jeanflop/post-ocr-correction huggingface dataset:
citri map. Carte physique et génomique du procaryote Spiroplasma melliferum et sa comparaison avec celle de Spiroplasma citri. Fengchun Ye, Frédéric Laigret, Joseph-Marie Bové 399 On small genomes in eukaryotic organisms: molecular karyotypes of two microsporidian species (Protozoa) parasites of vertebrates. Recherches sur les petits génomes chez les eucaryotes : caryotypes moléculaires de deux espèces de microsporidies (protozoaires) parasites de vertébrés. Corinne Biderre, Michel Pages, Guy Metenier, Danièle David, Jacqueline Bâta, Christian P. Vivares ▲ Biologie du développement/Development biology 405 Production de blastulas partielles par parthénogenèse chez le xénope. Parthénogenèse production of partial blastulas in Xenopus. Frédéric Tournier, Anne Joly,
production of partial blastulas in Xenopus. Frédéric Tournier, Anne Joly, Michel Bornens A Biologie et pathologie animale/Animal biology and pathology 411 Effets d'extraits de glandes du sinus sur la physiologie osmorégulatrice de branchies perfusées du crabe Pochygraspus marmoratus. Effects of sinus gland extracts on the osmoregulatory physiology of perfused gills from the crab Pachygraspus marmoratus. Céline Pierrot, Erik Eckhardt, François van Herp, Mireille Charmantier-Daures, Guy Charmantier, Jean-Paul Trilles, Pierre Thuet ▲ Biologie et pathologie végétale/Plant biology and pathology 419 Les espèces moléculaires de phosphatidylinositol et de phosphatidylinositolphosphate présentes dans les coléoptiles et premières feuilles de maïs (Zea mays L.). The
coléoptiles et premières feuilles de maïs (Zea mays L.). The molecular species of phosphatidylinositol and phosphatidylinositolphosphate evidenced in the coléoptiles and the first leaves of maize (Zea mays L.). Ahmed Chicha, Chantal Demandré, Anne-Marie Justin, Paul Mazliak 424 Sélection clonale d'iris à parfum. Clonal selection of iris species for parfumery. Huguette Jehan, Didier Courtois, Charles Ehret, Konrad Lerch, Vincent Petiard ▲ Pharmacologie/Pharmacology 430 Potentiation of 2',3'-dideoxycytidine (ddc) by hydroxyurea and thymidine on the moloney murine leukemia virus (MoMLV) early replicative steps. Potentiation de l'effet du 2',3'-didéoxycytidine (ddc) par l'hydroxyurée et la thymidine sur les étapes précoces du cycle réplicatif du
la thymidine sur les étapes précoces du cycle réplicatif du virus de la leucémie murine de Moloney (MoMLV). Hélène Goulaouic, Frédéric Subra, Jean-François Mouscadet, Sandrine Carteau, Christian Auclair ▲ Sciences médicales/Medical sciences 437 Hb Taybe (a 38 or 39 THR deleted): an a-globin defect, silent in the heterozygous state and producing severe hemolytic anemia in the homozygous. L'Hb Taybe (a. 38 ou 39 THR délitée) : une anomalie de la chaîne a de la globuline, muette à l'état hétérozygote mais responsable d'une anémie hémolytique sévère à l'état homozygote. Frédéric Galacteros, Emmanuelle Girodon, Amel M'Rad, Josiane Martin, Michel Goossens, Lutfi Jaber,
Emmanuelle Girodon, Amel M'Rad, Josiane Martin, Michel Goossens, Lutfi Jaber, lan J. Cohen, Hannah Tamary, Yakov Goshen, Rina Zaizov, Henri Wajcman 445 Do microglial cells phagocyte the p7A4-amyloid senile plaque core of Alzheimer disease? Les cellules microgliales phagocytent-elles la substance $/A4 amyloïde du coeur de la plaque sénile de la maladie d'Alzheimer ? Khalid Hamid El Hachimi, Jean-François Foncin 452 Intérêt de la linsidomine (SIN 1) pour l'évaluation de la viabilité myocardique par scintigraphie au 2°lTl après effort. Value of linsidomine (SIN 1) for assessment of myocardial viability by exercise 201 Tl scintigraphy. André Vacheron, Pierre de Vernejoul Écologie/ Ecology
