[Gamma-glutamyltransferase activity in the human red blood cell membrane (author's transl)]. / Activité gamma-glutamyltransferase dans la membrane du globule rouge humain

A gamma-glutamyltransferase activity is found in the human red blood cell membrane. Membrane isolation was carried out according to the method of Dodge et al. (Dodge, J. T., Mitchell, C. and Hanahan, J. (1963) Arch. Biochem. Biophys. 100, 119-130) (modified) and proteins were solubilized either with 1% sodium deoxycholate or 5 mM EDTA or 10 mM of its disodium salt, under various conditions of time and temperature. The gamma-glutamyltransferase activity of the membrane preparations was investigated using two substrates, gamma-L-glutamyl-p-nitroanilide and gamma-L-glutamyl-alpha-naphthylamide. The specific enzymatic activities of the various preparations, expressed in m units per mg of protein, were found to have similar values under similar technical conditions. The chelating agents seem to allow a more specific isolation than the detergent. The presence of a gamma-glutamyltransferase activity in the erythrocyte membrane is discussed in relation to the membrane association of this enzyme in other tissues.

Fluorescence studies of the interactions of serum albumin and rat alpha1-fetoprotein with aflatoxin B1. Specificity and binding parameters.

The interaction of bovine serum albumin and rat alpha1-fetoprotein with aflatoxin B1 has been followed by the fluorescence quenching of the protein in presence of the ligand. The binding parameters (n, number of sites and Kd, dissociation constant) have been determined for the bovine serum albumin-alflatoxin B1 system: n = 3.5 and Kd = 3.1 +/- 0.5 . 10(-5) M and for the alpha-fetoprotein-aflatoxin system: n = 4 and Kd = 3.7 +/-0.5 . 10(-5) M. The competition of anilino-naphthalene-sulfonate and aflatoxin B1 for the same hydrophobic sites on bovine serum albumin has been demonstrated. The fluorescence quenching of various proteins (lysozymes, egg-albumin, gamma-globulin) by aflatoxin B1 have shown that there is not a strict specificity of aflatoxin towards proteins.

[Determination, at equilibrium, of association constants of labelled or unlabelled ligands by a non-graphical method (author's transl)]. / Determination, à l'équilibre, des constantes d'association de ligands radioactifs ou non radioactifs par une méthode non graphique

The present work deals with the determination of association constants at equilibrium by a non-graphical method in binding systems containing one specific receptor. Equations have have been derived from that originally described by Lea (Biochim. Biophys. Acta, 322, 68--74), the terms of which are obtained from the data of simple displacement curves of a bound radioactive ligand by unlabelled competitors identical or different in nature. By knowing the function relating the variations of the bound ligand (B) to the affinity constant (Ki) and the quantity (Mi) of competitor for a given system, it is possible to calculate any of these parameters when the two others are measured. Thus, it becomes easy to compare the relative affinities of differents receptors for the same ligand or that of one receptor for various labelled or unlabelled ligands. Furthermore, theoretical displacement curves can be drawn and compared to experimental data, when only knowing the affinity constant of a specific binding system in given conditions. These modes of calculation have been tested in a study of interactions between various steroids and a fraction of human serum proteins precipitated by ammonium sulfate (30-45%) and containing the sex hormone-binding globulin. Association constants thus obtained agree well with those reported in the literature and determined by graphical procedures.

Isolation of two forms of rat alpha-fetoprotein and comparison of their binding parameters with estradiol-17beta.

In polyacrylamide gels, highly purified rat alpha1-fetoprotein shows a molecular heterogeneity, i.e. a "slow" and a "fast" moving fraction. We have isolated by electrophoretic fractionation and subsequent elution these two forms of alpha1-fetoprotein, and we have studied comparatively the binding parameters for estradiol-17beta of whole alpha1-fetoprotein preparations and of the isolated forms. We have shown that the number of binding sites per molecule of whole alpha1-fetoprotein is always, in our experimental conditions, a fractional number, inferior to unity (0.3). Furthermore, the analysis of the binding parameters of the "two forms" of alpha1-fetoprotein allows discrimination between different classes of binding sites. For the "slow" fraction, the number of predominant binding sites per molecule of protein is close to unity (0.7-0.9), whereas for the "fast" fraction, a very low fractional value is found (0.1). The corresponding association constants are reproducibly different for the two fractions: Ka = 0.1.10(8) M-1 for the "slow" alpha1-fetoprotein, and Ka = 0.7.10(8) M-1 for the "fast" alpha1-fetoprotein. Traces of a very high affinity (10(9) M-1) minor class of binding sites are demonstrated in the "slow" fraction. These results point to the existence of a molecular population of alpha1-fetoprotein, some forms of which have a strong or very strong affinity, and some a negligible affinity, for estrogens.

