Formation of mixed disulfide of cystatin-beta in cultured macrophages treated with various oxidants.
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.
Tsukahara
T
T
Department of Enzyme Chemistry, School of Medicine, University of Tokushima.
Kominami
E
E
Katunuma
N
N
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
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