Purification and characterization of the untransformed androgen receptor in rat prostate.
The isolation and characterization of the untransformed form of androgen receptors has not yet been successful, owing to their inherent lability as well as to their ready proteolysis. In this study, we have stabilized rat prostate androgen receptors by sodium molybdate and by rapid filtration on phosphocellulose. Proteases were inhibited by bacitracin, aprotinin, leupeptin and PMSF. Under these conditions the untransformed complex was purified approx 3000-fold, corresponding to 18% yield, by differential chromatography on DEAE cellulose and phosphocellulose gels. The partially purified receptor has the same ionic characteristics as the original untransformed receptor of crude cytosol; in addition, it possesses a Stokes' radius of 75 A, as determined by Sephacryl S-300 gel filtration, a sedimentation coefficient of 8.8S, a calculated molecular weight of 275 kDa and a friction coefficient of 1.6. The [3H]R1881 receptor complex was specific to androgens since unlabelled R1881 and dihydrotestosterone were able to completely displace bound [3H]R1881, whereas estradiol, cortisol, and triamcinolone acetonide did not compete. The purified complex was a multimer dissociable by 0.6 M KCl, resulting in a form migrating in the 4S area on sucrose density gradient. After treatment with 0.5% formaldehyde, three forms were obtained, migrating in the areas of 8-9, 5-6 and 3-4S respectively, of a sucrose density gradient containing 0.6 M KCl. This is the first step towards the purification to homogeneity of the untransformed androgen receptor.
Radwan
F
F
Department of Biochemistry, Faculty of Medicine, Sherbrooke University, Montreal, Canada.
Carmel
M
M
Elhilali
M
M
Bouthillier
F
F
Lehoux
J G
JG
D000818:Animals; D001667:Binding, Competitive; D002499:Centrifugation, Density Gradient / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D007700:Kinetics; D008297:Male; D011467:Prostate / Q000378:metabolism*; D051381:Rats; D011944:Receptors, Androgen / Q000302:isolation & purification* / Q000378:metabolism
D011944:Receptors, Androgen