Triiodothyronine binding to isolated liver cell nuclei.
Nuclei of euthyroid rat liver have been prepared from homogenates by sedimentation through 2.3M sucrose with or without a 0.25% Triton wash. Triiodothyronine is accumulated by these nuclei during incubation in vitro in solutions containing 0.32M sucrose, 1mM MgCl2 and 0.02M Tris-Cl buffer at pH 7.4 or 7.85. Specific T3 binding sites occupied at 10-1,000 pM T3 are saturated by excess unlabeled T3 (0.15 muM). Specific T3 binding at 20 C is maximal at 203 hr nad is proportional to amount of nuclei. Calcium ion enhances nuclear integrity by reduces T3 accumulation. EDTA and phosphate ion cause nuclear damage but increase T3 accumulation. Binding is unaffected by inhibition of energy dependent reactions or of RNA synthesis. It is markedly increased under certain conditions by addition of dithiothreitol (DDT). Binding does not require mediation of a cytosol protein. T3 binding is not prevented by RNAse or DNAse, but is obliterated by pronase. Te binds to a nuclear iodothyronine binding protein (NTBP) to form an NTBP-T3 complex similar to that form-d after in vivo administration of the hormone. The complex can be extracted from the nuclei by 0.4M KC-. T3 present in the NTBP-T3 complex resists accumulation by anion exchange resin at 0-2C, but is bound by resin after 20 min at 37C or after addition of 0.1 mM p-hydroxymercuribenzoate. At pH 7.85 and with 5 mM DTT, the apparent Ka for isolated nuclei is 0.2 times 10-10M-1 and the capacity is 508 pg T3/g wet tissue or 53 times 10-15 moles T3/100 mug DNA. The data may not represent total capacity, but rather the amount of T3 dissociated during the period of incubation so that NTBP-T3 can be exchanged with labeled T3. Among analogues tested, triiodothyroacetic acid appears to bind with four times the affinity of L-TX, D-T3 binds with equal affinity, and L-T4 with one-fourth the affinity of T3.
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000494:pharmacology; D004226:Dithioerythritol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D007470:Monoiodotyrosine; D009698:Nucleoproteins / Q000096:biosynthesis; D010710:Phosphates / Q000494:pharmacology; D011402:Pronase / Q000494:pharmacology; D011485:Protein Binding; D012313:RNA / Q000096:biosynthesis; D051381:Rats; D011956:Receptors, Cell Surface; D012260:Ribonucleases / Q000494:pharmacology; D013974:Thyroxine / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism*
D009698:Nucleoproteins; D010710:Phosphates; D011956:Receptors, Cell Surface; D014284:Triiodothyronine; D012313:RNA; D004226:Dithioerythritol; D004492:Edetic Acid; D004254:DNA Nucleotidyltransferases; D012260:Ribonucleases; D011402:Pronase; D007470:Monoiodotyrosine; D013974:Thyroxine; D002118:Calcium