The activated glucocorticoid receptor of rat liver. Purification and physical characterization.
The activated form of the hepatic glucocorticoid receptor has been purified 60 000-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 40 000 +/- 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 (S20, 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.
D000818:Animals; D002499:Centrifugation, Density Gradient / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008099:Liver / Q000378:metabolism*; D008297:Male; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification*; D011987:Receptors, Steroid / Q000302:isolation & purification*
D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid