PROTEIN S-THIOLATION IN HEPATOCYTES STIMULATED BY T-BUTYL HYDROPEROXIDE, MENADIONE, AND NEUTROPHILS

In order to examine potentially important S-thiolated proteins, S-35-l abeled hepatocytes were exposed to oxidative stress. A similar group o f S-thiolated proteins including carbonic anhydrase III was observed i n cells treated with t-butyl hydroperoxide, menadione, or stimulated n eutrophils. The radioactive thiols bound to hepatocyte proteins were i dentified by HPLC and more than 85% was glutathione. In menadione-trea ted hepatocytes, proteins were gradually S-thiolated over 30 min and 2 5% of the cellular glutathione pool became protein-bound. In t-butyl h ydroperoxide-treated cells, S-thiolation was more transient and 11% of the glutathione was protein-bound. Neutrophil-treated hepatocytes had nearly the same amount of protein S-thiolation (8% after 25 min). Two major proteins that were S-thiolated in untreated hepatocytes did not increase during any form of oxidative stress. In neutrophil-treated h epatocytes protein S-thiolation was not accompanied by either formatio n of glutathione disulfide or a measureable change in the total amount of glutathione. In both t-butyl hydroperoxide- and menadione-treated cells there was extensive formation of glutathione disulfide and in me nadione-treated cells a significant increase in the total hepatocyte g lutathione pool was observed. This result suggests that protein S-thio lation may occur by mechanisms that do not result from thiol/disulfide exchange between glutathione disulfide and protein sulfhydryls. It is suggested that a thiyl radical intermediate is important in neutrophi l-mediated protein S-thiolation. (C) 1994 Academic Press, Inc.