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Structural and functional analysis of a cystine/glutamate-specific carrier protein of plasma membrane

质膜胱氨酸/谷氨酸特异性载体蛋白的结构和功能分析

基本信息

批准号：
02670103
负责人：
ISHII Tetsuro
金额：
$ 1.28万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

The amino acid transport activity designated System x_C^- is a Na^+-independent system and is highly specific for cystine and glutamate. In cell culture systems, the uptake of cystine mediated by this system is usually important in maintaining intracellular glutathione levels, because cystine and/or cysteine is the rate limiting precursor amino acid of glutathione synthesis. This transport activity can be induced in mouse peritoneal macrophages during culture by diethylmaleate, a sulfhydryl-reactive agent. We prepared mRNA from those macrophages and injected into Xenopus oocytes and demonstrated the expression of System x_C^-, i. e., a Na^+-independent, glutamate-inhibitable cystine transport system. The expressed cystine transport activity depended on the assay temperature, in that cystine uptake measured at 37ﾟC was several-fold higher than that measured at 20ﾟC. Injection of size- fractionated mRNA indicated that the System x_C^--transporter of the mouse macrophage is encoded by mRNA of 1.5 to 2.9 kb.In the next step, we constructed a lambdaZAPII cDNA library from the macrophage mRNA. Using a differential hybridization technique, we have isolated hundreds of cDNA clones which appeared to be induced under diethylmaleate treatment in macrophages. We are examining those selected cDNA clones expecting to find out a cDNA clone encoding cystine transporter.

被命名为系统x_C^-的氨基酸转运活性是一个不依赖Na^+的系统，对胱氨酸和谷氨酸具有高度特异性。在细胞培养系统中，由该系统介导的胱氨酸的摄取通常在维持细胞内谷胱甘肽水平方面是重要的，因为胱氨酸和/或半胱氨酸是谷胱甘肽合成的限速前体氨基酸。这种转运活动可以诱导小鼠腹腔巨噬细胞培养过程中的马来酸二乙酯，巯基反应剂。我们从这些巨噬细胞中制备mRNA，并注射到非洲爪蟾卵母细胞中，证明了系统x_C^-的表达，即：例如，一种不依赖于Na^+的、谷氨酸可降解的胱氨酸转运系统。表达的胱氨酸转运活性取决于测定温度，因为在37 ℃下测量的胱氨酸摄取比在20 ℃下测量的高数倍。注射大小分级的mRNA表明，小鼠巨噬细胞的系统x_C^-转运蛋白由1.5至2.9 kb的mRNA编码。使用差异杂交技术，我们已经分离出数百个cDNA克隆，这些克隆似乎是在马来酸二乙酯处理的巨噬细胞中诱导的。我们正在对这些cDNA克隆进行检测，希望能找到编码胱氨酸转运蛋白的cDNA克隆。