Characterization of human retroplacental blood plasma proteins isolated by biospecific chromatography on immobilized cortisol]

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.     

Studies of human transcortin at different pHs: circular dichroism, polymerisation and binding affinity.

The transcortin we have used in this work is extremely pure. This was shown by the polymerisation observed at pH 4. This polymerisation is never observed with an impure form of transcortin [4]. Moreover, since it is known that the presence of cortisol in the binding site is an essential condition to the activity of purified transcortin [5], it appears that a correlation between the secondary structure and the biological activity of the transcortin exists. The results we have obtained are summarized below: (1) The inhibition of the transcortin binding capacity essentially takes place between pH 5 and 4. (2) A reorganisation of the structure of the protein moiety is observed between pH 6.5 and 5.9. (3) A decrease of the helicity ratio is observed between pH 5 and 4. It appears therefore that, in the limits of experimental accuracy of CD measurements to determine the amount of beta-structure, no appreciable change of binding activity is taking place after the appearance of a large percentage of beta-structure between pH 6.5 and 6. On the other hand, the sudden decrease of protein activity at low pH is likely to be correlated with the disappearance of a well-defined helical region. Other biochemical and physical experiments would be of course necessary, in order to precise this first observation of a structure-function relationship in transcortin.     

Human liver nuclear transcortin its postulated role in glucocorticoid regulation of genetic activity

Highly purified human transcortin was injected into rabbits, and the antibody subsequently obtained was employed for the demonstration of transcortin-like molecules within various subcellular fractions of the human liver cell. Results obtained via quantitative double diffusion ouchterlony procedures indicate that proteins extracted from the nucleus or from chromatin form continuous precipitin lines of identity with those of transcortin. Fluorescein-tagged anti-transcortin permitted the visual localization of this molecule within isolated nuclei. Cortisol binding studies of all the subcellular fractions, particularly that extracted from the chromatin, suggest that such proteins do indeed bind cortisol specifically, as well as responding to exogeneous additions to the buffer (sulfhydryl reagents) as does purified transcortin. Purified transcortin when dialyzed exhaustively loses its cortisol binding ability, although the latter can be restored after its incubation with chromatin at 4°C. The restoration of such activity is dependent upon a dialyzable, heat-resistant chromatin component which itself lacks cortisol binding activity and which increases the sedimentation characteristics of dialyzed transcortin. The effect of transcortin on the in vitro synthesis of RNA in an Escherichia coli RNA polymerase human liver chromatin system is also presented. All of the above results are interpreted to indicate that transcortin is involved directly in the regulation of that genetic activity observed subsequent to the administration of cortisol.     

The influence of transcortin on adrenocorticotropin-stimulated corticosterone production in monolayer cultured rat adrenal cells

To verify the influence of the protein binding status of steroids adjacent to adrenal cells on steroidogenesis, the effect of transcortin, a specific binding protein of cortisol or corticosterone, on adrenocorticotropin (ACTH)-stimulated corticosterone production in monolayer cultured rat adrenal cells was studied. The transcortin in concentration of 5 x 10(-7) M was loaded with 0, 2.5, 5 and 10 pg/ml ACTH-(1-24), and the cells were incubated for 2 and 4 hours. Since molar concentrations of corticosterone produced in the medium were below the transcortin concentration at all levels of stimulation, protein-unbound corticosterone in the medium may have been largely reduced by the addition of transcortin. However, the total corticosterone production was not influenced by the transcortin added to the medium. It was speculated that protein-unbound steroid within the concentration range modulated by transcortin in the area surrounding the adrenal cells may not affect adrenal steroidogenesis.     

