Traish A M, Muller R E, Wotiz H H
J Biol Chem. 1981 Dec 10;256(23):12028-33.
Experimental conditions for the measurement of prostatic androgen receptors occupied with unlabeled hormone are described. The assay allows quantitation of cytoplasmic and nuclear receptors occupied with unlabeled ligand by exchange with 17 beta-hydroxy-17 alpha-methyl [3H]estra-4,9,11-trien-3-one at 0 degrees C. The mercurial reagent mersalyl acid (0.2 mM) is used to promote dissociation of more than 90% receptor-bound steroid. The half-time of this reaction is 10-15 min. The reaction is reversible by addition of dithiothreitol or monothioglycerol. Treatment of the receptor with mersalyl and displacement of this reagent by thiol reagents does not alter the steroid binding affinity of the receptor or the sedimentation characteristics. Binding is measured by the hydroxylapatite procedure and all buffers contain 10 mM sodium molybdate. This assay circumvents many of the difficulties associated with conventional exchange assays performed either at elevated temperature or at 16 degrees C for 20-24 h, such as receptor degradation or incomplete exchange.
本文描述了测量被未标记激素占据的前列腺雄激素受体的实验条件。该测定法通过在0℃下与17β-羟基-17α-甲基[3H]雌甾-4,9,11-三烯-3-酮交换，对被未标记配体占据的细胞质和核受体进行定量。汞试剂硫柳汞酸（0.2 mM）用于促进超过90%受体结合类固醇的解离。该反应的半衰期为10-15分钟。通过添加二硫苏糖醇或单硫甘油可使反应逆转。用硫柳汞处理受体并通过硫醇试剂取代该试剂不会改变受体的类固醇结合亲和力或沉降特性。通过羟基磷灰石程序测量结合，所有缓冲液均含有10 mM钼酸钠。该测定法避免了许多与在高温或16℃下进行20-24小时的传统交换测定法相关的困难，例如受体降解或不完全交换。