201 Tl scintigraphy. André Vacheron, Pierre de Vernejoul Écologie/ Ecology Incidence écophysiologique de l’absorption épithéliale d’acides aminés dissous par Crassostrea gigas. Ecophysiological relevance of dissolved amino acid transepidermal uptake in Crassostrea gigas. Solange Le Gall, Christian Bechemin Évolution/Evolution Widespread occurrence of the proteobacteria Wolbachia and partial cytoplasmic incompatibility in Drosophila melanogaster. Infection par la protéobactérie Wolbachia et incompatibilité cytoplipoparticielle sont fréquentes chez Drosophila melanogaster. Michel Solignac, Dominique Vautrin, François Rousset Les Tropidophiidae (Serpentes ; Alethinophidia) sont-ils réellement monophylétiques ? Arguments en faveur de leur polyphylétisme. Are the Tropidophiidae (Serpentes; Alethinophidia) really monophyletic? Arguments in favor of their polyphyletic status. Hussam
really monophyletic? Arguments in favor of their polyphyletic status. Hussam Zaher C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, vol. 317 n° 5, mai 1994. COMPTES RENDUS DE L’ACADÉMIE DES SCIENCES SÉRIE III SCIENCES DE LA VIE/LIFE SCIENCES Biologie et pathologie animale/ Animal biology and pathology Biologie et pathologie végétale/Plant biology and pathology Biologie du développement/Development biology Biologie cellulaire/ Cell biology Biologie du développement/Development biology Epidémiologie/Epidemiology Écologie/Ecology Évolution/Evolution Taxonomie/Taxonomy Agronomie/Agronomy Anthropologie/Anthropology Biochimie/Biochemistry Microbiologie/Microbiology Pharmacologie/Pharmacology Sciences médicales/Medical sciences Biologie des populations/Populations biology Biologie et génétique animale/Animal biology and genetics Biologie et génétique végétale/Plant biology and genetics Biologie et génétique
Biologie et génétique végétale/Plant biology and genetics Biologie et génétique moléculaire/Molecular biology and genetics Biophysique/Biophysics Immunologie/Immunology Physiologie/Physiology C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317: 381-385 Biochimie/Biochemistry A catalytic antibody isomerizing a A5-3-ketosteroid Un anticorps catalytique isomérisant un A5 -3-cétostéroïde BACHRA KHETTAL, SOLANGE de LAUZON, BERNARD DESFOSSES, SATOSHI USHIDA, ANDRÉE MARQUET URA 400, CNRS, UFR Biomédicale des Saints-Pères, 45, rue des Saints-Pères, 75270 Paris Cedex 06, France. 2' Mukogawa Women's University, Ikeda-Cho, Nishinomiya 663, Japon. 3) URA 493, CNRS, Université Paris-VI, 2, place Jussieu, 75005 Paris, France. Tirés à part : S. de Lauzon. RÉSUMÉ Des
France. Tirés à part : S. de Lauzon. RÉSUMÉ Des anticorps ont été produits à partir du 3-fluoro-4-aza-estradiol-17-hémisuccinate afin d’obtenir des abzymes capables d’isomériser les A5-3-cétostéroïdes. L’haptène choisi possède un noyau A plan présentant une analogie avec l’intermédiaire réactionnel dienol et une polarité susceptible d’induire des groupes catalytiques dans l’anticorps. Les anticorps liant fortement l’haptène ont été clones et purifiés soit à partir d’ascites soit à partir de surnageant d’hybridomes. L’activité catalytique a été évaluée vis-à-vis de l'androst-5-ene-3,17-dione. Parmi les cinq anticorps monoclonaux différents obtenus, l’un s’est révélé capable d’augmenter significativement la vitesse d’isomérisation du substrat (kcat/kuncat = 830). La
significativement la vitesse d’isomérisation du substrat (kcat/kuncat = 830). La réaction d’isomérisation suit une cinétique michaelienne et est inhibée de manière compétitive par l’haptène. Mots clés : anticorps catalytique, A5-3-cétostéroïde, isomérisation, cinétique. ABSTRACT Antibodies were raised against 3-fluoro-4-aza-estradiol-17-hemisuccinate to elicit abzymes capable of isomerizing A'-3-ketosteroids. The hapten was designed to présent a planar A ring showing some analogy with the intermediate dienol and a polarity capable of inducing catalytic groups. Antibodies binding the hapten tightly were cloned and purified from either ascites or hybridoma supernatants. Catalytic activity was tested with androst-5-ene-3,17-dione. Among the five different monoclonal antibodies binding the hapten,
