[Studies on cytosol thyroid hormone binding proteins in the rat liver: Part III. Partial purification and binding characteristics of thyroxine-binding protein in hypothyroid rat liver cytosol and serum]

Abstract

Cytosolic thyroxine-binding protein (CT4BP) was partially purified from rat liver cytosol obtained 10 days after thyroidectomy using Sephadex G-200 gel filtration, and its binding characteristics were analyzed in displacement experiments using a charcoal binding method to separate bound and free hormones. Serum T4-binding proteins were also partially purified, and their binding characteristics were similarly determined. Sephadex G-200 gel filtration of liver cytosol from thyroidectomized rats revealed that CT4BP had an apparent molecular weight of 100 X 10(3) daltons. CT4BP had a very high affinity constant (Ka) of 2.2 +/- 10(10) M-1 and a small maximum binding capacity (MBC) of 5.1 X 10(-9) g/mg. protein for T4. Relative affinities of T4 analogues for CT4BP (if the affinity of L-T4 was assigned a value of 100, then D-T4 would have a value of 25.3; L-T3, 16.6; D-T3, 2.3; reverse T3, 1.4 and both Tetrac and Triac less than 1) showed that CT4BP had a rigid specificity for alanine-side chain of T4-molecule. This CT4BP was not demonstrated when cytosol from normal rat liver was used. Sephadex G-200 gel filtration of rat serum obtained 10 days after thyroidectomy revealed two T4-binding proteins. The faster peak (Peak I; MW about 100 X 10(3) daltons) was eluted before the albumin peak, and the slower peak (Peak II; MW about 56 X 10(3) daltons) appeared after the albumin peak. Peak I was barely detectable when normal rat serum was used. Peak I had a higher Ka of 2.0 X 10(10) M-1 and a smaller MBC of 3.9 X 10(-9) g/mg. protein than Peak II (Ka; 8.9 X 10(8) M-1, MBC; 3.7 +/- 10(-7) g/mg. protein). Relative affinities of T4 analogues for Peak I (L-T4 100, D-T4, 34.9, L-T3 11.1, D-T3 1.8, reverse T3 6.8, Tetrac 0.25 and Triac 0.1) showed that Peak I had a rigid specificity to alanine-side chain of T4 molecule, but Peak II had little specificity to this side chain (L-T4 100, D-T4 9.2, L-T3 2.1, D-T3 1.0, reverse T3 14.3, Tetrac 69 and Triac 26.3). Thus, Peak I had a similar binding characteristics to those of human thyroxine-binding globulin (TBG), and Peak II was comparable to human thyroxine-binding prealbumin (TBPA). The results that both molecular weight and binding characteristics were similar between CT4BP and Peak I suggest that both proteins are identical, being comparable to human TBG. This must be clarified in future.