Production of anti-human thyroglobulin and anti-thyroid hormone antibodies in rabbits immunized with homogenate of human papillary cancer tissue

Papillary cancer tissue of the thyroid gland removed from each of three patients was homogenized in phosphate buffer followed by centrifugation. Each of three rabbits was immunized with each of the supernatants (TC-1, TC-2, TC-3). These rabbits were immunized on days 0, 7, 14, and 21, and serum from each rabbit, obtained 4 weeks after the first immunization, was examined for the presence of anti-human thyroglobulin (HTg), anti-thyroxine (T4), and anti-triiodothyronine (T3) antibodies. Production of anti-HTg antibodies was observed in all three rabbits. In addition, despite the low content of iodine, T3, and T4 in thyroglobulin that had been purified from the papillary cancer tissues (p-HTg), production of anti-T4 and anti-T3 was observed in two of the three rabbits, and the other immunized with TC-1 showed anti-T4 but no anti-T3 antibodies. The significance of the production of anti-thyroid hormone antibodies in rabbits with respect to the antigenic structure of p-HTg with low content of iodine and thyroid hormone is discussed.     

Autoimmune recognition of thyroglobulin and thyroid hormones in rabbits

Two rabbits (RG-1, RG-2) were immunized with rabbit thyroglobulin (RTg) purified from thyroid glands of four other normal rabbits of the same strain, and bled serially. Antisera were obtained at different times after the first immunization and kept separately and studied. Production of anti-RTg as well as anti-thyroid hormone antibodies such as anti-thyroxine (T4) and anti-triiodothyronine (T3) antibodies was observed in both rabbits. Physicochemical parameters of anti-RTg antibodies with RTg, T4, and T3 were calculated in two selected antisera (70-day and 253-day) of each of the rabbits, using a Scatchard plot. Extraction of serial sera from both rabbits disclosed the presence of larger amounts of T3 and T4 in immune sera than in preimmune serum. Examination of pathology of thyroid glands and kidneys in both rabbits was negative for the lesions of autoimmune thyroiditis and immune nephritis. These results indicate that anti-Tg as well as anti-thyroid hormone autoantibodies can be raised without thyroid pathology in rabbit by immunization with autologous Tg.     

Characterisation of monoclonal antibodies raised against testosterone

Using the antigens testosterone-17 beta-hemisuccinate and testosterone-3-(o-carboxymethyl) oxime, each coupled to bovine serum albumin, we have produced 44 monoclonal antibodies to testosterone. Of the 17 monoclonal antibodies raised against the 17 beta-linked antigen 8 showed extremely low affinity for testosterone (Ka less than or equal to 8 X 10(7) M-1) and none had an affinity greater than 5 X 10(9) M-1. Of the 27 monoclonal antibodies raised against the 3-linked antigen 2 had affinities less than 8 X 10(7) M, 7 had affinities greater than 5 X 10(9) M-1 and one had an affinity (Ka = 9 X 10(10) M-1) greater than that of a high affinity rabbit antiserum (Ka = 6 X 10(10) M-1). The affinity constant (Ka = 5 X 10(9) M-1) measured in the serum of the mouse whose spleen gave rise to the greatest number of high affinity antibodies, was significantly higher than those measured in the sera of the remaining mice (Ka = 0.7 - 3 X 10(8) M-1). The cross-reactions of the monoclonal antibodies varied widely but none showed an overall improvement in specificity when compared with the corresponding rabbit antisera. Results suggest that as well as the structure of the steroid antigen careful selection of the spleen donor facilitates the development of monoclonal antibodies with good binding characteristics.     

Androgen and estrogen binding in rat skeletal and perineal muscles.

