Affinity chromatography of thyroid hormone receptors. Biospecific elution from support matrices, characterization of the partially purified receptor.

Abstract

In the preceding paper we reported the synthesis of affinity matrices containing 3,5,3'-triiodo-~-thyonine (T3) Linked via its amino group and the diactivated ester of glutaric acid to the free amino groups of diaminohexane-Sepharose.This report describes the optimization of this system for the purification of the intranuclear thyroid hormone receptor.The thyroid hormone receptors (Kd for T3 -50 to 200 PM) were solubilized from nuclei obtained from rat, sheep, or steer liver.These receptors were partially purified by Sephadex G-100 chromatography and then bound to the affinity gel.After the receptor-containing gel was washed, the receptors were eluted with free TI.An exchange assay was developed for measuring the binding capacity of the eluted receptor; after most of the unlabeled T3 in the affinity-gel eluate had been removed by chromatography on Sephadex G-26, the small amount of nonradioactive Ta remaining was displaced from the receptor by incubation with ["'1]T3.The recovery of the receptors from the affinity gel was stimulated approximately 5-fold by the addition of purified core histones (HzA, HZB, H3, and H4, themselves devoid of T3-binding activity) to the wash, elution, and assay buffers, but was not enhanced by the addition of several other acidic or basic proteins.In the presence of 25 to 50 pg/ml of core histones, recovery of receptor from the gel was 10 to 35%, with a purification of more than 500-fold.Scatchard analysis of hormone binding by the affinity-purified material showed an apparent Kd of 50 p~ for Ta and 1 n M for thyroxine (3,5,3',5'-tetraiodo-~-thyronine).The relative binding affinities of the affinitypurified receptor €or various hormones and analogs