High efficiency covalent radiolabeling of the human androgen receptor. Studies in cultured fibroblasts using dihydrotestosterone 17 beta-bromoacetate.
High efficiency covalent radiolabeling of the human androgen receptor. Studies in cultured fibroblasts using dihydrotestosterone 17 beta-bromoacetate.
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人类雄激素受体的高效共价放射性标记。
DOI：
10.1172/jci113326
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Turney,MK
中科院分区：
文献类型：
--
作者：
Kovacs,WJ;Turney,MK
由于靶组织中这些蛋白质的丰度低且不稳定，对影响人类细胞中雄激素受体蛋白质的突变的分析受到限制。迄今为止使用的所有方法都是基于放射性标记的雄激素与受体的配体结合位点的非共价相互作用。我们在此报告亲电亲和标记二氢睾酮 17 β-溴乙酸酯的合成和使用。这种配体是一种高比活性放射性化合物，可快速共价结合正常生殖器皮肤成纤维细胞胞浆中 58,000 道尔顿的蛋白质。该蛋白是配体的高亲和力、可饱和的特异性结合位点，并且在雄激素抵抗受试者的培养细胞或受体阴性非生殖器成纤维细胞中检测不到。根据在完整细胞测定中使用非共价配体对受体含量的估计，共价放射性标记掺入 58-kD 蛋白质的效率大于 80%。这些研究表明，二氢睾酮 17 β-溴乙酸酯可用于高效共价标记培养细胞粗胞浆提取物中的人雄激素受体。
Analysis of mutations affecting the androgen receptor protein in human cells has been limited because of the low abundance and lability of these proteins in target tissues. All methods used to date have been based on the noncovalent interaction of radiolabeled androgens with the receptor's ligand binding site. We report here synthesis and use of the electrophilic affinity label dihydrotestosterone 17 beta-bromoacetate. This ligand, prepared as a radioactive compound of high specific activity, rapidly and covalently binds to a protein of 58,000 daltons in cytosol from normal genital skin fibroblasts. This protein is a high affinity, saturable specific binding site for the ligand and was not detectable in cultured cells from a subject with androgen resistance or in receptor-negative nongenital fibroblasts. The efficiency of incorporation of the covalent radiolabel into the 58-kD protein is greater than 80% based on estimates of receptor content using noncovalent ligands in intact cell assays. These studies demonstrate that dihydrotestosterone 17 beta-bromoacetate is useful for high efficiency covalent labeling of the human androgen receptor in crude cytosolic extracts from cultured cells.Images