Gitler C, Zarmi B, Kalef E
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Anal Biochem. 1997 Oct 1;252(1):48-55. doi: 10.1006/abio.1997.2294.
Some 5% of the soluble proteins of L1210 murine leukemia lymphoblasts contain surface vicinal thiols (Kalef, E., Walfish, P. G., and Gitler, C. (1993) Anal. Biochem. 212, 325-334). Redox dithiol to intraprotein disulfide conversion could regulate the cellular function of these proteins. A general method is presented to identify and enrich vicinal thiol proteins existing in cells in their oxidized, disulfide state. The method is based on the in situ blockage by cell permeable N-ethylmaleimide (NEM) of readily accessible cellular protein sulfhydryls. Following removal of the excess NEM, disulfide-containing proteins were identified by reduction with DTT and specific labeling with N-iodoacetyl-[125I]-3-iodotyrosine. The vicinal thiol proteins formed could also be enriched, prior to labeling with [125I]IAIT, by their selective binding to Sepha-rose-aminohexanoyl-4-aminophenylarsine oxide. Exponentially growing L1210 lymphoblasts contain more than 20 proteins with thiols in the oxidized, disulfide state. The majority derive from vicinal thiol proteins. The fraction oxidized, in some proteins, represents almost the totality of the protein present in the cell. Exposure of lymphoblasts to diamide increases the number and concentration of proteins with intraprotein disulfides. This method allows sensitive direct identification of vicinal thiol proteins that participate in redox regulation and those that are targets to oxidative stress conditions.
L1210小鼠白血病淋巴母细胞中约5%的可溶性蛋白质含有表面邻位硫醇（卡莱夫，E.，瓦尔菲什，P.G.，和吉特勒，C.（1993年）《分析生物化学》212卷，325 - 334页）。氧化还原过程中二硫醇向蛋白质内二硫键的转化可能调节这些蛋白质的细胞功能。本文提出了一种通用方法，用于鉴定和富集细胞中以氧化二硫键状态存在的邻位硫醇蛋白质。该方法基于细胞可渗透的N - 乙基马来酰亚胺（NEM）对细胞内易于接近的蛋白质巯基进行原位阻断。去除过量的NEM后，通过用二硫苏糖醇（DTT）还原并用N - 碘乙酰 - [125I] - 3 - 碘酪氨酸进行特异性标记来鉴定含二硫键的蛋白质。在用[125I]IAIT标记之前，形成的邻位硫醇蛋白质也可以通过它们与琼脂糖 - 氨基己酰 - 4 - 氨基苯胂氧化物的选择性结合来富集。指数生长的L1210淋巴母细胞含有20多种处于氧化二硫键状态的含硫醇蛋白质。大多数来自邻位硫醇蛋白质。在某些蛋白质中，氧化部分几乎代表了细胞中存在的蛋白质的全部。将淋巴母细胞暴露于二酰胺会增加蛋白质内二硫键的蛋白质数量和浓度。这种方法能够灵敏地直接鉴定参与氧化还原调节的邻位硫醇蛋白质以及那些是氧化应激条件下靶点的蛋白质。
