Denis M, Wikström A C, Gustafsson J A
Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, Sweden.
J Steroid Biochem. 1988;30(1-6):271-6. doi: 10.1016/0022-4731(88)90105-7.
A monoclonal IgG 2a antibody directed against the activated rat liver glucocorticoid receptor (GR) was used to prepare an immunoaffinity matrix of high capacity. The molybdate-stabilized GR from rat liver cytosol was immunoadsorbed on this gel. A non-hormone-binding protein of Mr approximately 90,000, as determined after denaturing gel electrophoresis, was eluted from this matrix following removal of molybdate and exposure to heat (25 degrees C) and salt (0.15 M NaCl). Subsequently, the Mr approximately 90,000 protein was purified to homogeneity using high-performance ion-exchange chromatography, covalently radiolabelled, and analyzed by high-performance size-exclusion chromatography and sucrose gradient ultracentrifugation. Hydrodynamic characterization indicates that, under our experimental conditions, the molybdate-stabilized rat liver GR (Rs approximately 7.4 nm, s20,w approximately 9.1 S, calculated mol. wt Mr approximately 285,000) includes one steroid-binding unit (Rs approximately 5.5 nm, S20,w approximately 4.3 S, calculated Mr approximately 100,000) and a dimer of Mr approximately 90,000 non-hormone-binding protein (Rs approximately 6.9 nm, S20,w approximately 6.1 S, calculated native Mr approximately 180,000).
一种针对活化大鼠肝脏糖皮质激素受体（GR）的单克隆IgG 2a抗体被用于制备高容量免疫亲和基质。来自大鼠肝脏胞质溶胶的钼酸盐稳定化GR被免疫吸附到该凝胶上。在变性凝胶电泳后测定，一种分子量约为90,000的非激素结合蛋白，在去除钼酸盐并暴露于热（25℃）和盐（0.15 M NaCl）后从该基质上洗脱下来。随后，使用高效离子交换色谱将该分子量约为90,000的蛋白纯化至同质，进行共价放射性标记，并通过高效尺寸排阻色谱和蔗糖梯度超速离心进行分析。流体动力学表征表明，在我们的实验条件下，钼酸盐稳定化的大鼠肝脏GR（Rs约为7.4 nm，s20,w约为9.1 S，计算分子量Mr约为285,000）包括一个类固醇结合单元（Rs约为5.5 nm，S20,w约为4.3 S，计算Mr约为100,000）和一个分子量约为90,000的非激素结合蛋白二聚体（Rs约为6.9 nm，S20,w约为6.1 S，计算天然Mr约为180,000）。
