The Activated Glucocorticoid Receptor of Rat Liver

Abstract

The activated form of the hepatic glucocorticoid receptor has been purified 60000‐fold taking advantage of the differential affinity for phosphocellulose of the activated and the nonactivated forms of the receptor. Rat liver cytosol was incubated with [³H]triamcinolone acetonide at low temperature and low ionic strength and adsorbed to a large excess of phosphocellulose. The unbound fraction was heat‐activated, passed through a column of DEAE‐cellulose and precipitated with ammonium sulfate. The activated receptor was then bound to a small phosphocellulose column and eluted with a gradient of NaCl. Additional steps included chromatography on DNA‐cellulose, precipitation with ammonium sulfate and centrifugation through a sucrose density gradient. The final preparation was identified as the steroid‐receptor complex by electrophoresis on polyacrylamide gels in the presence of heparin; it exhibits a single band of molecular weight 40000 ± 4000 in gels containing sodium dodecyl sulfate. An independent calculation of the molecular weight under nondenaturing conditions based on the Stokes' radius (2.7 nm) and the sedimentation coefficient (s_20,w= 3.0 S) yields similar results, indicating that the activated form of the receptor we have isolated is composed of a single polypeptide chain and contains a single steroid‐binding site per molecule. Since this homogenous preparation of receptor has retained its affinity for DNA it may be useful for cell‐free studies.