Specific in vitro binding of [3H]testosterone (T), 5ALPHA[3H]dihydrotestosterone (DHT), and [3H[estradiol (E2) was demonstrated in the 30 000 X g supernatant (cytosol) of thigh muscles (TM) and of the levator ani - bulbocavernosus muscle complex (LA-BC) by gel filtration through Sephadex G-25 columns. In TM cytosol, T and E2 [are bound with high affinity (Ka = 1.1 X 10(9) M-1, and 2.3 X 10(9) M-1 respectively) whereas DHT binding is of lower affinity (Ka = 5.0 X 10(7) M-1).] In LA-BC cytosol, T, E2, and DHT are bound with high affinity (Ka = 1.9 X 10(9) M-1, 0.3 X 10(9) M-1, and 0.5 X 10(9) M-1, respectively). Competition experiments suggest that the binding of the three hormones (T, E2, and DHT) is due to different proteins. In addition to TM and LA-BC, T and E2 binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm, and heart.     

Fractionation of polyclonal antibody by isoelectric focusing and chromatofocusing: separation of high-affinity rabbit clonotype anti-thyroxine antibody

Immunoglobulin G fractions prepared from conventional rabbit anti-thyroxine (T4) antisera were fractionated by agarose gel isoelectric focusing (IEF) in the range of pH 3 to 10, and by chromatofocusing using a Fast Protein Liquid Chromatography (FPLC) system. The clonotype antibodies were recovered from the fractions and subjected to Scatchard plot analysis. The highest affinity constants of the initial antibody (shown in parentheses) and those of the antibodies recovered were IEF, 1.8 X 10(9) to 8.3 X 10(9) M-1 (2.2 X 10(9) M-1); FPLC, 2.4 X 10(9) to 6.0 X 10(9) M-1 (2.5 X 10(9) M-1). A sensitive radioimmunoassay of T4 was achieved with the isolated high-affinity anti-T4 antibody. The minimum detectable concentration of T4 was 6.3 X 10(-15) to 1.5 X 10(-14) mol/tube, which was three to five times lower than detectable with the initial antibodies.     

A human monoclonal IgG1 with anti-idiotypic activity against anti-human thyroglobulin autoantibody

Sixty-one human myeloma proteins (HMP) from patients with multiple myeloma and Waldenstr?m macroglobulinemia were tested for anti-idiotypic (Id) activity against autoantibodies to double-stranded DNA, small nuclear ribonucleoproteins, and human thyroglobulin (HTg), by competitive radioimmunoassays and enzyme immunoassays. An IgG1, lambda HMP from patient BEN with anti-Id activity against antibodies to HTg is reported. IgG1 BEN was not directed toward human Fc fragments and its activity was not related to allotypic determinants. IgG1 BEN molecules recognized Id determinants (idiotopes) on F(ab')2 anti-HTg fragments, but not idiotopes of F(ab')2 fragments of antibodies of other specificities. This observation supports the general significance of Id network interactions in regulation and diversification of immune responses in man.     

Thyroid hormone receptors in human cultured fibroblasts: evidence for cellular T4 transport and nuclear T3 binding

In cultured normal human skin fibroblasts specific and saturable binding sites for triiodothyronine (T3) have been revealed. In fact radiolabelled T3 binds rapidly to intact cells with maximum uptake after 1 hour, while nuclear binding is delayed, the equilibrium being reached after 2 hours. In intact cells it is possible to identify a single binding site for 125I-T3, with a Ka = 1.8 X 10(10)M-1 and Ro = 1.25 X 10(-11)M, similarly in nuclei it was possible to identify a single binding site of Ka = 8.8 X 10(9)M-1 and Ro = 2.3 X 10(-11)M. Intact human fibroblasts take up thyroxine (T4) even more rapidly than T3, with maximum after 5 min, showing a lower affinity for T4 than for T3 and a negligible specific and saturable binding sites for T4, the presence of a cellular transport system for T4 may be hypothesized, considering that iodothyronine cellular binding is increased by preincubation with low doses of T4.     

