Dissertations / Theses on the topic 'Malate'
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1
Smith, K. "Enzymes of L -malate metabolism : Malate dehydrogenase from porcine heart, mesophilic bacteria and thermophilic bacteria and malate synthase from thermophilic bacteria." Thesis, University of Manchester, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356707.
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Cheffings, Christine. "Expression of the vacuolar malate channel in Xenopus oocytes." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361741.
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Jeffery, David. "Studies on citrate and malate metabolism in Lycopersicon esculentum." Thesis, University of Bath, 1985. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.353243.
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The specific activities of citrate synthase and malate dehydrogenase extracted from mature green fruit of Lycopersicon esculentum, fell 60% during the first two weeks of a twelve week experiment in which the fruit were stored in an atmosphere designed to inhibit ethylene synthesis. Throughout the remainder of the experiment, the specific activities were relatively constant. In the initial two week period, the specific activity of NADP-linked malic enzyme rose by 400%, malic acid concentration fell by 50%, while the concentration of citric acid rose by 20%. Those features of ripening such as the de novo synthesis of lycopene and polygalacturonase, which were thought to depend on ethylene for initiation of response, could not be detected until the fruit were removed to a normal atmosphere. Additionally, citrate synthase and malate dehydrogenase from mature green tomato fruit stored in the presence or absence of ethylene, showed similar trends in specific activity, and the presence of the olefin made no significant difference to the rate of loss of enzyme specific activity. The purification and partial characterisation of citrate synthase from Lycopersicon esculentum is described. The enzyme is a dimer with sub-units of similar size and a total Mr of approximately. 100,000. The characterisation revealed no obvious regulatory features that would easily account for the fall in specific activity. Sub-cellular fractionation studies demonstrated unequivocally that the site of organic acid metabolism was the mitochondrion. Citrate synthase, NAD-dependent isocitrate dehydrogenase and NAD-dependent malic enzyme were shown to be located exclusively in the mitochondrion, while malate dehydrogenase was located both in the cytosol and the mitochondrion. All these enzymes including cytosolic malate dehydrogenase exhibited the co-ordinated fall in specific activity described above. A hypothesis is proposed which includes a novel coarse control of the citric acid cycle and related enzymes, as an early indicator of senescence.
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Howard, Bruce Riley. "The crystal structure of malate synthase and mechanistic implications /." view abstract or download file of text, 1999. http://wwwlib.umi.com/cr/uoregon/fullcit?p9948022.
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Thesis (Ph. D.)--University of Oregon, 1999.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 67-71). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9948022.
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Costenaro, Lionel. "Interactions faibles protéine – protéine en solution : La malate déshydrogénase halophile." Phd thesis, Université Joseph Fourier (Grenoble), 2001. http://tel.archives-ouvertes.fr/tel-00007698.
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La cellule est un milieu très concentré où les interactions entre macromolécules, même faibles, jouent un grand rôle dans leur solubilité et dans l'assemblage des complexes labiles assurant les fonctions biologiques. Les interactions faibles entre protéines déterminant la non-idéalité de leurs solutions vont aussi gouverner leur cristallisation.Dans quelle mesure les interactions protéine – solvant influencent-elles les interactions protéine – protéine ? Nous avons mis en relation ces deux types d'interactions pour la malate déshydrogénase (Hm MalDH) de Haloarcula marismortui, protéine halophile très acide qui a des solvatations variées et très riches en eau et en sel.
Nous avons développé une nouvelle méthode de détermination du second coefficient du viriel A2 par la modélisation des profils de vitesse de sédimentation en ultracentrifugation analytique, qui permet l'étude de solvants complexes.
Les interactions protéine – protéine de la Hm MalDH en divers sels ont été caractérisées par diffusion de neutrons ou de rayons X aux petits angles. Les A2 et les facteurs de structure en solution ont été modélisés par des potentiels d'interaction de type DLVO. Les interactions répulsives sont principalement dues au terme de volume exclu et dans une moindre mesure au terme électrostatique. Les interactions attractives sont qualitativement corrélées à des valeurs positives ou négatives des paramètres d'interaction préférentielle avec le sel. Ces résultats permettent d'expliquer l'adaptation moléculaire des protéines halophiles qui doivent ainsi avoir une solvatation riche en sel pour rester soluble à haut sel.
La cristallisation par dilution de la Hm MalDH dans des mélanges sel – MPD (méthyl-2-pentanediol-2,4) résulte d'une lente évolution des interactions protéine – protéine, de répulsives à modérément attractives. Le MPD modifie les interactions protéine – protéine en divers sels en ajoutant une attraction qui est liée à la répulsion du MPD par les charges de la protéine.
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Dymov, Sergiy. "Isolation and characterization of malate dehydrogenase mutant of Sinorhizobium meliloti." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=31224.
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A Sinorhizobium meliloti (S. meliloti ) mutant, Rm30O49, deficient in malate dehydrogenase (MDH) activity was isolated via random Tn5tac1 mutagenesis. DNA sequence analyses revealed 60 the inaction is within the mdh gene. Rm30049 lacks MDH activity under all growth conditions, but shows increased or decreased activities of the TCA cycle enzymes 2-oxoglutarate dehydrogenase and succinate dehydrogenase in the presence or absence, respectively, of IPTG (isopropyl beta-D-thiogalactoside). The symbiotic phenotype of the mutant is an inability to fix nitrogen. Alfalfa seedlings inoculated with Rm30049 produced small white root nodules, but were chlorotic and failed to reach a wild-type shoot dry weight. Cosmid clone pDS15 was isolated by heterologous complementation of a Rhizobium leguminosarum sucD mutant by the S. meliloti pLAFR1 clone bank. This cosmid also restored MDH activity to Rm30049, and complemented the mutant growth and symbiotic phenotypes. Three Tn5 insertions isolated in pDS15 within sucA failed to complement Rm30049. DNA sequence analyses indicate that the mdh gene is part of the TCA cycle operon with sucCD, and that downstream and upstream of this, are operons encoding sucAB and sdhCDAB, respectively.
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Ingram, J. "Developmental and environmental regulation of malate decarboxylation in CAM plants." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357414.
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Mohan, Anand. "Myoglobin redox form stabilization : role of metabolic intermediates and NIR detection." Diss., Manhattan, Kan. : Kansas State University, 2009. http://hdl.handle.net/2097/2310.
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Rentsch, Doris. "The vacuolar malate/citrate carrier of Hordeum vulgare and Hevea brasiliensis /." [S.l.] : [s.n.], 1993. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10455.
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Jackson, Richard Michael. "A theoretical investigation into the properties of lactate and malate dehydrogenases." Thesis, University of Bristol, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282473.
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Saayman, Maryna. "Characterisation of the malate transporter and malic enzyme from Candida utilis." Thesis, Stellenbosch : University of Stellenbosch, 2011. http://hdl.handle.net/10019.1/16520.
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Dissertation (PhD)--University of Stellenbosch, 2005.ENGLISH ABSTRACT: Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. The fission yeast Schizosaccharomyces pombe effectively degrades L-malate, but only in the presence of an assimilable carbon source. In contrast, the yeast Saccharomyces cerevisiae is unable to effectively degrade L-malate, which is ascribed to the slow uptake of L-malate by diffusion. In contrast, the yeast Candida utilis can utilise L-malate as the only source of carbon and energy, but this is subject to substrate induction and catabolite repression. Very little research has been done on a molecular level in C. utilis and only a few of its genes have been studied. In this study, we have shown that the yeast C. utilis effectively degraded extracellular L-malate and fumarate, but in the presence of glucose or other assimilable carbon sources, the transport and degradation of these dicarboxylic acids was repressed. The transport of both dicarboxylic acids was shown to be strongly inducible by either L-malate or fumarate and kinetic studies suggest that the same transporter protein transports the two dicarboxylic acids. In contrast, S. pombe effectively degraded extracellular L-malate, but not fumarate, only in the presence of glucose or other assimilable carbon sources. The S. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of L-malate. In order to clone the C. utilis dicarboxylic acid transporter, a cDNA library from C. utilis was constructed using a number of strategies to ensure representativeness and high transformation frequencies. The cDNA library was transformed in a S. cerevisiae strain carrying a plasmid containing the S. pombe malic enzyme gene (mae2) to allow screening for a malate-degrading S. cerevisiae clone. However, no positive clones that would indicate the successful cloning of the C. utilis malate transporter were obtained. The C. utilis malic enzyme gene, CuME, was subsequently isolated from the cDNA library based on conserved sequence homologies with the genes of S. cerevisiae and S. pombe, and characterised on a molecular and biochemical level. Sequence analysis revealed an open reading frame of 1926 bp, encoding a 641 amino acid polypeptide with a predicted molecular weight of 70.2 kDa. The optimum temperature for the C. utilis malic enzyme was 52°C and the enzyme was stable at 50°C for 2 hours. The inferred amino acid sequence showed significant homology with the malic enzymes of S. pombe and S. cerevisiae. Expression of the CuME gene is subject to glucose repression and substrate induction, as was observed for the dicarboxylic acid transporter from C. utilis. The CuME gene was successfully coexpressed with the S. pombe malate permease gene (mae1), resulting in a recombinant strain of S. cerevisiae able to effectively degrade L-malate.