androst-5-ene-3,17-dione. Among the five different monoclonal antibodies binding the hapten, one proved to enhance significantly the substrate isomerization rate (kcat = 830). Initial rates followed Michaelis kinetics and the activity was competitively inhibited by the hapten. Key words : catalytic antibody, A5-3-ketosteroid, isomerization, kinetics. Voir Version abrégée (p. 384) The Pauling theory of enzymatic catalysis [1], throwing light on the close structural complementarity of the enzymatic site with the transition state of the reaction, has been the starting point in the generation of catalytic antibodies elicited against stable analogs of the transition state of an enzymatic reaction. Moreover, adequate basic
the transition state of an enzymatic reaction. Moreover, adequate basic or acidic, nucleophilic or electrophilic amino acid side chains should be présent and precisely positioned into antibody combining sites [2]. The mammalian A5-3-ketosteroid isomerase plays an essential biological role since it is implicated in cholesterol hepatic detoxification and in steroid hormone biosynthesis. A great deal is known about this enzyme due to intensive study of bacterial A5-3-ketosteroid isomerase of Pseudomonas testosteroni which promotes the highly efficient isomerization of a variety of A5 or A5-6-3-ketosteroids to A4-3-ketosteroids at a rate approaching the diffusion limit. This reaction has been shown to occur
the diffusion limit. This reaction has been shown to occur via the enolization of the A5-3-ketosteroid to form a dienolic intermediate [3, 4]. In the active site of the enzyme, Tyr-14 acts as an acid to protonate the 3-keto group and Asp-38 acts as a base to remove the axial proton at position 4 (Fig. 1). The présent challenge was to induce antibodies able to catalyze this transformation of A5-3-ketosteroids into A4-3-ketosteroids. Note présentée par Marianne Grunberg-Manago. Note remise le 7 mars 1994, acceptée après révision le 11 avril 1994. B. Khettal et al. Figure 1. Reaction mechanism of A5-3-ketosteroid
B. Khettal et al. Figure 1. Reaction mechanism of A5-3-ketosteroid isomerase of Pseudomonas testosteroni. In this study we présent preliminary data showing the unequivocal catalytic activity of a monoclonal antibody raised against 3-fluoro-4-aza-estradiol-17-hemisuccinate. Material and methods Synthesis of hapten and substrates The structures of hapten, substrates and products are given in Figure 2. 3-Fluoro-4-aza-estradiol-17-hemisuccinate (1) was synthesized from 19-nor-testosterone [5]. 5(10)-Estradiol-17-one (2) was a gift from Roussel UCLAF and androst-5-ene-3,17-dione (3) was obtained by oxidation of 3|3-hydroxy-17-oxo-androst-5-ene [6]. Preparation of conjugates, immunization, immunoassays, and monoclonal antibody production The mixed anhydride method [7] was used to couple the hapten to the
method [7] was used to couple the hapten to the carrier proteins, bovine serum albumin (BSA) and ß-lactoglobulin. Mice were immunized with the BSA conjugate. After fusion of mouse spleen cells with myeloma cells according to standard protocols, the resulting hybridomas were screened by an enzyme-linked immunosorbent assay (ELISA) for binding to the hapten ß-lactoglobulin conjugate using peroxidase-linked rabbit anti-mouse antibodies [8]. Hybridoma cells that secreted IgG spécifique for the hapten were cloned at least twice before further sélection by compétition ELISA [9] using hapten (1), substrate (2) and product (4) competitors. Large amounts of selected monoclonal antibodies were produced
(4) competitors. Large amounts of selected monoclonal antibodies were produced and purified from either ascites or from hybridoma supernatants. Purification included a 45 % saturated ammonium sulfate précipitation and protein A affinity chromatography. Purity of immunoglobulins was examined by SDS-polyacrylamide gel electrophoresis. Enzymatic assays The purified antibodies were tested for catalytic activity by measuring the rate of isomerization of 5,10-estren-3-one-17β-ol (2) and androst-5-ene-3,17-dione (3). The release of 19-nor-testosterone (4) or of androst-4-ene-3,17-dione [5] was monitored by UV spectroscopy at 248 nm at 20 °C. The assay cell contained defined concentrations of substrate in 1 ml of 100 mM phosphate buffer