A case of systemic lupus erythematosus (SLE) and Sj?gren's syndrome associated with anti-T3 autoantibodies

A case of systemic lupus erythematosus (SLE) associated with Sj?gren's syndrome had extremely low serum triiodothyronine (T3) with normal levels of serum thyroxine (T4) measured by single antibody radioimmunoassays (RIAs) and thyroid stimulating hormone (TSH) during steroid treatment. Measurement of serum T3 and T4 with double antibody RIAs showed unusually high T3 and normal T4 concentrations. Examination of her serum revealed the presence of IgG class anti-T3 autoantibodies whose Scatchard plot was analyzed in two components; one with a higher associate constant (8.6 X 10(8)M-1) and a lower binding capacity (5.6 X 10(-7) mol/ml serum); the other a lower associate constant (3.5 X 10(7)M-1) and a higher binding capacity (2.1 X 10(-6) mol/ml serum). Antithyroglobulin (Tg) autoantibody has been positive throughout the seven year observation period. A significant positive correlation between titers of anti-Tg autoantibodies indicated that the antigen of anti-T3 antibodies in the patient could be T3 containing antigenic site(s) on the Tg molecule.     

Seasonal changes in oestrogen receptor affinity in the domestic rabbit, Oryctolagus cuniculus

Over the course of 1 year the Ka value of uterine oestrogen receptor for oestradiol varied 10-fold from a low of 0.259 +/- 0.065 X 10(9) M-1 in early spring to 2.21 +/- 0.21 X 10(9) M-1 in fall. There were no significant changes in receptor number. In addition, administration of oestradiol or progesterone to ovariectomized rabbits resulted in significant reductions in measureable oestrogen receptor affinity. Although these variations in oestrogen receptor Ka values parallel reproductive success in the domestic rabbit, no causal relationship has been established.     

Monoclonal antibody for calcitriol (1 alpha,25-dihydroxyvitamin D3)

Hybridoma cell lines secreting antibodies for vitamin D3 metabolites have been generated by fusing splenocytes from BALB/c mice immunized with 3 beta-glutaryl-25-hydroxyvitamin D3 conjugated to bovine serum albumin (3 beta-glu-25-OH-D3-BSA) and Sp2/O-Ag14 myeloma cells. Purification of monoclonal antibodies from culture media or ascites fluids was accomplished by procedures including affinity chromatography on Protein A-Sepharose 4B. Each monoclonal antibody was analyzed as to its affinity and specificity by equilibrium dialysis and an enzyme immunoassay (EIA) based on a double antibody system. It was demonstrated that clone 1C2-60 produced an antibody highly specific to 1 alpha,25-dihydroxyvitamin D3 (calcitriol), and the clone 2B3-66 antibody was reactive to 25-hydroxyvitamin D3 and similar structural compounds. These two monoclonal antibodies produced by 1C2-60 and 2B3-66 were determined to belong to the IgG2a class, and their affinity constants (Ka) with 3 beta-glu-25-OH-D3 were demonstrated to be 3.6 X 10(9) M-1 and 2.9 X 10(9) M-1, respectively, at 4 degrees C. The characteristics of these monoclonal antibodies were compared with those of conventional antibodies raised in mice and rabbits. Finally, by using monoclonal antibody 1C2-60, a sensitive EIA has been developed that can detect 10 pg of calcitriol.     

Rabbit mammary prolactin receptors. Demonstration of a late puerperal increase in affinity.

Crude receptor preparations of rabbit mammary gland were made by differential centrifugation and reacted with lactoperoxidase-iodinated ovine prolactin (oPRL) in order to determine their binding characteristics. Receptors prepared from the mammary glands of animals less than 4 days postpartum bound oPRL with high affinity (Ka = 3.50 X 10(9) M-1), in good agreement with previous results of other investigators. The binding capacity of these preparations was 107 +/- 16.3 fmol/mg of protein. In contrast, receptors prepared from the mammary glands of late lactating rabbits (Days 25 to 30 of lactation) showed a 2.5-fold increase in binding affinity (Ka = 8.63 X 10(9) M-1, p less than 0.001) without a significant increase in binding capacity (135 +/- 21.4 fmol/mg, p greater than 0.2). Kinetic experiments revealed that the rates of association of hormone and receptor were identical in early and late receptor preparations, and that the 2.5-fold decrease the dissociation rate observed in the late preparations was fully explanatory of the differences in equilibrium binding. The mechanism of this affinity increase is not known. Such a change in binding characteristics, which would tend to enhance tissue responsiveness, may underlie the well characterized maintenance of full lactation in women despite falling concentrations of prolactin.     

