Search

The structural characteristics and topography of the thyroxine T4-binding site on the human immunoglobulin M (IgM) molecule have been studied using affinity binding with T4 structural analogs blocking the formation of the T4-IgM complex with proteins displaying an affinity for individual structural components of IgM and proteolytic fragmentation followed by determination of the T4-binding activity of the isolated fragments. It has been found that IgM has a poor selectivity towards the binding of T4 structural analogs which is characteristic of transport proteins but not of autoantibodies. The T4-binding region of IgM lies outside the variable portion of the Fab-fragment and is apparently constituted by the Cmu 1-domain of the heavy chain and the constant part of the light chain linked via a disulphide bridge. The T4-binding site located within this region possesses no stereospecificity and contains structural elements complementary to the iodine atoms of the outer ring and the aliphatic chain of the iodothyronine molecule.

The kinetic and equilibrium characteristics of the interaction of thyroxine (T4) with immunoglobulins (Ig) A, G and M as well as with Bence-Jones proteins purified from human blood serum have been investigated. The formation of complexes between T4 and human immunoglobulins has been found to be time-dependent, reversible, saturable and sensitive to specific inhibitors. The L-chain (ae or lambda) is a component of the immunoglobulin molecular structure which appears to be essential and sufficient for the T4 binding. The covalent attachment of the H-chain can increase dramatically the affinity for the thyroid hormone (the mu-chain in IgM) or alter the sensitivity of the binding region to chemical agents and pH (the mu-chain in IgM, the gamma-chain in IgG). The experimental data suggest that the T4-binding IgM does not belong to a pathological type of proteins- anti-T4 autoantibodies-because: (i) the dependence of the T4 binding reaction on the physico-chemical conditions of the environment is typical of normal transport proteins; (ii) the prevalence of the T4-binding IgM in random individual serum samples from healthy subjects is 100%; (iii) the IgM-T4 complex differs structurally from the antigen-antibody complex, being unable to interact with the first complement component. The specific T4-binding properties of normal human serum immunoglobulins could remain so far unrecognized due to the inability of the conventional analytical methods to detect the weak manifestations of the T4-binding activity of these proteins in physiological fluids containing the endogenous inhibitor (Cl-), and/or the exogenous inhibitor (8-anilino-1-naphthalene sulphonic acid).

Binding processes in in vitro systems modelling specific interactions between transport and receptor proteins, thyroid hormones of human placental tissue and the washing blood, have been studied. These systems included syncytiotrophoblast villous membranes, thyroxine (T4), triiodothyronine (T3) and iodothyronine-binding proteins: transthyretin, albumin, immunoglobulins (Ig) M and G, apolipoprotein A-I, and the T4-binding globulin purified from human retroplacental serum. All of the transport proteins at concentrations close to the Ka values of their complexes with thyroid hormones produced inhibitory effects on the binding of [125I]T3 or [125I]T4 to the thyroid hormone membrane receptor. The dependence of [125I]T4 membrane binding on IgM concentration in the system was characteristic of all proteins studied. In the case of T3 such dependence was unique for IgM, and included the phase of the IgM stimulatory effect (10(-11)-10(-9) M) and the phase of inhibition (10(-8)-10(-7) M). In the presence of 30 pM IgM, the concentration of the membrane T3-binding sites increased by 75% with a 2.2-fold decrease of the association constant (Ka). It was shown that IgM interacts specifically with two classes of binding sites on the plasma membranes with Ka(1) = 5.0 x 10(9) M-1, Bmax(1) = 34 fmol/mg of membrane protein and Ka(2) = 2.7 x 10(7) M-1, Bmax(2) = 2.0 pmol/mg of membrane protein. It is suggested that the stimulatory effect of IgM is caused by increases in the number of T3-binding sites on the placental villous membranes as a result of complex formation between IgM and its membrane receptor which displays an enhanced T3-binding activity.

