The putative thyroid hormone receptor(s) from rat liver nuclei could be covalently photolabeled with underivatized hormone. When 0.4 M NaC1 extract from purified nuclei is labeled, two major binding proteins (57 and 46 kDa) are resolved on SDS gel. However, radioactivity ratios vary considerably, ranging from predominance to absence of 57 kDa protein. Limited proteolysis of these two major binding proteins yielded virtually identical patterns. This is indicative of a high sensitivity of 57 kDa protein to breakdown in the course of sample preparation. To eliminate such a possible breakdown, we introduced the following modifications to our previously used procedure: 1) use of glycerol in all buffers; 2) increase of Mg2+ concentration; 3) reduction of irradiation time from 30 min to 1 min; 4) introduction of an acetone precipitation of labeled nuclear proteins. These modifications resulted in incorporation of radioactivity in the range of 57 kDa protein (resolved as a doublet) while incorporation in the 46 kDa protein became minimal. Thus, it appears that a high concentration of glycerol, magnesium and a shorter time of irradiation stabilize the doublet (60, 57 kDa). The presence of this doublet was also detected in nuclear extracts from heart and brain while testis and spleen showed only a trace of it. This is in agreement with the absence of high affinity binding sites for thyroid hormone in testis and spleen as determined by Scatchard analysis. Hence, the endogenous thyroid hormone receptor has a molecular weight of about 60 kDa. Furthermore, preliminary results showed that the high molecular weight thyroid hormone binding protein binds to calf thymus DNA and is eluted by a relatively high concentration of salt.
