Dissertations / Theses on the topic 'Metabolism; Cytosol'

Der Citratzyklus (TCA) ist einer der bedeutendsten Stoffwechselwege für alle lebenden Organismen. Trotz der zentralen Rolle dieses Prozesses im Pflanzenmetabolismus ist er nur relativ wenig untersucht worden. In dieser Arbeit berichte ich über die Produktion und die funktionale Analyse von Tomatenpflanzen (Solanum lycopersicum), die unabhängig eine leicht eingeschränkte Aktivität der mitochondrialen Citrat-Synthase (CS) und zweier Isocitrat-dehydrogenasen (mitochondriale NAD-IDH und cytosolische NADP-ICDH) zeigen. Die transgene Pflanzen wiesen mehrheitlich keine erkennbare Veränderung eines Wachstumphänotyps auf. Obwohl die photosyntetische Leistung keine Änderungen gezeigt hatte, war die mitochondriale Respiration gestiegen, begleitet von einem reduzierten Kohlenstoff-fluss durch den Citratzyklus. Darüber hinaus waren die CS Pflanzen charakterisiert durch wesentliche Änderungen im Blattmetabolismus, einschließlich eines eingeschränkten Niveaus des photosynthetischen Pigments und Zwischenprodukten des Citratzyklus zusammen mit einer Akkumulation von Nitraten, verschiedenen Aminosäuren und Stärken. Zusammengefasst deuten diese Ergebnisse auf eine Einschränkung der Nitrat-Aufnahme hin. Das mit Hilfe von TOM1 Mikroarrays und quantitativer RT-PCR durchgeführte Transcript-profiling hat gezeigt, dass die fehlende Aktivität der mitochondrialen CS teilweise von einer gestiegenen, peroxisomalen CS Isoform ausgeglichen wird. Die metabolische Verschiebung ergab eine Verstärkung der photorespiratorischen Leistung, die vermutlich eine ausgleichende Rolle in der Produktion organischer Säuren und der Wiederherstellung der Redox-Balance spielt. Interessantenweise war die metabolische Antwort von Blättern auf Stickstoffmangel in NADP-ICDH Pflanzen dramatischer als in NAD-IDH Pflanzen, was darauf hindeutet, dass die cytosolische Isoform der Hauptlieferant von 2-Oxoglutarat im Tomatenmetabolismus sein könnte.
Although the TCA cycle is a respiratory metabolic pathway of central importance for all living organisms, relatively few molecular physiological studies of plants were performed to date. Here, I report the generation and functional analysis of tomato plants (Solanum lycopersicum) independently displaying mildly limited activity of mitochondrial citrate synthase (CS) and two isocitrate dehydrogenases, namely mitochondrial NAD-IDH and cytosolic NADP-ICDH. The transgenic plants revealed minor phenotypic alterations. Although the leaf photosynthetic performance was largely unaltered, the changes in mitochondrial respiration and carbon flux through the TCA cycle were observed. Moreover, the plants were characterized by significant modifications in the leaf metabolic content and in maximal catalytic activities of several enzymes involved in primary C and N metabolism. These results hint towards limitations in nitrate assimilation pathway. The transcript profiling performed by utilizing TOM1 microarrays and quantitative RT-PCR approach revealed that the deficiency in mitochondrial CS activity was partially compensated by up-regulation of peroxisomal CS isoform. The limitations in the activities of isocitrate dehydrogenases resulted in up-regulation of the photorespiratory pathway, which presumably played a compensatory role in supporting organic acid production and re-establishing redox balance in the transgenic leaves. Interestingly, the leaf metabolic response towards nitrogen starvation conditions was far more dramatic in NADP-ICDH transgenic plants than NAD-IDH plants, hinting that the cytosolic isoform may be the major 2-oxoglutarate supplier in tomato metabolism.

One-carbon metabolism (1CM) is compartmentalized in the mitochondria and cytosol and generates a host of metabolites critical to tumor propagation. Although drug-targeting of cytosolic 1CM remains a clinically-relevant mainstay, development of clinically-useful agents targeting mitochondrial 1CM remains elusive. Of particular pharmacological interest is the mitochondrial 1CM enzyme, serine hydroxymethyltransferase2 (SHMT2). SHMT2 expression correlates with the oncogenic phenotype in lung, colon, breast, glioma, and liver cancer and, overall, is the fifth-most differentially expressed metabolic enzyme in cancer cell versus normal tissue. Despite the unequivocal oncogenic importance and therapeutic potential of SHMT2, there are no clinically relevant (i.e. active in vivo) inhibitors of this enzyme. In this dissertation work, we sought to design, synthesize, and characterize pharmacodynamics of our 5-substituted pyrrolo[3,2-d]pyrimidine antifolates synergistically dual-targeting mitochondrial SHMT2 and cytosolic 1CM, the latter specifically at the purine nucleotide biosynthesis enzymes glycinamide ribonucleotide formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase). By depleting SHMT2-derived formate, these compounds potentiated their own inhibition of the downstream formate-dependent GARFTase and AICARFTase. We generated these compounds (AGF291, AGF320, and AGF347) by melding structures of SHMT2 cofactor 5,10-methylene tetrahydrofolate with our previously reported purine inhibitors and confirmed enzyme targets with in vitro targeted metabolomics in H460 (large cell lung carcinoma), HCT-116 (colorectal carcinoma), and MIA PaCa-2 (pancreatic ductal adenocarcinoma) human tumor cell lines as well as in vitro cell-free assays. Transport assays revealed significant uptake by both the proton-coupled folate transporter (narrow physiological niche, but commonly expressed in many solid tumors) and the reduced folate carrier (major tissue folate transporter). Subcellular fractionation of MIA PaCa-2 and GlyB Chinese hamster ovary cells revealed AGF347 to be heavily (>98%) polyglutamylated in both cytosol and mitochondria with mitochondrial uptake partially mediated by the mitochondrial folate transporter. Intracellular glycine depletion secondary to SHMT2 inhibition by all compounds also depleted cellular ROS scavenging capacity as reflected in decreased GSH/GSSG ratio. In vivo, AGF347 demonstrated potent antitumor efficacy against MIA PaCa-2 xenografts in SCID mice with tumor growth delay (T-C) of 61 days and one out of five treated mice tumor-free 120+ days after treatment. In vivo metabolomics on these xenografts confirmed inhibition of purine biosynthesis. Collectively, the work in this dissertation establishes the exceptional therapeutic potential of dual-targeting mitochondrial and cytosolic 1CM.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
In tropical regions, where there is a high incidence of light, electrons can accumulate in the transport chain (PET) producing large quantities of H 2 O 2 and other ROS, which might generate photodamage and photoinhibition. To survive to these challenges, plants have developed several mechanisms to mitigate the excess energy in photosystems, besides having an efficient machinery for removal of excess H 2 O 2 , which includes cytosolic APX (cAPX). However, double - silenced rice plants for cAPX (OsAPX1/2) do not show large differences in morpho - phenotype when compared to non - transformed (NT), although OsAPX1/2 presents induction of expression on several proteins re lated to photosynthesis. The physiological implications of this induction, as well as its consequences for OsAPX1/2 resistance against stresses of high light (HL), are still poorly known. Aiming to clarify the role of cAPx in pho tosynthesis, OsAPX1/2 plant s were produced, subjected to 24 hours of HL (2 , 000 μ mol m - 2 s - 1 ) and studied for the expression and activity of proteins related to photosynthesis , ph otorespira tion and redox homeostasis . The amount of several PET proteins (Lhcb1, PsbO, Psb P, PsbQ, PSAC, P C, FNR and FDX) and Chl and Pheo were increased in OsAPX1/2 in normal growth conditions, however without causing changes in the in vivo photochemistry activity parameters (Fv /Fm and ΔFm/Fm'). In contrast, expression of proteins associated with Calvin - Benso n cycle (Rls, ativase RBC) and rubisco carboxylation activity ( in vivo and in vitro ) were not altered in mutants under normal growth conditions . In HL, the expression of proteins related to photosynthesis was strongly repressed in all genotypes , as well as gas exchange parameters and Fv/Fm, the latter being strong indication of photoinhibition. Moreover, proteins related to photorespiration showed increased expression/activity in response to HL in NT and maintenance of already high levels in OsAPX1/2. In OsAPX1/2 t h e expression and activity of chloroplastic Cu/Zn - SOD showed a similar response exhibited by photorespiration - related proteins, although the activity of thylakoid APX has been greatly reduced, meaning deficiency in water - water cycle. Taken togeth er, these data demonstrate that induction of expression of proteins related PET in OsAPX1/2 plants may represent a compensatory mechanism for maintaining the photosynthetic activity levels similar to NT. Moreover, in HL, it is possible that the increased e xpression of photorespiration - related proteins in OsAPX1/2 acts as alternative electron sink, compensating the deficiency in the water - water cycle from these plants
Em regiÃes tropicais, onde existe alta incidÃncia luminosa, os elÃtrons podem se acumular na cadeia transportadora (PET) produzindo grandes quantidades de H 2 O 2 e outras ROS, que podem gerar fotodano e fotoinibiÃÃo. Para sobreviver a esse desafio, plantas desenvolveram vÃrios mecanismos de atenuaÃÃo do excesso de energia nos fotossistemas, alÃm de contar com uma eficiente ma quinaria de remoÃÃo do excesso de H 2 O 2 , da qual fazem parte as APX citosÃlicas (cAPX). Entretanto, plantas duplamente silenciadas para as cAPX (OsAPX1/2) nÃo apresentam grandes diferenÃas morfo - fenotÃpicas quando comparadas Ãs nÃo transformadas (NT), embor a OsAPX1/2 apresente induÃÃo de expressÃo de diversas proteÃnas relacionadas com a fotossÃntese, comparadas com as NT. As implicaÃÃes fisiolÃgicas dessa induÃÃo, assim como suas consequÃncias para a resistÃncia de OsAPX1/2 contra estresses de alta luz (HL) , ainda sÃo pouco conhecidas. Objetivando clarificar o papel das cAPx na fotossÃntese, plantas de arroz OsAPX1/2 foram produzidas, submetidas a 24 horas de HL (2000 μ mol m - 2 s - 1 ) e estudadas quanto à expressÃo e atividade de proteÃnas relacionadas com a fotossÃntese, fotorespiraÃÃo e homeostase redox. A quantidade de diversas proteÃnas da PET (Lhcb1, PsbO, PsbP, PsbQ, PSAC, PC, e FDX FNR), bem como teores de Chl e Pheo foram aum entadas em OsAPX1/2 em condiÃÃes normais de crescimento sem causar alteraÃÃes nos parÃmetros de atividade fotoquÃmica in vivo (Fv/Fm e Δ Fm/Fm'). Em contraste, as proteÃnas relacionadas com expressÃo ciclo de Calvin - Benson (Rls, ativase de Rbc) e a ativida de de carboxilaÃÃo da rubisco ( in vivo e in vitro ) nÃo foram alterados nos mutantes em condiÃÃes normais de crescimento. Em HL, a expressÃo de proteÃnas relacionadas com fotossÃntese foi fortemente reprimida em ambos os genÃtipos, assim como os parÃmetros de trocas gasosas e Fv/Fm, sendo esse Ãltimo forte indÃcio de fotoinibiÃÃo. Por outro lado, as proteÃnas relacionadas com a fotorespiraÃÃo, ou mostraram aumento na expressÃo/atividade em resposta à luz elevada (NT) ou manutenÃÃo de nÃveis jà elevados (OsAP X1/2) . A expressÃo e atividade de Cu/Zn - SOD de cloroplastos mostrou resposta similar a exibida pelas proteÃnas da fotorespiraÃÃo, embora a atividade de APX de tilacÃides tenha sido fortemente reduzida em OsAPX1/2, evidenciando deficiÃncia no ciclo Ãgua - Ãgu a. Tomados em conjunto, estes dados demonstram que a induÃÃo da expressÃo de proteÃnas relacionadas com o PET em OsAPX1/2 pode representar um mecanismo compensatÃrio para a manutenÃÃo da atividade fotossintÃtica aos nÃveis da NT. Por outro lado, sob HL, à possÃvel que o aumento da expressÃo de proteÃnas associadas com fotorespira ÃÃo em OsAPX1/2 atue como dissipador alternativo de elÃtrons, compensando a deficiÃncia no ciclo da Ãgua - Ãgua dessas plantas

L’augmentation de la disponibilité des espèces actives de l’oxygène, comme le H2 O2 est une caractéristique commune aux réponses aux stress biotiques et abiotiques. L’accumulation du H2 O2 est contrôlée par les catalases ou par les peroxydases. Ces derniers systèmes dépendent des réducteurs cellulaires comme l’ascorbate et le glutathion, dont les teneurs sont soutenues par le NADPH. Le flux augmenté via ces voies peut provoquer des perturbations (comme celles qui peuvent affecter le statut thiol-disulfure) qui prennent le relais pour la transduction des signaux oxydatifs. Bien que plusieurs enzymes puissent réguler le système NADPH-glutathion dans le cytosol, l’importance physiologique de chacune de ses enzymes reste à élucider. Cette étude est basée sur la combinaison d’approches génétiques, biochimiques, transcriptomiques et métaboliques dans le but d’analyser les rôles de la glutathion réductase cytosolique (GRI) et des déshydrogénases NADP-dépendantes dans le métabolisme, les réponses induites par le H2 O2 et la résistance aux pathogènes. L’analyse comparative des mutants T-ADN des trois catalases d'Arabidopsis a révélé que seulement cat2 montre des perturbations redox et un stress oxydatif conditionnel au niveau foliaire. Cette lignée a été donc utilisée comme un fond génétique pour examiner l'importance du système NADPH-glutathion cytosoliquedans la réponse au H₂ O₂ produit au niveau intracellulaire par une voie physiologiquement pertinente (la photorespiration). L'analyse comparative des mutants gr1, cat2 e(t cat2 gr1 a révélée que GR1 joue un rôle déterminant dans les réponses des feilles au H₂ O₂intracellulaire et qu'elle asure une expression génique appropriée via les voies de signalisation par l'acide salicylique (AS) et par l'acide jasmonique. Une strtégie similaire a été utilisée pour explorer les fonctions de l'isocitrate déshydrogène cytosolique à NADP (ICDH) et des deux glucose-6-phosphate déshydrogénases cytosoliques (G6PPD5 et G6PD6). Cette analyse a montré que ces trois enzymes contribuent au maintient du statut glutathion dans le fond cat2. L'étude a aussi révélée que la mutation icdh et les mutations g6pd produisent des effets distincts où antagonistes par rapport à la mort cellulaire déclenchée par H2 O2 aux réponses aux pathogènes SA dépendantes et à la résistance aux bactéries. Ainsi, l'étude montre que la GR ne peut être remplacée par la deuxième GR ou par le système thiorédoxine dans les conditions de sur-disponibilité de H2 O2 et met en évidence les fonctions spécifiques des systèmes cytosoliques producteurs de NADPH dans la détermination des résultantes du stress oxdatif
Increased availability of ractive oxygen species asuch as H2 O2 is a feature of biotic and abiotic stresses. H2 O2 accumulation is controlld either by catalases or by peroxidases. The second depend on cellular reductants such as ascorbate and glutathione, both of which are supported by NADPH pools. Increased flux through these pools may cause perturbations (eg. In thiol-disulfide statud) that act in the relay of H2 O2-dependent redox signals. Although several enzyme systems are known that could regulate the NADPH-glutathione system in the cytosoln the significance of each remians largely unknown. This study look a combined genetic, biochemicaln and transcript and metablotie profiling approach to analyzing the roles of cytosolic glutathione reductase (GR) and NADP-dependent dehydrogenases in metabolism, H2 O2-triggered responses, and biotic stress. Comparative analysis of T-DNA mutants for the three Arabidopsis catalases revealed that only cat2 showed conditional redox pertubation and stress in rosettes. This line was therefore used as a gnentic background to investigate the importance of the cytosolic NADPH-glutathione system in response to H₂ O₂ produced intracellularly through a physiologically relevant pathway (photorespiration). Comparative analysis of gr1, cat2 and cat2 gr1 mutant lines revealed that GR1 plays a crucial role in leaf reponses to intracellular H2 O2 and is required to ensure appropriate gene expression throug both salicylic acid (SA) and jasmonic acid signaling pathways. Using a similar strategy, the functions of cytosolic NADP-isocitrate dehydrogenase (ICDH) an the two cytosolic glucose-6-phosphate dehydrogenases (G6PD5 and G6PD6) were explored. This analysis provided evidence that all three dehydrogenases contribuate to maintaining glutathione status under conditions of increased H₂ O₂ availability but revealed that the icdh and g6pd mutations produce distinct or opposing effects on H2 O2-triggered cell death, SA-dependent pathogenesis reponses and bacterial resistance. Thus, the study shows that GRI cannot be replaced by the second GR or by the thioredoxin system under conditions of increased H2 O2 and provides evidence for the specificity of cytosolic NADPH-producing systems in determining the outcome of oxidative stress

Les mitochondries jouent un rôle majeur dans le métabolisme de l'ATP des cellules eucaryotes. Le maintien de l'ADN mitochondrial (ADNmt) est fondamental pour la production d'énergie chez les organismes aérobie stricte. De grandes délétions de ADNmt sont à l'origine d'anomalies mitochondriales entrainant des maladies chez l'homme. Plusieurs gènes nucléaires impliqués dans le métabolisme de l’ADNmt ont été identifiés et caractérisés chez l'homme. Cependant, l’ensemble des facteurs et leurs activités requis pour le maintien de l'ADNmt reste largement inconnu. L'identification de ces facteurs et la détermination de leurs activités dans des systèmes modèles simples peuvent contribuer à l’étude du maintien de l'ADNmt et à la compréhension des mécanismes induisant des délétions de l’ADNmt chez l'homme. Le champignon filamenteux Podospora anserina est un modèle d'étude du maintien de l’ADNmt. Chez P. anserina, l’accumulation de délétions région-spécifiques de l’ADNmt (Δmt) est corrélée à la présence de la mutation AS1-4 dans le gène nucléaire codant la protéine cytosolique ribosomale S15. L'altération de la protéine S15 pourrait modifier la traduction de transcrits codant des protéines impliquées dans le maintien de l'ADNmt et indirectement causer l'accumulation des Δmt. Par une approche globale (translatome), nous avons analysé l’ensemble des transcrits associés aux ribosomes AS1-4 en cours de traduction. A partir des données obtenues, deux gènes candidats, PaIML2 et PaYHM2 potentiellement impliqués dans le maintien de l'ADNmt, ont été identifiés et étudiés. L'analyse fonctionnelle a été principalement développée pour PaYHM2. La protéine PaYHM2 partage 68% d’identité avec la protéine mitochondriale bi-fonctionnelle Yhm2 de levure, impliquée dans le transport de métabolites dans la mitochondrie et possèdant un domaine de liaison à l'ADN. J'ai démontré que le gène PaYHM2 est essentiel pour P. anserina, un organisme aérobie stricte et que la protéine PaYHM2 est mitochondriale. Par mutagénèse, j'ai montré que c'est la fonction de transport qui est essentielle à la survie du champignon et non pas la putative capacité à se lier à l'ADN. Les résultats obtenus suggèrent également que PaYHM2 participe au métabolisme de l'acétyl-CoA chez P. anserina
Mitochondria play main role as adenosine triphosphate (ATP)-energy factories of the eukaryotic cells. To ensure energy production, mitochondrial DNA (mtDNA) maintenance is essential for all obligate-aerobe eukaryotic organisms. Large-scale mtDNA deletions are major causes of mitochondrial dysfunction in human diseases. Several nuclear genes implicated in mtDNA metabolism were identified and characterized in human. Nuclear-encoded factors and their activities required for mtDNA maintenance are, however largely unknown. Identification of these factors and discovery of their activities in simple model systems can contribute to the comprehension of mtDNA maintenance and of the mechanisms leading to mtDNA deletions in human. The filamentous fungus Podospora anserina is a useful model system for studying mtDNA maintenance. An S15 cytosolic ribosomal protein mutant in P. anserina, named AS1-4 mutant, shows a positive correlation with the accumulation of specific large mtDNA deletion (Δmt) at the time of death. Alteration of S15 protein might modify translation of transcripts encoding proteins related to mtDNA maintenance and indirectly cause Δmt accumulation. Polysome profiling (called translatome), a global approach giving genome-wide informations about modified transcripts on translation, was performed on AS1-4 mutant. From the data of this translatome, two candidate genes potentially related to mitochondrial DNA maintenance, the PaIML2 gene and PaYHM2 gene has been identified and functionally analyzed. The function of the PaYHM2 gene has been especially characterized in this project. This gene encodes a protein sharing 68% of identity with yeast Yhm2, a bi-functional protein as a mitochondrial carrier and as a protein with DNA-binding activity. I demonstrated that the PaYHM2 gene is essential for P. anserina, an obligate-aerobe organism and that the PaYHM2 protein localizes to mitochondria. Through mutagenesis approach, I showed that the transport function decides the essentiality of mitochondrial carrier PaYHM2 while the putative DNA binding activity of PaYHM2 protein is important for P. anserina. Furthermore, I found that the function of PaYHM2 probably participates in the cytosolic acetyl-CoA metabolism

Phenylalanine (Phe) is a proteinogenic aromatic amino acid that also serves as a precursor for numerous primary and secondary metabolites in plants. Phe is synthesized from chorismate, the final product of the shikimate pathway. In plants, Phe is predominantly synthesized in the plastids via the arogenate pathway, while most Phe-derived compounds are produced in the cytoplasm, requiring exportation of Phe from plastids to the cytosol. Here, we provided genetic evidences that a Petunia hybrida plastidial cationic amino acid transporter (PhpCAT) participates in the exportation of Phe from plastids, as well as regulation of carbon flux through Phe biosynthesis.

