Efficacy of five methods for the bound-free separation of gluco- and mineralocorticoids from type I, II and III receptors found in hepes- and tris-buffered mouse brain cytosol.
We compared the efficacy of G-25 and LH-20 column chromatography, dextran-coated charcoal adsorption, and DEAE-cellulose and glass fiber filter disc assays to separate unbound steroids from three classes of brain cytosolic receptors prepared in HEPES and TRIS buffers and labeled selectively as follows: Type I = [3H]aldosterone + unlabeled RU26988, Type II = [3H]triamcinolone acetonide and Type III = [3H]corticosterone + unlabeled Prorenone and RU26988. Prorenone and RU26988 were added to reduce unwanted [3H]steroid binding to Type I and Type II receptors, respectively. In each case total, non-specific and specific binding and free steroid were compared individually. No single assay was found to be best for all three receptor classes, but both buffers and most assays could be used with appropriate correction factors. Variations between the results with different assays suggest fundamental differences between the three classes of adrenosteroid receptors and their ligands.
D000327:Adsorption; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002021:Buffers; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D005938:Glucocorticoids / Q000302:isolation & purification*; D006531:HEPES; D051379:Mice; D008901:Mineralocorticoids / Q000302:isolation & purification*; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification*; D011987:Receptors, Steroid / Q000302:isolation & purification*; D014325:Tromethamine
D002021:Buffers; D005938:Glucocorticoids; D008901:Mineralocorticoids; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D014325:Tromethamine; D006531:HEPES