Nuclear thyroxine and triiodothyronine binding in mononuclear cells in dependence of age

Nuclear binding of thyroxine (T4) and triiodothyronine (T3) in mononuclear blood cells was investigated in 12 young (age 16-30 years) healthy subjects (group A), in 12 middle-aged (age 31-60 years) healthy subjects (group B) and in 12 elderly (61-90 years) healthy subjects. Serum free T3 was depressed in group C as compared to the younger age groups, whereas serum free T4 and TSH did not differ between the groups. Maximal specific nuclear binding capacity for both T4 and T3 decreased with increasing age, T4 group A: 1.2 fmol T4/100 micrograms DNA, group B: 1.2 fmol T4/100 micrograms DNA, group C: 0.7 fmol T4/100 micrograms DNA; T3 group A: 1.7 fmol T3/100 micrograms DNA, group B: 1.0 fmol T3/100 micrograms DNA, group C: 0.9 fmol T3/100 micrograms DNA. The equilibrium association constant (Ka) for T4 increased with age, group A: Ka = 3.3 X 10(9) l/mol, group B: Ka = 3.2 X 10(9) l/mol, group C: Ka = 6.4 X 10(9) l/mol, whereas Ka for nuclear binding of T3 decreased with age group A: Ka = 3.9 X 10(9) l/mol, group B: Ka = 5.9 X 10(9) l/mol, group C: Ka = 1.8 X 10(9) l/mol. We conclude that, whereas the opposite variations of nuclear capacity and binding affinity for T4 tend to preserve the nuclear T4 concentration, the nuclear T3 concentration definitely decreases with age. The unaltered serum levels of TSH suggest that the decrease of both serum levels of free T3 and the nuclear T3 concentration might represent physiologically changes in old age.     

Protease inhibitors from the parasitic worm Parascaris equorum

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 10(9) M-1, and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are approximately 3.3 X 10(8) M-1 (inhibitor I) and approximately 2 X 10(6) M-1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka approximately 5 X 10(7) M-1), while inhibitor II is inactive towards this enzyme.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Comparison of variable region primary structures within an anti-fluorescein idiotype family

Previous reports described the properties of a high affinity (Ka = 1.7 X 10(10) M-1) prototype anti-fluorescein monoclonal antibody 4-4-20, an intermediate affinity (Ka = 3.7 X 10(7) M-1) prototype 9-40, and Ig members of the 9-40 idiotype family (comprised of 3-24, 5-14, 5-27, 10-25 and 12-40). Although the seven monoclonal anti-fluorescein antibodies expressed similar active site structural determinants (idiotypes) as determined serologically, each was characterized by different affinities for fluorescein and fine specificity binding patterns. Partial heavy (H)- and light (L)-chain N-terminal amino acid sequence analyses revealed all antibodies (except 5-27) were composed of highly homologous VHIII(C) and V kappa II subgroup genes, respectively. Antibody 5-27 utilized a VHIII(B) and a V kappa V subgroup genes and shared low V-region sequence homology with 4-4-20, 9-40 and the remaining 9-40 idiotype family. In addition, complete 4-4-20, VH- and VL-region primary structures were determined to better understand antibody-antigen interactions. Antibody 4-4-20 utilized a VHIII(C) subgroup VH-gene, a truncated Sp2 D gene segment, JH4, a V kappa II subgroup VL-gene, and J kappa 1. Antibody 4-4-20 VH and VL complementarity-determining regions contained many basic and aromatic amino acid residues capable of interaction with fluorescein. Results are discussed in terms of idiotypic and fluorescein-binding characteristics as well as antibody structural and functional diversity in the immune response.     

