Triiodothyronine Binding to Isolated Liver Cell Nuclei

Abstract

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.

Relationship of receptor affinity to the modulation of thyroid hormone nuclear receptor levels and growth hormone synthesis by L-triiodothyronine and iodothyronine analogues in cultured GH1 cells.

“…These results with isolated GH, cell nuclei are similar to those previously reported with isolated rat liver nuclei (21,23). Furthermore, the affinity of triac and L-T4 as compared with L-T3 are also almost identical with isolated nuclei to that determined in the serumfree intact-cell study indicating no apparent differences in cell permeability of L-T3, triac, and L-T4.…”

“…This is based primarily on the good agreement between the relative affinity of thyroid hormone analogues for isolated liver nuclei in vitro and their reported in vivo biologic effects in terms of anti-goiter activity and 02 consumption (14)(15)(16)(17)(18)(19), and induction of a-glycerophosphate dehydrogenase in rat liver (20). Two hormonal analogues which show a higher affinity for the receptor, using isolated nuclei in vitro (21)(22)(23)) as compared with their in vivo biological activity, are triiodothyroacetic acid (triac) and D-triiodothyronine (D-T3). It has been argued that this discrepancy indicates that the cell nucleus is not the only site of action of thyroid hormone in the cell (24).…”

“…Studies with isolated liver nuclei by Koemer et al (21) and DeGroot and Torresani (23) have demonstrated that triac has a 164 (21) to 400% (23) greater affinity than L-T3, whereas the relative affinity of D-T3 was equal to (23) or 60% (21) of that of L-T3. These results differ significantly from the relative affinity determined in intact cells with 10% hypothyroid calf serum medium in which triac had an affinity which was slightly less then L-T3, and D-T3 had only 5.8% of the affinity as compared with L-T3.…”

Binding of insulin to isolated nuclei.

“…Nonspecific binding of insulin (binding in the presence of excess unlabeled insulin) ranged from 40 to 50% of total binding. Similar high ratios of nonspecific binding to total binding have been reported in studies of TS binding to nuclei prepared under similar conditions (25,26), and in studies of the binding of insulin to isolated fat cells (39). In the present investigation nonspecific binding was due in part to the trapping of insulin in the cell pellet, since either modifying the method of centrifugation or employing filtration reduced nonspecific binding to 30% of total ( Table 2).…”

The Action of Thyroid Hormone

“…This would involve promotion of DNA transcription, into the messenger and ribosomal M A S (see below). A notable difference between the binding of thyroid and steroid hormones to the nucleus is the lack of requirement of a cytosol receptor protein for thyroid hormone-nucleus interaction (DeGroot & Torresani, 1975).…”