Steroid-affinity purification of the rat liver glucocorticoid hormone receptor complex.
The molybdate-stabilized GHRC was isolated from rat liver cytosol with a 9000-fold purification and 46% yield. The major purification step was achieved using an affinity matrix consisting of an agarose support coupled to a dexamethasone ligand via an aliphatic spacer arm. Spacer arms containing disulfide bridges were found to be unsuitable due to their instability in cytosol. To reduce the non-specific binding properties of the affinity matrix, underivatized amino groups were acetylated, since the receptor was found to bind avidly to such groups thus evading elution by the ligand. Sodium molybdate present during biospecific elution from the gel stabilized the steroid-binding activity of the receptor. The use of denaturing and sulfhydryl modifying reagents (NaSCN, DMSO, Mersalyl) during elution led to partial or complete irreversible loss of steroid-binding activity of the unoccupied receptor. Efficient biospecific elution occurred at competing concentration of high affinity steroid in the presence of sodium molybdate. The ligand specific eluate was further purified by DEAE-Sephacel chromatography resulting in additional purification of 3.2-fold. The GHRC eluted from the DEAE-Sephacel column at a salt concentration characteristic of the untransformed GHRC. Molybdate was removed from the purified untransformed GHRC in the ligand eluate by DEAE-Sephacel chromatography in the absence of molybdate, for subsequent heat transformation.
UCT-CSIR Research Centre for Molecular Biology, Department of Biochemistry, Republic of South Africa.
J P
JP
Hapgood
C
C
von Holt
D000107:Acetylation; D000818:Animals; D002846:Chromatography, Affinity; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D008099:Liver / Q000032:analysis*; D008297:Male; D008634:Mersalyl / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification*; D013861:Thiocyanates / Q000494:pharmacology
D011965:Receptors, Glucocorticoid; D013861:Thiocyanates; C024553:sodium thiocyanate; D008634:Mersalyl; D004121:Dimethyl Sulfoxide