Conjugates of thyroxine (T4) and triiodothyronine (T3) with rhodamine B in which the hormone and the fluorescent dye are linked via a thiourea bond have been synthesized. These conjugates possess an ability to inhibit in a competitive manner the binding of [125I]T4 to three protein preparations: T4-binding globulin (TBG), apolipoprotein A-I (ApoA-I), and high density lipoprotein particles (ApoA-I-HDL) isolated from human serum by T4-Sepharose 4B chromatography and further purified. The following values of association constants have been estimated: for the T4 derivative-3 x 10(7) M-1 (TBG), 4.1 x 10(5) M-1 (ApoA-I), and 4.2 x 10(5) M-1 (ApoA-I-HDL); for the T3 derivative-1.6 x 10(7) M-1 (TBG), 5.3 x 10(5) M-1 (ApoA-I), and 5.4 x 10(5) M-1 (ApoA-I-HDL). The binding of rhodamine B-labeled thyroid hormones to TBG or ApoA-I do not alter significantly the parameters of rhodamine B chromophore absorption and fluorescence. The interaction of the conjugates with ApoI-HDL leads to a significant enhancement of the absorption intensity and a 3 nm blue shift in the absorption maximum as well as to a 1.5-fold increase in the fluorescence band amplitude at 586 nm. Biological and fluorescent properties of T4 and T3 derivatives suggest that these compounds may be a useful tool in fluorescence studies of plasma binding protein-driven transport of thyroid hormones in model biological systems.

A protein having a molecular mass of about 25 kWa was isolated by thyroxin (T4)-Sepharose affinity chromatography from human blood serum; its properties were found to be distinct from those of known T4-binding proteins. Using immunodiffusion, radioimmunoassay, lipid analysis, differential precipitation and electrophoresis, it was shown that the isolated protein is a component of high density lipoprotein (HDL) particles and represents an apolipoprotein A-1 (apoA-1). Using cholate-Sepharose chromatography apoA-1 was separated from the lipid moiety and contaminant proteins, and apoA-1 was effectively isolated directly from the blood serum. Apo-A-1-HDL and apoA-1 obtained by affinity chromatography as well as the HDL3 fraction isolated by a standard ultracentrifugation technique, all displayed a T4-binding activity, the affinities for the hormones being of the same order of magnitude. The T4 interaction with these preparations induced difference UV-absorption signals, altered the characteristics of apoA-1 intrinsic fluorescence without affecting the circular dichroism of the protein-hormone system. The binding of spin-labelled T4 to apo-1, apoA-1-HDL or HDI3 caused substantial changes in the shape of the ESR spectrum and an increase in the apparent rotational correlation time. The mobility of the radical fragment of spin-labelled T4 depended on the composition and properties of the protein preparation. The electron spectroscopy data suggest that the T4-HDL interaction occurs via specific mechanisms and that the molecular structures of the complexes formed thereby are not characteristic of other known T4-binding proteins.

A molecular variant of human serum thyroxine-binding globulin (TBG), containing only triantennary oligosaccharide chains and having a more prolonged in vivo survival (TBG-1), was first detected in normal pregnancy and then in the postpartum period. Serum TBG-1 was measured in normal and in some pathological conditions associated with normal or increased TBG biosynthesis using a combination of Con A-Sepharose 4B microcolumn affinity chromatography and a highly sensitive TBG radioimmunoassay. TBG-1 was shown to be present in the sera of healthy subjects (0.21 +/- 0.04 micrograms/ml, n = 60; 1.15% of total TBG). The proportion of TBG-1 in total serum TBG was significantly increased (up to 9.5%) in conditions with serum TBG excess (cancer, hypothyroidism and liver diseases). It has been assumed that a selective enhancement of biosynthesis of the TBG molecular variant with a prolonged half-life in the circulation during the posttranslational modification of the polypeptide chain is a response to the body requirements in a high TBG concentration.

The kinetic and equilibrium characteristics of interaction of thyroxine (T4) and its structural analogs with a high density lipoprotein (HDL) fraction isolated from human serum by T4-Sepharose affinity chromatography and containing apolipoprotein A-I (apo A-I) as a sole protein component, were studied. The binding of [125I]T4 to apo A-I-HDL reached a maximum after 40 min and did not change during the next 80 min of incubation at 0 degrees--22 degrees C. Dissociation of [125I]T4 induced by the addition of excess unlabeled T4 to the complex solution proceeded more intensely on a time scale at 0--2 degrees C than at 22 degrees C. Incubation of apo A-I-HDL with increasing concentrations of T4 showed that the binding is saturable. The data analysis using different computer programs revealed the presence in apo A-I-HDL of a single class of binding sites with K alpha = (4.0 +/- 2.1).10(-7) M- and Bmax = 1.7 +/- 0.8 nmol T4/mg of protein. Naturally occurring iodothyronines, their analogs and D-isomers of thyroid hormones competed with [125I]T4 for the binding sites on apo A-I-HDL with the following inhibitory potencies: L-T4 = D-T4 greater than or equal to 3,3',5-triiodo-L-thyronine = 3,3',5-triiodo-D-thyronine greater than 3,5-diiodo-L-thyronine = 3,3',5- triiodothyroacetic acid greater than 3,3',5-triiodothyropropionic acid greater than or equal to 3,5-diiodo-L-thyrosine.(ABSTRACT TRUNCATED AT 250 WORDS)

