Application of high-performance liquid chromatofocusing to the study of prostatic androgen receptors. Effect of stabilizing agents on the heterogeneity of receptor structure.
Cytosolic androgen receptors from normal rat prostate were analyzed by high-performance liquid chromatofocusing. Two ion-exchange columns, AX-300 and AX-500, and two equilibration systems, Tris-HCl and imidazole-HCl, were used. pH gradients ranged between 8.3 and 3.5 for Tris-HCl and from 7.7 to 3.5 for imidazole-HCl. In the absence of sodium molybdate and inhibitors of proteolytic enzyme, six specific radioactive fractions (pH: 7.9, 7.7, 7.0, 5.1, 4.7 and 4.4) were eluted from AX-300 equilibrated with Tris-HCl in a ratio of 28:20:52 for acidic, intermediary and basic forms, respectively; similar results were obtained with AX-500 although this column was less effective in resolving basic forms of the receptor. The buffering capacity of imidazole-HCl was lower than that of Tris-HCl, resulting in a steeper elution pH profile. The resolution between acidic and basic forms was thus diminished and only four specific radioactive fractions at pH 7.2, 7.1, 6.5 and 3.6, were observed on AX-500 in a ratio of 23:10:67 for acidic, intermediary and basic forms. In the presence of sodium molybdate, two acidic fractions were found with Tris-HCl at pH 4.3 and 4.7 (47%) on AX-300, whereas the radioactivity of fractions at pH 7.0 and 5.1 (32%) was considerably lowered and intermediary forms remained unchanged (21%). With imidazole-HCl on AX-500, the peak at pH 7.2 disappeared and the acidic form shifted from pH 3.6 to 4.3. In the presence of inhibitors of proteolytic enzyme and sodium molybdate, specifically bound radioactivity was found mostly in a broad acidic fraction (75%) at pH 4.5 on columns equilibrated with Tris-HCl; radioactivity at pH 7.6 disappeared completely but a small amount (15%) remained at pH 7.9. In imidazole-HCl, a main radioactive fraction was eluted at pH 7.1 and two other fractions were collected at pH 6.8 and 4.3 respectively. In conclusion, multiple forms of the rat prostate androgen receptor were evinced by high-performance liquid chromatofocusing. Tris-HCl proved to be a more efficient equilibration system than imidazole-HCl for the resolution of rat prostate cytosolic binding proteins. Under the experimental conditions used, sodium molybdate and inhibitors of proteolytic enzyme greatly favored the acidic form to the detriment of the intermediary and basic entities.
Bouthillier
F
F
Department of Biochemistry, Faculty of Medicine, Sherbrooke University, Québec, Canada.
Carmel
M
M
Elhilali
M
M
Radwan
F
F
Lehoux
J G
JG
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000032:analysis; D007202:Indicators and Reagents; D008297:Male; D008982:Molybdenum / Q000494:pharmacology; D011467:Prostate / Q000032:analysis*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011944:Receptors, Androgen / Q000302:isolation & purification*
D007202:Indicators and Reagents; D011480:Protease Inhibitors; D011944:Receptors, Androgen; D008982:Molybdenum; C024687:sodium molybdate(VI)