Nath R, Bhakta A, Moudgil V K
Department of Biological Sciences, Oakland University, Rochester, Michigan 48309-4401.
Arch Biochem Biophys. 1992 Jan;292(1):303-10. doi: 10.1016/0003-9861(92)90083-9.
We have examined steroid binding characteristics of a newly synthesized antisteroid, ZK98299 [onapristone, 11 beta-(4-dimethylaminophenyl)-17 alpha-hydroxy-17 beta-(3-hydroxypropyl)- 13 alpha-methyl-4,9-gonadien-3-one], in the calf uterus cytosol and compared the nature of this interaction with the binding of progesterone receptor (PR) agonist R5020 [promegestone, 17,21-dimethylpregna-4,9-diene-3,20-dione]. In the freshly prepared cytosol, [3H]ZK98299 interacted specifically with a macromolecule: the binding was abolished in the presence of excess progestins (R5020 and progesterone) and the antiprogesterone ZK98299. The high affinity (Kd = 2.5 nM) interaction between [3H]ZK98299 and PR was temperature- and time-dependent, reaching an optimum by 2-3 h at 0 degrees C, and was facilitated by 20 mM Na2MoO4. Under nontransforming conditions, [3H]ZK98299-receptor complexes sedimented as 8 S species in 8-30% linear glycerol gradients. Upon salt or thermal transformation, there was a loss of the 8 S form, with only a small fraction of total complexes (5-7%) binding to DNA-cellulose. In contrast, transformed [3H]R5020-receptor complexes exhibited a greater extent of binding (25-55%) to DNA-cellulose. [3H]ZK98299-receptor complexes could be resolved into two ionic species over DEAE-Sephacel following incubation of the complexes at 0 or 23 degrees C. [3H]ZK98299 binding was sensitive to sulfhydryl group modification as beta-mercaptoethanol increased the extent of steroid binding. Although treatment with iodoacetamide (IA) abolished [3H]R5020 binding, there was a significant (nearly twofold) increase in the [3H]ZK98299 binding. The results of this study point to similarities and differences between the steroid binding properties of the uterine PR occupied by R5020 and ZK98299: both steroids appear to bind the same 8 S receptor but exhibit differential DNA binding and sensitivity to IA. The reported antagonist properties of ZK98299 may, therefore, be explained on the basis of a distinct receptor conformation induced by the antisteroid.
我们检测了一种新合成的抗类固醇药物ZK98299[奥那司酮，11β-(4-二甲基氨基苯基)-17α-羟基-17β-(3-羟丙基)-13α-甲基-4,9-孕二烯-3-酮]在小牛子宫胞质溶胶中的类固醇结合特性，并将这种相互作用的性质与孕激素受体(PR)激动剂R5020[普美孕酮，17,21-二甲基孕-4,9-二烯-3,20-二酮]的结合进行了比较。在新鲜制备的胞质溶胶中，[3H]ZK98299与一种大分子特异性相互作用：在过量孕激素(R5020和孕酮)和抗孕激素ZK98299存在的情况下，结合被消除。[3H]ZK98299与PR之间的高亲和力(Kd = 2.5 nM)相互作用是温度和时间依赖性的，在0℃下2-3小时达到最佳，并且20 mM Na2MoO4可促进这种相互作用。在非转化条件下，[3H]ZK98299-受体复合物在8-30%线性甘油梯度中以8 S形式沉降。在盐或热转化后，8 S形式消失，只有一小部分总复合物(5-7%)与DNA-纤维素结合。相比之下，转化后的[3H]R5020-受体复合物对DNA-纤维素的结合程度更高(25-55%)。在0或23℃孵育复合物后，[3H]ZK98299-受体复合物可以在DEAE-葡聚糖凝胶上分离为两种离子形式。[3H]ZK98299的结合对巯基修饰敏感，因为β-巯基乙醇增加了类固醇结合的程度。虽然用碘乙酰胺(IA)处理可消除[3H]R5020的结合，但[3H]ZK98299的结合却显著增加(近两倍)。这项研究的结果表明，R5020和ZK98299占据的子宫PR的类固醇结合特性既有相似之处，也有不同之处：两种类固醇似乎都结合相同的8 S受体，但表现出不同的DNA结合能力和对IA的敏感性。因此，ZK98299所报道的拮抗剂特性可能是基于抗类固醇诱导的独特受体构象来解释的。
