Evidence for distinct sulfhydryl groups associated with steroid- and DNA-binding domains of rat thymus glucocorticoid receptors.
We have found that nonactivated and activated forms of the rat thymus glucocorticoid-receptor complex (GRC) will react with reactive sulfhydryl matrices to form covalently immobilized complexes that can subsequently be eluted with reducing agents. The interaction of GRCs with these matrices depends on the nature of both the immobilized sulfhydryl group and the type of leaving group attached. One matrix, agarose CL-4B-diaminoethyl-succinyl-thioethylamine-2-thiopyridyl+ ++ (DSTT), binds total receptor-bound steroid. A second matrix, agarose CL-4B-diaminoethyl-succinyl-cysteinyl-2-thiobenzoic acid (DSCT), binds activated but not nonactivated complexes. The reaction of activated complexes with the DSCT matrix is apparently through a sulfhydryl group located near the DNA binding domain, as soluble DNA interferes with the reaction. This sulfhydryl group(s) appears to be located in a portion of the GRC that is resistant to degradation, since proteolytic digestion of activated GRC to a point where DNA binding is lost results in only a moderate decrease in binding with the DSCT matrix. Purified receptor, covalently labeled with [3H]dexamethasone to the sulfhydryl associated with the steroid binding domain, was able to bind to DSCT matrix, providing evidence for distinct sulfhydryl groups associated with the steroid and DNA binding domains.
D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism*; D003907:Dexamethasone / Q000378:metabolism; D010450:Endopeptidases / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000032:analysis; D013439:Sulfhydryl Reagents; D013950:Thymus Gland / Q000378:metabolism*
D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D003907:Dexamethasone; D004247:DNA; D010450:Endopeptidases