Binding activities of thyroxine binding globulin versus thyroxine binding prealbumin in rat sera: differential modulation by thyroid hormone ligands, oleic acid and pharmacological drugs

We use gel equilibration and electrophoretic techniques to compare the binding properties of thyroxine binding globulin and thyroxine binding prealbumin in rat sera. The evidence indicates that TBG bears the serum lowest capacity highest affinity sites for thyroxine (T4) and triiodothyronine (T3) (Ka1 greater than or equal to 10(9) M-1) as well as weaker saturable T3 sites (Ka2 approximately 10(8) M-1). TBPA bears for T4 only Ka2 approximately 10(8) M-1 sites and for T3 only Ka approximately 10(6) M-1 sites. Consistent with these parameters are the specific responses of TBG and TBPA binding activities to varying serum concentrations of T4, T3, oleic acid, the drugs diphenylhydantoin or salicylate. The primary attack of these compounds is aimed at TBG. Small T4, oleate or DPH doses chase the TBG-bound T4 to TBPA, high doses of T4 or oleate but not of DPH inhibiting the T4 binding to both proteins. In the T3-serum interactions, all tested compounds displace the TBG-bound hormone without chasing it to TBPA. The high reactivity of TBG sites designates the protein as crucially involved in modulating the free vs bound serum levels of T4 and T3 against physiological or pathological variations of binding competitors.     

Quantitative autoradiographic determination of angiotensin-converting enzyme binding in rat pituitary and adrenal glands with 125I-351A, a specific inhibitor

We describe a quantitative autoradiographic technique which allows measurement of angiotensin-I-converting enzyme [ACE] (kininase II, peptidyldipeptide hydrolase, EC 3.4.15.1) levels in discrete areas of pituitary and adrenal glands in individual animals. Tissue sections were incubated with 125I-351A, a specific ACE inhibitor, and results were obtained with computerized densitometry and comparison to 125I standards. There were high levels of ACE in both the anterior and posterior lobes of the pituitary, with no detectable binding in the intermediate lobe. The maximum binding capacity (Bmax) was 920 +/- 62 fmol/mg protein for the anterior pituitary and 1162 +/- 67 fmol/mg protein for posterior pituitary. The binding affinity constant (Ka) was 0.95 +/- 0.11 X 10(9) M-1 and 1.20 +/- 0.19 X 10(9) M-1 for the anterior and posterior lobes, respectively. In the adrenal gland, there were two distinct areas of specific binding, the adrenal medulla and the adrenal capsule-zona glomerulosa area. The Bmax for the adrenal medulla was 652 +/- 80 fmol/mg protein and 294 +/- 53 fmol/mg protein for the adrenal capsule-zona glomerulosa. The Ka for 351A was 1.04 +/- 0.19 X 10(9) M-1 and 1.74 +/- 0.40 X 10(9) M-1 for medulla and adrenal capsule-zona glomerulosa respectively. The results support the existence of local ANG systems active in both the pituitary and adrenal glands.     

Carbohydrate-structure-dependent recognition of desialylated serum glycoproteins in the liver and leucocytes. Two complementary systems.

Oligosaccharides with four different types of branching were prepared from purified human transferrin, alpha 2-macroglobulin, caeruloplasmin and alpha 1-acid glycoprotein and labelled with NaBH3 3H. Binding of these oligosaccharides to rat liver plasma membrane, rat leucocytes, pig liver plasma membranes and pig leucocyte plasma membranes was investigated. A striking dependence of binding on oligosaccharide branching was observed. The values of apparent association constants Ka at 4 degrees C vary from 10(6) M-1 (biantennary structure) to 10(9) M-1 (tetra-antennary structure) in the liver, whereas in the leucocytes the Ka values were found to be of reversed order, from 1.8 X 10(9) M-1 for biantennary to 2.2 X 10(6) M-1 for tetra-antennary structures. The binding is completely inhibited by 150 mM-D-galactose, but 150 mM-D-mannose has almost no effect on binding. Leucocyte plasma membranes bind preferentially 125I-asialoglycoproteins with biantennary oligosaccharides, thus completing the specificity pattern of the hepatic recognition system for desialylated glycoproteins. Possible physiological roles of these two complementary recognition systems under normal and pathological conditions are discussed.     

