S-THIOLATION AND IRREVERSIBLE OXIDATION OF SULFHYDRYLS ON CARBONIC-ANHYDRASE-III DURING OXIDATIVE STRESS - A METHOD FOR STUDYING PROTEIN MODIFICATION IN INTACT-CELLS AND TISSUES
S-THIOLATION AND IRREVERSIBLE OXIDATION OF SULFHYDRYLS ON CARBONIC-ANHYDRASE-III DURING OXIDATIVE STRESS - A METHOD FOR STUDYING PROTEIN MODIFICATION IN INTACT-CELLS AND TISSUES
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DOI：
10.1006/abbi.1994.1033
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发表时间：
1994-01-01
影响因子：
3.9
通讯作者：
HENDRICH, S
中科院分区：
文献类型：
--
作者：
LII, CK;CHAI, YC;HENDRICH, S
用等电聚焦凝胶硝酸纤维素膜免疫印迹法研究了氧化应激条件下培养的大鼠肝细胞中碳酸酐酶III(CA III)的S硫基化。在甲奈二酮处理的肝细胞中，检测到三种S硫化的CAⅢ形式，而在叔丁基氢过氧化氢处理的肝细胞中，仅观察到两种形式的CA III。硝酸纤维蛋白印迹上也检测到两种“不可还原”的CA III氧化形式。这些形式随着应激量的增加而增加，并且是丁硫氨酸亚砜处理的肝细胞中唯一修饰的CA III形式，其谷胱甘肽含量比对照肝细胞少10倍。这些实验支持S硫基化在氧化应激过程中保护CA III免受不可逆氧化的概念。免疫印迹法可在雄性和雌性肝细胞中检测到部分或全部S硫代修饰的CAⅢ，但雌性肝细胞中CA III的含量比雄性肝细胞少15倍。在大鼠骨骼肌和心脏中也检测到了S硫化形式的CAⅢ，表明该方法可用于在体内测定氧化应激对几种组织中特定的S硫化蛋白的影响。
S-thiolation of carbonic anhydrase III (CA III) in cultured rat hepatocytes under oxidative stress was studied by immunodetection on nitrocellulose blots of isoelectrofocusing gels. In cells treated with menadione, three S-thiolated forms of CA III were detected, whereas only two forms were observed in hepatocytes treated witht-butyl hydroperoxide. Two "nonreducible" oxidized forms of CA III were also detected on nitrocellulose blots. These forms increased with the amount of stress and were the only modified forms of CA III in buthionine sulfoxide-treated hepatocytes containing 10-fold less glutathione than control hepatocytes. These experiments support the concept that S-thiolation protects CA III from irreversible oxidation during oxidative stress. Partly and fully S-thiolated forms of CA III were easily detected in both male and female hepatocytes by the immunoblotting method, although female cells contained 15-fold less CA III than did male liver. S-thiolated forms of CA III were also detected in rat skeletal muscle and heart showing the utility of this method for determining the effect of oxidative stress on specific S-thiolatable protein in several tissuesin vivo.