Triiodothyronine binding to liver nuclear solubilized proteins in vitro.
Nuclear proteins extracted from purified nuclei with 0.4M KCl at pH 7.4 OR 8.5 are able to bind L-triiodothyronine (T3) giving rise to nuclear thyroid hormone binding protein-T3 (NTBP-T3) complexes. Binding is maximum in 3 h at 20 C. It is thermolabile even at 36 C, inhibited by p-hydroxymercuribenzoate and markedly enhanced by dithiothreitol. Optimum pH is between 7.8 and 8.5. Divalent cations are not necessary. The NTBP-T3 complex exhibits similar anodal electrophoretic migration in polyacrylamide gel at pH 8.5, whether formed in vivo or in vitro. Scatchard plots obtained with various amounts of T3 from 0.15 nM TO 0.15 MUM and either unlabeled nuclear proteins or in vivo formed NTBP-[125I]-T3 complexes, give apparent association constants K-a of 0.2 X 10-10 M minus at pH 7.4 and 0.8 X 10-10 M minus 1 at pH 8.5. Capacity is about 0.5 pmol T3 per mg protein or 800 pg/g liver. The presence of dithiothreitol markedly enhances the Ka. The nuclear binding sites are not highly specific for L-T3 since they are able to bind D-T3 with almost equal affinity and triiodothyroacetic acid with a higher affinity. L-thyroxine (T4) can also displace L-T3 but with about 10-fold lesser effectiveness. Nuclear binding proteins of low capacity and high affinity have been demonstrated in vitro. The NTBP-T3 complexes formed in vivo, with whole nuclei, or in vitro are indistinguishable.
D000818:Animals; D001665:Binding Sites; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008626:Mercuribenzoates / Q000494:pharmacology; D007470:Monoiodotyrosine / Q000031:analogs & derivatives; D009698:Nucleoproteins / Q000378:metabolism*; D051381:Rats; D013974:Thyroxine / Q000378:metabolism; D013997:Time Factors; D014284:Triiodothyronine / Q000378:metabolism*
D008626:Mercuribenzoates; D009698:Nucleoproteins; D014284:Triiodothyronine; D007470:Monoiodotyrosine; D013974:Thyroxine; D004229:Dithiothreitol