Characterization of the partially purified, ligand-free glucocorticoid receptor.
A new method was developed to synthesize a cortexolone-substituted affinity matrix, based on the fast, mild and quantitative reaction between alpha-ketomesylates and thiols. The resulting cortexolone-Sepharose absorbed easily the cytosolic chick thymus glucocorticoid receptor. Owing to the relatively fast dissociation of the glucocorticoid receptor-cortexolone complex, glucocorticoid receptor could be eluted with cortexolone as well as with triamcinolone acetonide from the affinity gel with similarly good yields. We obtained 75-150-fold purification factors (yield: 20-30%) using this column procedure. The partially purified glucocorticoid receptor was obtained in non-activated form. It had a Stokes radius of 6.2 +/- 0.1 nm. It could be activated to DNA-cellulose binding form by heat or 0.3 M KCl. KCl treatment activated 30-50% of the partially purified glucocorticoid receptor. Heat activation, however, was rather poor. Cortexolone-complexed, partially purified glucocorticoid receptor dissociated easily, and partially purified free glucocorticoid receptor, capable of steroid binding, could be obtained. Binding properties of the partially purified glucocorticoid receptor were then analyzed using different steroids. Dissociation rate constants were similar to those of the cytosolic glucocorticoid complexes. Association rate constants were consistently smaller than in the case of cytosolic glucocorticoid receptor, but the relative order of rates for different steroids was basically the same for glucocorticoid receptor in the two studied systems.
D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003350:Cortodoxone / Q000378:metabolism; D004247:DNA / Q000031:analogs & derivatives / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*; D013950:Thymus Gland / Q000032:analysis; D014222:Triamcinolone Acetonide / Q000378:metabolism
C033190:DNA-cellulose; D011965:Receptors, Glucocorticoid; D002482:Cellulose; D004247:DNA; D014222:Triamcinolone Acetonide; D003350:Cortodoxone