Interaction of blood sex steroid-binding globulin with cell membranes of human decidual tissue

The interaction of sex steroid-binding globulin (SHBG) from human blood with plasma membranes of human decidual tissue cells (estradiol target tissue) was investigated. It was shown that SHBG complexed with estradiol specifically interacts with these membranes. The dissociation constant (Kdis) for this interaction is (3.5 +/- 2.0) X 10(-12) M. The interaction of the SHBG-estradiol complex with the membranes is characterized by a high selectivity; such serum globulins as albumin, orosomucoid, transferrin, transcortin and the thyroxine-binding globulin do not compete with SHBG for the binding sites on the membranes. The SHBG-testosterone complex and SHBG alone do not interact with the membranes either.     

Effect of transcortin on the corticosterone-transforming activity of cytosol from the rat liver

The study of transcortin role in 3H-corticosterone metabolism has shown that transcortin of blood plasma from rats bearing Walker carcinosarcoma preserves the hormone conversion to dihydrocompounds 4 time less intensively than transcortin taken from healthy rats. Inactivated transcortin exerts no effect on the rate of formation of 5 beta-metabolites. Under the influence of homogeneous transcortin samples, a decrease in the content of 5 beta-reduced corticosterone metabolites is revealed to occur depending on transcortin concentration in the system. It is shown that in incubation systems where hormone is in the bound state the metabolism preserving capacity of transcortin depends on the temperature degree. The transcortin activity on corticosterone metabolism is supposed to be closely related to the intensity of its complexing with transcortin.     

Transcortin in rat kidney: subcellular distribution of transcortin-synthesizing polyribosomes

The [3H]corticosterone-transcortin complexes from kidney cytosol show elution positions on DEAE-cellulose identical to serum transcortin. The incorporation of 14C-labeled amino acids into anti-transcortin-precipitable material of kidney slices has been measured and compared with that of serum transcortin. It was established that kidney synthesized transcortin with an apparent molecular weight of 66 kDa on SDS-electrophoresis which resembles serum corticosteroid-binding globulin. Studies on the binding of [125I]anti-transcortin-IgG to membrane-bound rat kidney polyribosomes revealed an association of [125I]anti-transcortin-IgG with a discrete polyribosome fraction in the heavy polyribosome region; free polyribosomes were devoid of antigenic material able to bind antibodies to transcortin.     

Transcortin in rat kidney tissue: distribution in microsomal fractions

During chromatography of renal tissue cytosolic proteins on DEAE-cellulose the protein specifically binding [3H]corticosterone is eluted within the potassium phosphate concentration range of 0.08-0.10 M. Analysis of kidney slices revealed the synthesis of [3H]transcortin whose electrophoretic mobility was close to that of the blood plasma protein. Using radioimmunochemical methods, it has been found that transcortin-specific [125I]IgG antibodies interact with growing polypeptide chains of membrane-bound polyribosomes. Free polyribosomes do not bind antibodies against transcortin.     

3-O-methoxyimino group inhibits interactions between progestins and blood transcortin

The interactions between E- and Z-isomers of 3-O-methoxyimino-pregn-4-ene-20-one and its 17α-hydroxy derivative and transcortin from human blood were investigated. The substitution of the progesterone 3-oxo group for a 3-O-methoxyimino group was shown to diminish the affinity of the steroid for transcortin by approximately one order of magnitude irrespective of the substituent’s orientation. The data suggests that progesterone derivatives substituted thereby must have higher bioavailability compared to progesterone and must not significantly affect the biodynamics of glucocorticoid in vivo.     

Comparative study of primates' transcortin: immunoreactivity and steroid-binding activity