By using reverse genetics, we demonstrated that a petunia phenylpyruvate aminotransferase (PhPPY-AT) is able to convert phenylpyruvate to Phe in the cytosol in vivo, and that a cytosolic chorismate mutase (CM2), which converts chorismate to prephenate, directs carbon flux from the plastidial Phe biosynthesis pathway towards the cytosolic pathway. Downregulation of PhPPY-AT and PhCM2 resulted in significant decreases in Phe levels and emission of Phe-derived volatiles in petunia flowers, respectively. Metabolic flux analysis showed that the carbon flux through the cytosolic Phe biosynthesis pathway is significantly lower in PhCM2 RNAi petunia flowers relative to wild type control. We also demonstrated that the conversion of prephenate to phenylpyruvate in the cytosol is catalyzed by a cytosolic prephenate dehydratase (PDT) produced from an alternative transcription start site of a known plastidial arogenate dehydratase (ADT). These results suggest that a microbial-like phenylpyruvate pathway for Phe biosynthesis operates in the cytosol of plant cells and the cytosolic pathway splits from the plastidial pathway at chorismate.

To evaluate the metabolic potential of the cytosolic phenylpyruvate pathway, PhCM2 overexpressing transgenic petunia plants were generated. Unexpectedly, Phe levels and emission of Phe-derived volatiles were both reduced, even though the flux through the cytosolic pathway was increased relative to wild type control. Electron microscopy, metabolic profiling and metabolic flux analysis revealed that the number of leucoplasts, starch levels and flux through the plastidial pathway were all reduced in PhCM2 overexpression lines, while the concentrations of auxin and its biosynthetic intermediate, indole-3-pyruvic acid (IPA), were elevated. Overexpression of Arabidopsis aminotransferase VAS1, which converts IPA to Trp, in PhCM2 overexpression petunia background recovered Phe levels and Phe-derived volatiles emission. These results indicate that there exists a metabolic crosstalk between cytosolic Phe production and Trp-dependent auxin biosynthesis .

Our research completed the post-chorismate cytosolic Phe biosynthesis pathway in plants and revealed possible metabolic crosstalk between cytosolic Phe production and auxin biosynthesis in plant cells, providing targets for future genetic modification of metabolites in plants.

碩士
國防醫學院
生物化學研究所
91
Cytosolic ALDH1A1 and mitochondrial ALDH2 are the principal enzymes responsible for oxidation of acetaldehyde, an immediate metabolite of ethanol, in the human liver. Approximately 50% of Oriental populations exhibit ALDH2 deficiency. In ALDH2-deficient individuals, ALDH1A1 is the sole functional form for acetaldehyde metabolism. Recombinant human ALDH1A1 was expressed in E. coli and isolated to apparent homogeneity via DEAE-Sepharose and 5’AMP-Sepharose chromatographic procedures. Subunit molecular mass was determined to be 55 kDa. Initial velocity and product inhibition studies suggest that acetaldehyde oxidation follows an ordered sequential mechanism with binding of NAD+ and release of NADH being the first and last steps, respectively, in the ternary complex reaction. Dead-end inhibition studies suggest that the reaction is predominantly consistent with an ordered sequential mechanism but tendency of a rapid-equilibrium random addition of substrate and coenzyme cannot be completely ruled out. Substrate saturation kinetic studies indicate that recombinant ALDH1A1 exhibits negative cooperativity toward oxidation of formaldehyde and acetaldehyde, and even more interestingly both negative and positive cooperativity, i.e. mixed cooperativity, toward propionaldehyde. Mechanism-based computer numerical simulations suggest that ALDH1A1 highly effectively contributes to acetaldehyde oxidation in the hepatocytes of ALDH2-active individuals following ethanol consumption. This is mainly attributed to the negative cooperativity of the cytosolic ALDH1A1.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1988.
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