AFRIKAANSE OPSOMMING: Daar is ’n merkwaardige verskil in die vermoë van verskillende gisspesies om ektrasellulêre dikarboksielsure af te breek en dit as enigste bron van koolstof te benut. Die splitsingsgis Schizosaccharomyces pombe kan L-malaat effektief afbreek, maar slegs in die teenwoordigheid van ’n ander benutbare koolstofbron. In teenstelling hiermee is dit vir die gis Saccharomyces cerevisiae onmoontlik om L-malaat effektief af te breek en te benut, wat hoofsaaklik toegeskryf kan word aan die stadige opname van L-malaat deur middel van diffusie. Die gis Candida utilis kan egter L-malaat as die enigste bron van koolstof en energie benut, maar dit is onderhewig aan substraat-induksie en kataboliet onderdrukking. Baie min navorsing op molekulêre vlak is tot hede in C. utilis uitgevoer en slegs ’n paar gene in hierdie gis is al bestudeer. In hierdie studie het ons aangetoon dat die gis C. utilis L-malaat en fumaraat effektief afbreek, maar dat glukose of ander benutbare koolstofbronne die opname en afbraak van hierdie dikarboksielsure onderdruk. Die opname van beide dikarboksielsure is sterk induseerbaar deur L-malaat óf fumaraat, terwyl kinetiese studies toon dat beide dikarboksielsure deur dieselfde transporter-proteïen vervoer word. In teenstelling hiermee kan S. pombe ekstrasellulêre L-malaat, maar nie fumaraat nie, in die teenwoordigheid van glukose of ’n ander benutbare koolstofbron effektief afbreek. Die S. pombe L-malaat transporter was nie in staat om fumaraat te vervoer nie, alhoewel fumaraat die opname van L-malaat onderdruk het. Ten einde die dikarboksielsuur transporter van C. utilis te kloneer, is verskeie strategieë gevolg ten einde ’n cDNA-biblioteek van C. utilis te konstrueer wat verteenwoordiging en hoë transformasie-frekwensies kan verseker. Die cDNA-biblioteek is getransformeer in ’n S. cerevisiae ras wat die S. pombe malaatensiem geen (mae2) bevat om die sifting van ’n S. cerevisiae kloon wat malaat effektief kan afbreek, moontlik te maak. Geen positiewe klone wat dui op die klonering van die C. utilis malaat transporter kon egter gevind word nie. Die C. utilis malaatensiem geen, CuME, is vervolgens van uit die cDNA biblioteek geïsoleer deur van gekonserveerde DNA-homologie met S. cerevisiae en S. pombe gebruik te maak, en op molekulêre en biochemiese vlak gekarakteriseer. DNA-volgordebepaling het ’n oopleesraam van 1926 bp onthul, wat kodeer vir ’n 641 aminosuur polipeptied met ’n verwagte molekulêre gewig van 70.2 kDa. Die optimale temperatuur van die C. utilis malaatensiem was 52°C en die ensiem was vir 2 ure stabiel by 50°C. Die afgeleide aminosuurvolgorde het beduidende homologie met die malaatensieme van S. pombe en S. cerevisiae getoon. Die CuME geen is suksesvol saam met die S. pombe malaat permease geen (mae1) uitgedruk om ’n rekombinante S. cerevisiae ras te genereer wat in staat is om L-malaat effektief af te breek.
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Kirstetter, Anne-Sophie. "Etude de la fixation du carbone inorganique chez la levure pour la production industrielle de molécules d’intérêt." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLC015/document.
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Ces dernières années ont vu un grand développement des biotechnologies blanches et de l'ingénierie métabolique avec l'objectif de remplacer les procédés de synthèse de molécules d’intérêt de l’industrie chimique classique par des voies de synthèse biologique. Dans ce contexte, les réactions anaplérotiques, qui produisent les acides dicarboxyliques, sont particulièrement intéressantes puisqu'au delà de la production de ces molécules d’intérêt elles permettent une fixation nette de carbone, réduisant ainsi l’impact environnemental des procédés. Ce travail de thèse a donc porté sur l'élaboration d'une stratégie d'ingénierie métabolique faisant appel à des réactions de fixation de carbone inorganique chez la levure pour la production d'acide malique, une molécule plateforme ayant de nombreuses applications industrielles. La levure Saccharomyces cerevisiae a été choisie comme hôte pour sa commodité d’utilisation dans les procédés industriels et ses nombreux outils génétiques. L'approche développée repose sur la mise en place d'une voie de production d'acide malique par surexpression de la phosphonéolpyruvate carboxylase d'Escherichia coli (PEPC), de la malate déshydrogénase peroxysomale de S. cerevisiae relocalisée dans le cytosol (MDH) et du transporteur d'acides dicarboxyliques de Schizosaccharomyces pombe. La souche de levure recombinante obtenue a été caractérisée lors d'essais en fioles, en présence notamment de carbonate de calcium pour assurer un apport de carbone inorganique. Ces essais ont permis de mettre en évidence un effet stimulant de l'apport de carbone inorganique sur la production de malate et d'obtenir des concentrations de malate de l'ordre de 2,5 g/L à partir de 50 g/L de glucose, pour un rendement maximal de 0,046 gramme de malate par gramme de glucose. Des essais en bioréacteur de 5 L en présence d'air ou d'air enrichi à 5% de CO2 ont montré un effet positif de l'apport de carbone inorganique sous forme de dioxyde de carbone sur la production de malate. La concentration maximale de malate obtenue est de 1,46 g/L à partir de 50 g/L de glucose, soit un rendement de 0,029 gramme de malate par gramme de glucose. Des souches intermédiaires exprimant la PEPC et la MDH obtenues pour la production de malate ont également été caractérisées pour la production d'éthanol, car elles semblaient présenter une augmentation du rendement de production d'éthanol par effet transhydrogénase par rapport à la souche sauvage. Les essais n'ont cependant pas permis de confirmer cette augmentation de rendementWhite biotechnologies have been developing quickly during the last decades, aiming at replacing chemical syntheses by biological processes for the industrial production of target compounds. In this context, the implementation of anaplerotic reactions in the metabolism is of great interest, since those reactions allow both production of dicarboxylic acids and direct fixation of inorganic carbon. This work is about the development of a metabolic engineering strategy using inorganic carbon fixation reactions to produce malic acid, a compound with various industrial applications. The yeast Saccharomyces cerevisiae was chosen as a host for its convenient use in industrial processes and the availability of genetic tools. The approach developed to produce malic acid is based on the overexpression of Escherichia coli phosphoenolpyruvate carboxylase (PEPC), S. cerevisiae peroxysomale malate dehydrogenase relocated in the cytosol (MDH) and Schizosaccharomyces pombe dicarboxylic acid carrier. A recombinant yeast strain expressing those three genes was obtained and characterised in shake-flasks experiments, involving more specifically calcium carbonate as an inorganic carbon source. Those experiments showed an enhancement of the malate production in the presence of calcium carbonate and allowed to obtain a concentration of 2.5 g/L from 50 g/L glucose, for a maximal yield of 0.046 gram malate per gram glucose. Fermentation experiments were performed in a 5 L bioreactor in the presence of air or 5% CO2 enriched air; they confirmed the positive effect of inorganic carbon addition as CO2 on malate production. A malate concentration of 1.46 g/L from 50 g/L glucose was obtained, giving a yield of 0.029 gram malate per gram glucose. Intermediate recombinant strains expressing PEPC and MDH were also characterised, for ethanol production, as they seemed to give increased ethanol yields, probably due to a transhydrogenase effect. Shake flasks and bioreactors experiments did unfortunately not confirm the yield improvement
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Anstrom, David Michael. "Structural, mutagenic, and kinetic studies on the reaction mechanism of malate synthase /." view abstract or download file of text, 2005. http://wwwlib.umi.com/cr/uoregon/fullcit?p3181081.
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Thesis (Ph. D.)--University of Oregon, 2005.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 90-101). Also available for download via the World Wide Web; free to University of Oregon users.
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Nobbs, Timothy J. "Protein engineering of E. coli malate dehydrogenase and B. stearothermophilus lactate dehydrogenase." Thesis, Open University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293546.
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Lemaire, Martine. "Etude du site actif de la malate deshydrogenase a nadp de sorgho." Paris 11, 1995. http://www.theses.fr/1995PA112447.
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Chez le sorgho, la malate deshydrogenase a nadp (mdh a nadp, ec 1. 1. 1. 82) est un enzyme clé de la voie d'incorporation du co#2 atmosphérique. Le sujet de cette thèse a consisté à étudier son site actif par mutagenese dirigée et par dérivation chimique. Le site de fixation du nadph a été étudié par une approche chimique en utilisant des analogues arylazido-aminobutyryl du cofacteur. Les expériences réalisées ont montré que ces analogues sont des inhibiteurs compétitifs du nadph et que, après une forte irradiation, ils se fixent de façon covalente au site du cofacteur. Des expériences préliminaires de localisation de ce site de fixation ont été réalisées. L'effet du diethylpyrocarbonate (depc) a été testé sur l'activité de la mdh a nadp de sorgho. Les résultats obtenus montrent qu'une histidine est localisée au site actif cet enzyme. Apres coupure trypsique de la protéine traitée au depc, des analyses par séquençage et par spectrométrie de masse ont permis d'identifier l'histidine dérivée (his229). Afin de préciser le rôle de ce résidu dans l'activité de l'enzyme, il a été remplace par mutagenèse dirigée. Les protéines mutées sont totalement inactives démontrant que l'his229 joue un rôle primordial dans la catalyse de la mdh a nadp de sorgho. Une interaction de l'histidine du site actif avec un aspartate ayant été suggérée par analogie avec les mdh a nad, le résidu candidat a ce rôle (asp201) a été remplace par une alanine ou une asparagine. Les protéines mutées ont des paramètres cinétiques fortement modifies, indiquant que l'asp201 participe a la catalyse en interaction avec l'his229. Par ailleurs, il avait été propose que la cystéine 175 forme une triade catalytique avec l'his229 et l'asp201. Une mdh mutée dans laquelle la cys175 a été remplacée par une alanine a donc été produite, mais elle présente des paramètres cinétiques peu différents de ceux de l'enzyme non mute, ce qui indique que ce résidu ne participe pas à la catalyse
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Charbonnier, Teddy. "Transport du pyruvate et régulations du métabolisme central par le malate chez Bacillus subtilis." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS076/document.
