Physical Properties of a Dog Liver and Kidney Cytosol Protein That Binds Thyroid Hormone

Abstract

Abstract An acidic protein in dog kidney and liver cytosols (105,000 x g supernatants) which binds thyroid hormone with high affinity has been examined by polyacrylamide gel electrophoresis, chromatography on Sephadex G-100, and isoelectric fractionation. The cytosol thyronine-binding protein (CTBP) binds both thyroxine and triiodothyronine, has a molecular weight of about 70,000, and is distinct from thyroxine-binding proteins in dog serum in terms of electrophoretic mobility, immunospecificity, and ability to bind congeners of thyroid hormone. CTBP appears to be qualitatively identical in kidney and liver. Scatchard analysis of thyroxine-binding in kidney cytosol, with bound/free hormone partition made by charcoal adsorption, reveals the presence of two classes of binding sites: the high affinity sites have a Kassoc thyroxine of 2 x 109 m-1 and the low affinity sites have a Kassoc of 9.2 x 107 m-1. Only a single class of triiodothyronine binding sites on CTBP has been observed and these have a relatively low Kassoc (2.3 x 107m-1). In comparison to certain receptor proteins which bind sex steroids in target organs, CTBP has a relatively high capacity for its ligand (0.15 to 0.20 nmole of thyroxine per mg of crude cytosol protein; 0.75 nmole of triiodothyronine per mg of crude cytosol protein). Binding of thyroxine by CTBP is unaffected by dexamethasone and bromosulfophthalein, but d-thyroxine, tetraiodothyroacetic acid, and tetrachlorothyronine do compete with thyroxine for sites on CTBP. Unlabeled triiodothyronine displaces radioactive thyroxine from the protein and radioactive triiodothyronine is displaced from CTBP by unlabeled thyroxine. Isoelectric fractionation of kidney cytosol containing radioactive triiodothyronine results in the emergence of radioactivity peaks at pH 3.0, 3.8, and 4.5 to 5.5, similar to those obtained with labeled thyroxine. Each peak contains other proteins in addition to CTBP; when subjected to electrophoresis or gel chromatography, each peak contains CTBP activity which is identical in terms of mobility or elution pattern with the single CTBP species observed in unfractionated cytosol. These observations are consistent with microheterogeneity of CTBP or with aggregation of CTBP molecules.