Purification and characterization of androgen receptor from steer seminal vesicle
- 17 August 1982
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 21 (17), 4102-4109
- https://doi.org/10.1021/bi00260a029
Abstract
The androgen receptor was 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 .apprx. 3 .mu.g 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 .apprx. 540,000-fold. A single band, migrating at 60,000 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-estratien-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 60,000 daltons by NaDodSO4 gel electrophoresis. The receptor had an estimated Stokes radius of 35 .ANG. and a sedimentation coefficient of 3.8 S in the presence of 0.3 M NaCl. The calculated MW and frictional ratio for the androgen binding activity were 57,000 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 methyltrienolone > testosterone > 5.alpha.-dihydrotestosterone .mchgt. 3.beta.-hydroxy-.DELTA.5-androsten-17-one .mchgt. 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. The androgen receptor from steer seminal vesicle was substantially purified without significant modification of its physicochemical or steroid binding properties.Keywords
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