Surface behaviour of oestradiol-17beta.

The absorption of [3H]oestradiol-17beta from its aqueous solutions has been measured in the range 0-10mug/ml. It is found that the first adsorbed molecules are parallel to the interface and occupy 100 A2. Those adsorbed in the range 6-10 mug/ml occupy 21 A2. They are presumably associated. When the adsorption occurs in the presence of a synthetic lecithin monolayer, the molecular area is equal to 16 A2. Surface tension measurements of the solutions of oestradiol-17beta and a parallel study of their fluorescence have been performed. No association of the hormone molecules has been observed in bulk. It is concluded that surfaces and liquid monolayers may favour molecular association of the oestradiol-17beta.

[In vitro aromatization of androgens in the presence of testosterone binding proteins from human placenta or serum]. / Aromatisation in vitro des androgènes en présence de protéines humaines, placentaire ou sérique, liant la testostérone.

Two related 17 beta hydroxy steroid binding proteins of serum and placental origin respectively are shown to inhibit significantly the aromatization of testosterone and androstenedione by isolated human placenta microsomes. Quantitative correlations suggest that this inhibitory effect is due to the fixation of the testosterone substrate (or androstenedione previously converted to testosterone) on the binding proteins. A possible role for these macromolecules in the steroidogenic activity of the placenta is discussed.

[Isolation and physical chemical properties of rat hemopexin]. / Isolement et propriétés physico-chimiques de l'hémopexine de rat

Rat hemopexin was purified by a procedure involving three different steps : ammonium sulfate precipitation, rivanol precipitation and DEAE-cellulose chromatography with concave gradient of molarity. Purity of the preparation was checked by three different methods : analytical ultracentrifugation, immunoelectrophoresis and acrylamide gel electrophoresis. The principal physical properties were studied. The amino acid and carbohydrate composition was determined and compared with that of human and rabbit hemopexin.

[In vitro comparative metabolism of 6,7-3H estrone and 6,7-3H estrone-3-sulfate in maternal and fetal guinea-pig liver]. / Etude comparée du métabolisme, in vitro, de la [6, 7-3H-A1 estrone et du [6, 7-3H] estrone-3-sulfate par le foie maternel et foetal du cobaye.

The metabolism of [6,7-3H] estrone and of [6,7(3)H] estrone-3-sulfate have been comparatively studied in the maternal and fetal guinea-pig livers. The appearance of estradiol-17 beta resulting from the activity of the 17 beta-hydroxysteroid-dehydrogenase is more important in the fetal than in the maternal hepatic tissue. This suggests the direct transformation of estrone-3-sulfate into estradio-3-sulfate in the fetus. After incubation of the [3H] estrone, there is an abundant hepatic conjugation. The glycuroconjugated components are predominant, as well in the maternal as in the fetal hepatic tissue. For the latter-one the sulfoconjugation is inexistant. The sulfatasic activity shown after the incubation of [3H] estrone-3-sulfate is very low in the fetal hepatic tissue; in contrast, this activity is higher in the maternal tissue.

[Preparation and immunoephelometric quantitation of fibrinogen in rabbits]. / Préparation et dosage immunonéphélémétrique du fibrinogène de lapin

Pure rabbit fibrinogen was prepared by a method involving two ammonium sulfate precipitations, one 2 M phosphate buffer precipitation, one DEAE cellulose chromatography and lastly one Sepharose 6 B chromatography. The aminoacid composition was determined and an immunonephelemetric assay was proposed. This assay followed an accurate determination of fibrinogen concentration in a rabbit with inflammatory reaction.

