Formation of Mixed Disulfide of Cystatin-β in Cultured Macrophages Treated with Various Oxidants1

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Abstract
Macrophage cell cultures were treated with menadione, zymosan, or phorbol myri-state acetate (PMA), and changes in productions of superoxide anion and hydro-peroxide, and in glutathione oxidation and S -thiolation of cystatin-β (formation of a mixed disulfide of cystatin-β 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-β 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-β induced by menadione. Inhibition of catalase by amino-triazol caused slight increase in the GSSG content in both menadione- and zymosan-treated cells, but not in S-thiolation of cystatin-β 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-β occurs in cells in response to oxidative challenge by menadione but not by zymosan or by the tumor promoter PMA. Dethiolation of cystatin-β 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-β 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-β in cells.

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