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 ≈ 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 Mr ≈ 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 ≈ 7.4nm, s20,w ≈9.1 S, calculated mol. wt Mr ≈ 285,000) includes one steroid-binding unit (Rs ≈ 5.5 nm, s20,w ≈ 3 S, calculated Mr ≈ 100,000) and a dimer of Mr ≈ 90,000 non-hormone-binding protein (Rs ≈ 6.9nm, s20,w ≈ 6.1 S, calculated native Mr ≈ 180,000).
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