of substrate in 1 ml of 100 mM phosphate buffer pH 7.0, containing 5 % ethanol (v/v). Reactions were initiated by addition of purified immunoglobulins. The reference cell contained either the buffer (Fig. 3) or the buffer and substrate (Fig. 4). Figure 3. Isomerization of androst-5-ene-3,17-dione at 140 μM concentration, catalyzed by 9D5H12 at a final concentration of 3 μM in 100 mM phosphate buffer, pH 7.0 (•), and in 100 mM phosphate buffer alone (o). Results Screening of antibodies elicited against 3-fluoro-4-azaestradiol-17-hemisuccinate-BSA (1-BSA) led to 5 different monoclonal purified antibodies binding tightly to the hapten (1), the substrate (2)
antibodies binding tightly to the hapten (1), the substrate (2) and product (4) to various degrees. The subtype study of the selected antibodies showed that all antibodies were IgG1. The antibodies were judged to be homogeneous by SDS-polyacrylamide gel electrophoresis (SDS/PAGE). However, in the case of IgGs purified from hybridoma supernatants, two closely spaced bands could be observed for the non-reduced IgGs and for the heavy chain of mercaptoethanol-reduced IgGs. These double bands were not observed in the case of ascites IgGs. The 5 purified IgG antibodies (3 μM) have been tested for their ability to catalyze the isomerization of
been tested for their ability to catalyze the isomerization of substrates (2) and (3) (100 μM). The catalytic activity was monitored spectroscopically by measuring the increase of absorbances at 248 nm. None of the 5 antibodies produced any measurable isomerization of 5,10-estren-17β-ol-3-one (2). However, one of them (9D5H12) proved to enhance significantly the rate of isomerization of androst-5-ene-3,17-dione (3) (Fig. 3). This enhancement was inhibited by the hapten. The initial rates of isomerization of androst-5-ene-3,17-dione (3) were measured as a function of substrate concentrations (7-35 mM) in the presence of 0.44 μM purified 9D5H12 immunoglobulin. The reaction followed Michaelis-Menten kinetics
0.44 μM purified 9D5H12 immunoglobulin. The reaction followed Michaelis-Menten kinetics (Fig. 4). Kinetic parameters (Table I) were determined using a Lineweaver-Burk analysis. As expected, the spontaneous reaction followed first order kinetics (kuncat = 2.7 x 10−3 min−1). Isomerization of (3) by the antibody was thus accelerated by a factor of 830. Inhibition studies performed at two fixed concentrations of the inhibitor (1) using the same substrate and antibody concentrations demonstrated a competitive inhibition with a Ki of 130 μM. The catalytic activity associated with 9D5H12 monoclonal antibody was destroyed by thermal denaturation of the antibodies at 95 °C for 30
thermal denaturation of the antibodies at 95 °C for 30 min. Figure 4. Lineweaver-Burk plot for isomerization of androst-5-ene-3,17-dione by 9D5H12. Velocities were determined as described in Table I. (•) no inhibitor present (A) inhibited by 1.33 μM hapten (o) inhibited by 1.87 μM hapten. Discussion The first step in the production of catalytic antibodies is the design of the hapten. Steroids with planar A or A rings are competitive inhibitors of the isomerase and have been considered as analogs of the high-energy dienol intermediate [10]. We thus chose a molecule with an aromatic A ring. The electronegative fluorine at
molecule with an aromatic A ring. The electronegative fluorine at position 3 should induce an acidic side-chain in the antibody leading to a strong hydrogen bond. We initially planned to quaternize the 4-nitrogen to induce a complementary basic group. As the corresponding compound could not be obtained, we used (1) as a hapten. The hybridoma selection procedure is the second crucial phase in the successful production of catalytic monoclonal antibodies [11]. We selected antibodies by competition ELISA [9] measuring their relative affinities (a) for the hapten (b), for molecule (2) which is a potential substrate, (c) for the corresponding product
