In the preceding paper we reported the synthesis of affinity matrices containing 3,5,3'-triiodo-L-thyronine (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 (K(d) 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 T3. 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-25, the small amount of nonradioactive T3 remaining was displaced from the receptor by incubation with [125I]T3. The recovery of the receptors from the affinity gel was stimulated approximately 5-fold by the addition of purified core histones (H(2A), H(2B), 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 μg/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 K(d) of 50 pM for T3 and 1 nM for thyroxine (3,5,3',5'-tetraiodo-L-thyronine). The relative binding affinities of the affinity-purified receptor for various hormones and analogs was T3 ≥isopropyl T2 (3'-isopropyl-3,5-diiodothyronine) >thyroxine>reverse T3 (3,3',5'-triiodo-L-thyronine), which is the same as observed for whole nuclei or crude nuclear extract. The affinity-purified receptor also behaved the same as the crude receptor during chromatography on Sephadex G-100 or DEAE-Sephadex. Thus, characteristic properties of the thyroid hormone receptors are retained after affinity chromatography purification, and, importantly, the affinity-purified receptors retain the capacity for reversible hormone binding.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1981|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology