Binding Of L-triiodothyronine Research Articles

Effect of methylxanthines on binding of L-triiodothyronine (T3) to cytosolic and nuclear extract receptors of human leukocytes.

The effect of 15 mM methylxanthines (theophylline, coffeine) and 15 mM papaverine on T3 binding in the cytosol or nuclear extract receptors of human leukocytes was investigated by charcoal-dextran assay. In the cytosol, the presence of all three compounds increases the apparent association constants (KA) without affecting the total number of binding sites (N). Binding capacity was practically the same, only coffeine slightly decreases the number of binding sites. In the nuclear extract receptor protein, only theophylline increases its apparent affinity for T3.

Specific binding of L-triiodothyronine modulates Na(+)-K(+)-ATPase activity in adult rat cerebrocortical synaptosomes.

Interactions of L-triiodothyronine (T3) in adult rat cerebrocortical synaptosomes were studied in vitro. Scatchard plot analysis revealed two sets of T3 binding sites. The degree of saturation of T3 binding sites (putative receptor) correlated well with the dose-dependent inhibition of Na(+)-K(+)-ATPase activity in synaptosomes. The relative binding affinities and relative inhibition of enzyme activities for different TH analogues were L-T3 > T3-amine > TRIAC = L-T4 > r-T3 > T2 and L-T3 > T3-amine > TRIAC > L-T4 > r-T3 > T2, respectively. The present study demonstrates the nature of inhibition of synaptosomal Na(+)-K(+)-ATPase activity may be as a function of T3 occupancy of synaptosomal receptor sites in adult mammalian brain.

Triiodothyronine binding sites in the rat erythrocyte membrane: involvement in triidothyronine transport and relation to the tryptophan transport System T

The binding of l-triiodothyronine (T 3) to rat erythrocyte membranes (ghosts and peripheral protein-depleted vesicles) was studied under equilibrium conditions. Ghosts contained high-affinity T 3 binding sites whose dissociation constant (21 nM) was similar to the equilibrium-exchange Michaelis constant of T 3 transport measured in ghosts. Each ghost contained about 8·10 3 high-affinity binding sites. The high affinity T 3 binding was stereospecific and was inhibited by l-tryptophan (Trp) but not by l-leucine. The iodothyronine and amino acid specificity of binding is therefore similar to that of System T, the crythrocyte T 3/Trp transporter. These Trp-inhibitable high-affinity T 3-binding sites were also present in peripheral protein-depleted membrane vesicles, indicating that they are integral part of the membrane. Ghosts prepared from human erythrocytes, which have very low System T transport activities, contained no detectable Trp-inhibitable high-affinity T 3-binding sites. In rat erythrocyte ghosts, N-ethylmaleimide inactivated both the binding and the transport of T 3. This inactivation was blocked by T 3 and Trp with similar efficiencies. Phenylglyoxal, an arginine residue modifier, also inhibited both high-affinity T 3 binding and System T transport activity. It is concluded that the Trp-inhibitable high-affinity T 3-binding sites in the rat erythrocyte membrane are likely to be associated with System T.

Thionamides and arsenite inhibit specific T3 binding to the hepatic nuclear receptor

Methimazole (MMI) and propylthiouracil (PTU) are widely used for the treatment of Graves' disease. However, no studies have been reported on the action of these drugs on binding of L-triiodothyronine (T3) to the nuclear receptor. T3 receptors of rat liver nuclei, prepared by differential centrifugation, were extracted with 0.4 M KCl and 5 mM dithiothreitol (DTT). In the assessment of T3 binding to the DTT-reduced receptor, the hepatic nuclear extract was chromatographed on Superose 6 to remove DTT and isolate proteins of relative mass approximately 50,000 (chromatographed nuclear receptors (CNRs)), prior to the addition of [125I]T3 of high specific activity (3300 microCi/micrograms; 1 Ci = 37 GBq). MMI or PTU at 2 mM reduced specific T3 binding to CNR by 84% and 85%, respectively. The inhibitory effects of these reagents and 2 mM sodium arsenite (which complexes dithiols) were additive. Scatchard analyses indicated that neither MMI nor PTU (at 2 mM) significantly altered the affinity constant (Ka) (from 2.41 x 10(9) to 1.74 x 10(9) M-1 for PTU and 1.79 x 10(9) M-1 for MMI), while they both decreased (p less than 0.02) maximal binding capacity (from 0.36 +/- 0.02 to 0.19 +/- 0.02 pmol/mg protein for MMI and 0.17 +/- 0.02 pmol/mg protein for PTU). Dose-response curves showed that 50% inhibition was attained at 0.6 mM PTU or 1.0 mM MMI with approximately 25% inhibition by both at 0.1 mM. Artefactual binding effects by MMI and PTU on [125I]T3 were excluded by chromatography experiments. Similar results were obtained using nuclear receptors prepared from livers of hyperthyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)

High affinity L-triiodothyronine binding to right-side-out and inside-out vesicles from rat and human erythrocyte membrane

3,5,3'-Triiodo-L-thyronine (L-T3)-binding sites from rat and human red cells were characterized as to their distribution between the two surfaces of the membrane. Analysis of L-T3 binding to sealed right-side-out and inside-out vesicles from erythrocyte membrane revealed that high affinity L-T3-binding sites are located on the external side in rat erythrocytes and on the internal side in human red cells. These results were further confirmed by preincubation of intact red cells with p-chloromercuribenzoate, a slowly permeant reagent that interacts reversibly with SH groups of proteins. Following this treatment only the SH groups of L-T3 sites from rat erythrocytes were found to be blocked. Scatchard analysis of the binding data for rat right-side-out and human inside-out vesicles showed high affinity sites with Kd values of 0.2 x 10(-10) and 2 x 10(-10) M, respectively. The results suggest that the orientation of L-T3-binding sites in the erythrocyte membrane is species-dependent.

Thioredoxin and glutaredoxin enhance the binding of L-triiodothyronine to its hepatic nuclear receptors.

The influence of thioredoxin and glutaredoxin on binding of L-triiodothyronine (T3) to the rat hepatic nuclear T3 receptor was compared with that of the exogenous activator dithiothreitol. Specific [125I]T3 binding, the affinity constant, Ka, and the maximal binding capacity, MBC, were measured using whole nuclei, solubilized preparations of receptor, and chromatographed nuclear receptor. Both the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH) and the glutaredoxin system (glutaredoxin, glutathione reductase, glutathione, and NADPH) increased specific binding of T3 to nuclei, solubilized receptor, and chromatographed receptor significantly. Compared with the values obtained in the absence of added thiol (Ka = 1.6 +/- 0.1 x 10(9) M-1, MBC = 1.7 +/- 0.06 pM), the thioredoxin and glutaredoxin systems increased Ka by 147 and 112%, respectively, while decreasing MBC by 51 and 45%, respectively, when chromatographed receptor was used. The same tendency was observed with solubilized receptor. However, dithiothreitol increased Ka without affecting MBC when solubilized receptor was used. These results, the first demonstration of endogenous disulphide reductant systems enhancing binding of T3 to its receptor, suggest that the thioredoxin and (or) glutaredoxin systems may modulate the physiological effects of thyroid hormone.

Rat pancreatic nuclear thyroid hormone receptor: Characterization and postnatal development

To document the presence of 3,5,3′-triiodothyronine nuclear receptor in the rat pancreas, the solubilized pancreatic nuclear fraction was characterized for its binding to 3,5,3′-l-triiodothyronine. Binding was found to be reversible, saturable, and specific. Scatchard analysis revealed a single class of 3,5,3′-l-triiodothyronine receptors in the pancreata of adult rats. The dissociation constant was 0.29 ± 0.02 × 10−9 M, and maximal binding capacity was 242 ± 10 fmol of T3 bound per milligram of non-histone protein at 30 °C (n = 20). Both parameters are comparable with those of adult rat liver nuclei. The degree of receptor occupancy was 30%–40% of the total sites in the adult rat pancreas. Developmentally, 3,5,3′-l-triiodothyronine binding was not detectable at birth and very low in pups < 5 days old. It reached a peak at the third to fourth weeks. The degree of receptor occupancy changed with development from a low (19.2% ± 2.0%) in 5–10-day-old pups to a high (50.5% ± 3.0%) in 11–20-day-old pups, then returned to a moderate level (36.9% ± 3.0%). The dissociation constants remained unchanged at various postnatal ages. Our data suggest that the rat pancreas is a target tissue for thyroid hormone and that thyroxine plays a role in pancreatic development.

