I. Molecular mechanism of thyroid hormone nuclear receptor-thyroid hormone- DNA interaction. A. The effects of Zn+ 2 on the binding of 3,3',5-triiodo-L-thyronine (T3) to the purified human T3 nuclear receptor (h-TRbeta1) was studied. In the presence of Zn+2, T3 binding to h-TRbeta1 was reduced in a concentration- and temperature-dependent manner. The inhibition of T3 binding was within minutes and fully reversible. Analysis of the binding data indicated that Zn+2 is a noncompetitive inhibitor of T3 binding to h-TRbeta1. The inhibitory activity of other divalent cations was found to be in the following order: CO+2>Cd+2>Ni+2>Zn+2>Cu+2>Mn+2. However, Mg+2 and Ca+2 had no effect. The rapid and reversible effects of Zn+ 2 suggest that Zn+2 may play a role in modulating hormone binding to h-TRbeta1 in vivo. B. Three monoclonal antibodies (mAbs) recognizing different domains of h-TRbeta1 were developed. J51, J52, and J53 are specific against the beta form of thyroid hormone nuclear receptor. J51 is human specific and J52 and J53 cross reacted with rat TRbeta1 with about 1% and about 30% of the activity, respectively. The epitopes for J51 and J52 and J53 are located in the DNA binding domain, D domain and hormone binding domain, respectively. These mAbs are useful to understand the structure and the inter-relationship of domains of TRbeta1 at the molecular level. II. Regulation of thyroid hormone binding to its cytosolic binding protein (p58) by L-alpha-alanine. The human cytosolic thyroid hormone binding protein (p58) was recently shown to be a monomer of pyruvate kinase, subtype PKM2, and have intrinsic pyruvate kinase activity. The effect of L-alpha-alanine on the binding and enzymatic activity of p58 was evaluated. Analysis of the competitive binding data indicated that alanine, at the physiological concentration, is a non-competitive inhibitor of T3 binding to p58. Furthermore, alanine was found to be a """"""""mixed"""""""" inhibitor of the substrate phosphoenol pyruvate. These data suggested that alanine might play a role in the regulation of T3 binding to p58 in vivo.
