Dissociable and non-dissociable cytoplasmic protein-thyroid hormone interactions

Abstract

Dog kidney cytosol contains a high molecular weight (50 000–70 000) and a low molecular weight (approx. 6000) thyronine-binding protein. Low molecular weight cytosol thyronine-binding protein has not been previously recognized in cytoplasm. Binding of thyroxine (tetraiodothyronine, T 4) by the low molecular weight protein has a half-time of association of more than 24 h and accounts for 32% of bound cytoplasmic tetraiodothyronine after 48 h of incubation. Binding of labeled tetraiodothyronine and triiodothyronine by this moiety is non-dissociable in the presence of 1 · 10 −5 M unlabeled tetra- or triiodothyronine. The low molecular weight protein exists in a dispersed and apparently aggregated form; the latter elutes in the void volume on Sephadex G-100 and its generation is minimized by 2 mM Ca 2+. This binding protein elutes in a fraction which has a high A 260nm : A 280nm ratio, is pentose enriched (orcinol method) and which, because of these characteristics and low susceptibility to digestion by nuclease, is postulated to be a ribosylated cytoplasmic protein or polypeptide. Binding of tetra- and triiodothyronine by the high molecular weight protein has a half-time of association of 2 h and is saturable. Displacement of labeled triiodothyronine from this cytosol thyronine-binding protein is more readily effected with excess unlabeled tetra- than with triiodothyronine, indicating the absence of a triiodothyronine-specific cytosol thyronine-binding protein site. 3,3′,5′-Triiodothyronine (reverse triiodothyronine) is bound with low avidity. Uptake of high molecular weight protein by isolated kidney cell nuclei cannot be demonstrated. Binding of tetraiodothyronine by cytosol proteins is independent of pH in the pH range 6.8–8.9, but binding of triiodothyronine is minimized at pH 7.4 and enhanced at alkaline pH to the point of equivalency of tetra- and triiodothyronine binding at pH 8.9. At concentrations of tetraiodothyronine calculated to exist intracellularly, essentially all soluble fraction tetraiodothyronine is bound to cytosol thyronine-binding protein, restricting access of this iodothyronine to binding sites in nucleus and mitochondria. Cytosol removes labeled tetra- and triiodothyronine previously reacted in vitro with isolated cell nuclei; such removal is a linear function of cytosol protein concentration and is blocked by saturation of cytosol thyronine-binding protein with unlabeled iodothyronines. Only the high molecular weight protein accounts for unbinding by cytosol of nuclear hormone.