Radwan F, Carmel M, Elhilali M, Bouthillier F, Lehoux J G
Department of Biochemistry, Faculty of Medicine, Sherbrooke University, Montreal, Canada.
J Steroid Biochem. 1988;30(1-6):251-5. doi: 10.1016/0022-4731(88)90101-x.
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.
由于雄激素受体固有的不稳定性及其易于被蛋白水解，未转化形式的雄激素受体的分离和特性鉴定尚未成功。在本研究中，我们通过钼酸钠并在磷酸纤维素上进行快速过滤来稳定大鼠前列腺雄激素受体。杆菌肽、抑肽酶、亮抑酶肽和苯甲基磺酰氟抑制蛋白酶。在这些条件下，通过在DEAE纤维素和磷酸纤维素凝胶上的差异色谱法，未转化的复合物被纯化了约3000倍，产率为18%。部分纯化的受体具有与粗胞质溶胶中原始未转化受体相同的离子特性；此外，通过Sephacryl S-300凝胶过滤测定，其斯托克斯半径为75 Å，沉降系数为8.8S，计算分子量为275 kDa，摩擦系数为1.6。[3H]R1881受体复合物对雄激素具有特异性，因为未标记的R1881和双氢睾酮能够完全取代结合的[3H]R1881，而雌二醇、皮质醇和曲安奈德不竞争。纯化的复合物是一种可被0.6 M KCl解离的多聚体，产生一种在蔗糖密度梯度上4S区域迁移的形式。用0.5%甲醛处理后，在含有0.6 M KCl的蔗糖密度梯度上分别得到三种形式，分别在8 - 9、5 - 6和3 - 4S区域迁移。这是朝着将未转化的雄激素受体纯化至同质迈出的第一步。
