Affinity labeling of rat liver thyroid hormone nuclear receptor.

VM Nikodem, SY Cheng… - Proceedings of the …, 1980 - National Acad Sciences
VM Nikodem, SY Cheng, JE Rall
Proceedings of the National Academy of Sciences, 1980National Acad Sciences
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 …
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.
National Acad Sciences