[Semi-automated determination of urinary 17-oxosteroids in a continuous flow system (author's transl)]. / Dosage semi-automatique en flux continu des 17-cetosteroides urinaires

A semi-automated determination of 17-oxosteroids in urinary extracts is described using a modified Zimmerman reaction in aqueous phase according to Epstein. In order to eliminate the interfering chromogens a double manifold and a double-beam colorimeter are used in continuous flow. The spectra and the chromogenecity of seven different 17-oxosteroids are presented. The specificity and the reproducibility of this technique are good and the comparison with gas-liquid chromatography and manual Zimmerman reaction shows a good correlation.

Plasma diethylstilboestrol binding proteins of rat, mouse and man in the course of development: relations with the binding of estradiol.

High diethylstilboestrol (DES) binding has been demonstrated in fetal and adult sera from man, rat and mouse by equilibrium dialysis and electrophoretic techniques. In the adults of the three species and in the human fetus only albumin shows an elevated binding capacity for DES. By contrast, in the case of rat and mouse embryos there are two proteins, namely albumin and alpha-fetoprotein, which afford major and quantatively similar contributions to the binding. Human alpha-fetoprotein does not bind DES. These phenomena are analysed in relation to the estrogen binding characteristics of the alpha-fetoproteins of the three species.

Simultaneous radioimmunoassay of 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol unconjugated and conjugated in human serum.

The simultaneous determinations of both 3alpha and 3beta epimers of 5alpha-androstane-3,17beta-diol as their glucuronides, sulfates and in their unconjugated forms are described. The diol estimation is carried out by radioimmunoassay with two specific immune sera after purification of the serum by use of chromatography on Sephadex LH-20. The values obtained (mean +/- S.D.) in pg/ml for the unconjugated 3alpha and 3beta epimers were, respectively, 267 +/- 67 and 816 +/- 76 for men; 114 +/- 33 and 515 +/- 177 for women; 142+/- 77 and 779 +/- 200 for hirsute women. Among the conjugates, the most important were the sulfoconjugates, their rates being, respectively (men +/- S.D. in ng/ml 41.6 +/- 9.5 and 103+/- 40 for men; 12.4 +/- 3.1 and 51.2 +/- 14.9 for women and 36 +/- 22 and 72 +/- 36 for hirsute women. Differences in the conjugation of both epimers were also noticed.

The preparation of three fluorescence-labeled derivatives of testosterone.

Three fluorescence-labelled derivatives of testosterone were prepared consisting of the steroid separated from the fluorochrome by a hydrocarbon "bridge". "Bridges" of different lengths (C2 to C7) were used as the length required to avoid steric hindrance effects by the fluorochrome in studies on steroid-protein binding was unknown. The three derivatives prepared were: 17 beta-hydroxy--4-androsten-3-one 3-(O-(N-(2'-mercapto)ethyl)carbamoylmethyl)oxime, 17 beta-hydroxy-4-androsten-3-one 3-(O-(N-(3'-amino)propyl)carbamoylmethyl)oxime and 17 beta-hydroxy-4-androsten-3-one 3-(O(N-(7'-amino)heptyl)carbamoylmethyl)oxime. These were then coupled with either a dansyl or a fluorescein molecule. Overall yields were sufficient and the products immunoreactive with anti-testosterone antiserum.

Gas chromatography profile of estrogens: application to pregnancy urine.

A method for the simultaneous quantitation of 7 estrogens in pregnancy urine is described. It involves enzymatic hydrolysis extraction of free steroids, ion-exchange column chromatography and quantitation of the trimethylsilyl derivatives by gas chromatography on OV 1. Data obtained from normal and twin pregnancies and from women with anencephalic fetus or intra uterine fetal death are analysed. The sensibility of the method is about 40 mug of each estrogen by liter of urine.

The conjugation of testosterone with horseradish peroxidase and a sensitive enzyme assay for the conjugate.

The formation of a horseradish peroxidase-testosterone conjugate for the enzyme-linked immunoassay of testosterone was investigated, using tritiated testosterone to follow the reaction. The formation of testosterone-3-(carboxymethyl) oxime-peroxidase by the mixed anhydride method was found to give a conjugate of high enzymatic activity and with three molecules of testosterone per molecule of peroxidase. The optimum conditions for the assay of peroxidase activity were studied and an assay capable of measuring 1 to 5 ng of the conjugate developed; the standard curve being virtually linear. The stability of the conjugate in solution and the effect of lyophilisation on enzymatic activity are also described. The peroxidase-testosterone conjugate was suitable for enzyme-linked immunoassay and the quantities measurable with the peroxidase assay covered the range necessary for a plasma testosterone assay. The stability of the conjugate was such that no particular precautions were necessary for its storage.

