Preparation of unoccupied thyroid-hormone receptor.
Thyroid hormone (3,5,3'-tri-iodothyronine; T3) regulates gene expression through binding to its specific receptor in the nucleus. In euthyroid animals, roughly half of all receptors are occupied by the hormone. Nuclear extracts thus yield mixtures of occupied and unoccupied receptors. We present here a simple method for transforming occupied receptors into unoccupied ones. In vitro, the T3-receptor complex dissociated in a half-dissociation time exceeding 100 h at 0 degrees C, and at temperatures that accelerated the dissociation the receptor was quickly inactivated. Long-chain-fatty-acyl-CoAs, on the other hand, greatly accelerated the dissociation of T3-receptor complex at 0 degree C. The receptor was extracted from rat liver nuclei, incubated with oleoyl-CoA to release the bound hormone, and passed through a small column of Lipidex, which strongly adsorbed both oleoyl-CoA and the dissociated hormone. The receptor was recovered in the flow-through fraction in its unoccupied form, as seen by the results of DEAE-Sephadex column chromatography and the loss of all previously bound [125I]T3. The maximum T3-binding capacity of the unoccupied receptor was about 1.5-fold that of the untreated sample, and the dissociation constant was unaltered. The results suggest that most nuclear thyroid-hormone receptors occupied by the hormone were transformed into unoccupied ones. From the T3-binding capacity before and after oleoyl-CoA treatment, the in vivo T3 occupancy of the receptor was estimated. The procedure is easy to perform, and the method should be useful for studies of unoccupied receptors.
Li
Q
Q
Department of Biochemistry, Osaka City University Medical School, Japan.
Inoue
A
A
D000214:Acyl Coenzyme A / Q000378:metabolism; D000327:Adsorption; D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000737:chemistry; D003911:Dextrans; D008099:Liver / Q000737:chemistry* / Q000648:ultrastructure; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000302:isolation & purification* / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism
D000214:Acyl Coenzyme A; D003911:Dextrans; D011988:Receptors, Thyroid Hormone; C010599:lipidex; D014284:Triiodothyronine; C017585:oleoyl-coenzyme A