Characterization of canine triiodothyronine (T3) autoantibodies and their effect on total T3 in canine serum

A study of 3,5,3'-L-triiodothyronine autoantibody (T3 AA) in 18 dogs revealed an average apparent affinity constant for T3 of 2.24 +/- 1.78 X 10(10) M-1, an average T3 binding capacity of 639.3 +/- 666.5 ng/dl and a low thyroxine (T4) cross-reactivity (less than 1%) in all samples tested. A valid radioimmunoassay (RIA) procedure which involved heat treatment of samples for 1 hr at 70 degrees C and assay on Sephadex minicolumns was developed for measuring T3 in the presence of T3 AA. Total T3 was elevated (mean = 374.8 +/- 158.4 ng/dl) in samples in which T4 was in the normal canine range, but T3 was lower (mean = 96.1 +/- 63.3 ng/dl) in samples with T4 values in the hypothyroid range. For each sample the concentration of T3 not bound by T3 AA was calculated from the total T3 concentration, the affinity constant, and the binding capacity. In dogs with normal total T4 concentrations the average calculated T3 not bound by T3 AA was 147.2 +/- 144.4 ng/dl while in dogs with low total T4 the value was 15.7 +/- 26.3 ng/dl (normal canine range is 45-150 ng/dl). Canine samples containing T3 AA were compared to serum from three rabbits actively immunized against T3 to provide anti-T3 for commercial RIA. The rabbit T3-antisera had an average T3 affinity constant similar to those of the canine samples (1.57 X 10(10) M-1), but had average titer, T3 binding capacity, and total T3 values more than 10-fold higher. Our findings indicate that, in dogs with serum containing T3 AA and normal total T4 concentrations, a compensatory mechanism appears to exist to maintain non-T3 AA bound T3 within the range of normal total T3. This compensatory mechanism does not operate in those dogs with insufficient thyroid activity to maintain normal total T4 values.     

3,5,3'-triiodo-L-thyronine binding sites in nuclei of human trophoblastic cells

Nuclear binding sites of T3 in human trophoblastic cells were biochemically characterized. Nuclei were isolated by a combination procedure with mild homogenization of the freshly obtained trophoblastic tissue aged term gestation, centrifugations and Triton X-100 treatment. The isolated nuclei were incubated with various concentrations of 125I-T3 at 20 degrees C for 3 h. The total number of T3 binding sites per nucleus was approximately 650. The apparent association constant (Ka) was 6.0 X 10(9)M-1. Nuclear proteins extracted from purified nuclei with 0.4M KCl were able to bind T3 giving rise to nuclear thyroid hormone binding protein-T3 complexes and they were precipitated with bovine IgG, as a carrier protein, by 12.5% polyethylene glycol. Binding was maximum in 3 h incubation at 20 degrees C or in 18 h at 0 degrees C, while it dropped quickly at 37 degrees C. The binding characteristics were analyzed by Scatchard plots. In nuclear proteins obtained from 8 term placentae there was a single set of high affinity-low capacity T3 binding sites with Ka of 7.0 X 10(9)M-1. The capacity is about 62.7 fmol T3/mg DNA. The binding sites were found to be specific for L-T3, while L-T4 was about 100-fold less effective, rT3 ineffective, and D-T3 and D-T4 were roughly 1/8 and 1/5 as active as L-T3 and L-T4, respectively in displacing 125I-T3 from the binding sites. These data confirmed that human placenta is a target organ of thyroid hormones; trophoblastic cells contain T3 nuclear receptors which are biochemically similar to those isolated from liver, although the capacity is low.