A transcortin-binding protein in the plasma membrane of human syncytiotrophoblast.

Using affinity chromatography on immobilized transcortin of 125I-labeled, cholate-solubilized plasma membranes of human syncytiotrophoblast, a transcortin-binding protein with a minimal Mr of about 20 kDa has been isolated. It was found to be a sialoglycoprotein with an isoelectric point at pH 4.4 (about 5.0 after the treatment with neuraminidase). We assume that this protein is a component of membrane recognition system for transcortin-steroid complexes.     

Testosterone destroys the transcortin-receptor complex.

Dissociation of the complex of transcortin receptor with immobilized transcortin in the presence of 10(-5) M testosterone has been shown with the use of affinity chromatography on transcortin-Sepharose. The specificity of this effect is confirmed by its abrogation in the presence of cortisol. The testosterone effect has been used for the elution of transcortin receptor from affinity column. The receptor retained transcortin-binding capacity after the elution and removal of testosterone. Characteristics of the receptor obtained by testosterone elution were identical with those of the transcortin eluted preparation.     

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.     

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.     

Physico-chemical properties and evidence for electrophoretic variants of rat transcortin

Isolation of rat plasma transcortin was carried out by affinity chromatography, as previously described for human. The protein was shown to be pure by PAGE and one single N-terminal amino acid was identified (Ser), which suggested that the protein molecule has a single polypeptide chain. This assumption is supported by SDS-PAGE. The amino acid composition was reported and compared with the one of human transcortin. The purified protein always migrated in PAGE (with or without SDS) as a double band; the faster component being more intense than the slower one. Whether transcortin was free or bound to corticosterone, the same aspect was observed. Molecular weight of these two variants were determined by SDS-PAGE as 65,900 and 75,800. Polymers only appeared after irreversible denaturation of the protein, as previously described for human transcortin. Various other physical parameters were determined: a sedimentation coefficient of 3.71 S +/- 0.18 was calculated by ultracentrifugation in sucrose gradient, association constants at 4 degrees C for corticosterone and cortisol (2.7 X 10(9) M-1 and 4.2 X 10(8) M-1, respectively).     

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 chemistry of human transcortin: Improved affinity matrices for the purification of transcortin

While spacer arms have been shown to play an important role in affinity chromatography, no systematic investigations of spacer arms in the purification of transcortin have been reported. Among the five cortisol-agaroses, cortisol-21-succinyl-1,6-hexanediamidoagarose achieved the highest extraction efficiency of transcortin from plasma. The optimal length of the spacer arm for extraction is ca. 12–13 Å. Cortisol-succinyl-agaroses having hydrophobic spacer arms extract transcortin better then those having hydrophilic arms of approximately equal length. Affinity supports are usually synthesized sequentially; cortisol-agaroses thus prepared were found to complicate the purification of transcortin. The problems of nonspecificity and instability associated with these agaroses were eliminated by using reverse addition. A complete ligand-spacer arm, synthesized in a single step by displacing the tosyl group from cortisol-21-tosylate with 1,6-hexanediamine, was coupled with cyanogen bromide-activated agarose. Although the 21-deoxy-21-(ω-amidohexyl) aminocortisol-agarose ranked second in extraction efficiency, its superior stability and low nonspecific adsorption of other proteins make it the prime choice for affinity chromatography of transcortin.     

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.     

The role of sialic acid in determining the survival of transcortin in the circulation

The effect of desialylation on the survival of human transcortin and of its ligand cortisol has been investigated using the isolated perfused rat liver preparation. In contrast with native transcortin, sialic acid-free transcortin was promptly cleared from the perfusate. The hepatic uptake was accompanied by a significant reduction of the cortisol half-life.     

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.     

Phylogenetic study of transcortin using monoclonal antibodies

We produced monoclonal antibodies that recognise three distinct epitopes of human transcortin. These epitopes are present on transcortin of humans with normal and altered transcortin levels, as well as on a variant with lower affinity for cortisol. One epitope is present on transcortin of Old World Monkeys and apes, the others are only present on transcortin of apes. The epitopes are not present on transcortin of other species. These results indicate that human transcortin contains a highly evolved and a more conserved part.     

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.     

Red cell membrane sialoglycoprotein beta in homozygous and heterozygous 4.1(-) hereditary elliptocytosis

Sialoglycoprotein beta, a minor sialoglycoprotein of the red cell membrane, was studied in homozygous and heterozygous 4.1(-) hereditary elliptocytosis, a variety of hereditary elliptocytosis characterized by total or partial absence of protein 4.1. Erythrocytes were treated with the periodic acid-NaB3H4 procedure. Following polyacrylamide gel electrophoresis in the presence of SDS, labelled sialoglycoproteins were revealed by fluorography. (i) In the ghosts from the 4.1(-) homozygote, sialoglycoprotein beta was sharply decreased. It is not sure whether the residual material is sialoglycoprotein beta itself, or a distinct sialoglycoprotein migrating in the same place. In long exposure fluorograms, sialoglycoprotein gamma (a sialoglycoprotein related to sialoglycoprotein beta) also turned out to be reduced. In the homozygote's Triton-shells, sialoglycoprotein beta and gamma appeared completely absent. (ii) In the 4.1(-) heterozygote, sialoglycoprotein beta appeared slightly reduced, whereas sialoglycoprotein gamma appeared normal. Both of these proteins were extracted in seemingly normal amounts in the Triton-shells. These observations bring further support to the view that there is an interaction between skeletal membrane protein 4.1 and sialoglycoprotein beta, that is additional to other interactions between the former protein and the lipid bilayer and/or other transmembrane proteins.     

Purification and characterization of human transcortin.

Human transcortin was purified to apparent homogeneity from plasma by a two-step procedure involving affinity and hydroxyapatite chromatography. The affinity gel incorporated denatured bovine serum albumin as the spacer and cortisol hemisuccinate as the ligand. Although isolated transcortin showed a propensity for spontaneous polymerization according to a geometric progression (1, 3, 9) only one band was observed on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Cortisol-binding activity of the isolated protein gave an apparent association constant of 2.5 X 10(8) M-1 at 4 degree C in equilibrium dialysis. Isoelectric focusing of purified native transcortin showed six discrete bands, five between pH 3.75 and 4.15 and another, possibly desialylated, at pH 6.15. Desialylated transcortin also gave six bands on isoelectric focusing, with pI values ranging from 4.90 to 6.30.     

Human transcortin synthesis by a cell-free translation of hepatic mRNA

To evaluate the site of synthesis and to characterize the translated transcortin, poly (A)-containing RNA (mRNA) from human liver was translated in a cell-free system derived from rabbit reticulocyte lysate. The in vitro synthesized product was identified as transcortin by immuno-precipitation with its specific antiserum. This translated transcortin could be displaced from the antibody by unlabeled purified transcortin obtained from plasma. Furthermore, when the translation mixture was applied to a cortisol-Sepharose column, the translated transcortin was bound to the matrix in a specific manner, indicating that this product binds to cortisol. The molecular weight of the translated transcortin was estimated to be 45,700 by its mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis, while that of plasma transcortin was 53,800. The difference in molecular weight between the translated transcortin and plasma transcortin was probably due to the presence of pre-sequence (signal peptide) in addition to the absence of carbohydrate moiety in the former. In conclusion, human liver mRNA directed the synthesis of transcortin, and the translated transcortin binds to cortisol in spite of the absence of carbohydrate moiety.     

Transcortin and alpha 2u-globulin messenger RNA activities during turpentine-induced inflammation in the rat

Previously we have shown that the serum concentration of transcortin and alpha 2u-globulin markedly decreases during turpentine-induced inflammation. In the present study transcortin and alpha 2u-globulin mRNA from healthy rats and from animals with inflammation was translated in a rabbit reticulocyte lysate system. Female rats had higher levels of translatable transcortin mRNA than male animals and the level of mRNA for transcortin and alpha 2u-globulin decreased rapidly during inflammation. These results indicate that the sex difference in the serum level of transcortin and the changes in serum transcortin and alpha 2u-globulin during inflammation are mainly determined by differences in the mRNAs in the liver.     

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.     

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.     

Comparative study of human transcortin isolated from normal donor blood and retroplacental blood

It was demonstrated that the physico-chemical properties of human transcortin, i.e., electrophoretic, hydrodynamic and immunochemical characteristics, amino acid composition, steroid binding parameters, do not depend on the source of the glycoprotein (male or female blood, retroplacental blood). Conversely, the retroplacental blood serum was shown to contain a transcortin form whose carbohydrate component is structurally different from that of the normal donor blood transcortin. It was found that this form interacts with the sites of specific binding of transcortin in liver cell plasma membranes in a weaker degree than the donor blood 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.