Izawa M, Satoh Y, Yoshida A, Ichii S
Endocrinol Jpn. 1985 Jun;32(3):385-97. doi: 10.1507/endocrj1954.32.385.
To elucidate the relationship between binding parameters and biopotencies of glucocorticoids, we partially purified the receptor from the liver cytosol of rats in a dexamethasone-bound and unactivated form by precipitation with protamine sulfate, gel filtration and DEAE-cellulose chromatography (approximately 100-fold) and examined the interaction of the preparation with 3 glucocorticoids of different biopotencies (dexamethasone; Dex, corticosterone; Cort and prednisolone; Pred). The partially purified receptor (PPR) was stable at -20 degrees C for at least 2 months in the presence of bovine serum albumin, glycerol, molybdate and dithiothreitol. Treatment of the PPR with p-hydroxymercuribenzoate liberated the ligands and the treated PPR reassociated 3H-glucocorticoids efficiently following the addition of dithiothreitol. The reassociated PPR was bound to the DNA-cellulose after a brief heating. Metabolic activity on ligands and inactivation of the binding sites in the PPR were insignificant under the conditions used. Kd's were approximately 0.9, approximately 3 and approximately 6 nM for Dex, Cort and Pred, respectively (at 0 degree C). Relative binding affinity of ligands to the PPR which was estimated by competitions was higher in the order of triamcinolone acetonide greater than Dex greater than Cort greater than Pred greater than progesterone greater than cortexolone. Association of Dex and Cort was relatively rapid and significantly accelerated by raising the incubation temperature, while the association of Pred was slower and effects of the temperature was moderate. The rate of dissociations was also varied with ligands. The rate of dissociation of Dex was the lowest among the 3 ligands and was elevated by raising the temperature. Because the effect of temperature was more pronounced in the dissociation than in the association, apparent Ka's decreased at higher temperature. Thermodynamic examinations of glucocorticoid binding in the PPR revealed that the binding reaction proceeds at a higher rate in the order of Dex greater than Cort greater than Pred. Because the relative biopotencies of these 3 glucocorticoids in vivo is higher in the order of Dex greater than Pred greater than Cort, from the results obtained in the present study, it appears that biopotency of glucocorticoids in vivo does not correlate with the affinity of the binding to the receptor estimated in vitro.
为了阐明糖皮质激素的结合参数与生物活性之间的关系，我们通过硫酸鱼精蛋白沉淀、凝胶过滤和DEAE -纤维素色谱法（约100倍），从大鼠肝脏胞质溶胶中以地塞米松结合且未活化的形式部分纯化了受体，并研究了该制剂与3种具有不同生物活性的糖皮质激素（地塞米松；Dex、皮质酮；Cort和泼尼松龙；Pred）的相互作用。部分纯化的受体（PPR）在牛血清白蛋白、甘油、钼酸盐和二硫苏糖醇存在下，于-20℃至少稳定2个月。用对羟基汞苯甲酸处理PPR可释放配体，加入二硫苏糖醇后，处理过的PPR能有效地重新结合³H -糖皮质激素。短暂加热后，重新结合的PPR与DNA -纤维素结合。在所使用的条件下，配体的代谢活性和PPR中结合位点的失活不明显。在0℃时，Dex、Cort和Pred的解离常数（Kd）分别约为0.9、约3和约6 nM。通过竞争估计的配体与PPR的相对结合亲和力按以下顺序更高：曲安奈德＞Dex＞Cort＞Pred＞孕酮＞皮质酮。Dex和Cort的结合相对较快，提高孵育温度可显著加速结合，而Pred的结合较慢，温度的影响适中。解离速率也因配体而异。Dex的解离速率在这3种配体中最低，且温度升高会使其升高。由于温度对解离的影响比对结合的影响更明显，因此在较高温度下表观解离常数（Ka）降低。对PPR中糖皮质激素结合的热力学研究表明，结合反应以Dex＞Cort＞Pred的顺序以更高的速率进行。由于这3种糖皮质激素在体内的相对生物活性按Dex＞Pred＞Cort的顺序更高，从本研究获得的结果来看，糖皮质激素在体内的生物活性似乎与体外估计的与受体结合的亲和力无关。
