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Physicochemical characterization of specific and nonspecific polyaromatic hydrocarbon binders in rat and mouse liver
Abstract:
The aryl hydrocarbon hydroxylase inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene, and benzo(a)pyrene were all shown to bind in a saturable manner to a distinct component in cytosol from both rat and C57BL/6J mouse liver. This component was analyzed by gel permeation chromatography on Sephacryl S-300 and by sucrose density gradient centrifugation and was found to have a Stokes radius of 61 +/- 1.5 A and a sedimentation coefficient of 4.4 S under high salt conditions. Based on these parameters, which were identical for the rat and mouse receptor, a molecular weight of 111,000 was calculated. The same Stokes radius and sedimentation coefficient were observed regardless of the ligand used for labeling of the receptor protein [(3H]TCDD, 3-[3H]methylcholanthrene or [3H]benzo(a)pyrene). On the other hand, 3-[3H]methylcholanthrene and [3H]benzo(a)-pyrene exhibited much more nonspecific binding than [3H]TCDD, at least partially due to contaminating serum components, and it cannot be excluded that some previous findings on low molecular weight hepatic "receptors" for these polyaromatic hydrocarbons may actually be explained in this way. Clearly, use of thoroughly perfused livers would seem to be a prerequisite for investigations on specific binding of aryl hydrocarbon hydroxylase inducers to liver cytosol. The rat hepatic TCDD receptor was also shown to be retained on DEAE-Sepharose (eluted at 0.2-0.3 M NaCl), hydroxylapatite (eluted at 0.15-0.17 M phosphate), and heparin-Sepharose (eluted at 0.3-0.4 M NaCl). In conclusion, the TCDD receptor showed similar physicochemical and chromatographic characteristics to those previously reported for the androgen and glucocorticoid receptors. However, ligand competition experiments indicated that the TCDD receptor is not identical to any steroid receptor. In line with this, monoclonal anti-glucocorticoid receptor-IgG antibodies did not react with the TCDD receptor.