Sarup J C, Rao K V, Williams R E, Fox C F
Department of Microbiology, University of California, Los Angeles 90024-1489.
J Biol Chem. 1988 Apr 25;263(12):5624-33.
Each of four independent experimental approaches showed that human breast carcinoma T47D cells contain both high and low affinity progesterone receptors. (i) Equilibrium-specific [3H]progesterone binding to adherent cultured cells revealed dissociation constants (Kd) of 1.5 and 60 nM and 0.33 and 2.4 X 10(6) sites/cell, respectively. Both the high and low affinity receptors were specific for progestins as demonstrated by steroid binding competition studies conducted at 5 and 50 nM [3H]progesterone. (ii) Equilibrium [3H]progesterone binding to the resolved soluble and particulate fractions from a cell homogenate sedimented at 40,000 X g.min revealed Kd = 1.4 nM high affinity binding sites exclusively in the supernatant fraction and Kd = 24 nM low affinity sites exclusively in the particulate fraction. Extraction of the particulate fraction with a high ionic strength buffer solubilized the low affinity receptors stoichiometrically; but once solubilized, they displayed Kd = 2.4 nM high affinity progesterone binding. Characterizations of 3H-ligand bound specifically to progesterone receptors in intact cells or resolved subcellular fractions revealed no [3H]progesterone metabolites that could account for the low affinity binding. (iii) Calculations based on the rate constants of [3H] progesterone association with or dissociation from adherent cells revealed the same dissociation constants for both high and low affinity binding as those determined by equilibrium measurements. (iv) Nonionic detergent extraction of cells incubated with a wide range of [3H]progesterone concentrations revealed high affinity progesterone binding to receptors in the detergent-soluble fraction and low affinity binding associated primarily with the particulate residue, consistent with the data on equilibrium progesterone binding to resolved cell homogenate fractions. The rate of extraction of the high affinity receptor-progesterone complex with nonionic detergent (t1/2 = 1 min at 0 degrees C) equaled the rate of extraction of a representative lysosomal enzyme, beta-acetylglucosaminidase.
四种独立的实验方法均表明，人乳腺癌T47D细胞同时含有高亲和力和低亲和力的孕酮受体。（i）特异性[3H]孕酮与贴壁培养细胞的平衡结合显示，解离常数（Kd）分别为1.5和60 nM，以及0.33和2.4×10^6个位点/细胞。在5 nM和50 nM [3H]孕酮下进行的类固醇结合竞争研究表明，高亲和力和低亲和力受体均对孕激素具有特异性。（ii）平衡[3H]孕酮与以40,000×g.min离心沉淀的细胞匀浆中分离出的可溶性和颗粒性组分的结合显示，Kd = 1.4 nM的高亲和力结合位点仅存在于上清液组分中，而Kd = 24 nM的低亲和力位点仅存在于颗粒性组分中。用高离子强度缓冲液提取颗粒性组分可化学计量地溶解低亲和力受体；但一旦溶解，它们就显示出Kd = 2.4 nM的高亲和力孕酮结合。对完整细胞或分离的亚细胞组分中特异性结合孕酮受体的3H配体的表征显示，没有[3H]孕酮代谢产物可解释低亲和力结合。（iii）基于[3H]孕酮与贴壁细胞结合或解离的速率常数进行的计算显示，高亲和力和低亲和力结合的解离常数与通过平衡测量确定的解离常数相同。（iv）用广泛的[3H]孕酮浓度孵育细胞后进行的非离子去污剂提取显示，高亲和力孕酮与去污剂可溶组分中的受体结合，低亲和力结合主要与颗粒性残余物相关，这与平衡孕酮与分离的细胞匀浆组分结合的数据一致。用非离子去污剂提取高亲和力受体 - 孕酮复合物的速率（0℃下t1/2 = 1分钟）等于代表性溶酶体酶β-乙酰氨基葡萄糖苷酶的提取速率。
