A hydroxylapatite microassay for receptor binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene in various target tissues

Abstract

A "batch" hydroxylapatite assay for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor that does not require detergents is described. The receptor could be assayed in rat target tissues using either of the cytochrome P1-450 inducers [3H]TCDD or [3H]3-methylcholanthrene as radioligands. A phosphate buffer washing procedure was developed on the basis of chromatographic data and optimized to separate nonspecifically and specifically bound ligand. The assay was characterized with respect to washing efficiency, binding specificity, competition, adsorption time, amount of hydroxylapatite required to bind receptor complexes, sensitivity, and effects of detergents. Equilibrium binding parameters were determined. Receptor extracted with phosphate from hydroxylapatite was analyzed on sucrose gradients and was found to exhibit the same sedimentation properties as the receptor in crude cytosol. Furthermore, the applicability of the assay has been demonstrated in cytosolic preparations from three different target tissues: liver, lung, and thymus.