Search

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.

Human retroplacental blood plasma proteins with affinity for cortisol were isolated by biospecific chromatography and identified by electrophoretic and immunochemical methods as alpha1- and beta1-globulins and IgG. IgM and IgA immunoglobulins. A high specific affinity for cortisol (Kas = 1,5 . 10(8) M-1 at 23 degrees C) and progesterone (Kas = 2,0 . 10(8) M-1 at 23 degrees C) was observed only for alpha-globulin; other proteins had a low affinity for cortisol. The molecular weight of alpha1-globulin (transcortin) was found to be 50,000-55,000. The amino acid and monosaccharide compositions of this glycoprotein were studied. Its N-terminal and C-terminal amino acid sequences are: Met-Asx-Pro-Asx-Ala- and (Val, Gln)-Leu, respectively. It was concluded that under normal physiological conditions and during pregnancy transcortin is the only specific corticosteroid-binding plasma protein. A complete removal of bound cortisol from the protein mixture and subsequent hydroxylapatite chromatography resulted in homogeneous transcortin retaining more than 90% of its binding capacity. The formation of the transcortin-steroid complex and its complete dissociation are accompanied by conformational changes of the protein globule. Significant changes of the spectral properties of the tryptophane residue of protein and the steroid delta4-3-keto group are indicative of the possibility of their direct interaction.