which is a potential substrate, (c) for the corresponding product (4). Antibody should recognize the hapten with a high affinity, the substrate with a medium affinity and the product with a much lower affinity to avoid product inhibition. Among known substrates of A5-3-ketosteroid isomerase, (2) was preferred to (3) for this selection step since the spontaneous isomerization of (3) is much faster. Five antibodies exhibited the required relative affinities and were purified in larger amounts from 2 ascitic fluids and 3 hybridoma supernatants. Though antibodies were virtually the only proteins present in the purified preparations from the SDS-electrophoresis gels stained
present in the purified preparations from the SDS-electrophoresis gels stained with Coomassie blue, very close double bands could be observed when IgGs arose from hybridoma supernatants. This double band concerned the heavy chain of IgGs only. Horse serum present in culture medium of hybridomas was specified low-IgG serum. It is however likely that the remaining horse IgGs were not perfectly separated from mouse IgGs on protein A. These horse IgGs cannot be involved in the catalytic activity observed for antibody 9D5H12 since other antibodies purified in the same manner from hybridoma supernatants did not show any catalytic potency. When initial
hybridoma supernatants did not show any catalytic potency. When initial rates of isomerization of substrate (3) by 9D5H12 antibody as a function of substrate concentration were measured, saturation kinetics were observed. This finding is consistent with the formation of a Michaelis complex between the substrate and the antibody prior to isomerization. At 20 °C the value of kcat and Km were determined to be respectively 2.25 min−1 and 160 μM. The observed rates were corrected automatically for the uncatalyzed isomerization of androst-5-ene-3,17-dione since the substrate was present in the two spectrophotometric cells (differential measurements). However, spontaneous reaction in the absence
spectrophotometric cells (differential measurements). However, spontaneous reaction in the absence of antibodies was measured at the same temperature for the determination of kcat/kuncat ratio, which gave evidence of a substantial enhancement of the catalysis by the antibody. Convincing evidence that substrate reacts in the induced binding pocket of the antibody was obtained by examining the kinetics in the presence of the hapten molecule. As shown in Figure 3, the isomerization of androst-5-ene3,17-dione to androst-4-ene-3,17-dione is competitively inhibited by hapten with a Ki value of 130 μM. This Ki value is relatively high and reveals that the structure of the hapten
relatively high and reveals that the structure of the hapten is likely to be far different from that of the transition state. The activity is probably rather due to the presence in the binding site of the proper acidic and basic residues. No significant isomerization of substrate (2) could be detected with 9D5H12 antibody, which may be explained by the non-flexibility of amino acid residues in the binding site of the antibody. In conclusion, using a rationally designed hapten, we have successfully elicited an antibody that accelerates the rate of A5-3-ketosteroid isomerization. Preparation of larger amounts of this immunoglobulin purified
A5-3-ketosteroid isomerization. Preparation of larger amounts of this immunoglobulin purified from ascites is in progress. This will allow a more complete study of the mechanism of this abzyme. This paper is dedicated to the memory of Nicole Cittanova (1938-1993) who initiated this work. VERSION ABRGÉE La A5-3-cétostéroïde isomerase joue un rôle essentiel au niveau des glandes endocrines pour la biosynthèse des hormones aussi bien qu'au niveau du foie pour le catabolisme du cholestérol. Le mécanisme d'action de cette isomerase, très étudié avec l'enzyme de Pseudomonas testosteroni fait intervenir essentiellement un acide et une base du site enzymatique, la tyrosine 14
