title: Formation of mixed disulfide of cystatin-beta in cultured macrophages treated with various oxidants.
abstract: Macrophage cell cultures were treated with menadione, zymosan, or phorbol myristate acetate (PMA), and changes in productions of superoxide anion and hydroperoxide, and in glutathione oxidation and S-thiolation of cystatin-beta (formation of a mixed disulfide of cystatin-beta and glutathione) were examined. All three compounds promoted production of superoxide anion and hydroperoxide, but only menadione caused extensive oxidation of glutathione. Menadione caused S-thiolation of cystatin-beta in a dose-dependent fashion, but the other two compounds did not. Removal of menadione promptly reduced the oxidation of glutathione and S-thiolation of cystatin-beta induced by menadione. Inhibition of catalase by aminotriazol caused slight increase in the GSSG content in both menadione- and zymosan-treated cells, but not in S-thiolation of cystatin-beta in zymosan-treated cells. None of the three compounds influenced appreciably the activity of glutathione peroxidase, glutathione reductase, or superoxide dismutase in cultured cells. These results indicate that S-thiolation of cystatin-beta occurs in cells in response to oxidative challenge by menadione but not by zymosan or by the tumor promoter PMA. Dethiolation of cystatin-beta by purified thiol transferase and protein disulfide isomerase in the presence of different concentrations of GSH was examined in vitro. Both enzymes catalyzed dethiolation of cystatin-beta at a much lower level of GSH than that required for the non-enzymatic reaction, suggesting the importance of enzymatic catalysis of S-thiolation and dethiolation of cystatin-beta in cells.
authors.affiliation: Department of Enzyme Chemistry, School of Medicine, University of Tokushima.
authors.forename: T
authors.initials: T
authors.lastname: Tsukahara
authors.forename: E
authors.initials: E
authors.lastname: Kominami
authors.forename: N
authors.initials: N
authors.lastname: Katunuma
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D055313:Cystatin B; D015891:Cystatins; D004220:Disulfides / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects*; D011480:Protease Inhibitors / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014812:Vitamin K / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
chemical_list: D015891:Cystatins; D004220:Disulfides; D011480:Protease Inhibitors; D011506:Proteins; D013438:Sulfhydryl Compounds; D013481:Superoxides; D014812:Vitamin K; D055313:Cystatin B; D015054:Zymosan; D006861:Hydrogen Peroxide; D005978:Glutathione; D013755:Tetradecanoylphorbol Acetate