Subunit composition of the molybdate-stabilized non-activated glucocorticoid receptor from rat liver

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 M_r ≈ 90,000, as determined after denaturing gel electrophoresis, was eluted from this matrix following removal of molybdate and exposure to heat (25°C) and salt (0.15 M NaCl). Subsequently, the M_r ≈ 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 (R_s ≈ 7.4nm, s_20,w ≈9.1 S, calculated mol. wt M_r ≈ 285,000) includes one steroid-binding unit (R_s ≈ 5.5 nm, s_20,w ≈ 3 S, calculated M_r ≈ 100,000) and a dimer of M_r ≈ 90,000 non-hormone-binding protein (R_s ≈ 6.9nm, s_20,w ≈ 6.1 S, calculated native M_r ≈ 180,000).