Quantitative aspects of iodothyronine binding by cytosol proteins of rat liver and kidney

The binding of L-triiodothyronine (T₃), L-thyroxine (T₄), 3’-isopropyl-3, 5-diiodothyronine, 3, 5, 3’-triiodothyroacetic acid, 3, 3’, 5’-triiodothyronine, and sulfobromophthailine (BSP) by rat liver and T₃, T₄ and BSP by kidney cytosol was analyzed in displacement experiments using tracer ¹²⁵I-T₃, graded doses of nonradioactive ligand, and dextrancoated charcoal to separate bound and free ligands. Plots of the ratio of ¹²⁵I-T₃ bound to cytosol (bound) to the ¹²⁵I-T₃ in charcoal (free) against the concentration of cytosol (bound) ¹²⁵I-T₃ revealed two classes of T₃ binding sites: (A) a set of nonspecific sites with low affinity and apparently unlimited capacity, and (B) a set of limited capacity sites with higher affinity. In liver cytosol the equilibrium constant of the latter set was 4.3 × 10⁷M-¹ and the binding capacity was 5.3 × 10⁷M. The corresponding values in kidney cytosol were 2.5 × 10⁷M^-1 and 2.9 × 10-⁶M. Thyroxine, all of the T₃ analogues used, and BSP appeared to bind to the same sites with a lesser affinity. The observed T³ binding characteristics of cytosol protein appeared to differ markedly from those recently described for the nucleus. First, the apparent association constant of the cytosol T₃ binding sites was less than l/200th that of the nucleus. Secondly, the nuclear T₃ binding sites appear to be at least 70% saturated at endogenous T₃ concentrations in euthyroid animals, whereas less than 1% of cytosol sites are occupied by T₃. Finally, the relative strength of binding of T₃ analogues to cytosol differs markedly from the corresponding pattern at the nuclear site. These differences indicate that nuclear binding of T₃ cannot represent simple translocation of cytosol T₃ receptor sites to the nucleus. Either different sites are involved or the binding characteristics of the cytosol protein T₃ complex are markedly altered once the putative T₃ receptor enters the nucleus.