mutant is the neutral thiol (e.g. EY6F.GSH) which is in contrast to the native enzyme in which the thiol is substantially deprotonated (e.g. E.GS-). Spectrophotometric titration suggests that the pKa of the thiol is 6.9 +/- 0.3 in the E.GSH complex and greater than or equal to 8 in the EY6F.GSH binary complex. In addition, the pH dependence of kcat/KmCDNB reveals that the reactions catalyzed by the native enzyme and the Y6F mutant are dependent on a single ionization in the E.GSH and EY6F.GSH complexes with pKa = 6.2 +/- 0.1 and 7.8 +/- 0.3, respectively. The results suggest that the hydrogen bond between Tyr6 and the enzyme-bound nucleophile helps to lower the pKa of GSH in the binary enzyme-substrate complex.
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PMID:Contribution of tyrosine 6 to the catalytic mechanism of isoenzyme 3-3 of glutathione S-transferase. 153 22

A soluble glutathione S-transferase isoenzyme, designated SmGST-4 was purified to apparent homogeneity in a single step from the cytosol of adult Schistosoma mansoni by selective elution of the enzyme from a glutathione-agarose affinity column using glutathione disulfide. SmGST-4, which comprised about 5% of the bound glutathione S-transferase activity, could be distinguished from the previously characterized glutathione S-transferase isoenzyme family (SmGST-1/2/3), by its unique chromatographic behavior, lower subunit M(r) (26,000), differences in substrate specificity and inhibitor sensitivity, and a lack of reactivity with antiserum to SmGST-3. The purified isoenzyme catalyzed the conjugation of several model xenobiotics including 1-chloro-2,4-dinitrobenzene, ethacrynic acid, and trans-4-phenyl-3-buten-2-one. Like the SmGST-1/2/3 isoenzyme family, SmGST-4 failed to catalyze the conjugation of a model epoxide substrate, 1,2-epoxy-3-(p-nitrophenoxy)propane. Because glutathione S-transferases from other organisms play a role in protecting cells against the toxic products of lipid peroxidation, SmGST-4 and the members of the SmGST-1/2/3 isoenzyme family were tested for their capacity to reduce cumene hydroperoxide and to catalyze the conjugation of 4-hydroxyalk-2-enals. Although all four isoenzymes catalyzed both reactions, the specific activity of SmGST-1, SmGST-2, and SmGST-3 toward cumene hydroperoxide was at least 10-fold greater than that of SmGST-4. In contrast, the latter more effectively conjugated a homologous series of 4-hydroxyalk-2-enal isomers.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Schistosoma mansoni: single-step purification and characterization of glutathione S-transferase isoenzyme 4. 163 64

1. The major glutathione S-transferase (GST) from the common squid Loligo vulgaris has been purified and shown to be a homodimer of subunit molecular mass 24,000 and pI 6.8. 2. It has high activity towards 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 4-hydroxynon-2-enal and linoleic acid hydroperoxide, low activity with 1,2-dichloro-4-nitrobenzene and no activity with ethacrynic acid, trans-4-phenyl-3-buten-2-one and 1,2-epoxy-3-(p-nitrophenoxy)propane. 3. The L. vulgaris GST did not cross-react with any of the available polyclonal antibodies raised against mammalian GSTs. 4. Forty amino acids of its N-terminal sequence have been determined. 5. Its activities and primary structure are compared with related proteins from other species.
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PMID:The isolation and characterization of the major glutathione S-transferase from the squid Loligo vulgaris. 186 85