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Chez Bacillus subtilis comme pour toutes les bactéries, le métabolisme central du carbone est essentiel pour la croissance de la cellule. Elle utilise le glucose (source de carbone glycolytique) et le malate (source de carbone gluconéogenique) comme sources de carbone préférentielles. Ces deux sources de carbone sont capables d'induire la répression catabolique au travers de la protéine régulatrice CcpA et ainsi d'établir une hiérarchie dans l'utilisation des sources alternatives de carbone. Au centre du métabolisme du carbone se trouve le pyruvate que B. subtilis est capable d'utiliser comme seule source de carbone, mais son transporteur reste inconnu.Des analyses transcriptomiques ont montré que seul l'opéron ysbAB était spécifiquement induit en présence de pyruvate, et nous avons montré que sa délétion entraînait une perte de croissance presque totale sur pyruvate. En utilisant des protéines étiquetées, nous avons mis en évidence qu'YsbA et YsbB formaient un complexe se localisant à la membrane. Nous avons ensuite montré que ce complexe est le transporteur principal du pyruvate et fonctionne comme un transporteur par diffusion facilitée. A l'aide d'une fusion rapportrice, nous avons démontré que l'opéron lytST situé en amont d'ysbAB, et codant pour un système à deux composants, était responsable de l'induction d'ysbAB. Nous avons également montré qu'en plus d'une répression par CcpA en présence de glucose ou de malate, une régulation dépendante de l'activité enzyme malique de MaeA s'exerce sur ysbAB. Cette régulation est due à l'accumulation de pyruvate dans la cellule qui perturbe l'activation d'ysbAB par LytST.Nous avons aussi montré qu'une régulation indépendante de CcpA s'exerce sur dctP, le gène codant pour le transporteur du succinate et du fumarate en présence de malate, suggérant un mécanisme similaire à celui observé pour ysbAB. Enfin, nous avons montré que le flux métabolique traversant MaeA était également impliqué dans la régulation par CcpA de l'entrée des sources glycolytique par le malateIn Bacillus subtilis like for all the bacteria, the central carbon metabolism is essential for growth. It uses glucose (a glycolytic carbon source) and malate (a gluconeogenic carbon source) as preferential carbon sources. These two carbon sources are able to induce carbon catabolite repression through the transcription factor CcpA and thus establishing a hierarchy in the use of alternative carbon sources. The pyruvate is in the middle of the carbon metabolism, and can be used by B. subtilis as sole carbon source; however its transporter remains unknown.Transcriptome analyses revealed that the only operon specifically expressed in cells grown on pyruvate is ysbAB, and we showed that its deletion led to a strong growth defect on pyruvate. Using tagged proteins, we highlighted that YsbA and YsbB formed a complex localized at the membrane. We next showed that this complex is the major pyruvate transporter, and operates as a facilitated transporter. Using a reporter fusion, we showed that the operon lytST located upstream of ysbAB, and coding for a two-component system, is responsible for the induction of ysbAB. We also showed that besides the CcpA-mediated repression by both glucose and malate, an additional regulation mechanism through the malic enzyme activity of MaeA is acting on ysbAB. This regulation is due to the accumulation of pyruvate in the cell which hinders the LytST-mediated induction of ysbAB.We also showed that a CcpA-independent repression is exerted on dctP, the gene coding for the succinate and fumarate transporter, in the presence of malate, suggesting a regulation mechanism similar to the one observed for ysbAB. Finally, we showed that the metabolic flux going through MaeA is also involved in the CcpA-dependent repression of the genes coding for glycolytic transporter in presence of malate
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Erivalda, Farias de Aragão Maria. "Purification de l'enzyme malique à NAD et étude du métabolisme du malate chez Eucalyptus citriodora en situation de contrainte saline." Nancy 1, 1995. http://www.theses.fr/1995NAN10356.
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L’enzyme malique à NAD (EM NAD) a été purifiée à partir d'hypocotyles Etoiles de Vigna unguiculata. Les étapes de purification de l'enzyme comportent : l'obtention de mitochondries purifiées, une chromatographie échangeuse d'ions (DEAE-cellulose) et deux chromatographies d'affinité (Blue-sepharose et CoA-agarose). L’enzyme purifiée est présente sous la forme dimérique, composée de deux sous-unités de masse moléculaire très proche (64 et 61 kDa). L’enzyme présente un mécanisme cinétique séquentiel au hasard et l'affinité pour chaque substrat augmente quand l'autre substrat est déjà lié à l'enzyme. L’enzyme est inhibée par les sels monovalents (NaCL, KCl et NaNO3) et par les produits de la réaction. Des anticorps polyclonaux, obtenus à partir de l'enzyme purifiée, ont été utilisés pour un suivi des modifications de l'enzyme en situation de contrainte saline. Pour cette deuxième partie, des plants d'Eucalyptus citriodora, âgés de 3 mois, sont cultivés en serre en ajoutant du NaCL (50 et 100 mm) à la solution nutritive. Les différences de croissance des plants ne sont observées qu'après 8 semaines de traitement. Cependant, dès la 3ème semaine, le sodium s'accumule dans les parties aériennes des plants traités alors que la teneur en potassium diminue. Ces phénomènes sont plus accentués pour le traitement NaCL 100 mm. Parallèlement, la teneur en acides aminés, en particulier en proline, augmente dans les feuilles, tandis que les teneurs en ions nitrates et en malate diminuent. Dans les feuilles des plants traites, les activités spécifiques des enzymes maliques (NAD et NADP) augmentent après 3 semaines de traitement, notamment avec NaCL 50 mm. L’étude immunologique a permis de montrer que l'augmentation de l'activité de L'EM NAD est associée à une augmentation de la quantité de protéine enzymatique. Par contre, pour l'EM NADP, l'augmentation de l'activité est plutôt associée à une forme plus active de la protéine
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Yoon, Heejeong. "Purification and characterization of malate dehydrogenase and 6- phosphogluconate dehydrogenase from Haemophilus influenzae." Diss., Virginia Polytechnic Institute and State University, 1988. http://hdl.handle.net/10919/54472.
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Haemophilus influenzae, the primary causative factor in bacterial meningitis, displays a unique growth requirement for intact NAD. Selective inhibitors of the pyridine nucleotide-requiring enzymes from H. influenzae could have a pronounced effect on growth of the organism.
Haemophilus malate dehydrogenase was purified 109-fold with a 26% recovery through a 4-step procedure involving salt fractionation, and hydrophobic and dye affinity chromatography. The purified enzyme was demonstrated to be a dimer of M,= 61,000. Initial velocity, product, and dead-end inhibition studies were consistent with an ordered bi-bi mechanism in which NAD is the first substrate bound to the enzyme. Several NAD analogs structurally altered in either the pyridine or purine moiety functioned as coenzymes in the reaction catalyzed. Selective interactions occurring at the coenzyme binding sites were investigated. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate moiety of the coenzyme. Positive chain length effects in the coenzyme-competitive inhibition by aliphatic carboxylic acids indicated the presence of a hydrophobic region close to the pyrophosphate region at the coenzyme binding site. Several structural analogs of NAD and malate were evaluated as selective inhibitors of the enzyme. The enzyme was inactivated by incubation with diethylpyrocarbonate whereas no inactivation was observed with sulfhydryl reagents.
Haemophilus influenzae 6-phosphogluconate dehydrogenase was purified 308-fold with a 16% recovery through a 4-step chromatographic procedure involving a PhenylSepharose hydrophobic column, and affinity chromatography on Matrex gel Green A, Matrex gel Red A, and 2',5’ADP-Sepharose resin. The purified enzyme was demonstrated to be a dimer of M,= 70,000. Initial velocity studies of 6-phosphogluconate oxidation indicated a sequential reaction mechanism. Although certain product and dead-end inhibition studies were consistent with an ordered mechanism, the direct binding of 6-phosphogluconate in protection experiments did not support a strictly ordered reaction sequence. Inhibition by adenosine derivatives indicated that the 2’-phosphate is important in binding to the coenzyme binding site of the enzyme.
The 3-acetylpyridine analogs of NAD and NADP which support growth of H. influenzae were demonstrated to function as coenzymes with the two dehydrogenases studied. The most effective inhibitors of the purified malate dehydrogenase and 6-phosphogluconate dehydrogenase were observed to inhibit the growth of Haemophilus influenzae. However, the most potent inhibition of growth by 3-aminopyridine analogs of NAD and NADP could not be explained on the basis of interactions of these analogs with the two dehydrogenases studied.Ph. D.
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Augagneur, Yoann. "Métabolisme du L-malate et du citrate chez Oenococcus oeni et Lactococcus lactis." Dijon, 2007. http://www.theses.fr/2007DIJOS021.
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L’objectif de la thèse est d’étudier les effets du métabolisme du L-malate et du citrate chez Oenococcus oeni et Lactococcus lactis. L’étude menée chez O. Oeni a mis en évidence une inhibition de la croissance en présence de citrate à pH bas, laquelle serait liée principalement à la production d’acide acétique. Dans une deuxième partie du travail, la construction d’une souche de L. Lactis déficiente pour l’oxaloacétate décarboxylase a mis en évidence un ralentissement de la croissance en présence de citrate avec un effet plus important à pH bas. De plus, ce ralentissement ne serait pas liée en une altération du maintien du pH intracellulaire mais plus vraisemblablement en une accumulation transitoire de l’oxaloacétate. L’étude plus approfondie de la toxicité de l’acétate et de l’effet de la mutation dans le gène codant l’oxaloacétate décarboxylase permettra une meilleure connaissance et compréhension du métabolisme des acides organiques chez les bactéries lactiquesThe objective of the thesis is to study the effects of the metabolism of L-malate and citrate on Oenococcus oeni and Lactococcus lactis. The study performed on O. Oeni highlighted an inhibition of the growth in the presence of citrate at low pH, which would be mainly related to the production of acetic acid. In a second part of the work, the construction of a deficient strain of L. Lactis for the oxaloacetate decarboxylase highlighted an impairment of the growth in the presence of citrate that was more efficient under low pH conditions. Moreover, this impairment would not be dependent in an alteration of the maintenance of the intracellular pH but more probably in a transitory accumulation of the oxaloacetate. The thorough study of the toxicity of the acetate and the effect of the mutation in the gene encoding the oxaloacetate decarboxylase would allow a better knowledge and comprehension of the metabolism of organic acids in lactic acid bacteria
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TAILLADE, MARIE-PIERRE. "La preparation biologique : meilleure arme contre le dopage." Toulouse 3, 1990. http://www.theses.fr/1990TOU31178.
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Graham, Ian Alexander. "Structure and function of the cucumber malate synthase gene and expression during plant development." Thesis, University of Edinburgh, 1989. http://hdl.handle.net/1842/12057.
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Ismail, Ismanizan. "Sugar regulation of malate synthase and isocitrate lyase gene expression in cucumber (Cucumis sativus)." Thesis, University of Edinburgh, 1997. http://hdl.handle.net/1842/14159.
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The glyoxylate cycle is known to take part in the net conversion of storage lipids to sugar germinating oilseeds. Two enzymes are exclusive for this cycle, malate synthase (MS) and isocitrate lyase (ICL) and their synthesis is coordinately regulated. These enzymes are active during postgerminative growth of seeds but are repressed in mature plants. However, they appear again when plants senesce. Genes for both enzymes are regulated by carbohydrate status. The aim of this study was to examine carbohydrate regulation. Expression of Ms and Icl genes in cucumber roots was low but increased upon excision and dark-incubation during a six day period in the absence of exogenous sugar. However, when sucrose was added to the incubation medium their expression was repressed. Hairy roots obtained using Agrobacterium rhizogenes strain A4 showed the same pattern of expression. Transgenic hairy roots containing Ms and Icl promoters fused to the GUS reporter gene, had a low level of GUS activity. This GUS activity increased dramatically when roots were excised and incubated in the absence of sugar, indicating regulation at the transcriptional level. Histochemical staining showed that GUS activity is concentrated in root tips and lateral root primordia where demand for carbohydrate is presumably greatest. Defoliation and shading experiments were carried out to examine the expression of Ms and Icl in roots of whole plants under natural conditions. In both cases, MS and ICL mRNA increased and roots showed a decline in sugar content. Thus, indication of Ms and Icl expression takes place in roots when supply of carbohydrate from the shoot is impaired. Results are consistent with the hypothesis that gene expression in the roots is controlled by carbohydrate supply from the shoot. Identification of regulatory elements in the Icl promoter required for the sugar response were made possible using transgenic cucumber hairy roots. Deletions of the Icl gene were assayed, which located a 200 bp region necessary for the sugar response more than 1 kbp upstream of the transcriptional start.
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Pracharoenwattana, Itsara. "Functions of peroxisomal citrate synthase and peroxisomal malate dehydrogenase in lipid catabolism in Arabidopsis." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/15658.
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It is proposed that peroxisomal citrate synthase (CSY) is required for carbon transfer from peroxisomes to mitochondria during respiration of fatty acids in Arabidopsis seedlings. Two genes encoding peroxisomal CSY are expressed in Arabidopsis seedlings. Double mutant seeds in which both genes are knocked out are dormant, and unable to utilise their stored lipid. Germination can be achieved by removing the seed coat and supplying sucrose. The seedlings are resistant to 2,4-dichlorophenoxybutyric acid (2,4DB), indicating a defect in peroxisomal β-oxidation. Beyond the seedling stage, double mutants also show arrested growth phenotype, and crucially are unable to produce seeds. The double mutant phenotypes can be restored by complementation with a cDNA encoding CSY with either its native PTS2 targeting sequence or a heterologous PTS1 sequence. These results suggest that peroxisomal CSY is not just a glyoxylate cycle enzyme but is also required for fatty acid respiration and to break seed dormancy. It is hypothesised that peroxisomal malate dehydrogenase (PMDH) serves to oxidise NADH produced by β-oxidation, and does not oxidise malate to provide oxaloacetate for the glyoxylate cycle. The Arabidopsis genome encodes eight putative NAD+-dependent malate dehydrogenase enzymes, two of which are predicted to be PMDHs. Double mutant seeds in which these two PMDH genes are knocked out are unable to establish as seedlings unless exogenous sucrose is supplied. In this respect the double mutant is similar to a range of β-oxidation mutants. Seedlings are impaired in breakdown of stored lipid and insensitive to 2,4-DB, showing that β-oxidation is defective. The metabolism of [2-14C]-acetate into sugars and organic acids is normal in double mutant seedlings, indicating that the glyoxylate cycle is still active.
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Flores, Pérez Cristóbal. "Improving performance of sheep using fibrolytic enzymes in dairy ewes and malate in fattening lambs." Doctoral thesis, Universitat Autònoma de Barcelona, 2004. http://hdl.handle.net/10803/5651.
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Coquelle, Nicolas. "Mécanismes moléculaires d'adaptation aux conditions physico-chimiques extrêmes dans la famille des lactate-malate déshydrogénases." Université Joseph Fourier (Grenoble), 2008. http://www.theses.fr/2008GRE10259.
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La vie est présente partout, ou presque, sur Terre, et notamment dans des environnements considérés comme extrêmes. Ces organismes extrêmophiles, non contents de subsister sous ces contraintes physico-chimiques extrêmes, s'y complaisent. Plusieurs mécanismes adaptatifs, à divers niveaux de l'organisation cellulaire, ont été mis en place. Les études présentées ici s'intéressent aux mécanismes d'adaptation moléculaire des protéines en utilisant la famille des lactate-malate déshydrogénases comme modèle. Il semblerait qu'une réorganisation des interactions au sein de la structure assure la stabilité, la solubilité et l'activité de la protéine sous cette contrainte physico-chimique extrême. Dans une première partie, les propriétés biochimiques et structurales des lactates déshydrogénases de la bactérie thermophile Thermus thermophilus (TtLDH1 de la bactérie mésophile Deinococcus radiodurans (DrLDH) et du poisson psychrophile de Champsocepha/us gunnari (CgLDH) ont été déterminées. Les deux premières ont été comparées pour étudier la transition thermophile/mésophile. La dernière a été comparée à la LDH de Squa/us acanthias (SaLDH) pour comprendre la transition mésophile/psychrophile. Peu de substitutions semblent être à l'origine des différences de propriétés thermiques entre ces enzymes. Cette hypothèse a pu être vérifiée par la caractérisation d'un mutant de TtLDH. Cinq substitutions judicieusement choisies ont permis d'altérer ses propriétés thermiques. L'adaptation moléculaire aux fortes concentrations en sel a été, pendant longtemps, étudiée en utilisant la malate désydrogénase de Ha/oarcu/a marismortui (HmMalDH). Nous présentons ici les caractéristiques biochimiques et structurales de la malate désydrogénase issue de Sa/inibacter ruber (SrMaIDH), la seule bactérie halophile extrême connue à ce jour. Les propriétés de cette enzyme semblent intermédiaires entre une enzyme non halophile et une enzyme totalement efficace aux fortes concentrations en sel. Cela a permis, pour la première fois, de proposer un découplage des différents effets affectant la stabilité conformationnelle,l'activité enzymatique et la solubilité. Enfin, l'irradiation des cristaux de protéine provoque des dommages au sein de la structure de la macromolécule, et touche notamment les groupements carboxyle des chaînes latérales des résidus acides. La forme apo (enzyme seule) et le complexe ternaire (enzyme :NADH :analogue de substrat) de TtLDH ont été irradiés et, à dose absorbée équivalente, l'amplitude des dommages entre les deux formes a été comparée. La fixation du cofacteur et du substrat ne protège pas des radiations le résidu acide présent dans le site actifLife is found everywhere, or almost, on earth and particularly in environments considered as extreme. These extremophilic organisms do not only subsist, but thrive in these conditions. Several adaptative mechanisms, at different cellular levels, have beendevelopped. Our studies focus on molecular mechanisms of proteins adaptation, using the lactate-malate dehydrogenases family as model. An intramolecular reorganization of interactions seems to be sufficient to en sure conformational stability, enzymatic activity and solubility of the protein in the se eXtreme conditions. First, biochemical and structural properties of lactate dehydrogenases from the thermophilic bacterium Thermus thermophilus (TtLDH), the mesophilic bacterium Deinococcus radiodurans (DrLDH) and the psychrophilic fish Champsocepha/us gunnari (CgLDH) have been determined. The first two have been compared to understand the thermophilic/mesophilic transition. The latter has been compared to the LDH from Squa/us acanthias (SaLDH) to study the mesophilic/psychrophilc transition. Few substitutions seems to be responsible for the differences in thermal properties between these enzymes. This hypothesis has been validated using a molecular variant of TtLDH. Five amino acid substitutions have modified its thermal properties. Molecular adaptation to high salt concentrations has been studied for a long time using the Ha/oarcu/a marismortui malatedehydrogenase (HmMaIDH). We present here the biochemical and structural properties of the malate dehydrogenase tTom Sa/inibacter ruber (SrLDH), the only extreme halophilic bacterium known to date. Properties of this enzyme appear to be 'intermediate' between a non halophilic enzyme and a fully active enzyme in high salt. For the first time, different effects that modiry conformational stability, enzymatic activity or solubility have been uncoupled
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Cendrin, Fabrice. "Clonage, séquençage et expression dans Escherichia coli du gène de la malate deshydrogénase de Haloarcula marismortui." Grenoble 1, 1992. http://www.theses.fr/1992GRE10058.
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L'enzyme malade deshydrogenase (hl-mdh) de la bacterie halophile extreme haloarcula marismortui, a ete sequencee dans sa partie n-terminale pour pouvoir synthetiser une sonde oligonucleotidique homologue du gene, permettant de cribler une banque genomique de la bacterie sus-citee. Un clone s'hybridant avec la sonde a ete isole de la banque, et la partie codante du gene de la hl-mdh a ete sequencee. La sequence en acides amines a montre des homologies plus importantes avec les lactates deshydrogenases (l-ldh) qu'avec les malates deshydrogenases de differentes sources, et apporte des lumieres interessantes sur l'evolution de cette famille d'enzymes. L'enzyme active est maintenant consideree comme un tetramere. Le modele de stabilisation de la hl-mdh propose anterieurement, dans lequel differents mecanismes de stabilisation dominent suivant la nature du solvant est confirme. Dans un milieu proche des conditions physiologiques, de fortes interactions entre la proteine et les molecules d'eau et de sel du solvant peuvent expliquer la stabilite observee. Le gene a ete clone dans un vecteur d'expression et sur-exprime dans la bacterie e. Coli. La proteine produite sous forme soluble a ete purifiee, caracterisee et montre une etonnante facilite a etre reactivee, indiquant un etat partiellement folded dans la cellule malgre la faible concentration en sel. Le gene de l'enzyme a ete la base de mutagenese dirigee qui a permis de creer une enzyme capable d'utiliser le pyruvate comme substrat en plus de l'oxaloacetate, et fait apparaitre une activite enzymatique ldh en plus de l'activite mdh
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Johansson, Kenth. "Structural studies of four nucleotide binding proteins : aldehyde dehydrogenase, NADP-malate dehydrogenase and two deoxynucleoside kinases /." Uppsala : Swedish University of Agricultural Sciences, 2000. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=009416200&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Li, Youzhong, and Youzhong Li@health gov au. "Respiration and nitrogen fixation by bacteroids from soybean root nodules : substrate transport and metabolism in relation to intracellular conditions." The Australian National University. Faculty of Science, 2003. http://thesis.anu.edu.au./public/adt-ANU20040630.114138.
