Isoelectric focusing of androgen receptors from wild-type and Tfm mouse kidneys

Putative androgen receptors from wild-type mice and the androgen-resistant mutant with testicular feminization (Tfm) were analyzed sequentially by DNA-cellulose chromatography, isoelectric focusing, and sucrose density gradient sedimentation. Wild-type kidney receptors labeled with [3H]testosterone or [3H]dihydrotestosterone were partially purified by single step elution from DNA-cellulose. For these eluates, two isoelectric focusing peaks were obtained, with approximate pI values of 5 (pH 4.9 +/- 0.16; n = 10) and 6 (pH 5.7 +/- 0.09; n = 10), respectively. In isoelectric focusing of Tfm mice eluates, a single step DNA-cellulose eluate appeared predominantly at pH 7-8. The complete complement (10-15% of the wild-type level) of Tfm receptors elutes only as a higher salt form in DNA-cellulose chromatography, while the wild-type yields both lower salt and higher salt forms. Accordingly, for comparison to Tfm mice, we examined separated DNA-cellulose peaks of wild-type mice by isoelectric focusing. For isoelectric focusing, as for differential DNA-cellulose chromatography, the ratio of the two wild-type peaks differed when [3H]testosterone and [3H]dihydrotestosterone were used as the bound ligand, with [3H]testosterone favoring the pH 6 peak and the lower salt eluting peak. As predicted from this correlation, when the lower salt fraction was focused, a peak was still detected at pH 6, with less radioactivity at pH 5. However, when subjected to isoelectric focusing, the higher salt fraction appeared predominantly at pH 7-8. Thus, a characteristic pattern was obtained during isoelectric focusing for both the higher salt eluting fraction of wild-type mouse androgen receptor and the single step, complete eluate of the Tfm mouse.