Protein S-thiolation and regulation of microsomal glutathione transferase activity by the glutathione redox couple.
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.
Dafré
A L
AL
Institut für Physiologische Chemie I, Heinrich-Heine Universität Düsseldorf, Federal Republic of Germany.
Sies
H
H
Akerboom
T
T
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
D010545:Peroxides; D011506:Proteins; D013438:Sulfhydryl Compounds; D005982:Glutathione Transferase; D005978:Glutathione; D019803:Glutathione Disulfide