Tsukahara T, Kominami E, Katunuma N
Department of Enzyme Chemistry, School of Medicine, University of Tokushima.
J Biochem. 1987 Jun;101(6):1447-56. doi: 10.1093/oxfordjournals.jbchem.a122014.
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.
巨噬细胞培养物用甲萘醌、酵母聚糖或佛波酯（PMA）处理，检测超氧阴离子和过氧化氢生成的变化，以及谷胱甘肽氧化和胱抑素-β的S-硫醇化（胱抑素-β与谷胱甘肽形成混合二硫键）的变化。这三种化合物均促进超氧阴离子和过氧化氢的生成，但只有甲萘醌引起谷胱甘肽的大量氧化。甲萘醌以剂量依赖的方式引起胱抑素-β的S-硫醇化，而其他两种化合物则不会。去除甲萘醌可迅速降低甲萘醌诱导的谷胱甘肽氧化和胱抑素-β的S-硫醇化。氨基三唑抑制过氧化氢酶导致甲萘醌和酵母聚糖处理的细胞中氧化型谷胱甘肽（GSSG）含量略有增加，但酵母聚糖处理的细胞中胱抑素-β的S-硫醇化没有增加。这三种化合物均未明显影响培养细胞中谷胱甘肽过氧化物酶、谷胱甘肽还原酶或超氧化物歧化酶的活性。这些结果表明，胱抑素-β的S-硫醇化发生在细胞中，是对甲萘醌氧化应激的反应，而不是对酵母聚糖或肿瘤启动子PMA的反应。在体外，研究了在不同浓度谷胱甘肽存在下，纯化的硫醇转移酶和蛋白质二硫键异构酶对胱抑素-β的脱硫醇作用。两种酶催化胱抑素-β脱硫醇所需的谷胱甘肽水平远低于非酶反应所需的水平，这表明细胞中胱抑素-β的S-硫醇化和脱硫醇化的酶催化作用很重要。