Decreased hindlimb skeletal muscle weight and nuclear T 3 receptor binding of obese (ob/ob) mice

Hindlimb muscle weights and binding of L-triiodothyronine (T 3) to isolated nuclei of hindlimb skeletal muscle were investigated in obese (ob/ob) and lean mice. At 12 weeks of age, although weighing substantially more, obese mice had only 55% as much muscle mass as their lean littermates. There was no phenotype effect observed for the equilibrium dissociation constant (Kd). However the apparent binding capacity (Bmax) was significantly less (P<0.001) for obese mice as compared to lean, thus confirming the preliminary results of Khan, et al. (7) which showed a trend for lower T 3 receptor concentration in skeletal muscle nuclei of obese mice.

Ribonucleic acid efflux from isolated mouse liver nuclei is altered by diet and genotypically determined change in nuclear envelope composition

Differencecs in immunological abnormalities like autoimmunity, abnormal T cell proliferative disorders and accelerated ageing occur between MRL/Mp-Ipr/Ipr(Ipr/Ipr) and MRL/Mp-+/+(+/+) mice as a consequence of one gene. The present study was designed to assess the effect of these differences in genotype and diet on the composition and function of the liver nuclear envelope. Mice of both stains were fed nutritionally adequate diets differing only in fatty acid composition for 4 weeks. Phospholipid fatty acid composition of the liver nuclear envelope was determined and the effect of altering the lipid composition of the nuclear membrane on nucleoside-triphosphatase (NTPase) activity, ribonucleic acid (RNA) efflux and binding of l-triiodothyronine ( l-T 3) was determined. Strain of mouse and level of dietary linoleic acid exhibited significant effects on the phospholipid fatty acid composition of the nuclear envelope. Levels of 18:1(n−9) and 18:2(n−6) were lower and 20:4(n−6) content was higher in nuclear envelope phospholipids of Ipr/Ipr mice compared with mice of the +/+ strain. Mice fed the high linoleic acid diet exhibited higher levels of 18:0, 18:2(n−6) and 20:4(n−6) and lower levels of 16:0 and 18:1(n−9) in liver nuclear envelope phospholipids, compared with mice fed the low linoleic acid diet. These changes in membrane composition were reflected in alteration of NTPase activity and efflux of RNA from isolated mouse liver nuclei. Nucleoside triphosphatase activity and efflux of ribonucleic acid from isolated nuclei were significantly higher in livers of the Ipr/Ipr strain. NTPase activity and RNA efflux from isolated nuclei were higher in the high linoleic acid fed group compared with the low linoleic acid group. A single class of binding sites for l-T 3 was present in liver nuclear envelopes of these mice and K d values were not influenced by strain or dietary linoleic acid levels. Nuclear envelopes prepared from +/+ animals exhibited a significantly higher number of binding sites for l-T 3 compared with the Ipr/Ipr group. These observations indicate that the single gene difference characterizing Ipr/Ipr mice from +/+ mice results in alterations in the composition and function of the nuclear envelope. This genetic differencec also alters the response of this membrane to dietary factors known to modulate characteristics and functions of the nuclear envelope.

Modification of l-triiodothyronine binding sites from rat erythrocyte membrane by heating and by proteinase treatments

The number of binding sites for l-triiodothyronine in rat erythrocyte membranes was increased 2-fold by incubation at 37°C for 60 min. An increase of approximately 3-fold was found when the incubation was carried out at 50°C. The proteinase inhibitor phenylmethylsulfonyl fluoride abolished the effect. Similar increments in the number of binding sites were obtained by treatment of the membranes with proteinases. The K d values (0.09 · 10 −10 M and 3.6 · 10 −10 M for the high-affinity and the low-affinity binding sites, respectively) remained unchanged after the treatment, as did the free-SH group requirements, storage stability and stereospecificity. Our results suggest that endogenous proteolytic activity could be involved in the increase of the number of membrane latent sites for l-triiodothyronine.

