title: Protein S-thiolation and regulation of microsomal glutathione transferase activity by the glutathione redox couple.
abstract: Microsomal glutathione transferase (GSTm) is activated up to fivefold by incubation with glutathione disulfide (GSSG). The process is reversed by the addition of an NADPH-regenerating system consisting of glutathione reductase and glucose 6-phosphate/glucose-6-phosphate dehydrogenase. By treating the microsomes at different GSH/GSSG ratios a Kox value of 0.047 is found, i.e., 21 times more GSSG than GSH is necessary to produce half-maximal activation. The Kox is independent of the total glutathione concentration, indicating that S-thiolation by GSH rather than interchain or intrachain disulfide bridge formation is responsible for activation. Further evidence for S-thiolation of GSTm comes from SDS-PAGE under nonreducing conditions and Western blotting. Treating microsomes with GSSG or with GSH and t-butyl hydroperoxide or cumene hydroperoxide results in the appearance of a second GSTm band at approximately 17.7 kDa in addition to the native band at 17.3 kDa, the size difference approximately corresponding to the molecular mass of glutathione. The 17.7-kDa band is not seen in the presence of mercaptoethanol. Microsomal preparations from rat livers perfused with t-butyl hydroperoxide or cumene hydroperoxide also contain both GSTm forms. We suggest that under oxidative stress the microsomal GST in the cell can be activated through direct hydroperoxide-mediated S-thiolation of the enzyme with GSH, its reversal occurring via a thiol exchange-mediated dethiolation imposed by the intracellular glutathione redox state.
authors.affiliation: Institut für Physiologische Chemie I, Heinrich-Heine Universität Düsseldorf, Federal Republic of Germany.
authors.forename: A L
authors.initials: AL
authors.lastname: Dafré
authors.forename: H
authors.initials: H
authors.lastname: Sies
authors.forename: T
authors.initials: T
authors.lastname: Akerboom
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D019803:Glutathione Disulfide; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010477:Perfusion; D010545:Peroxides / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D010545:Peroxides; D011506:Proteins; D013438:Sulfhydryl Compounds; D005982:Glutathione Transferase; D005978:Glutathione; D019803:Glutathione Disulfide