To test the proposition that a histidine residue is essential in the catalytic mechanism of glutathione S-transferase, rat liver isoenzyme 3-3 specifically labeled with [ring-2-13C]histidine was prepared. The 13C NMR spectrum of the labeled enzyme revealed four resonances corresponding to the 4 histidine residues in the mu gene class type 3 subunit. Titration of the four resonances in the range of pH 4-9 both in the presence and absence of glutathione gave pK alpha values of much less than 4, 5.2, 7.1, and 7.8 for the four side chains that were identified by site-specific mutagenesis as His14, His83, His84, and His167, respectively. The magnetic resonance properties and titration behavior of His14 suggest that this residue is buried in a hydrophobic environment. Conservative replacement of each histidine with asparagine results in mutant enzymes that have catalytic properties very close to the native protein as assessed with three different substrates, 1-chloro-2,4-dinitrobenzene, 4-phenyl-3-buten-2-one, and phenanthrene 9,10-oxide. The results indicate clearly that none of the histidine residues of isoenzyme 3-3 is essential for stabilization of the bound glutathione thiolate or for any other aspect of catalysis.
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PMID:Are the histidine residues of glutathione S-transferase important in catalysis? An assessment by 13C NMR spectroscopy and site-specific mutagenesis. 191 58

The effect of aflatoxin B1 (AFB1) on the glutathione S-transferase activity (GST) and on non-protein thiol levels of different tissues was studied in adult male Wistar rats. Animals received a single dose of the toxin (100 or 500 micrograms/kg body wt., p.o.), and were studied 6 or 24 h after administration. GST was determined in liver, renal cortex, duodenum, jejunum-ileum and distal ileum, using 3 substrates: 1-chloro-2,4-dinitrobenzene (CDNB), trans-4-phenyl-3-buten-2-one (PBO) and 1,2-epoxyethylbenzene (STOX). The non-protein thiol content of all tissues tested increased with the lowest dose at 6 h, returning to normal values at 24 h, while the higher dose produced a significant decrease in reduced thiol levels at 6 h, returning to normal values at 24 h. AFB1 administration induced, independently of dose and tissue, total GST (CDNB) and epoxide-transferase activity (STOX) while A--C-type transferases (PBO) were inhibited. Almost all activities returned to normal values at 24 h. In cases of enzyme induction there was in general an increase in Vmax and a decrease in apparent Km. The opposite was seen in cases of inhibition. In conclusion, the results provide evidence that extrahepatic GST could be important in the overall process of detoxification of AFB1. The behavior seen in hepatic and extrahepatic tissues revealed the functions of catalysis (B-type transferases) and covalent bond formation, as well as inactivation by probable AFB1 metabolites (A--C-type transferases).
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PMID:Effect of aflatoxin B1 treatment in vivo on the in vitro activity of hepatic and extrahepatic glutathione S-transferase. 210 96

1. The influence of the antifungal agent clotrimazole on cytosolic glutathione S-transferase activities was studied in male Wistar rats. 2. Animals received clotrimazole by gastric lavage for 3 days (75 mg/kg per day). Hepatic glutathione S-transferase activity was determined with five different substrates: 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), p-nitro-benzyl chloride (PNBC), ethacrynic acid (EA) and trans-4-phenyl-3-buten-2-one (TPBO). 3. The largest increases in glutathione S-transferase activity were found with CDNB, DCNB and PNBC (+61%, +50% and +50%, respectively, when expressed per mg of cytosolic protein). Enzyme activity toward EA was induced to a lower extent (+33%). Changes in the formation of the conjugate of TPBO were relatively small (+22%). 4. These data indicate a differential induction of glutathione S-transferase isoenzymes and suggest that clotrimazole is a phenobarbital-type inducer of enzyme activity.
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PMID:Changes in hepatic cytosolic glutathione S-transferase enzymes induced by clotrimazole treatment in rats. 261 64