Solubilization of L-triiodothyronine binding site from human erythrocyte membrane

A thyroid binding peripheral membrane protein(s) has been characterized in human red cell. Two classes of affinity sites for triiodothyronine have been demonstrated. The high affinity, low capacity site showed values for dissociation constant of 2 × 10 −10M. The binding activity depended on the presence of free -SH group and showed a high stereospecificity for L-triiodothyronine, L-thyroxine was less potent (about 1,000-fold) than L-triiodothyronine in competing for this site. The results are discussed with respect to their cellular significance.

High affinity L-triiodothyronine binding sites on washed rat erythrocyte membranes.

Two orders of saturable binding sites for L-triiodothyronine were found on washed rat erythrocyte membranes. The high affinity, low capacity site showed values of Kd 0.19 X 10(-10) M. This value was in the range of concentration of free L-triiodothyronine found in the plasma and was several orders of magnitude higher than the Kd values previously reported for other L-triiodothyronine membrane-binding systems. The binding site also showed a high stereospecificity for L-triiodothyronine. D-3,5,3'-triiodothyronine and L-thyroxine were less potent (about 1000-fold) than L-triiodothyronine in competing for these sites. L-3,3,5'-triiodothyronine and triiodothyroacetic acid were inactive. The physiological relevance of this site is considered.

In vivo demonstration of the presence of specific saturable binding sites for triiodothyronine in nuclei of tadpole liver

The binding of l-triiodothyronine (T 3) by subcellular fractions of tadpole liver has been studied following in vivo administration of [ 125I]T 3. A steady state between T 3 in the nuclei and T 3 in the extranuclear fraction of liver was reached 2 hr after intraperitoneal injection of a low dose of [ 125I]T 3 into animals maintained at 25°. Saturable binding sites for T 3 were not detectable in mitochondrial and microsomal fractions but were definitely present in nuclei and possibly cytosol. As determined by a loading experiment, the maximum binding capacity of the T 3 binding sites in nuclei was 350 fmol/mg DNA or about 2700 molecules of T 3 per nucleus. Injection of a high dose of an unlabeled iodothyronine simultaneously with a tracer dose of [ 125I]T 3 indicated that 3′-isopropyldiiodothyronine competed most effectively for the nuclear T 3 binding sites followed closely by triiodothyronine and thyroxine, while 3,5-diiodothyronine had the least competitive effect. Analysis of subnuclear fractions for distribution of radioactivity, after in vivo labeling of nuclei with a small dose of [ 125I]T 3, indicated that 63% of the hormone was associated with chromatin. Extraction of nuclei with 0.8 M KCl removed about 50% of the macromolecular bound [ 125I]T 3. Chromatographic analysis of the 0.8 M KCl extract on Sephadex G-200 indicated a molecular weight in the range 70–80,000 for the T 3 binding macromolecule(s) in tadpole liver nuclei.

277 TRIIODOTHYRONINE NUCLEAR BINDING IN RABBIT LUNG AND CULTURED LUNG CELLS

To investigate the possible mechanism of thyroid hormone effects in lung development and function, we examined rabbit lung and two cell lines apparently derived from type II alveolar cells for specific nuclear binding of L-triiodothyronine (T3). Nuclei isolated from lung were incubated at 37 C for 1.5 h with various concentrations of [125I]-T3 in the presence and absence of excess non-labelled T3, and then washed in buffer with 0.2% Triton X-100. In fetal lung, the concentration of T3 binding sites and the dissociation constant (Kd) were constant between 24-30 days gestation with mean±SE values (n=17) of 0.67±0.05 fmol/μg DNA (2400 sites/cell) and 500±52 pM, respectively. Adult lung (n=3) bound 0.31±0.03 fmol T3/μg DNA (1120 sites/cell, p<.01 vs. fetal) and the Kd = 540±144 pM (NS). The L2 and A549 cell lines contained 2280 and 1580 nuclear sites/cell, and had Kd values of 200 and 280 pM, respectively. In fetal lung, the ability of analogs to compete for L-T3 binding (100%) was: 3, 5-diiodo-3′-isopropylthyronine 81%, D-T3 73%, L-T4 6.7%, L-T2 0.19%, 3, 5-dimethyl-3′-isopropylthyronine 0.15%, and reverse T3 0.08%. We conclude that rabbit lung and cultured epithelial lung cells contain nuclear binding sites with a high affinity and specificity for thyroid hormones. This suggests that both fetal and adult lung and their type II cells may be directly influenced by these hormones.

