[^3H]Cortisol及[^3H]Dexamethasone在大鼠肝细胞膜上的特异结合位点

SPECIFIC BINDING SITES OF [￣3H] CORTISOL AND [￣3H] DEXAMETHASONE IN RAT LIVER CELL MEMBRANE

本研究应用［３Ｈ］ｃｏｒｔｉｓｏｌ和［３Ｈ］ｄｅｘａｍｅｔｈａｓｏｎｅ（ＤＥＸ）两种配基，观察到大鼠肝细胞膜上存在一类糖皮质激素（ＧＣ）特异结合位点。这些位点与ＧＣ的结合具有饱和性、高亲和力及低容量。其平衡解离常数（Ｋｄ）分别为１２．８４±６．５８ｎｍｏｌ／Ｌ和４０．２７±２３．４４ｎｍｏｌ／Ｌ；最大结合容量（Ｂｍａｘ）分别为２．５７±１．８４ｐｍｏｌ／ｍｇ蛋白质与０．６４±０．１８ｐｍｏｌ／ｍｇ蛋白质（ｃｏｒｔｉｓｏｌ，ｎ＝４；ＤＥＸ，ｎ＝３；±ＳＥ）。动力学实验数据所得的Ｋｄ值与Ｓｃａｔｃｈａｒｄ分析所得的Ｋｄ值结果基本一致。［３Ｈ］ｃｏｒｔｉｓｏｌ和［３Ｈ］ＤＥＸ饱和结合实验数据用Ｓｃａｔｃｈａｒｄ作图分析，均显示为直线。Ｈｉｌｌ系数则分别为０．９８８０和０．９９９０．竞争抑制实验结果表明，ｃｏｒｔｉｓｏｌ对［３Ｈ］ｃｏｒｔｉｓｏｌ的结合位点有高度特异性竞争，比其它几种类固醇（强的松、黄体酮、ＲＵ４８６、ＤＥＸ）的竞争力至少强４０倍以上．用放射自显影技术进行研究，也提供了［３Ｈ］ｃｏｒｔｉｓｏｌ特异结合银粒位于大鼠肝细胞膜上的依据。

By using [3H] cortisol and [3H] dexamathasone (DEX), a class of specific binding sites of glucocorticoids (GC) in rat liver cell membrane was observed. These binding sites had saturability, high affinity and low capacity. The kinetic parameters of [3H] cortisol and [3H] DEX were: Kd=12. 84±6. 58 nmol/L (n= 4) and 40. 27±23. 44 nmol/L (n=3) respectively; Bmax= 2. 57±1. 84 pmol/mg protein and 0. 64±0. 18 pmol/mg protein respectively (±SE). The Kd obtained from the kinetic studies was corresponding to the results of Scatchard analysis. Scatchard plots from the equilibrium experiments showed the straight line. The Hill coefficients were 0. 9880 and 0. 9990 respectively. The competitive displacement experiments indicated that cortisol possessed the higher specificity for the [3H] cortisol binding sites than other tested steroids (prednisolone, progesterone, RU486, DEX). The competitive ability of cortisol was at least 40 folds higher than mentioned steroids. By means of autoradiography technique, the existence of [3H] cortisol labelled particles on the liver cell membrane was found.