Chemical demonstration of nuclear androgen receptor following affinity chromatography with immobilized ligands.
With increasing purification of the androgen receptor from nuclei of rat ventral prostate, a receptor-like protein could be demonstrated by chemical staining with silver nitrate. After sonication and digestion of nuclei with micrococcal nuclease, the solubilized receptor was applied to a column of Matrex Gel Green A and eluted with a linear gradient of 0-2 M NaCl. Characterized by specific binding of dihydrotestosterone, this form of the receptor was also androgen dependent and yielded an apparent Mr of 33,000 when analyzed by polyacrylamide gel electrophoresis and silver nitrate staining. To facilitate recovery following chromatography, the receptor was precipitated with 0-40% ammonium sulfate. Analysis of the 15-fold enriched fraction by sucrose density-gradient centrifugation confirmed the presence of a 3S androgen-binding protein. About 200 ng of the precipitated protein was applied to a column of dihydrotestosterone-17 beta-succinyl agarose (ligand concentration, 0.25 mumol/ml). The fractions eluted with 50 microM dihydrotestosterone were electrophoresed and stained as before; again, the presence of a 33,000 Mr protein sensitive to castration was demonstrated. Alternatively, when the precipitated protein was fractionated by fast protein liquid chromatography utilizing a Superose 12 HR 10/30 column, the receptor coeluted with nuclear proteins in the 29,000-36,000 Mr range as determined both by retention time and electrophoresis. In combination, the above methods may be used to obtain a receptor protein purified to near homogeneity with a yield of 5-10%. The amount of receptor afforded by the purification sequence is small but nevertheless sufficient for chemical detection. We anticipate that with modification, the procedures may prove suitable for the recovery of nuclear androgen receptor on a preparative scale.
D000818:Animals; D002467:Cell Nucleus / Q000032:analysis*; D002846:Chromatography, Affinity / Q000379:methods; D066298:In Vitro Techniques; D008297:Male; D011467:Prostate / Q000032:analysis*; D051381:Rats; D011944:Receptors, Androgen / Q000302:isolation & purification*
D011944:Receptors, Androgen