Observations Concerning the Binding of L-Triiodothyronine in the Human Polymorphonuclear Leukocyte

To examine the subcellular locus of L-triiodothyronine (T3) binding in the human polymorphonuclear leukocyte, intact leukocytes (greater than 90% polymorphonuclear) were incubated with small tracer concentrations of [125I]T3 with or without an excess of non-radioactive T3. Measurement of the [125I]T3 content of several subcellular fractions revealed that the non-radioactive T3 had led to significant displacement of [125I]T3 from the nuclear fraction alone. After correction for degradation and non-specific binding of the added T3 in intact leukocytes from hypothyroid patients in which the endogenous T3 concentration would be minimal, the T3-nuclear binding interaction was found to have an equilibrium dissociation constant of 1.1 X 10(-10)M and a binding capacity of 4.3 fmol/1 X 10(7) cells. Pre-incubation of intact cells with non-radioactive T3 did not increase the subsequent specific binding of [125I]T3 by the nuclei isolated therefrom. The data indicate that: 1) the human polymorphonuclear leukocyte possesses saturable nuclear binding sites for T3, and 2) the sites appear to bind T3 without the intermediation of a cytosol receptor.

Dose-dependent depletion of nuclear receptors by L-triiodothyronine: evidence for a role in induction of growth hormone synthesis in cultured GH1 cells.

The relationship between the binding of L-triiodothyronine (T3) to nuclear receptors and the induction of growth hormone synthesis was examined in cultured GH1 cells, a rat pituitary cell line. After 24 hr, T3 induced a maximal 4-fold increase in the rate of growth hormone wynthesis; the T3 concentration that induced a half-maximal increase was 0.22 nM. The biologic dose-response curve was shifted to the left of the receptor occupancy curve (Kd = 0.5 nM) by a factor of approximately 2 when receptor binding was examined for 4 hr but showed a significantly closer agreement when examined for the same 24-hr period as the biologic response. This shift in the fractional occupancy curve is not due to further equilibration but occurs as a result of a time- and dose-dependent depletion of the nuclear receptor by T3.

Thyroid hormone stimulates de novo growth hormone synthesis in cultured GH1 cells: evidence for the accumulation of a rate limiting RNA species in the induction process.

We have previously demonstrated with radioimmunoassay techniques that physiologic concentrations of thyroid hormone stimulate an increase in growth hormone production in GH1 cells in culture. In this study, with radiolabel techniques and selective immunoprecipitation, we demonstrate that L-triiodothyronine stimulates growth hormonw production solely by inducing an increase in the de novo synthesis of the polypeptide hormone. L-Triiodothyronine stimulated synthetic rates of growth hormone by 1.5-fold in 1.25 hr, 2-fold in 2.5 hr, to a maximal of 3- to 4-fold after 8.5 hr of incubation. The time interval between significant L-triiodothyronine binding to putative nuclear receptors and a detectable increase in growth hormone synthesis was 45-60 min. Studies on the effect of actinomycin D, 3'-deoxyadenosine, and cycloheximide support the thesis that L-triiodothyronine induces the accumulation of an RNA species which is rate limiting for growth hormone synthesis and lends further support for a primary action of thyroid hormone at the nuclear level.

Binding of thyroxine and triiodothyronine by nuclei of isolated tadpole liver cells.

