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

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).