Characterization of the in vitro stability of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Abstract

The in vitro stability of the Ah receptor from rat hepatic cytosol was evaluated by [³H]TCDD binding studies, gel filtration, and sucrose density gradient ultracentrifugation. Thermal inactivation of unoccupied receptor followed first-order kinetics between 5 and 40 °C, with an estimated E_a for inactivation of ~35 kcal/mol. Protease inhibitors did not reduce and dilution slightly increased the inactivation rate at 20 °C. Recovery and 20 °C stability decreased with increasing ionic strength. The TCDD-receptor complex was less susceptible to degradation at 20 °C, even in the presence of 0.4 m KCl. Specific binding was markedly pH dependent, with maximum recovery at 7.6. Analysis of the pH curve suggested that cysteine sulfhydryl groups may be involved in TCDD binding. Dithiothreitol (1 mm) maximized recovery and 20 °C stability, and addition of the thiol largely reactivated binding sites lost from cytosol prepared without it. Removal of low molecular weight components of cytosol by gel filtration resulted in a rapid 20 °C inactivation rate that could not be lessened by dithiothreitol. Glycerol (10% $\text{v}\text{v}$) and EDTA (1.5 mm) maximized recovery of specific binding, but both decreased 20 °C stability in a concentration-dependent manner. Calcium chloride (4 mm) increased stability at 20 °C by ~20%, and retarded the characteristic shift in sedimentation coefficient from ~9 to ~6 S in high-salt sucrose gradients. The fact that sodium molybdate (20 mm) decreased recovery and 20 °C stability when dithiothreitol was present but slightly increased stability in its absence suggested an antagonism between the two compounds. Molybdate mitigated the inactivation induced by 0.4 m KCl, an effect which may be related to the observation of dual peaks in molybdate-containing high-salt sucrose gradients. These data indicate that (i) thermal inactivation of the unoccupied rat hepatic Ah receptor primarily may be due to physical rather than enzymatic processes; (ii) sulfhydryl oxidation, removal of low molecular weight cytosolic components, and high ionic strength result in rapid rates of inactivation at 20 °C; and (iii) the large degree of protection conferred by TCDD binding implies a very tight ligand-receptor interaction, and as such accords with TCDDs extraordinary potency and persistence in producing its toxic and biochemical effects.