The binding of L-triiodothyronine (T3) and L-thyroxine (T4) to cytoplasm and nuclei has been studied in isolated Rana catesbeiana tadpole liver cells. Nuclear binding for both thyroid hormones occurred more slowly at 4 C than at 25 C, but reached the same level as at 25 S. Scatchard analyses suggest high affinity, saturable binding sites for both hormones in the nuclear but not in the cytoplasmic fraction. Apparent equilibrium dissociation constants were 6.8 X 10(-10)M and 4.6 X 10(-10)M for T3 and T4, respectively. The maximum number of binding sites per nucleus for T3 was about 12,300 and for T4 about 2,300. Unlabeled T3 competed for the binding of [125I]T3 to nuclei more effectively than unlabeled T4. No difference in the competition of [125I]T4 binding with non-radioactive T3 or T4 was found. Chromatographic analysis of the bound nuclear radioactivity demonstrated no chemical modification for either hormone.

Binding by thyroid hormones by nuclei of cells from bullfrog tadpole tail fins.

The regression of the tadpole tail is under the direct control of the thyroid hormones and offers a unique system for the study of the action of these hormones. We have examined the binding of L-triiodothyronine (T3) and L-thyroxine (T4) in vitro using tail fin bricks which included epidermal and connective cells. The binding of 125I-labeled hormones was followed in both nuclear and extranuclear fractions. High affinity and limited capacity sites for T3 and T4 were observed only for the nuclear fraction. Scatchard plots gave similar apparent dissociation constants for both hormones, about 10(-10)M. The maximum number of binding sites per nucleus for T3 was about 1500 and for T4 about 800. There was no significant change in the chemical identity of [125I]T3 and [125I]T4 which was associated with binding in the nuclei. Nuclear binding of [125I]T4 was inhibited competitively for both unlabeled T3 and T4, but unlabeled T3 displaced [125I]T3 significantly more than unlabeled T4. Thus, both binding and competition data support the conclusion that tadpole tail nuclei had more T3 than T4 binding sites.

In vitro binding of L-triiodothyronine to receptors in rat liver nuclei. Kinectics of binding, extraction properties, and lack of requirement for cytosol proteins.

Isolated hepatic nuclei from euthyroid rats were incubated with tracer (125I)L-triiodothyronine (T3) and increasing doses of nonradioactive T3 for 30 min at 37degrees C. The T3 bound specifically to nuclear sites increased with increasing T3 doses to a plateau, which represented the nuclear binding capacity, M. Addition of 1 mM KCN, NaF, dinitrophenol, oriodoacetate did not affect nuclear binding, indicating that active metabolism was not required. Kinetic studies showed that the nuclear sites were equilibrated with T3 within 30 min of incubation (one-half maximal binding at 3 min) and that the rate of release of T3 in vitro (0.058 min-1) was the same for endogenous T3 or for T3 bound to nuclei in vitro. Nuclear T3 resisted extraction with 0.14 M NaC1 buffered at pH 7.5, but both endogenous hormone and T3 bound in vitro were readily extracted by 0.4 M KC1 at pH 8.0. The elution profiles of endogenous and in vitro-bound T3 from Sephadex G-100 columns showed a common protein peak with a molecular weight of 60-65,000, assuming a globular protein. Scatchard analysis of in vitro displacement studies showed a single class of binding sites. Mean M equals 0.23 times 10-9 M or 0.85 ng T3 for nuclei isolated from 1 g of liver. Mean M closely corresponded to that anticipated from reported in vivo studies. The apparent association constant Ka for the nuclear sites, 5.55 times 108 M-1, was lower than in studies in vivo, probably attributable to the different ionic milieu of nuclei in the incubation buffer and in the intact cell. Thus, the identity of the nuclear T3 binding sites studied in vitro to those reported for endogenous hormone is demonstrated by similar binding capacities, release rates, analogue binding affinities (previously reported), and localization to chromatin nonhistone proteins of comparable molecular weight. The role of cytosol protein in nuclear binding was assessed by comparing binding parameters for extensively washed nuclei and nuclei incubated either with contaminating or added cytosol. No difference in Ka or M was found. Moreover, it was unlikely that specific cytosol proteins were already present in nuclei and functioned during incubation as a shuttle for T3, since Ka and M for nuclei obtained from athyreotic rats were similar to Ka and M for nuclei from euthyroid animals. Thus, an initial interaction between T3 and specific cytosol proteins does not appear to be a prerequisite for translocation of T3 to nuclear sites.