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Bacteroids of B. japonicum from nodules of soybean roots were isolated using differential centrifugation (the standard bench method) and density gradient centrifugation methods (either sucrose- or Percoll-) under anaerobic conditions in which N2 fixation was preserved. The relationships between N2 fixation and respiration, O2 supply, O2 demand, substrate (mainly malate) transport and metabolism in bacteroids were investigated using the flow chamber system. In related experiments, the primary products of N2 fixation which leave the bacteroids were investigated using a 15N-labelling technique in a closed shaken system and other biochemical methods.¶
In the flow chamber experiments, the rates at which O2 was supplied to bacteroids in the chamber were varied by (a) changing the flow rate of reaction medium through the chamber; (b) by changing the [O2 free] in the inflowing reaction medium by using either 3-5% (v/v) or 100% air in the gas mixture above the stirred reaction medium in two reservoir flasks; (c) by successively withdrawing bacteroids from the chamber, thus increasing the supply of O2 per bacteroid to those remaining in the chamber. The results showed that the rate of O2 supply regulates respiratory demand for O2 by bacteroids rather than the O2 concentration present in the reaction system. Respiration is always coupled to N2 fixation. ¶
Uptake of malate by bacteroids withdrawn from the flow chamber was measured under microaerobic conditions. Malate uptake by these N2-fixing bacteroids was lower than that by bacteroids isolated under aerobic conditions, which eliminate N2 fixation of bacteroids, but is closely correlated with bacteroid respiration rates. When respiration was increased by an increase in O2 supply, malate uptake by bacteroids was also increased. This suggested that transport of malate through the bacteroid membrane is also regulated by O2 supply, but indirectly. Higher uptake by bacteroids under aerobic conditions was observed because respiration was enhanced by the high availability of O2, but the fast uptake of malate by bacteroids driven by the abnormal respiration rates may not reflect the reality of malate demand in vivo by bacteroids when N2 fixation by bacteroids is fully coupled. ¶
The results of 15N labelling experiments and other biochemical assays once again demonstrated that ammonia is the principal significant 15N labelled product of N2 fixation accumulated during 30 min in shaken assays with 0.008-0.01 atm O2. Alanine although sometimes found in low concentrations in the flow chamber reactions, was not labelled with 15N in shaken closed system experiments. No evidence could be obtained from the other biochemical assays, either. Therefore, it is concluded that these and earlier results were not due to contamination with host cytosolic enzymes as suggested by Waters et al. (Proc. Natl. Aca. Sci. 95, 1998, pp 12038-12042). ¶
Malate transported into bacteroids is oxidized in a modified TCA cycle present in bacteroids. The results of flow chamber experiments with a sucA mutant (lacking a-ketoglutarate dehydrogenase) showed that respiratory demand for O2 by the mutant bacteroids is regulated by O2 supply in the same way as the wild-type. Despite differences in other symbiotic properties, rates of nitrogen fixation by the mutant bacteroids, based on the bacteroid dry weight, appeared to be the same as in the wild-type. Also N2 fixation was closely coupled with respiration in the same manner in both mutant bacteroids and wild type bacteroids. These results and other supporting data, strongly support the conclusion that there is an alternative pathway of the TCA cycle in bacteroids, which enables the missing step in the mutant to be by-passed with sufficient activity to support metabolism of transported malate.
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Hadjifilippou, Irineos. "Characterizing the determinants of berry acidity in the grapevine." Master's thesis, ISA/UL, 2018. http://hdl.handle.net/10400.5/17938.
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Mestrado Vinifera Euromaster - Viticulture and Enology - Instituto Superior de AgronomiaGlobal warming is expected to be a major issue for grapevine productivity and sustainability in the long-term future. Major grapevine characteristics at berry level (physiological development and com-position) but also at the whole plant level (e.g. sugar accumulation, malic acid respiration, photosyn-thesis rate etc) are expected to be changed by elevated temperatures. In order to further decipher major berry physiology and development traits, data from two different experimental conditions and V.vinifera genotypes were used. The data set from experiment 1 was obtained from three genotypes (Merlot, G7 and G14 which are interspecific crossings of V. vinifera x V. rotundifolia, (macrovine) and possess the trait VDQA – “Vins de qualité à teneur réduite en alcool”, which produce wines with lower alcohol content. Plants were grown in open field conditions and berry development was mon-itored every week since early stages to over-ripeness providing a full berry development curve. In a second trial,76 genotypes were tested at two key stages, at green stage (just before ripening onset) and at ripe stage (maximum berry volume) to explore the diversity of primary metabolites and cations that exist in a progeny of microvine deriving from a cross. The progeny derived from a crossing of V3 microvine (female dwarf plant) with G14. Data analysis provided important information on berry development at limited number (8 berries maximum) and at large number (hundreds) scale for all parameters (glucose + fructose, tartartic acid,malic acid,potassium). In addition, complex berry pa-rameters such as titratable acidity were calculated on the basis of simple parameters (e.g. anions and cations). Finally, our results showed that the genetic variability of V. vinifera is potentially inter-esting to identify QTLs that can be used in breeding programs to develop new grapevine genotypes more suitable to climate change conditions
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Costa, Fausto Guimarães. "Prospecção de inibidores para a enzima malato sintase do Paracoccidioides brasiliensis: uma avaliação por triagem virtual e dinâmica molecular." Universidade Federal de Goiás, 2015. http://repositorio.bc.ufg.br/tede/handle/tede/4841.
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Paracoccidioidomycose
A Paracoccidioidomicose...
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Ruelland, Eric. "Etude par mutagenese dirigee de l'activation par la lumiere de la malate-deshydrogenase a nadp de sorgho." Paris 11, 1998. http://www.theses.fr/1998PA112207.
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La malate deshydrogenase a nadp est une enzyme chloroplastique qui a la caracteristique d'etre totalement inactive a l'obscurite et active a la lumiere. Cette activation correspond a la reduction de l'enzyme par les electrons de la chaine de transfert photosynthetique via le systeme ferredoxine/thioredoxine. Des resultats anterieurs ont montre que 4 cysteines situees dans des extensions n- et c- terminales specifiques des mdh photomodulees etaient impliquees dans ce processus. Un modele d'activation a alors ete propose, selon lequel dans des conditions oxydantes l'enzyme possede deux ponts disulfures par sous-unite, situees dans les extensions de sequence. L'extension c-terminale bloque l'acces au site actif. En presence de thioredoxines reduites, le pont n-terminal est reduit, suite a quoi le site actif subit un changement de conformation conduisant a site de fixation pour l'oxaloacetate de haute affinite et a une activite catalytique plus grande. L'extension c-terminale est egalement reduite par les thioredoxines, ce qui a pour consequence le deplacement de l'extension qui ne bloque plus alors l'acces au site actif. Dans cette etude, le role des 4 cysteines conservees internes a ete etudie. Des experiences de mutagenese ont permis de proposer un modele d'activation en deux etapes faisant intervenir une isomerisation du pont 24-29 en 24-207. La seconde partie de ce travail a consiste a etudier comment l'extension c-terminale bloque l'acces au site actif dans l'etat oxyde. En continuant a utiliser une approche par mutagenese dirigee, les deux derniers residus c-terminaux ont ete identifies comme ceux ancrant l'extension c-terminale dans le au site actif. La synergie entre la reduction du pont n-terminal et la reduction du pont c-terminal a aussi ete etudiee. Ainsi, ces resultats representent les premieres preuves moleculaires que l'absence totale d'activite de la nadp-malate dehydrogenase dans des conditions oxydantes est en partie due a une inhibition intrasterique. Finalement, le role essentiel des arginines du site actif a ete demontre en mutant ces residus en glutamine ou aspartate. Les proteines obtenues montraient une importante perte d'activite.
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Bonnete, Françoise. "Stabilisation, interaction et structure en solution de la malate deshydrogénase halophile de Haloarcula marismortui : effet du D2O." Grenoble 1, 1992. http://www.theses.fr/1992GRE10057.
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La comprehension des processus de repliement, de stabilite et de solubilite des proteines est d'importance fondamentale en biologie moleculaire, ainsi que le developpement des techniques d'ingenierie des proteines. Les interactions proteine-solvant jouent un role important dans ces processus, et pour leur etude nous avons choisi comme modele des proteines qui fonctionnent dans des environnements extremes. La malate deshydrogenase de haloarcula marismortui (hmdh) est un enzyme qui n'est stable, soluble et active que dans des conditions extremes de salinite, semblables a son environnement physiologique (3-4m kcl). Elle a ete etudiee de facon intense, et un modele de stabilisation a ete propose en termes d'interactions avec le solvant en fonction des conditions (zaccai g. Et al, j. Mol. Biol. , 208, 491-500, 1989). Dans cette these, nous avons pris le modele de stabilisation comme hypothese de depart et etudie la hmdh dans des solutions contenant du d#2o (#2h#2o) et differents sels. De tels solvants ont des effets interessants sur les interactions proteine-solvant, tels que l'effet hydrophobe, les interactions d'hydratation et les interactions avec le sel. Les proprietes physiques et chimiques des solutions de sels en d#2o et h#2o ont ete caracterisees, d'abord par diffusion de neutrons et par densimetrie de haute precision et reliees aux differences connues entre les proprietes de d#2o et de h#2o pures. Ensuite les interactions de la proteine avec son solvant ont ete caracterisees par les memes methodes, dans les differents solvants, et les resultats relies aux mesures biochimiques d'activite et de stabilite dans les memes conditions. La proteine a ete soit purifiee a partir de h. Marismortui, soit exprimee dans e. Coli et renaturee. Les resultats de cette etude confortent le modele de stabilisation des proteines halophiles. La stabilisation repose de facon predominante sur les contributions de liaisons d'hydratation et de l'effet hydrophobe variant en fonction de l'environnement. La hmdh se place bien sur un schema general propose par d'autres auteurs pour la stabilisation des proteines solubles, montrant une stabilite dans un domaine limite de temperatures et la denaturation a la fois a haute et basse temperature. Cependant, pour la plupart des proteines, seule une zone restreinte de ce schema peut etre observee, en absence de fortes conditions denaturantes. Il a ete montre que ce n'est pas le cas de la hmdh; par des choix adequats de solvants, il est possible d'explorer un grand domaine du schema de stabilisation, faisant de ce modele un systeme particulierement adapte pour les etudes de repliement. Les resultats dans les differentes solutions ont ete utilises pour explorer des conditions de cristallisation de la proteine. Des resultats biophysiques publies precedemment sur la hmdh ont aussi ete reanalyses a la lumiere des resultats de cette etude et d'autres travaux recents sur la sequence de la sous-unite de l'enzyme, et un nouveau modele de la proteine est presente qui la montre tetramerique
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Mitsch, Michael James. "Characterization of the NADP+-dependent malic enzyme of Sinorhizobium (Rhizobium) meliloti and investigations into the requirements of malate uptake and malic enzyme activity in bacteroids /." *McMaster only, 2001.
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Steven, Blaire. "Regulation and expression of the mdh-sucCDAB operon of Sinorhizobium meliloti." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80880.
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The genes encoding malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD), and subunits of 2-oxoglutarate dehydrogenase (sucAB) constitute an operon in the order mdh-sucCDAB in Sinorhizobium meliloti. Regulation of the operon was studied using beta-galactosidase gene fusions. Expression of the operon was assayed in response to the carbon source provided, and over the growth of the culture. A promoter upstream of the mdh gene was identified, and although the promoter was active in S. meliloti it was not expressed in Escherichia coli. It was demonstrated that the role of 2-oxoglutarate dehydrogenase (OGD) is minimal in symbiosis, as nodules with no OGD activity formed nodules able to fix nitrogen. Alfalfa plants inoculated with strains of S. meliloti carrying extra-chromosomal copies of the mdh gene did not show any increase in shoot dry weight compared to plants inoculated with the wild-type strain.
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Silva, Neto Benedito Rodrigues da. "Perfil transcricional e proteômico de Paracoccidioides em resposta à itraconazol e anfotericina B e identificação de compostos com potencial antifúngico." Universidade Federal de Goiás, 2013. http://repositorio.bc.ufg.br/tede/handle/tede/3642.
