Naess O, Hansson V, Djoeseland O, Attramadal A
Endocrinology. 1975 Dec;97(6):1355-63. doi: 10.1210/endo-97-6-1355.
The specific androgen receptors for testosterone (T) and 5alpha-dihydrotestosterone (DHT) in the cytosol fraction of the anterior pituitary of rats have been further characterized using electrophoresis and isoelectric focusing in polyacrylamide gels. After labeling of the cytosol fraction in vivo and in vitro, we were able to demonstrate androgen-protein complexes moving with an electrophoretic mobility (Rf) of 0.5 in 3.25% acrylamide gels containing 0.5% agarose and 10% glycerol. This method was used for quantitative measurements of pituitary androgen receptors, allowing multiple samples to be run simultaneously with little or no non-specific binding. There was no measurable dissociation of the androgen-receptor complexes during electrophoresis. When radioactive testosterone (1 nM) was added to pituitary cytosol fractions in vitro, there was an increase in the binding up to 4 hours of incubation at 0 C and little or no increase between 4 and 24 hours. All the binding studies therefore were done by incubation overnight at 0 C. When cytosol fractions were incubated with increasing concentrations of radioactive testosterone, a typical saturation curve was found. Scatchard plot analysis showed a binding capacity of 12.0 femtomoles/mg protein and the equilibrium constant of dissociation was estimated to be 3.4 +/- 0.7 (SD) X 10(-10)M. Like other androgen-receptor complexes, the testosterone-receptor complex in the anterior pituitary gland had an extremely slow rate of dissociation at 0 C (t1/2 greater than 4 days). The steroid specificity of the cytoplasmic androgen receptors was tested in vitro by the competing efficiency of different unlabeled steroids for [3H] testosterone binding. T and DHT caused the same inhibition of [3H]T to the receptors. However, since metabolism, of DHT to 5alpha-androstane-3alpha,17beta-diol occurred even at 0 C, the affinity of DHT for the receptor is probably somewhat underestimated. Cyproterone acetate had approximately half the affinity for the receptor compared with T, whereas lower affinities were found for progesterone and 17beta-estradiol. Cortisol did not appear to have any affinity for the receptors. Isoelectric focusing in polyacrylamide gels showed a peak of bound radioactivity with an isoelectric point of 5.8. Thus, the characteristics of the cytoplasmic androgen receptors of the anterior pituitary gland are very similar to those of the androgen receptors described in the ventral prostate, epididymis, and testis.
利用聚丙烯酰胺凝胶电泳和等电聚焦技术，对大鼠垂体前叶胞浆部分中睾酮（T）和5α - 双氢睾酮（DHT）的特异性雄激素受体进行了进一步表征。在体内和体外对胞浆部分进行标记后，我们能够在含有0.5%琼脂糖和10%甘油的3.25%丙烯酰胺凝胶中，证明雄激素 - 蛋白复合物以电泳迁移率（Rf）为0.5移动。该方法用于垂体雄激素受体的定量测量，可同时检测多个样本，且非特异性结合很少或没有。在电泳过程中，雄激素 - 受体复合物没有可测量的解离。当在体外将放射性睾酮（1 nM）添加到垂体胞浆部分时，在0℃孵育4小时内结合增加，而在4至24小时之间几乎没有增加。因此，所有结合研究均在0℃过夜孵育后进行。当用浓度不断增加的放射性睾酮孵育胞浆部分时，发现了典型的饱和曲线。Scatchard图分析显示结合容量为12.0飞摩尔/毫克蛋白，解离平衡常数估计为3.4±0.7（标准差）×10⁻¹⁰M。与其他雄激素 - 受体复合物一样，垂体前叶中的睾酮 - 受体复合物在0℃时解离速率极慢（半衰期大于4天）。通过不同未标记类固醇对[³H]睾酮结合的竞争效率，在体外测试了细胞质雄激素受体的类固醇特异性。T和DHT对受体的[³H]T抑制作用相同。然而，由于即使在0℃时DHT也会代谢为5α - 雄甾烷 - 3α,17β - 二醇，DHT对受体的亲和力可能被略微低估。醋酸环丙孕酮对受体的亲和力约为T的一半，而孕酮和17β - 雌二醇的亲和力较低。皮质醇似乎对受体没有任何亲和力。聚丙烯酰胺凝胶等电聚焦显示结合放射性的峰值，等电点为5.8。因此，垂体前叶细胞质雄激素受体的特征与腹侧前列腺、附睾和睾丸中描述的雄激素受体非常相似。
