Purification of the glucocorticoid receptor from rat liver cytosol.
The [3H]-triamcinolone acetonide-labeled glucocorticoid receptor from rat liver cytosol was purified to 85% homogeneity according to sodium dodecyl sulfate gel electrophoresis. It consisted of one subunit with a molecular weight of 89,000 and had one ligand-binding site per molecule. The purification involved sequential chromatography on phosphocellulose, DNA-cellulose twice, and Sephadex G-200. Between the two chromatography steps on DNA-cellulose, the receptor was heat activated. The receptor was affinity eluted from the second DNA-cellulose column with pyrodixal 5'-phosphate. The purification achieved in the first three chromatographic steps varied between 60 and 95% homogeneity in different experiments. After chromatography on the second DNA-cellulose column, the steroid.receptor complex had a Stokes radius of 6.0 nm and a sedimentation coefficient of 3.4 S in 0.15 M KCl. In the absence of KCl, the sedimentation coefficient was 3.6 S. After concentration on hydroxylapatite, the steroid.receptor complex was analyzed by isoelectric focusing in polyacrylamide gel. The radioactivity was shown to focus together with the major protein band with pI 5.8. Following limited proteolysis with trypsin, the radioactivity, together with the major protein band, focused at pI 6.2 as previously described for the unpurified steroid.receptor complex.
D000818:Animals; D003600:Cytosol / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification* / Q000378:metabolism; D011987:Receptors, Steroid / Q000302:isolation & purification*; D014222:Triamcinolone Acetonide / Q000378:metabolism; D014357:Trypsin
D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D014357:Trypsin; D014222:Triamcinolone Acetonide