Studies on cellular thyroxine- and triiodothyronine-binding proteins. 3. Study, using Sephadex gel filtration, on the binding of thyroid hormones to serum and soluble liver proteins and on the thyroid hormone-binding proteins of the liver in rats

Abstract

Previous reports provided the confirmatory evidences for the existence of both cellular thyroxine-binding protein (C-T4BP) and cellular triiodothyronine-binding protein (C-T3 BP) in rat liver. The present paper describes further investigation concerning C-T4BP and C-T3BP by gel filtration on Sephadex G-25 and G-100. The polysaccharide matrix of Sephadex is known to adsorb phenolic compounds such as the thyroid hormones. It would be possible to take advantage of this phenomenon in order to eliminate the thyroid hormones bound nonspecifically to the cellular proteins and, further, to compare the affinity of the hormones for various proteins.Rat liver soluble proteins containing 131I-labeled L-thyroxine (T4-131I) and L-triiodothyronine (T3_131I) were filtered through a Sephadex G-25 column and fractionated with ammonium sulfate. 131I-labeled hormones bound loosely to liver proteins were eliminated by adsorption to Sephadex gel, and this has clarified the existence of C-T4BP and C-T3BP which bound the hormones more tightly. Both the C-T4BP and the C-T3BP were found to be precipitated between 0 and 30% saturation of ammonium sulfate.Rat serum and liver soluble proteins containing T4-131I and T3-131I were fractionated by gel filtration on Sephadex G-100. Both the C-T4BP and the C-T3BP were found to exist in the protein fraction non-diffusible into the gel grains, whereas serum T4BP emerged in the tailing position of the protein peak corresponding to albumin. These studies indicate the availability for separation of C-T4BP and C-T3BP from serum T4BP and other proteins. This finding also supported the previous suggestion that rat serum T4BP might not exactly correspond to albumin, and indicated that its molecular size might be a little smaller than that of albumin.The relative affinities of the thyroid hormones for serum and liver soluble proteins were compared by the use of Sephadex. The affinity of T3 for serum was much lower than that of T4whereas the affinity of T3 for liver soluble proteins was almost equal to that of T4. This may be due, in part, to the more rapid cellular penetration of T3 compared to T4.