Detection of S-glutathionylated proteins by glutathione S-transferase overlay

被引:35
作者
Cheng, G
Ikeda, Y
Iuchi, Y
Fujii, J
机构
[1] Yamagata Univ, Grad Sch Med, Dept Biomol Funct, Yamagata 9909585, Japan
[2] Saga Univ, Fac Med, Dept Biomol Sci, Div Cell & Mol Biol, Saga 8498501, Japan
基金
日本学术振兴会;
关键词
glutathione; S-glutathionylation; glutathione S-transferase; nitrosoalutathione; reactive oxygen species; reactive nitrogen oxide species;
D O I
10.1016/j.abb.2004.12.016
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Oxidative and nitrosative stress lead to the S-glutathionylation of proteins and subsequent functional impairment. Glutathione S-transferase (GST) from Schistosoma japonicum was found to bind to the glutathione moiety of S-glutathionylated proteins, thus establishing a convenient method for detecting S-glutathionylated proteins by biotinylated GST. Applications of this method to proteins that were prepared from Cultured cells and blotted onto a membrane exhibited numerous positive bands, which were abolished by treatment with dithiothreitol. Treatment of a cellular extract with nitrosoglutathione led to enhanced staining of the bands in a dose-dependent manner. The method was also applicable for the histochemical detection of S-glutathionylated proteins in situ. The positive staining by biotin-GST became faint in the presence of S-glutathionylated ovalbumin, Suggesting that the reaction is specific to S-glutathionylated proteins. Collectively, these data indicate that the method established here is simple and useful for detecting S-glutathionylated proteins on blotted membrane and in Situ. (C) 2005 Elsevier Inc. All rights reserved.
引用
收藏
页码:42 / 49
页数:8
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