Denis M, Wikström A C, Gustafsson J A
J Biol Chem. 1987 Aug 25;262(24):11803-6.
A glucocorticoid receptor-associated Mr approximately 90,000 non-hormone-binding protein was purified and characterized. The molybdate-stabilized nonactivated rat liver glucocorticoid-receptor complex (Mr approximately 300,000) was immunoadsorbed on cyanogen bromide-activated Sepharose 4B to which a monoclonal IgG 2a antibody directed against the activated rat glucocorticoid receptor (Mr approximately 94,000) had been coupled. Following removal of molybdate and thermal activation of the receptor immobilized on the immunoaffinity matrix, an Mr approximately 90,000 non-hormone-binding protein was specifically eluted. This protein was further purified to homogeneity using high performance ion exchange chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sucrose gradient ultra-centrifugation, and high performance size-exclusion chromatography. Hydrodynamic characterization under nondenaturing conditions revealed that the purified glucocorticoid receptor-associated protein represents a molecular species with a sedimentation coefficient of 6.1 S, a Stokes radius of 6.9 nm, and a calculated Mr approximately 184,000. These results, combined with analysis on denaturing electrophoresis indicate that, under certain conditions, the Mr approximately 94,000 steroid-binding protein is associated with a dimer of Mr approximately 90,000 non-hormone-binding protein.
一种与糖皮质激素受体相关的、分子量约为90,000的非激素结合蛋白被纯化并进行了表征。将经钼酸盐稳定的未活化大鼠肝脏糖皮质激素受体复合物（分子量约为300,000）免疫吸附到溴化氰活化的琼脂糖4B上，该琼脂糖已偶联了一种针对活化大鼠糖皮质激素受体（分子量约为94,000）的单克隆IgG 2a抗体。在去除钼酸盐并对固定在免疫亲和基质上的受体进行热活化后，一种分子量约为90,000的非激素结合蛋白被特异性洗脱。使用高效离子交换色谱将该蛋白进一步纯化至同质，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、蔗糖梯度超速离心和高效尺寸排阻色谱进行分析。非变性条件下的流体动力学表征显示，纯化的糖皮质激素受体相关蛋白代表一种分子物种，其沉降系数为6.1 S，斯托克斯半径为6.9 nm，计算分子量约为184,000。这些结果与变性电泳分析相结合表明，在某些条件下，分子量约为94,000的类固醇结合蛋白与分子量约为90,000的非激素结合蛋白二聚体相关。
