TY - JOUR
T1 - A hydroxylapatite microassay for receptor binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene in various target tissues
AU - Poellinger, Lorenz
AU - Lund, Johan
AU - Dahlberg, Erik
AU - Gustafsson, Jan Åke
PY - 1985/2/1
Y1 - 1985/2/1
N2 - 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.
AB - 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.
KW - 2,3,7,8-tetrachlorodibenzo-p-dioxin
KW - 3-methylcholanthrene
KW - cytochrome P-450
KW - hydroxylapatite
KW - polycyclic aromatic hydrocarbons
KW - TCDD receptor
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U2 - 10.1016/0003-2697(85)90130-7
DO - 10.1016/0003-2697(85)90130-7
M3 - Article
C2 - 2986476
AN - SCOPUS:0021928218
SN - 0003-2697
VL - 144
SP - 371
EP - 384
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -