Characterization of non-liganded glucocorticoid receptor in rat liver cytosol using indirect competitive enzyme-linked immunosorbent assay.
We have previously shown that the purified or unfractionated cytosolic, activated glucocorticoid receptor of rat liver consists of a polypeptide with a Stokes radius of approximately 6 nm, a sedimentation coefficient of 4S and a molecular mass of approximately 90,000 Daltons. We have confirmed previous observations by other authors that if sodium molybdate is introduced into the cytosol preparation buffer the non-activated glucocorticoid receptor appears as an 8 nm, 9S species with an apparent molecular mass of 330,000 Daltons. In order to study the physicochemical parameters of the glucocorticoid receptor prior to ligand binding, we have used an enzyme-linked immunosorbent assay (ELISA) based on antibodies raised in rabbits against the purified activated glucocorticoid receptor. In isotonic buffer, the non-liganded glucocorticoid receptor was shown to have a Stokes radius of 6 nm in the absence and 8 nm in the presence of molybdate. Furthermore, experimental conditions known to result in activation of the glucocorticoid receptor complex (increased ionic strength, increased temperature) did not lead to activation of the 6 nm non-liganded glucocorticoid receptor as judged from the lack of binding of the treated, non-liganded receptor to DNA-cellulose. The existence of both 6 and 8 nm forms of nonactivated, non-liganded glucocorticoid receptor in vitro suggests that dissociation of an 8 nm form to a 6 nm form, if it occurs in vivo, is probably not the only molecular event constituting the activation of the glucocorticoid receptor.
D000818:Animals; D001667:Binding, Competitive; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D003600:Cytosol / Q000032:analysis; D004247:DNA / Q000031:analogs & derivatives / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D008099:Liver / Q000032:analysis*; D008982:Molybdenum / Q000494:pharmacology; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D011987:Receptors, Steroid / Q000032:analysis*
C033190:DNA-cellulose; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D008982:Molybdenum; D002482:Cellulose; D004247:DNA; C024687:sodium molybdate(VI)