Partial purification of the triiodothyronine receptor from rat liver nuclei. Differences in the chromatographic mobility of occupied and unoccupied sites.

A 60to 125-fold purification of nuclear extracts from rat liver triiodothyronine receptor was achieved by dialysis of 0.4 M NaCl nuclear extracts followed by chromatography on DEAE-Sephadex. Extract was labeled either by injecting tracer [““Iltriiodothyronine into the animal or by direct addition of the tracer in. vitro prior to chromatography. Elution of the column with a linear NaCl gradient revealed a single peak of specific binding activity at 0.15 M NaCl and Scatchard plots of the solubilized receptors showed a single binding coinponent with an apparent K,, of approximately 2.0 x lo.-‘” M. The relative binding affinities of the solubilized receptors for triiodothyronine, thyroxine, 3,3’,5’-triiodothyronine, and 3’-isopropyl-3,5-diiodothyronine were similar to those previously observed in whole nuclei. By incubating nuclear extracts from euthyroid animals at O”, which minimizes dissociation of triiodothyronine, and at 30”, which facilitates such dissociation, it was possible to show that approximately 40% of the receptor sites were occupied. The binding capacity of extracts from hypothyroid animals was similar to that from euthyroid animals. Binding of triiodothyronine to the nuclear receptor altered the chromatographic mobility of the receptor. Occupied sites labeled with tracer triiodothyronine prior to DEAE-Sephadex chromatography eluted at 0.15 M NaCl. Unoccupied sites labeled at 0” after chromatographic separation showed a peak of specific binding activity at 0.18 M NaCl. Addition of tracer triiodothyronine to previously unlabeled chromatographic eluates which were subsequently warmed to 30” for 40 min