To investigate the phylogenic aspect of transcortin (corticosteroid-binding globulin, CBG), the immunoreactivity of transcortin with anti-human transcortin antiserum was studied in primates. The anti-human transcortin antibody was recognized by plasma proteins obtained from Catarrhini, taxonomically the most evolved monkey group. The immunoreactivity was not observed in plasma obtained from Platyrrhini and Prosimiae, classified as less evolved monkey groups than Catarrhini. Though comparison of immunoreactivity among different classes of Catarrhini was difficult because of non-parallelism of their displacement curves, displacement of 125I-labelled human transcortin from the antiserum by 1:10 and 1:100 diluted plasma was highest in human followed by Pongidae, Cercopithecoidea. The immunoreactivity of thyroxine-binding globulin (TBG) with anti-human TBG antiserum was also examined. The anti-human TBG antibody was only recognized in plasma from Pan (anthropoid ape) among Pongidae, highly evolved monkeys among Catarrhini. The existence of immunoreactive transcortin and TBG to respective human protein antibody in the highly evolved ape agreed well with the cladogenetic division of primate species delineated by Goodman and Moore (1971). Cortisol-binding activity of transcortin was detected in all monkeys except three, tafted capuchin monkey, night monkey and cotton-headed tamarin, which belong to Platyrrhini. The absence of cortisol-binding activity in these animals might be attributed to high levels of endogenous cortisol and low cortisol-binding capacity of transcortin. It is speculated that the structure of the immunoreactive site in transcortin could be modified by evolution without affecting the biologically important site, the site for cortisol binding.     

The presence of cortisol receptors in the human amnion

Cytosol extracts of human amnion tissue contained high affinity binding of cortisol (K_a = 2.48 ± 1.06 × 10⁹ M⁻¹; N = 30) and low capacity binding of cortisol (N_max = 279 ± 15.5 fmol mg⁻¹ protein). Kinetic studies of cortisol binding resulted in a similar value of K_a to that obtained by Scatchard analysis. Nuclear extracts of amnion tissue contained high affinity binding of cortisol (K_a = 5.8 ± 1.91 × 10⁷ M⁻¹) and low binding capacity (N_max = 91.4±21.4 fmol mg⁻¹ protein). K_a values were an order of magnitude higher in cytosol than in blood serum when amnion and blood were obtained from the same individuals. Differences in competitive ligand binding, especially dexamethasone, were observed between the amnion receptor and transcortin in serum. Gel permeation chromatography gave only one peak at 320 kDa for amnion receptor and only one peak at 48 kDa for transcortin from serum. When amnion tissue was incubated with or without cortisol, cytosol receptor activity was significantly lower in cortisol treated tissue than in control. The nuclear extracted receptor activity was significantly higher in cortisol treated tissue than control. The K_a values from cortisol treated tissue were significantly lower from control. Together the data support the presence of a specific cortisol receptor in the human amnion that is different from transcortin.     

Conditions affecting AtT-20 cell glucocorticoid uptake

Glucocorticoid uptake by intact AtT-20/D-1 cells was studied to determine if the extent of uptake was enhanced or retarded by binding components in serum. The results demonstrate that the uptake of corticosterone, which binds to transcortin, was reduced by addition of serum while uptake of triamcinolone acetonide, which is not bound by transcortin, was unaffected. Neither heat denatured serum nor bovine serum albumin affected corticosterone uptake, further emphasizing the specificity of the inhibition. The presence of serum also affected the apparent binding specificity, since steroids able to bind to transcortin became less effective competitors when serum was present in the incubation medium. In the absence of serum, the specificity of glucocorticoid uptake was qualitatively similar to that of the isolated cytosol receptor. These results emphasize that the selective inhibitory effect of serum transcortin on whole cell uptake of certain steroids should be considered when assaying steroid potency in intact cellular systems.     

Hormone-protein interactions and functional activity of the adrenal cortex during adaptation to pressure chamber hypoxia

Blood plasma and adrenals of rats subjected to the exposure of deep pressure-chamber hypoxia are studied. The method of high-performance liquid chromatography is used to determine the content of corticosterone and deoxycorticosterone as well as the binding ability of transcortin relative to hydrocortisone and corticosterone before and after ectomy of endogenic hormones. It is shown that hyperactivation of adrenals occurs already the first 20 min of the experiment, which is preserved during the whole experiment lowering, to some extent, by 150, 240 min. The binding ability of transcortin considerably lowers in the acute phase of stress and after the prolonged hypoxic exposure due to a 1.5-1.9-fold decrease in the number of the binding sites in the protein. The equilibrium redistribution is revealed in the plasma between free corticosterone and corticosterone bound to transcortin at the different stages of the hypoxic exposure.     

Reversible dissociation of cortisol-transcortin complex by sodium para-chloromercuribenzoate

Mercurials are considered as sulphydryl group specific reagents and one of them, sodium para-chloromercuribenzoate (PCMB), is currently used for SH titration. It has been shown that cellular steroid receptors are reversibly inactivated by mercurials even when the binding site is occupied by the steroid (Coty, W.A. (1980) J. Biol. Chem. 255, 8035-8037). This is a striking difference with alkylating SH reagents such as iodoacetic acid or N-ethylmaleimide, since these reagents inactivate only steroid-free receptors. In order to explain this discrepancy, we tested, in the present study, the specificity of PCMB on a blood plasma steroid binding protein: human transcortin. This protein presents the advantage, over cellular receptors, of being well characterized and to be available in a pure state. The transcortin-cortisol complex was also reversibly inactivated by PCMB when the reaction was carried out at a high excess of reagent over protein; such conditions are those previously used with steroid receptors. The reversibility was obtained not only with a reducing agent (dithiothreitol) but also with EDTA, which suggests a poor stability of the protein mercurial bond and therefore a nonspecific action. The decrease of activity was the result of a loss of binding sites and Scatchard plot analysis did not reveal any detectable decrease of the affinity constant for cortisol. Transcortin possesses two SH groups per molecule, one of these being buried in native conformation. After blockage of the accessible SH group by aminoethylation, transcortin kept the same activity, but when this aminoethylated transcortin was incubated with PCMB a loss of activity was obtained, although the residual buried SH group was again titrable with Ellman's reagent. Therefore, we can conclude that the action of PCMB on proteins must be interpreted with precaution, since it can induce an inactivation that is SH-independent.     

Protein binding glucocorticoid hormones (transcortin) during peri-and postnatal ontogenesis and pregnancy in rats]

The constants of association and the energy of interaction between transcortin and cortisol, the binding ability and other characteristics of transcortin have been studied in the embryos, sexually immature and mature young and old females, females on the 14th and 21st days of pregnancy, immature and mature males. The constant of association in all the groups amounted to ca. 10(8) and the energy of interaction ca. 10 Cal/mole. The embryos and immature rats of both sexes are characterized by relatively low levels of the binding ability of transcortin. During the sexual maturation, the level of transcortin increased--insignificantly in males and markedly in females. The level of transcortin in the latter remained almost invariable during pregnancy and senescence. By the electrophoretic and sedimentation properties transcortin was the same in different groups. The high level of transcortin during pregnancy corresponded to the high level of hormones bound by transcortin, the level of these hormones in the embryos being much lower than in the mother.     

Cortisol 17 beta acid, transcortin, and the heterogeneity of rat brain glucocorticoid receptors

Binding of tracer or competing steroids to transcortin can compromise specificity studies on receptors for adrenal steroids. Recently Alexis et al. have used cortisol 17 beta acid at high concentrations to prevent steroid binding to any transcortin possibly contaminating rat brain cytosol preparations. On the basis of limited specificity studies of [3H]dexamethasone and [3H]corticosterone binding under such conditions, it was claimed that binding sites for the two steroids are indistinguishable, and it is thus unnecessary to invoke distinct binding sites for each glucocorticoid. We have extended these competition studies in the presence of cortisol 17 beta acid, and shown that in rat hippocampus Type I, corticosterone-preferring glucocorticoid receptors can be clearly distinguished both from transcortin and from Type II, dexamethasone-binding glucocorticoid receptors.     

Studies on the interaction between human serum protein fractions and 18O-labeled oxosteroids

The nature of the interaction between progesterone or testosterone and human albumin as well as the interaction between progesterone and partially purified human transcortin has been studied. Modification of lysine residues of albumin with maleic anhydride resulted in a decreased binding of the steroid as judged from equilibrium dialysis experiments. This suggested that lysine residues in albumin interact with the oxosteroids. In order to check this hypothesis, steroids labeled with 18O in their oxo function (testosterone and progesterone) were synthesized for use as probes of the interactions. However, no loss of label was noted when testosterone or progesterone specifically 18O-labeled in their oxo functions were incubated with albumin. This suggested that no covalent interaction between the steroidal oxo group and albumin took place. This was in contrast to the results obtained with 3,20-18O-labeled progesterone and partially purified transcortin, where a complete loss of 18O label in the protein-bound steroid was found. The nonbound steroid showed an almost complete retention of label. These results indicate a participation of steroid oxo groups in the binding of progesterone to transcortin. Of the possible mechanisms discussed, imine bonds between the steroid and transcortin seem most likely although other types of interactions cannot be ruled out.     

Properties and serum levels of pregnancy-associated variant of human transcortin

The amino acid composition, N- and C-terminal amino acid sequences, and the basic physicochemical and immunochemical properties of the recently discovered pregnancy-associated molecular variant of human transcortin (Strel'chyonok, O.A., Avvakumov, G.V. and Akhrem, A.A. (1984) Carbohydr. Res. 134, 133-140) have been found to be identical to those of transcortin from normal donor serum. This suggests the identity of polypeptide moieties of the two glycoproteins. The transcortin variant has a lower isoelectric point (3.5-4.1) than normal transcortin (3.6-4.2), and different electrophoretic mobility in low-porosity polyacrylamide gel (one band versus two for normal transcortin). These differences can be reasonably explained by different organization of the carbohydrate moieties of these glycoproteins due to diverse post-translational modification of a single polypeptide chain. The levels of transcortin variant in the maternal venous serum throughout normal gestation (447 donors in all) and on the fifth day after delivery, as well as in umbilical cord serum and extracts of term placenta, have been measured by a radioimmune assay. Analysis of the data obtained allowed us to conclude that the biosynthesis of pregnancy-associated transcortin variant occurs in some organ of the maternal organism rather than in the feto-placental system, and it is a characteristic of pregnancy as a unique physiological state of the female organism rather than a phenomenon caused by individual features of certain women. We assume that the transcortin variant takes part in the guided transport of corticosteroids and/or progestins into some tissues that develop in the course of gestation.     

New aspects of isolation and purification of thyroxine-binding globulin from human biological fluids

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.     

Electron paramagnetic resonance study of the interactions between steroid hormones and binding proteins]

Interaction of a spin labeled corticosteroid (desoxycorticosterone nitroxyde: DOC -NO) with three purified proteins (albumin, transcortin, progesterone binding protein: PBG) was studied by electron spin resonance (ESR) spectroscopy. DOC-NO was competitive with natural corticosteroids and therefore bound at the same site to specific binding proteins. ESR spectra in the presence of each of the proteins showed an immobilized (bound) form of the spin labeled steroid and allowed the calculation of the corresponding association constant (Ka) at equilibrium. The three binding proteins could be characterized by the ESR parameters of the DOC-NO bound form. The thermodynamic parameters (deltaH, deltaS) of the steroid-protein interactions were calculated from the ESR data obtained within a wide temperature range (3--40 degrees C). The ESR spectra width (2T) was used to evaluate the polarity of the spin label environment within the steroid binding site: a hydrophobic character was observed for transcortin whereas PBG exhibited a more hydrophilic steroid binding sits. The rotational correlation time of the three protein DOC-NO complexes at equilibrium were calculated from ESR data; the results were correlated with the protein molecular size and suggested a non spherical shape for the binding macromolecule in solution. Spin labelling of biologically active steroids thus provides a novel approach for the study of the interaction of these hormones with their binding protein. Providing a suitable spin label, the ESR parameters may allow the characterization of several types of binding sites of different biological significance for the same hormone, in biological fluids as well as in target tissues.