The serum concentrations of the total thyroxine-binding globulin (TBGt), its pregnancy-associated molecular variant (TBG-1), thyroxine, progesterone, estradiol, and prolactin were measured by radioimmunoassay in the course of the postpartum period (0-40 days) in women. High serum TBG-1 concentrations (an average of 36% of the level observed at the time of delivery) were unexpectedly detected in the late postpartum period (days 36-40) when the concentrations of TBGt, thyroxine, estradiol and progesterone were within normal. The presence of TBG-1 in the maternal blood for a long time after delivery may be caused by estrogen-dependent synthesis and secretion rather than by its slow clearance from the blood.

Kinetic and equilibrium characteristics of the interaction of triiodothyronine (T3) and its structural analogues with plasmatic membranes of human placental syncytiotrophoblast were studied. The specific binding was temperature-dependent, saturable, reversible, selective and stereospecific. The following values of the binding parameters were obtained: association rate constant--1.0 x 10(10) M-1min-1, dissociation rate constant (fast phase)--2.4 x 10(-1) min-1, half-life time of the complex--2.9 min, equilibrium association constants for high- and low-affinity sites--2.0 x 10(10) M-1 and 4.8 x 10(6) M-1, respectively, concentrations of the corresponding sites--12.5 fmol and 36.0 pmol per mg of membrane protein, respectively. The affinity for membranes decreased in the following order: L = T3 greater than L = T4 greater than 3,3,5-triiodothyroacetic acid greater than 3,3,5-triiodothyropropionic acid greater than D = T3 greater than D = T4 greater than 3,5-diiodo-L-tyrosine.

The affinity matrix prepared by the attachment of L-thyroxine (T4) to epichlorohydrine-activated Sepharose 4B biospecifically absorbs the T4-binding globulin (TBG), 25K and 80/27K proteins, immunoglobulin G (IgG) and albumin (HSA) from human normal and retroplacental sera. The absorbed protein patterns were shown to depend on the immobilized T4 concentration, pH, temperature and incubation time. The potent eluents desorbing 85-100% of the protein are 1 mM NaOH, 3 M NH4SCN, 10(-5) M T4 or 3 mM 8-anilinonaphthalene-1-sulfonic acid (ANS) for TBG; NaOH, NH4SCN, 3 mM MgCl2 or 12mM sodium cholate for 25K protein and HSA; NaOH, NH4SCN or MgCl2 for the 80/27K and 25K proteins and IgG. Moreover, T4 desorbs small amounts (6-8%) of the 80/27K and 25K proteins, while sodium cholate elutes about 6% of TBG. The eluted from T4-Sepharose 4B and further purified TBG, 25K and 80/27K proteins display different [125I]T4-binding activities within the pH range from 2 to 9 and differ by their resistance to thermal inactivation at 50-80 degrees C. Double radial immunodiffusion analysis with the use of antisera to TBG, 25K, 80/27K, HSA and IgG demonstrated that the proteins share no common antigenic determinants. It was concluded that the novel 25K and 80/27K proteins represent endogenous components of the human blood thyroid hormone-binding protein system rather than fragments or aggregates of the known T4-binding proteins.

Two molecular variants, of thyroxin-binding globulin (TBG), TBG-1 and TBG-2, were obtained from human retroplacental blood by fractionation of pure TBG on concanavalin A-Sepharose. It was found that both variants are immunologically identical, have similar molecular weights, amino acid composition and spectral properties, and possess the same affinity for thyroid hormones. However, TBG-1 and TBG-2 differed in charge upon isoelectrofocusing and had different monosaccharide composition. The existence of two molecular variants of TBG in pregnancy is probably due to the peculiarities of the polypeptide chain glycosylation during TBG biosynthesis.

