Purification and characterization of androgen receptor from steer seminal vesicle.
The androgen receptor has been purified from steer seminal vesicle cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. The procedure produced about 3 micrograms of receptor protein from 35 g of steer seminal vesicle, with a yield of 48%. The receptor protein, as a complex with unlabeled testosterone, was purified approximately 540000-fold. A single band, migrating at 60000 daltons, was observed following electrophoresis on a polyacrylamide gel containing sodium dodecyl sulfate (NaDoDSO4). This was confirmed by affinity labeling of the partially purified receptor with both 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one and 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8)-5 alpha-androstan-3-one 17-(2-bromoacetate), which showed a peak of radioactivity migrating at 60000 daltons by NaDoDSO4 gel electrophoresis. The receptor had an estimated Stokes radius of 35 A and a sedimentation coefficient of 3.8 S in the presence of 0.3 M NaCl. The calculated molecular weight and frictional ratio for the androgen binding activity were 57000 and 1.42, respectively. Chromatofocusing of the purified receptor protein revealed an isoelectric point of 6.6. [3H]Methyltrienolone, bound to the purified receptor, was displaced with methyl-trienolone greater than testosterone greater than 5 alpha-dihydrotestosterone much greater than 3 beta-hydroxy-delta 5-androsten-17-one much greater than 5 alpha-androstane-3 alpha,17 beta-diol. The physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol. These results demonstrate that the androgen receptor from steer seminal vesicle was substantially purified without significant modification of its physicochemical or steroid binding properties.
D000345:Affinity Labels; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000032:analysis; D004963:Estrenes / Q000378:metabolism; D008297:Male; D015741:Metribolone; D008970:Molecular Weight; D011944:Receptors, Androgen / Q000302:isolation & purification* / Q000378:metabolism; D011987:Receptors, Steroid / Q000302:isolation & purification*; D012669:Seminal Vesicles / Q000032:analysis*; D013739:Testosterone / Q000378:metabolism
D000345:Affinity Labels; D004963:Estrenes; D011944:Receptors, Androgen; D011987:Receptors, Steroid; D015741:Metribolone; D013739:Testosterone