An enzyme-linked immunoassay of testosterone.

An enzyme-linked immunoassay of testosterone is described. The conjugation of testosterone with high specific activity horseradish per-oxidase and a highly sensitive assay for this enzyme having previously been studied, here we describe the immobilization of the anti-testosterone antibody and the development of assay conditions permitting the determination of 50 pg to 1.5 ng testosterone in one working day. In this work attention has been paid to keeping the assay method as simple as possible. The method is discussed in terms of other enzyme-linked immunoassays for steroids.

Conversion, in vitro, of (7n-3H) testosterone to estrone and estradiol-17beta and their 3-sulfate conjugate by the guinea-pig placenta.

Different cellular fractions of guinea-pig placenta were incubated in the presence of (7n-3H) testosterone. Microsomal aromatization of 3H-testosterone into estrone and estradiol-17beta was demonstrated in the presence of NADPH. The predominance of estrone after incubation with 17beta-hydroxylated precursors, (7n-3H) testosterone and (6,7-3H) estradiol-17beta, indicate that there is a microsomal 17beta-hydroxysteroid dehydrogenase activity. In this report, cytosolic sulfurylation of estrogens is demonstrated. This latter activity represents a quite original characteristic of the placental metabolism of estrogens in guinea-pigs. In contrast with the human placenta where there is considerable sulfatase activity, the guinea-pig placenta can sulfurylate estrogens.

Purification and interaction with estradiol-17beta.

The combination of polyacrylamide gel electrophoresis and Concanavalin-A-Sepharose affinity chromatography has permitted the isolation on a preparative scale, of four molecular forms of rat alpha1-fetoprotein: a "slow" and a "fast" fraction, each separable into Concanavalin-A-adorbed ("high carbohydrate", i.e. rich in accessible alphaD-Mannosyl and alphaD-Glucosyl residues) and a Concanavalin-A-non adsorbed ("low carbohydrate") fractions. These four iso-alpha-fetoproteins (iso-AFP) bind estradiol-17beta. However, they disclose differences in both their association constants and number of binding sites for this hormone. Very high affinity sites (10(9)) are mainly located on the "slow-low carbohydrate" form. Low affinity, high capacity sites are preferentially located on the "high carbohydrate" form. These results confirm the molecular and functional heterogeneity of rat AFP and suggest that the carbohydrate moiety of the protein may have a role in estrogen-AFP interactions.

Rat iso-alpha1-fetoproteins. Purification and interaction with estradiol-17beta.

The combination of polyacrylamide gel electrophoresis and Concanavalin-A-Sepharose affinity chromatography has permitted the isolation on a preparative scale, of four molecular forms of rat alpha 1-fetoprotein: a "slow" and a "fast" fraction, each separable into Concanavalin-A-adsorbed ("high carbohydrate", i.e. rich in accessible alphaD-Mannosyl and alphaD-Glu-cosyl residues) and a Concanavalin-A-non adsorbed ("low carbohydrate") fractions. These four iso-alpha 1-fetoproteins (iso-AFP) bind estradiol-17beta. However, they disclose differences in both their association constants and number of binding sites for this hormone. Very high affinity sites (10(9) are mainly located on the "slow-low carbohydrate" form. Low affinity, high capacity sites are preferentially located on the "high carbohydrate" form. These results confirm the molecular and functional heterogeneity of rat AFT and suggest that the carbohydrate moiety of the protein may have a role in estrogen-AFP interactions.

Rat and human embryo and post-natal sera contain a potent endogenous competitor of estrogen-rat alpha-fetoprotein interactions.

A highly active inhibitor of the binding of estrone and estradiol-17beta to rat alpha-fetoprotein is demonstrated for the first time in embryo, immature and adult rat sera as well as in fetal and adult human sera. The competitive character and the narrow specificity of this inhibition effect is shown. The major compound responsible for this activity is isolated by successive column Sephadex LH20 and thin layer chromatography: it is characterized as a nonpolar, nonphenolic, dialysable and thermostable substance, unreactive towards anti-estrone and anti-estradiol-17beta antibodies. The possible biological role of an endogenous non-estrogen ligand of rodent fetoproteins is discussed.