Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH
Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH
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DOI：
10.1006/abio.2000.4623
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发表时间：
2000-08-01
影响因子：
2.9
通讯作者：
Rhee, SG
中科院分区：
文献类型：
--
作者：
Kim, JR;Yoon, HW;Rhee, SG
开发了一种检测含有 H2O2 敏感半胱氨酸（或硒代半胱氨酸）残基的蛋白质的程序，作为研究细胞中 H2O2 蛋白质氧化的方法。该程序基于这样的事实：H2O2 和生物素缀合的碘乙酰胺 (BIAM) 选择性地竞争性地与表现出低 pK(a) 的半胱氨酸残基反应，并且可以通过链霉亲和素印迹分析监测由于细胞先前暴露于 H2O2 或诱导 H2O2 产生的试剂而导致的细胞裂解物蛋白用 BIAM 标记的减少。该程序适用于直接用 H2O2 处理的大鼠嗜铬细胞瘤 PC12 细胞、谷氨酸诱导产生 H2O2 的小鼠海马 HT22 细胞以及佛波醇肉豆蔻酸酯乙酸酯诱导产生 H2O2 的人红白血病 K562 细胞。研究表明，几种细胞蛋白含有半胱氨酸或硒代半胱氨酸残基，可被 H2O2 选择性氧化。其中三种 H2O2 敏感蛋白被鉴定为蛋白二硫键异构酶家族、硫氧还蛋白还原酶和肌酸激酶的成员，此前已知所有这些蛋白的催化位点均含有至少一个反应性半胱氨酸或硒代半胱氨酸。因此，该程序对于鉴定被 H2O2 氧化的蛋白质是有用的，H2O2 是响应各种细胞外物质而产生的。 (C) 2000 年学术出版社。
A procedure for detecting proteins that contain H2O2-sensitive cysteine (or selenocysteine) residues was developed as a means with which to study protein oxidation by H2O2 in cells. The procedure is based on the facts that H2O2 and biotin-conjugated iodoacetamide (BIAM) selectively and competitively react with cysteine residues that exhibit a low pK(a), and that the decrease in the labeling of cell lysate proteins with BIAM caused by prior exposure of cells to H2O2 or to an agent that induces H2O2 production can be monitored by streptavidin blot analysis. This procedure was applied to rat pheochromocytoma PC12 cells directly treated with H2O2, mouse hippocampal HT22 cells in which H2O2 production was induced by glutamate, and human erythroleukemia K562 cells in which H2O2 production was induced by phorbol myristate acetate. It revealed that several cell proteins contain cysteine or selenocysteine residues that are selectively oxidized by H2O2. Three of these H2O2-sensitive proteins were identified as a member of the protein disulfide isomerase family, thioredoxin reductase, and creatine kinase, all of which were previously known to contain at least one reactive cysteine or selenocysteine at their catalytic sites. This procedure should thus prove useful for the identification of proteins that are oxidized by H2O2 generated in response to a variety of extracellular agents. (C) 2000 Academic Press.