Hepatic glutathione S-transferase (GST) activities towards 1-chloro-3,4-dinitrobenzene (DNCB), 3,4-dichloronitrobenzene (DCNB), sulfobromophthalein (BSP), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), trans-4-phenyl-3-buten-2-one (TPBO) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) were determined in mice, rats, rabbits and guinea-pigs during ageing and after pretreatment with enzyme inducers. Variations were observed in the developmental patterns and in the phenobarbital-, benzo(a)pyrene-, pregnenolone-16 alpha-carbonitrile-, butylated hydroxyanisole-, trans-stilbene oxide-inducibility of hepatic GST activities in the same species towards different substrates. For example, in rats GST activities for EA, DCNB and TPBO increased respectively, 2.3-, 4.8- and 25-fold during age-development, and after treatment with TSO 1.2-, 3.6- and 1.3-fold. Species differences were found in the maturation and in the inducibility of GST activities. For instance, GST activity toward EA at birth is mature in guinea pigs but not in the other species; phenobarbital treatment increased GST activities in mice and rats but not in rabbits and guinea-pigs; treatment with trans-stilbene oxide enhanced GST activity for TPBO 4.5-fold in mice but not at all in rats. It is concluded that hepatic glutathione conjugation exhibits functional heterogeneity which may be due to species dependent variations in the responsiveness of GST isoenzymes to endogenous and exogenous influences.
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PMID:Age-development and inducibility of hepatic glutathione S-transferase activities in mice, rats, rabbits and guinea-pigs. 285 53

Glutathione transferase (GST) (EC 2.5.1.18) was purified from a cell extract of Issatchenkia orientalis, and two GST isoenzymes were isolated. They had molecular weights of 37,500 and 40,000 and were designated GST Y-1 and GST Y-2, respectively. GST Y-1 and GST Y-2 gave single bands with molecular weights of 22,000 and 23,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GST Y-1 and GST Y-2 were immunologically distinguished from each other. GST Y-1 showed specific activity 10.4-times and 6.0-times higher when 1-chloro-2,4-dinitrobenzene and o-dinitrobenzene were used as substrates, respectively, than GST Y-2. GST activity was not detected for either isoenzyme when other substrates such as bromosulfophthalein and trans-4-phenyl-3-buten-2-one were used. GST Y-1 and GST Y-2 had Km values of 0.51 and 0.75 mM for glutathione, respectively, and of 0.16 and 4.01 mM for 1-chloro-2,4-dinitrobenzene. GST Y-1 was significantly inhibited by Cibacron blue 3G-A, and GST Y-2 was significantly inhibited by bromosulfophthalein.
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PMID:Purification and properties of glutathione transferase from Issatchenkia orientalis. 291 66

A major isozyme of rat heart glutathione transferase was purified to homogeneity by Sephadex G-200 gel filtration, ammonium sulfate precipitation, CM-cellulose chromatography and affinity chromatography on S-hexylglutathione-linked Sepharose 6B. The purified isozyme was a dimer with an apparent relative molecular mass of 50 000 composed of two Yb-size subunits (Mr = 26 500). The isozyme is immunologically related to rat liver glutathione transferase X and 3-3, especially closely to transferase X, and no immunological cross-reactivity with subunits 1 and 2 of hepatic glutathione transferases was observed. The isoelectric point (pI = 6.9) of the isozyme was identical with and the substrate specificity was very similar to transferase X. Thus, the cardiac near-neutral isozyme is considered to be identical to glutathione transferase X recognized in rat liver. The amount of this near-neutral isozyme estimated to be present in heart tissue is 70 micrograms/g. The isozyme has relatively high activities towards alpha, beta-unsaturated carbonyl compounds such as trans-4-phenyl-3-buten-2-one and trans-4-hydroxynon-2-enal. The latter is a cytotoxic product resulting from lipid peroxidation of polyunsaturated fatty acids, and the cardiac isozyme may play a physiologically significant role with glutathione conjugation of this compound. In addition to the near-neutral isozyme, acidic forms with isoelectric points of 4.9, 5.2 and 5.5 were partially purified; some of them are considered to consist of subunits immunologically related to transferase X.
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PMID:The major isozyme of rat cardiac glutathione transferases. Its correspondence to hepatic transferase X. 308 Mar 15


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