Total thyroxine--binding globulin (TBGt) and its pregnancy-associated molecular variant (TBG-1) were detected by radioimmunoassay in human amniotic fluid collected at the time of delivery. TBGt purified from amniotic fluid by affinity chromatography and hydroxylapatite chromatography, displayed electrophoretic properties, immunoreactivity, a molecular mass, affinity for thyroxine and TBG-1 content identical to those of pure TBGt from human pregnancy serum. The concentrations of TBGt and TBG-1 in maternal venous blood serum were 49 +/- 7 mg/ml and 3.8 +/- 1.3 mg/ml (n = 23), respectively, and those in amniotic fluid were 2.0 +/- 0.5 mg/ml and 0.19 +/- 0.12 mg/ml (n = 30), respectively. The analysis of paired samples showed that the serum and amniotic fluid TBGt levels closely correlated (r = 0.91), whereas the correlation between the respective TBG-1 levels was poor (r = 0.63). The TBG-1/TBGt ratio in amniotic fluid was 20% higher than that in serum. These findings suggested a maternal origin of the most of the amniotic fluid TBG which may possibly enter amniotic fluid by passive diffusion through fetal membranes. An alternative mechanism can exist for TBG-1 transfer from maternal serum to amniotic fluid. It corresponds to a special role that TBG-1 may play in the fetoplacental system.

Using spectroscopic, electrophoretic and microcalorimetric techniques, the changes in the spatial structure of human thyroxine-binding globulin (TBG) induced by exposure of protein solutions to high temperatures (45-90 degrees C) and low pH (2.5-6.0) were studied. Simultaneously the biological activity and immunoreactivity of TBG samples were measured. The structural changes were manifested at 52 degrees C or at pH 4.0 and were then aggravated with a rise in temperature or a decrease of pH. The circular dichroism spectra showed that the molecular ellipticity had a maximum decrease (by 10%) at 218-222 nm. In fluorescence spectra excitable at 280 nm the band half-width increased by 4-6 nm; their intensity decreased by 30-40%, whereas the position of the maxima did not change significantly. After addition of an equimolar amount of thyroxine to inactivated TBG the protein fluorescence was quenched by 25-40%. The electrophoregrams of treated preparations contained additional protein bands possessing no biological activity, whose mobility was less than that of native TBG. Microcalorimetric assays of native TBG revealed a thermoabsorption peak with a maximum at 62.5 degrees C and a half-width of 7.1 degrees C. The thermodynamic parameters of melting of TBG spatial structure were consistent with a model of a two-domain structure of the molecule. The biological activity and immunoreactivity of TBG showed a coordinated decrease with a rise in the degree of protein denaturation, However, the formation of TBG complex with antibodies did not screen the thyroxine-binding center of TBG and did not alter its affinity. Possible mechanisms of structural transition of TBG and its effect on the biological properties of TBG are discussed.

The concentrations of the molecular variant of the thyroxine-binding globulin (TBG-1) and total TBG were measured in normal human sera (male and female), venous blood sera at various stages of normal pregnancy as well as in matched samples of retroplacental and venous sera of mothers and in cord sera of their newborns. It was shown that TBG-1 appeared on the 6th-11th week of pregnancy and its level increased up to the delivery. A TBG-1 portion in the total TBG content increased from 2.0% in the 3rd month of pregnancy to 8.8% in the last month. After delivery TBG-1 and TBG levels decreased on the first day from 3.82 +/- 0.52 micrograms/ml to 2.52 +/- 0.86 micrograms/ml (P less than 0.01) and from 43.3 +/- 7.2 micrograms/ml to 37.8 +/- 9.6 micrograms/ml (P less than 0.01), respectively. However on the 5th day a decrease in TBG-1 and TBG concentrations were not observed. TBG-1 was undetectable in normal adult and cord sera. TBG-1 concentrations in matched retroplacental and venous sera at term were similar. These results suggested that TBG-1 was related to pregnancy-associated proteins rather than to proteins of fetal or placental origin.

Based on the new data concerning the multicomponent system of thyroxine-binding proteins in human plasma, some methodological aspects of isolation and purification of thyroxine-binding globulin (TBG) were examined, and a simple two-step procedure for TBG purification was developed. Normal human blood serum, retroplacental serum and amniotic fluid were used as TBG sources. The procedure includes affinity chromatography and adsorption chromatography on a hydroxyapatite column. A biospecific adsorbent was synthesized by stepwise binding of epichlorohydrin and thyroxine to Sepharose. The yield of pure TBG varied from 60 to 80%, depending on the TBG source used. The properties of TBG preparations from retroplacental serum and amniotic fluid were identical; both preparations contained a pregnancy-associated molecular variant, TBG-1. Two novel serum thyroxine-binding proteins were detected, isolated and partly characterized.