acide et une base du site enzymatique, la tyrosine 14 et l'aspartate 38, qui catalysent l'enolisation de la cétone en 3 du stéroïde. La reprotonation du dienol intermédiaire conduit à la cétone conjuguée. Dans ce travail, nous avons cherché à obtenir des anticorps monoclonaux capables de mimer l'activité catalytique de la A -3-cétostéroïde isomerase. L'haptène choisi, le 3-fluoro-4-aza-estradiol-17-hémisuccinate, possède un noyau plan, présentant une analogie avec l'intermédiaire réactionnel dienol et une polarité susceptible d'induire des groupes catalytiques dans l'anticorps. L'immunisation des souries, la fusion cellulaire et les clonages ont été réalisés de manière habituelle. Les anticorps ont été sélectionnés d'après
réalisés de manière habituelle. Les anticorps ont été sélectionnés d'après leurs affinités relatives vis-à-vis de l'haptène. Les immunoglobulines purifiées ont été obtenues soit à partir d'ascites soit à partir de surnageants d'hybridome, par précipitation au sulfate d'ammonium et chromatographie d'affinité sur protéine A. L'électrophorèse en SDS/polyacrylamide en conditions réductrices et non-réductrices a confirmé la pureté des immunoglobulines provenant des ascites. Par contre, pour les immunoglobulines provenant des surnageants d'hybridomes, deux bandes très proches ont été observées, que ce soit avant réduction ou au niveau des chaînes lourdes. Une explication possible pourrait être une contamination par les immunoglobulines de cheval présentes
pourrait être une contamination par les immunoglobulines de cheval présentes en très faible pourcentage dans le milieu de culture des hybridomes et imparfaitement séparées des immunoglobulines de souris sur protéine A. Les tests d'activité catalytique, réalisés en présence de deux substrats de l'isomérase, le 5,10-estren-3-one-17S-ol et l'androst-5-ene3,17-dione, ont été réalisés en mesurant par spectrophotométrie l'augmentation des absorbances à 248 nm. Aucun des anticorps ne catalyse l'isomérisation du 5,10-estren-3-one-17P-ol. Par contre, la vitesse d'isomérisation de l'androst-5-ene-3,17-dione en présence de l'anticorps 9D5H12 est nettement augmentée par rapport à l'isomérisation spontanée mesurée en absence d'anticorps. La dénaturation thermique de l'anticorps fait disparaître cette
absence d'anticorps. La dénaturation thermique de l'anticorps fait disparaître cette activité catalytique. L'étude des vitesses initiales en fonction de la concentration du substrat a montré que la réaction suit une cinétique michaélienne. L'haptène est un inhibiteur compétitif par rapport au substrat, ce qui confirme que l'isomérisation du stéroïde a lieu dans le site de liaison de l'anticorps. Le rapport kat/kunat démontre une accélération de 830 fois de la vitesse d'isomérisation. La présence possible d'un artefact dans la préparation d'immunoglobulines, par exemple les immunoglobulines de cheval non séparées au cours de la purification, ne peut expliquer l'activité catalytique mesurée puisqu'aucune activité
la purification, ne peut expliquer l'activité catalytique mesurée puisqu'aucune activité n'a pu être observée pour trois autres anticorps liant l'haptène et qui ont été produits et purifiés dans les mêmes conditions. En conclusion, par le choix rationnel de la molécule d'haptène utilisée pour induire les anticorps, il a été possible d'obtenir un anticorps catalysant l'isomérisation des A -3-cétostéroïdes. La préparation d'une quantité plus importante d'immunoglobulines parfaitement purifiées à partir d'ascites est en cours. Ceci permettra d'étudier plus complètement le mécanisme de la catalyse. À jour 1. Pauling L. 1948. Nature of forces between large molecules of biological interest., Nature 161: