Affinity labeling of rat liver thyroid hormone nuclear receptor.
The thyroid hormone receptor from rat liver nuclei has been covalently labeled with the N-bromoacetyl derivatives of L-thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3). Displacement binding studies showed that, in the presence of 100-fold molar excess of unlabeled N-bromoacetyl-T3 or T4, binding of [125I]T3 or [125I]T4 was nearly totally inhibited. Heat inactivation of the receptor (55 degrees C for 15 min) resulted in parallel losses in the binding of T3 (95%) and N-bromoacetyl-T3 (93%). These results indicated that T3 and T4 and their bromoacetyl derivatives compete for the same binding site. The nuclear receptor showed identical behavior in high-pressure liquid chromatography (HPLC) whether bound to T3 or T4 or covalently labeled with their bromoacetyl derivatives. HPLC provided a single-step 100-fold purification of the nuclear receptor. Na-DodSO4 gel electrophoresis of the nuclear receptor labeled with N-bromoacetyl derivatives of [125I]T3 or [125I]T4 showed one major radioactive component with a molecular weight of 56,000. Furthermore, in the absence of denaturant, the nuclear receptor either bound to [125I]T3 or covalently labeled with N-bromoacetyl-[125I]T3 showed identical mobility. These results suggested that the nuclear receptor is a single polypeptide chain and binds either T3 or T4. Nuclear receptors covalently linked with N-bromoacetyl derivatives of [125I]T3 or [125I]T4 may be useful as a marker for the preparative purification of receptor.
D000345:Affinity Labels; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone; D013974:Thyroxine / Q000031:analogs & derivatives / Q000378:metabolism; D014284:Triiodothyronine / Q000031:analogs & derivatives / Q000378:metabolism
D000345:Affinity Labels; D011956:Receptors, Cell Surface; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D013974:Thyroxine