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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
The thermally dimorphic fungal pathogen Paracoccidioides is the agent of paracoccidioidomycosis. This disease is characterized by a granulomatous inflammation with clinical forms ranging from a benign localized infection to a disseminated one. The triazole drugs are broad-spectrum antifungal agents and are currently used to treat infections caused by various pathogenic yeast and molds. The mechanism of action of azoles has been elucidated in some fungi, although little is known in Paracoccidioides. Here we aim to investigate the mechanism of action of itraconazole on Paracoccidioides by using Representational Difference Analysis from Paracoccidioides yeast cells grown in the absence and presence of itraconazole for 1 and 2 h. Among the Paracoccidioides genes up-regulated by itraconazole were those mainly involved in cellular transport, metabolism/energy, transcription, cell rescue, defense and virulence. ERG11, ERG6, ERG3, ERG5 and ERG25 were up-regulated when evaluated in a timely manner. In vivo infection experiment in mice corroborated in vitro results. The glyoxylate cycle and its key enzymes isocitrate lyase and malate synthase (MLS) play a crucial role in the pathogenicity and virulence of various fungi such as the human pathogens. Here, we describe a study conducted to develop rational ligands as candidates to inhibit receptor PbMLS. The important step in the search for ligands for this receptor based on structural homology, molecular docking and molecular dynamics involving scanning virtual (virtual screening) through the program AutoDock Vina. Identified from the database of natural compounds (ZINC data bank) potential candidate ligands to inhibit the activity of PbMLS when compared to the original binder. This process led us to monoterpene indole alkaloids of the genus Palicourea (Rubiaceae) comprises about 230 species from shrubs and small trees distributed mainly in tropical regions. From the molecular docking fifteen compounds were tested as to its effectiveness in inhibiting the activity of PbMLS. The specific activity of PbMLS was affected by the compounds. Four indol alkaloids showed ability to reduce the enzyme activity. Since PbMLS is a linked surface protein that behaves as an anchorless adhesin, and PbICL is here described as adhesin, we also investigated if those compounds inhibit the adhesion of the protein to extracellular. Twodimensional gel electrophoresis we used to investigate the proteins expressed differentially during treatment with itraconazole and amphotericin B. Gels of three independent biological replicates were digitalized and the images were analyzed using the ImageMaster 2D Platinum 6.0 software (GE Healthcare). Spot intensities were normalized and the statistics analyses were estimated by one-way ANOVA. The spots of interest were excised, in-gel digested with trypsin, and the peptides were then analyzed by MS and/or MS/MS and and sequenced. The results obtained here should assist in understanding the mode of action of drugs in Paracoccidioides, and outline studies identifying compounds with antifungal activity.
O fungo patógeno termodimórfico Paracoccidioides é o agente da paracoccidioidomicose. Esta doença é caracterizada por uma inflamação granulomatosa onde as formas clínicas vão da infecção localizada benigna a uma uma disseminada. As drogas triazólicas são antifúngicos de amplo espectro e são usadas atualmente para tratar infecções causadas por vários fungos patogênicos e fungos. O mecanismo de ação dos azólicos foi elucidado em alguns fungos, embora pouco se sabe em Paracoccidioides. Aqui, em primeiro lugar pretendemos investigar o mecanismo de ação do itraconazol em Paracoccidioides usando análise de diferença representacional de Paracoccidioides células de levedura crescidas na ausência e na presença de itraconazol por 1 e 2 horas. Entre os genes Paracoccidioides up-regulados pelo itraconazol foram os envolvidos, principalmente no transporte celular, metabolismo/energia, transcrição, defesa e virulência. ERG11, ERG6, ERG3, ERG5 e ERG25 foram regulados quando avaliados de forma temporal. Experimentos de infecção em camundongos corroborou resultados in vitro. O ciclo do glioxilato e suas enzimas chave isocitrato liase (ICL) e malato sintetase (MLS) desempenham um papel fundamental na patogenicidade e virulência de vários fungos, assim como patogênese em humanos. Neste trabalho, descrevemos um estudo realizado para desenvolver ligantes racionais como candidatos pra inibir o receptor PbMLS. Apresentamos um passo importante na busca de ligantes para este receptor baseando-se em homologia de estruturas, dinâmica molecular e acoplamento molecular envolvendo varredura virtual (virtual screening) por meio do programa AutoDock Vina. Identificamos a partir de banco de compostos naturais (data bank ZINC) potenciais ligantes candidatos a inibir a atividade de PbMLS quando comparados ao ligante original. Este processo nos conduziu aos alcalóides indólicos monoterpênicos do gênero Palicourea (Rubiaceae) que compreende cerca de 230 espécies entre arbustos e pequenas árvores distribuídas, principalmente, nas regiões tropicais.A partir da ancoragem molecular quinze compostos foram testados quanto à sua eficácia na inibição da atividade de PbMLS. A atividade específica de PbMLS foi afetado pelos compostos. Quatro alcalóides indólico mostraram capacidade de reduzir a atividade da enzima. Desde que PbMLS é uma proteína associada à superfície que se comporta como uma adesina ancorada também foi investigado se os compostos inibem a adesão da proteína às matrizes extracelulares. O processo de eletroforese em gel bidimensional foi utilizado para investigar as proteínas diferencialmente expressas durante o tratamento com itraconazol e anfotericina B. Gel de três réplicas biológicas independentes foram digitalizadas e as imagens foram analisadas usando o software 6.0 Platinum 2D ImageMaster (GE Healthcare). Intensidades dos spots foram normalizados e foram estimadas as análises estatísticas por ANOVA one-way. Os spots de interesse foram excisadas do gel digerida com tripsina e os péptidos foram analisados por MS e / ou MS / MS e sequenciados. Os resultados obtidos aqui devem ajudar na compreensão do mecanismo de ação de drogas em Paracoccidioides, e delinear estudos de identificação de compostos com atividade antifúngica.
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ISSAKIDIS, EMMANUELLE. "Identification et caracterisation des domaines de photomodulation de la malate deshydrogenase a nadp de sorgho par mutagenese dirigee." Paris 11, 1993. http://www.theses.fr/1993PA112437.
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La malate deshydrogenase a nadp (mdh a nadp) est une enzyme chloroplastique activee a la lumiere par les thioredoxines. Les residus cysteine impliques dans le mecanisme de sa photomodulation ont ete recherches par une approche moleculaire. Tout d'abord, un systeme performant d'expression de l'adnc codant pour la mdh a nadp de sorgho a ete mis au point. Il permet d'obtenir en grande quantite une mdh a nadp recombinante dont les caracteristiques biochimiques sont identiques a celles de l'enzyme de plante. Les premieres experiences de mutagenese dirigee ont concerne les residus cysteine localises dans la partie n-terminale de la proteine. Les enzymes modifiees ont toujours besoin d'etre activees. Cependant, leur activation est beaucoup plus rapide que celle de la proteine sauvage. Leurs proprietes catalytiques restent pourtant inchangees. Ces resultats indiquant la necessite de reduire un deuxieme pont, des experiences complementaires de mutagenese dirigee ont cible les residus cysteine localises dans la partie c-terminale. Les proteines correspondantes necessitent un traitement d'activation pour atteindre une activite maximale, avec une cinetique comparable a celle de l'enzyme sauvage. Toutefois, elles presentent, a l'etat oxyde, une faible activite de base avec une affinite tres faible pour l'oxaloacetate. En cumulant les mutations des cysteines n- et c-terminales, une mdh a nadp dont l'activite n'est plus dependante de la reduction et dont les caracteristiques biochimiques sont celles de l'enzyme sauvage pleinement activee a ete produite. L'approche moleculaire a montre que deux ponts disulfures sont impliques dans le mecanisme d'activation de la mdh a nadp. En integrant l'ensemble des resultats de ce travail et les donnees structurales obtenues par modelisation, un modele pour le mecanisme d'activation par la lumiere de la mdh a nadp est propose
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Marchat, Laurence. "Enzymes du métabolisme énergétique de la filaire "Molinema dessetae" : caractérisation et évaluation comme cibles thérapeutiques." Paris 11, 1995. http://www.theses.fr/1995PA114832.
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McLaughlin, James Christopher. "The regulation and synthesis of malate synthase and isocitrate lyase in senescent and detached cotyledons of cucumber (C. sativus)." Thesis, University of Edinburgh, 1993. http://hdl.handle.net/1842/15348.
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The expression and role of MS and ICL during senescence and following detachment is investigated. After 7 weeks plant growth immunoblot analysis indicate the coordinate accumulation of MS and ICL. However, this increase in MS and ICL does not correlate with the decline in the thylakoid lipids MGDG and DGDG. A detectable decline in these lipid classes is observed after 2 weeks plant growth. Further analysis of changes in chlorophylls, carotenoids, protein and total RNA content confirm that senescence of cucumber cotyledons is initiated very soon after full greening. These data indicate that the glyoxylate cycle does not appear to play any significant role in the disassembly of chloroplast membranes during the earlier stages of senescence. In detached cucumber cotyledons the synthesis of MS and ICL is detectable in dark incubated cotyledons within 48 hours. This appears to be primarily due to an increase in transcripts that encodes these proteins. However, the demonstrable increase in MS and ICL levels occurs prior to any detectable decline in chlorophylls, carotenoids and galactolipids. This indicates that the glyoxylate cycle may play an additional role to the metabolism of products of chloroplast membrane degradation. The role sucrose may in controlling the synthesis of MS and ICL was also investigated. The presence of 25 mM was sufficient to greatly reduce the synthesis of MS and ICL under conditions where the synthesis of these proteins would normally occur in detached cucumber cotyledons and protoplasts.
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Chutia, Ranju [Verfasser], Steffen Gutachter] Abel, Ingo [Gutachter] [Heilmann, and Paul B. [Gutachter] Larsen. "Manipulation of malate biosynthesis and exudation in Arabidopsis thaliana / Ranju Chutia ; Gutachter: Steffen Abel, Ingo Heilmann, Paul B. Larsen." Halle (Saale) : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2019. http://nbn-resolving.de/urn:nbn:de:gbv:3:4-1981185920-322293.
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Chutia, Ranju [Verfasser], Steffen [Gutachter] Abel, Ingo [Gutachter] Heilmann, and Paul B. [Gutachter] Larsen. "Manipulation of malate biosynthesis and exudation in Arabidopsis thaliana / Ranju Chutia ; Gutachter: Steffen Abel, Ingo Heilmann, Paul B. Larsen." Halle (Saale) : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2019. http://d-nb.info/1210731223/34.
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Lin, Jie. "Etude des plasmides des bactéries du genre Leuconostoc et de leur relation avec le métabolisme du malate et du citrate." Dijon, 1991. http://www.theses.fr/1991DIJOS016.
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L'objectif principal de l'étude est de contribuer à une meilleure connaissance du contenu plasmidique et des fonctions métaboliques des plasmides chez les bactéries du genre Leuconostoc. Le métabolisme du malate et du citrate a été plus particulièrement étudié. Une méthode mini-préparation de l'ADN plasmidique adaptée aux bactéries du genre Leuconostoc a été proposée. Les contenus plasmidiques de 42 souches appartenant aux différentes espèces de ce genre ont été analysés. Les profils plasmidiques des souches étudiées sont assez hétérogènes chez Leuconostoc mesenteroides subsp. Mesenteroides ou plus homogènes chez Leuconostoc mesenteroides subsp. Cremoris. Les plasmides sont peu fréquents chez Leuconostoc oenos. Un milieu de sélection des mutants malate négatif a été mis au point. L'étude des mutants obtenus a permis de caractériser le rôle physiologique de l'acide L-malique qui est apparu comme indispensable pour la croissance à pH acide. Le co-métabolisme du malate et des sucres est dépendant du pH du milieu. L'analyse des mutants citrate négatif a montré que chez Leuconostoc mesenteroides subsp. Mesenteroides le citrate a un rôle énergétique qui modifie à la fois le taux de croissance, le rendement de biomasse et le bilan fermentaire. Pour Leuconostoc oenos 8413 nous n'avons pas observé de relation entre son unique plasmide (3,8 kb) et la perte de la capacité d'utilisation du malate ou du citrate. Chez 6 souches de Leuconostoc mesenteroides subsp. Mesenteroides, la disparition de la capacité d'utilisation du citrate est liée à la perte d'un plasmide d'environ 22 kb (pCIT) s'accompagnant ou pas de réarrangements d'autres plasmides. La technique d'hybridation par sonde froide a montré l'homologie de ce plasmide pour les souches analysées.
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Cretin, Claude. "Étude des mécanismes de photorégulation de la phosphoénolpyruvate carboxylase et de la malate déshydrogénase à NADP des feuilles de sorgho." Paris 11, 1987. http://www.theses.fr/1987PA112454.
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La phosphoénolpyruvate carboxylase (E. C. 4. 1. 1. 31) (PEPC) et la Malate Déshydrogénase à NADP (E. C. 1. 1. 1. 82) (MDH à NADP) sont deux enzymes clé du métabolisme photosynthétique des plantes de type C4. Dans le cytoplasme des cellules du mésophylle, la PEPC catalyse la réaction de β-carboxylation du PEP en acide oxaloacétique; celui-ci est réduit en malate dans les chloroplastes par la MDH à NADP. Le verdissement des feuilles de Sorgho (Sorghum vulgare F, cv. Tamaran FNK 140) s'accompagne d'une forte augmentation de l'activité enzymatique de ces deux biocatalyseurs. Ce travail a consisté en une étude des mécanismes de la photorégulation de la PEPC et de la MDH à NADP. Par traduction in vitro des ARNm poly (A+) des feuilles de Sorgho nous avons pu montrer, dans les deux cas, une augmentation de l'activité traductionnelle des ARNm spécifiques au cours du verdissement. De plus, dans le cas de la MDH, nous avons mis en évidence l'existence d'un précurseur possédant un peptide de transit d'environ 2,5 kDaltons intervenant dans le transport de la sous-unité de la MDH dans les chloroplastes. La construction et le criblage d'une banque d'ADN complémentaire dans le vecteur d'expression lambda gt11 à l'aide d'anticorps polyclonaux a permis d'isoler deux ADNc pour les deux enzymes. Ces clones ont été caractérisés par des expériences d'hybridation aux ARNm poly (A+) de feuilles vertes de Sorgho et traduction in vitro des ARNm sélectionnés. Grâce à ces sondes homologues, nous avons estimé la taille de l'ARNm de la PEPC à 3,4 kb et celui de la MDH à 1,6 kb, par la technique de Northern. De plus, nous avons pu montrer que le verdissement des feuilles s'accompagne d'une accumulation de ces ARNm. Enfin, une banque génomique a été construite dans le vecteur lambda EMBL4 à partir de fragments MboI de l'ADN de feuilles de Sorgho. 70 clones ont été sélectionnés par criblage de la banque à l'aide de la sonde d'ADNc PEPC. Un clone génomique (lambda CP46) a été plus particulièrement caractérisé. L'établissement de sa carte de restriction a permis de localiser une région de 7,5 kb couvrant tout le gène de la PEPC ainsi que les régions régulatrices et non codantes en 5' et en 3' du gènePhosphoenolpyruvate carboxylase (E. C. 4. 1. 1. 31) (PEPC) and NADP-Malate Dehydrogenase (E. C. 1. 1. 1. 82) (NADP-MDH) are two key enzymes involved in the photosynthetic pathway of C4 plants. In the cytoplasm of mesophyll cells, PEPC catalyses the β-carboxylation of PEP giving rise to oxaloacetate which is then translocated into chloroplasts and reduced to malate by NADP-MDH. During the greening process of Sorghum leaf (Sorghum vulgare F, cv. Tamaran FNK 140), a light-dependent increase in enzyme activity occurs for both enzymes. The present work is devoted to the study of photoregulation mechanisms of PEPC and MDH. By in vitro translation of Sorghum leaf poly (A+) mRNA and immunoprecipitation using specific antibodies, we have shown a light-dependent increase in the translational activity of both mRNAs. Furthermore, a precursor form was found for the MDH with a transit peptide of about 2. 5 kDaltons; results suggested that the peptide was processed during the subunit translocation into the chloroplast. We have constructed a cDNA library from Sorghum leaf poly (A+) mRNA in the expression vector lambda gt11. Using polyclonal antibodies raised against PEPC and MDH, we isolated two cDNA clones for these enzymes, which were subsequently characterized by hybrid-selection translation experiments. Sizes of PEPC and MDH mRNAs were estimated by Northern blotting to be 3. 4 kb and 1. 6 kb respectively. It was shown that a light-dependent accumulation of both PEPC and MDH mRNAs occurred during the greening of Sorghum leaves. Finally, we constructed a genomic library in the phage lambda EMBL4 from MboI restriction fragments of Sorghum leaf DNA. Using the PEPC cDNA, we isolated 70 positive clones from the library. One of them (lambda CP46) has been characterized by establishing its restriction map. We could localize a 7. 5 kb region in the clone containing the entire PEPC gene. We also showed that lambda CP46 genomic clone contained the putative regulatory and non-coding regions at 5’ and 3' ends of the gene
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Cretin, Claude. "Etude des mécanismes de photorégulation de la phosphoénolpyruvate carboxylase et de la malate deshydrogénase à NADP des feuilles de sorgho." Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb37604121k.
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Ousalem, Farès. "Rôles physiologiques et fonctionnels des facteurs de traduction appartenant à la famille ABC-F et leur implication dans la résistance aux antibiotiques." Thesis, Université de Paris (2019-....), 2020. http://www.theses.fr/2020UNIP7015.
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Les protéines de la famille ABC-F, universellement retrouvées chez les procaryotes et les eucaryotes, interagissent avec le ribosome. La souche Escherichia coli (MG1655) possède 4 ABC-F paralogues nommés EttA, YheS, YbiT et Uup. J’ai étudié ici le rôle physiologiques et fonctionnels des protéines ABC-F d’E. coli et la protéine MsrD de Streptococcus pneumoniae qui est synthétisée dans E. coli. Mes travaux ont montré que les protéines ABC-F sont importantes pour les bactéries dans des conditions de stress spécifiques. Les bactéries délétées du gène ettA sont plus sensibles au stress salin, les bactéries délétées du gène uup sont plus sensibles au stress acide tandis que la surexpression de la protéine YheS ou MsrD dans les bactéries leurs confèrent une résistance aux macrolides. J’ai également démontré que le gène ettA est important pour la croissance d’E. coli dans des conditions de stress salin et de faible concentration en phosphate quand les acides aminés, le malate ou fumarate sont utilisés comme seule source de carbone. La délétion du gène ettA rend les bactéries plus sensibles au stress salin et engendre une dérégulation de certains gènes liés au métabolisme du malate / glyoxylate ainsi que des gènes impliqués dans l'activité ribosomique. La souche délétée du gène ettA sous-exprime le gène de la malate synthase aceB par une régulation post-transcriptionnelle. La sous-expression de la malate synthase dans des bactéries favorise leurs croissances dans le milieu glyoxylate. La délétion du gène ettA a aussi induit une surexpression de facteur de modulation des ribosomes (RMF) dans le milieu LB, ce qui a provoqué une augmentation de la formation des ribosomes 100SProteins belonging to the ABC-F family are found in prokaryotes as well as eukaryotes. They interact with the ribosome and are considered as translation factors. The Escherichia coli strain (MG1655) has 4 ABC-F paralogs named: EttA, YheS, YbiT and Uup. Here I present a study of the physiological and functional role of E. coli ABC-F proteins and the Streptococcus pneumoniae MsrD protein. My work has shown that the ABC-F proteins studied are important for bacteria adaptation to stress conditions. Bacteria deleted from the ettA gene are more sensitive to salt stress, bacteria deleted from the uup gene are more sensitive to acid stress while the overexpression of the protein YheS or MsrD in bacteria provide resistance to macrolides. I also demonstrated that the ettA gene is important for the growth of E. coli under conditions of saline stress at low phosphate concentration when the amino acids, malate or fumarate are used as the only carbon source. The deletion of the ettA gene makes bacteria more sensitive to salt stress and causes dysregulation of certain genes linked to the malate / glyoxylate metabolism and genes involved in ribosomal activity. The deleted strain of the ettA gene under-expresses the malate synthase aceB gene by post-transcriptional regulation. The under- expression of malate synthase in bacteria promotes their growth in the glyoxylate medium. The deletion of the ettA gene also induced overexpression of ribosome modulation factor (RMF) in LB medium, which caused an increase in the formation of 100S ribosomes
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Lecomte, Catherine. "Le métabolisme intermédiaire chez le rat après exposition à l'hypoxie intermittente : étude de l'utilisation et des effets du malate de citrulline." Lyon 1, 1993. http://www.theses.fr/1993LYO10149.
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Les modifications metaboliques consecutives a l'exposition a l'hypoxie intermittente (ehi: 2. 2 h/j; 5 jours; 4500 m) chez le rat ont ete etudiees au niveau de l'organisme entier et au niveau cellulaire (hepatocytes isoles). Les effets de l'administration aigue ou chronique (5 jours) de malate de citrulline (mc), substance preconisee comme antiasthenique ont ete examines chez les animaux ehi et controles. Les productions de glucose et d'uree a partir de substrats (2 mm) sont legerement inferieures dans les hepatocytes isoles de rats ehi vs controles (ns); la difference devient significative avec le lactate (moins 20%) en tenant compte du contenu cellulaire en atp. L'ehi chez le rat aboutit a une reduction des capacites gluconeogeniques et ureogeniques des hepatocytes isoles et peut representer un modele experimental d'etude de gluconeogenese et d'ureogenese diminuees. Ces voies sont importantes a examiner dans certaines situations, en particulier lors de l'apparition de la fatigue. A cet egard, les rats ehi presentent un comportement explorateur diminuant leur temps de maintien sur le rota-rod (moins 46%, ns) par rapport aux controles, mais la reponse a des epreuves de nage, 18 heures apres la derniere exposition, n'est pas differente dans les deux groupes d'animaux. L'etude de la charge ip (3,2 mmol/kg) de mc montre une utilisation en 4 a 7 heures. A partir des courbes moyennes, les parametres cinetiques pour le malate et la citrulline ont ete obtenus. L'injection de mc s'accompagne d'une augmentation de la glycemie, de l'uremie et de la lactatemie. L'augmentation statistiquement significative au seuil de 5% du temps de nage (lest 5%) chez les rats traites au mc par rapport aux non traites (plus 25%) suggere un effet positif de l'administration de mc dans nos conditions experimentales qui pourrait etre la consequence de son utilisation metabolique
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Richard, Stéphane. "Etude de la solvatation d'une protéine halophile, la malate déshydrogénase de Haloarcula marismortui, par cristallographie des rayons X et diffusion centrale." Université Joseph Fourier (Grenoble), 1998. http://www.theses.fr/1998GRE10265.
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L'etude des interactions proteine-solvant est une question a la fois fondamentale et appliquee ; cependant, en cristallographie des rayons x, la modelisation du solvant est tres delicate en raison de son caractere peu ordonne. De recents developpements relatifs a la contribution du solvant au signal de diffraction permettent une meilleure determination des chaines laterales de surface de la proteine et ainsi, une meilleure localisation des molecules d'eau associees. Cette caracterisation des contributions respectives du solvant enfoui, associe a la surface (ordonne ou desordonne) ou libre, se trouvant dans les cristaux, est actuellement en plein developpement. La malate deshydrogenase de haloarcula marismortui (hmdh) est relativement instable dans une large gamme de conditions de sels et de temperature, ce qui en fait un tres bon candidat pour l'etude de l'effet du solvant sur la stabilite de la proteine (bonnete et al. , 1994). Il a ete demontre qu'une simple mutation ponctuelle de surface, e242r, accentue le caractere halophile de la malate deshydrogenase dans les sels physiologiques. La hmdh a ete largement etudiee dans notre laboratoire par des techniques tant biochimiques que biophysiques. Differents mecanismes de stabilisation dominent selon la nature de l'environnement salin : fixation des ions dans kcl et nacl, interactions hydrophobes dans le phosphate de potassium et (nh#4)#2so#4. En accord avec ces donnees, un modele de stabilisation des proteines halophiles a ete propose. La structure de la hmdh a ete determinee a une resolution de 3. 2 a en presence de son cofacteur, le nadh (dym et al. , 1995) ; or cette resolution n'etait pas suffisante pour une etude des interactions de la proteine avec l'eau et les ions impliques dans la stabilisation de la molecule. Pour optimiser la qualite des cristaux, la cristallogenese de proteines halophiles dans des conditions physiologiques a ete etudiee. Les structures cristallographiques de la hmdh sauvage et du mutant e242r, formes apo, et du mutant e100k, forme holo ont ete determinees par diffraction des rayons x a des resolutions respectives de 2,8 a, 2,6 a et 3,2 a. Ces structures ont permis la mise en evidence de motifs d'interactions proteine-proteine et proteine-solvant particuliers chez cette proteine halophile. De plus, des etudes en solution ont ete effectuees sur la proteine sauvage par diffusion centrale des neutrons et des rayons x, et comparees a des courbes obtenues par simulation, afin de caracteriser la couche d'hydratation de la hmdh.
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Rajapaksa, Ranjani 1949. "Pre-Steady State Kinetics of the NAD-Malic Enzyme from Ascaris suum in the Direction of Oxidative Decarboxylation of L-Malate." Thesis, University of North Texas, 1991. https://digital.library.unt.edu/ark:/67531/metadc500668/.
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Stopped-flow experiments in which the NAD-malic enzyme was preincubated with different reactants at near saturating substrate concentrations suggest a slow isomerization of the E:NAD:Mg complex. The lag is eliminated by preincubation with Mg˙² and malate suggesting that the formation of E:Mg:Malate either bypasses or speeds up the slow isomerization step. Circular dichroic spectral studies of the secondary structural changes of the native enzyme in the presence and absence of substrates supports the existence of conformational changes with NAD˙ and malate. Thus, a slow conformational change of the E:NAD:Mg complex is likely one of the rate-limiting steps in the pre-steady state.
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Lohman, Jeremy R. 1981. "Two-state conformational behavior in protein active centers." Thesis, University of Oregon, 2007. http://hdl.handle.net/1794/6198.
Full textAbstract:
xiv, 82 p., ill. (some col.)Cellular processes are carried out by proteins, which often utilize conformational changes for function. In theory, conformational changes can be harnessed to promote, prevent or monitor cellular processes. Such changes in protein active centers require perturbations through interactions with other proteins, small molecules or through energy input into the system, for example light. The work presented incorporates rational design and crystallographic elucidation of two-state conformational changes in two proteins, green fluorescent protein (GFP) and malate synthase (MS). GFP indicators were previously developed to quantitate the thiol/disulfide redox status within cells. Cysteine residues were introduced in close proximity on the surface of GFP and allow the formation of a disulfide bond. The indicators provide a fluorescent readout of the ambient thiol/disulfide equilibrium, however thermodynamic studies showed the resulting thiol/disulfide to be unusually stable (-287 mV) in comparison to the cellular redox buffer glutathione (-240 mV). In order to produce a family of redox indicators suitable for use in less reducing environments, amino acids were inserted near the introduced cysteine pair in order to destabilize the disulfide. The resulting family of redox indicators, termed roGFP-iX, exhibit midpoint potentials in the more desirable range of -229 to -246 mV. Crystallographic analysis indicates that roGFP-iX indicators undergo much larger two-state conformational changes than the original indicators. Surprisingly, a cis-peptide was discovered between the cysteine and the inserted residue which in combination with the conformational changes helps to explain the reduced stability of the disulfide. Malate synthase is an important virulence factor for certain microbes and carries out the Claisen condensation between glyoxylate and acctyl-CoA to produce malate. Crystal structures of Mycobacterium tuberculosis and Escherichia coli malate synthase isoform G had previously been determined with substrates or products bound. To determine the conformational changes necessary for substrate binding and product release, crystal structures of Escherichia coli malate synthase isoform A were determined in both the apo and acetyl-CoA/inhibitor bound forms. The crystallographic models revealed two-state conformational changes in the part of the active-site loop necessary for substrate binding, which has important implications for drug design. This dissertation includes my unpublished co-authored materials.
Adviser: S. James Remington
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Palmer, Antony James. "Production and characterization of ArAE family members including putative efflux transporters (PETs) from bacteria and aluminium activated malate transporters (ALMTs) from plants." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15292/.
Full textAbstract:
The focus of this study is proteins from the ArAE family, specifically two subfamilies: firstly, the ALMT family of plant membrane channels, which have numerous vital roles in plants, and secondly, the bacterial family first described as PET inner membrane exporters. Constructs were created for expression of firstly, the C-terminal domain (CTD) of wheat ALMT1 in E. coli and secondly, three full-length ALMTs (ALMT from wheat and ALMT5, ALMT9 from Arabidopsis) in Nicotiana benthamiana for structural and biochemical studies. Unfortunately, it appears that the CTD is not an independent soluble domain as originally thought, and this domain is now predicted to contain transmembrane helices, making it unsuitable for study in the manner planned. Similarly, production of full length ALMTs was unsuccessful as when extraction and purification was attempted, the protein degraded. Thirdly, a bacterial member of the ArAE family was expressed and characterised: AaeB from E. coli, along with its putative binding partner, AaeA. This was shown to form a complex with and be vital for the stability of AaeB. A strategy was devised for expression, solubilisation, and purification of these proteins and once they were obtained in pure form they were subjected to a range of biochemical and structural experiments. Microcrystals of AaeA were produced, towards a strategy for structural determination by X-ray crystallography. The first Electron Microscopy examination was performed on complexes of AaeB, and negative stain classes were produced. The first in silico homology model of AaeA has been produced and validated by CD spectroscopy, providing a range of insights. The complex formed by AaeA and AaeB has also been probed by crosslinking and SEC MALLS analysis, suggesting a 6:2 or 6:3 stoichiometry. Together this has furthered our understanding of this poorly characterised membrane protein family and provided a set of clones and protocols for future studies.
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Kim, Dae-Jae. "Cloning of cDNAs for glyoxysomal malate dehydrogenase and for phosphoenolpyruvate carboxykinase of cucumber (Cucumis sativus L.) and gene expression in cotyledons during development." Thesis, University of Edinburgh, 1994. http://hdl.handle.net/1842/12374.
Full textAbstract:
In higher plants a massive conversion of the storage lipid to carbohydrate takes place in germinating seedlings. The conversion involves β-oxidation, the glyoxylate cycle and gluconeogenesis. The key glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MS) are co-ordinately synthesised to high levels during germination and decline thereafter to undetectable levels. The enzymes reappear at the final stage of the growth, in senescent tissues. In this study the synthesis of other enzymes involved in the conversion of lipid to carbohydrate was investigated to determine if they are co-ordinately synthesised with ICL and MS, and whether the genes encoding them are subject to the same control of expression. Full length cDNA clones encoding glyoxysomal malate dehydrogenase (gMDH) and phosphoenolpyruvate carboxykinase (PEPCK) have been isolated from a Cucumis sativus senescent cotyledon cDNA library. gMDH is one of the glyoxylate cycle enzymes, and PEPCK is a key enzyme for gluconeogenesis, which had not previously been cloned from plants. The cDNA and predicted amino acid sequence of gMDH show very high homology (94% and 97% respectively) with watermelon counterparts. The amino acid sequence deduced from the cDNA encoding PEPCK shows 43 to 57% identity with bacterial, yeast and trypanosome enzymes, and includes a conserved ATP-binding domain. The sequence of a full length cDNA predicts a polypeptide of 74,397 Da. The cDNA was expressed in Escherichia coli and antibodies raised to the resultant protein. The cucumber genome was shown to contain single genes encoding gMDH and PEPCK, the expression of which was investigated by northern and western blotting.