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 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.     

The effect of catecholamines on ion transport in the toad bladder

Noradrenalin (8 · 10⁻⁶ M) and adrenalin (6 · 10⁻⁶ and 6 · 10⁻⁷ M) were found to cause marked stimulation of short-circuit current (S.C.C.) in isolated toad bladder, but isoprenalin (8 · 10⁻⁷ M) was found to be without effect. The percentage rise in S.C.C. due to noradrenalin was found to be inversely proportional to the initial S.C.C. or total conductance of the bladder. Again in the case of noradrenalin the rise in S.C.C. was almost completely abolished by α-adrenergic blockade but not by β-blockade. This rise in S.C.C. was found not to be significantly different from the rise in net Na⁺ flux. Bidirectional Cl⁻ fluxes were estimated using ⁸²Br as a companion radionuclide to ³⁶Cl. No significant net Cl⁻ flux was apparent, either before or after addition of any of the three catecholamines tested. However, in some cases the unidirectional Cl⁻ fluxes rose markedly following addition of noradrenalin or of adrenalin and this change was not reflected in a change in total conductance. This anomaly was noted to occur in bladders whose initial conductance was of the order of 0.5 kΩ⁻¹ · cm⁻² or greater. The evidence presented suggests that two actions of catecholamines on ion transport in toad bladder are (a) to increase Na⁺ transport via stimulation of α-adrenergic sites and (b) at the concentrations tested to cause an increase in passive Cl permeability in bladders whose initial conductance is high.     

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.     

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.     

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.     

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.     

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.     

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).     

清栓酶治疗缺血性脑血管病的经颅多普勒脑血流分析

本文选择缺血性脑血管病患者72例。随机分为两组:1治疗组(清栓酶组)36例。用清栓酶0.5~u 加入低分子右旋糖酐500ml 中静滴.每日一次.Ⅱ对照组(复方丹参组)36例。用复方丹参16ml,加入低分子右旋糖酐500ml 中静滴。每日一次,疗程均为十五天。治疗前、后分别行经颅多普勒脑血流分析(简称 TCD)。结果发现两种药物治疗缺血性脑血管病均有效。其总有效率清栓酶组为:94.4%,复方丹参组为61.1%。前者优于后者.有显著性差异(P＜0.01)。     

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.     

Characterization of the plasma membrane ATPase of Candida tropicalis

1) Plasma membrane vesicles from Candida tropicalis were isolated from protoplasts by differential centrifugation and purified in a continuous sucrose gradient. 2) The plasma membrane bound ATPase was characterized. It is highly specific for ATP and requires Mg2+. It is stimulated by K+, Na+ and NH4+. Lineweaver-Burk plots for ATPase activity are linear with a Vmax of 4.2 mumoles of ATP hydrolyzed min-1.mg-1 protein and a Km for ATP of 0.76 mM. The ATPase activity is inhibited competitively by ADP with a Ki of 1.7 mM and non competitively by vanadate with a Ki of 3 microM. The activity is unaffected by oligomycin or azide but is sensitive to DCCD.     

Relationship between the "phospholipid effect" and calcium in the thyroid. I. - Effects of calcium ions, E.G.T.A., ionophore A 23187, verapamil and chlorpromazine on resting and stimulate thyroid slices.

The "phospholipid effect" which is the enhanced turnover of the phosphorylinositol group of phosphatidylinositol (PI) occurs in the thyroid of response to thyreostimulin (TSH). The possibility that Ca2+ ions are involved in this stimulation has been investigated with pig thyroid slices. Experiments performed in media without Ca2+ or containing E.G.T.A. (2 mM), indicate that it is not the extracellular Ca2+ which is implied, but rather the intracellular Ca2+. The ionophore A23187 (6.10(-6) M) increases the specific radioactivity of the acid soluble precursors, but has also a specific effect on the PI turnover, which is additive with the effect of a high concentration of TSH (50 mU/ml). Washing and loading of slices with various Ca2+ concentrations show that 0.9 mM restores the TSH phospholipid effect. Verapamil (10(-3) M) and Chlorpromazine (10(-3) M) redirect glycerolipid metabolism by increasing PI and phosphatidic acid (PA) synthesis at the expense of other glycerolipids, as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). These results suggest that the "phospholipid effect" is not a result of Ca2+ entry into the thyroid cells. On the contrary, it seems that this increased turnover of PI in "long term" incubations (3 hr). An additive and acute effect of TSH effect is more pronounced when Ca2+ movements     

Characteristics of a transcortin-binding component of the plasma membrane of cells of the human decidual endometrium

A sialoglycoprotein was isolated by affinity chromatography on immobilized transcortin from plasma membranes of human decidual endometrium cells, whose components were labeled with 125I and solubilized with sodium cholate. The apparent molecular mass of the monomer is 20.0 +/- 1.5 kDa, pI is at pH 3.3. The sialoglycoprotein specifically binds transcortin complexed to progesterone with Kd approximately 10(-10) M.     

Demonstration of 2 phosphate transport systems in Candida tropicalis]

Candida tropicalis has two phosphate transport systems, one of which is constitutive and has a low affinity for its substrate (Kmapp 1.2.10(-3) M). The other one characterized by a high affinity for H2PO4- (Kmapp = 4.5.10(-6) M) appears only under phosphate starvation conditions. The regulation of the latter would act on the one hand on the synthesis of binding proteins for P1 (repression-derepression) and on the other on the activation or inactivation of the carriers.     

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.     

Structural organization of the carbohydrate moiety of human transcortin as determined by methylation analysis of the whole glycoprotein

Methylation analysis of human transcortin showed that this glycoprotein contains N-glycosidically linked oligosaccharide chains of N-acetyllactosamine type, most of the chains being biantennary and others tri- and/or tetraantennary. The carbohydrate chains of transcortin are also heterogenous with respect to the content of fucose and the position of the glycosidic linkages.