Freiburg (Breisgau), Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Thesis (doctoral)--University of Göteborg, 1995. / Includes bibliographical references.

Düsseldorf, Universiẗat, Diss., 2004.

Frankfurt (Main), Univ., Diss., 2002.

Tetrahydrobiopterin (BH4) is a limiting cofactor for nitric oxide synthase (NOS) catalysed conversion of L-arginine to nitric oxide and citrulline. Content of BH4 in mammalian cells is regulated at many levels, but most important is de novo biosynthesis from GTP. GTP cyclohydrolase (GTPCH) is the rate-limiting enzyme for the de novo synthesis of BH4. While various immunostimulants, hormones and growth factors have been reported to increase GTPCH mRNA levels and intracellular biopterin (BH4 degradation product), it is not known whether these factors act at the level of GTPCH gene transcription. To test this I utilised 1, 3 and 6 kb 5'upstream GTPCH gene sequence in a secreted alkaline phosphatase reporter vector (SEAP). These constructs were stably transfected in PC-12 cells and rat aortic smooth muscle cells, and the cells were treated with various immunostimulants and growth factors in order to determine whether these factors could enhance GTPCH gene transcription. Intracellular biopterin levels were also measured to confirm that the upregulation of the SEAP-reporter correlated with a rise in biopterin. Our investigations conclude that transcriptional regulation of the GTPCH gene is indeed a major site for control of intracellular BH4 levels. In further experiments, we have characterised the binding of [3H]BH4 to endothelial NOS (eNOS) and examined influences of the substrate, arginine, on the BH4 binding. In addition we selected tetrahydropterins (that support NOS catalysis) and dihydropterins (that are catalytically incompetent) to determine the extent to which modifications of BH4 alter pterin binding affinity to eNOS. Dihydropterins are unable to support NOS catalysis. Studies showed for the first time that dihydropterins, but not tetrahydropterins, support superoxide generation by eNOS. We also have determined that eNOS may be able to produce NO in the absence of BH4 cofactor from the reaction intermediate hydroxyarginine. We have characterised this reaction and are able to provide a plausible mechanism for the NOx generation from eNOS in the absence of BH4 cofactor.

abstract: Adenosine triphosphate (ATP) is the universal chemical energy currency in most living cells, used to power many cellular reactions and generated by an enzyme supercomplex known as the ATP synthase, consisting of a hydrophilic F1 subcomplex and a membrane-bound FO subcomplex. Driven by the electrochemical gradient generated by the respiratory or photosynthetic electron transport chain, the rotation of the FO domain drives movements of the central stalk in response to conformational changes in the F1 domain, in which the physical energy is converted into chemical energy through the condensation of ADP and Pi to ATP. The exact mechanism how ATP synthesis is coupled to proton translocation is not known as no structure of the intact ATP-synthase nor the intact FO subcomplex has been determined to date. Structural information may shed light on these mechanisms and aid in understanding how structural changed relate to its coupling to ATP synthesis. The work in this thesis has successful established a defined large-scale CF1FO isolation procedure resulting in high purity and high yield of this complex from spinach thylakoid membranes by incorporating a unique combination of biochemical methods will form the basis for the subsequent structural determination of this complex. Isolation began from the isolation of intact chloroplasts and the separation of intact thylakoid membranes. Both native and denaturing electrophoresis analyses clearly demonstrated that the purified CF1FO retains its quaternary structure consisting of the CF1 and CFO subcomplexes and nine subunits (five F1 subunits: α, β, γ, δ and ε, and four FO subunits: a, b, b' and c). Moreover, both ATP synthesis and hydrolysis activities were successfully detected using protein reconstitution in combination with acid-base incubation and in-gel ATPase assays, respectively. Furthermore, the ATP-synthase of H. modesticaldum, an anaerobic photosynthetic bacterium, was also isolated and characterized at the biochemical level. These biochemical characterizations directly influenced recent studies on the high-resolution structure determination of intact CF1FO using electron crystallography on two-dimensional crystals. The availability of the functionally intact CF1FO purified at a large scale will lead to studies that investigate the possible crystallization conditions to ultimately determine its three-dimensional structure at atomic resolution. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2015

No description available.

La F1FO-ATP synthase mitochondriale produit la majorité de l’énergie cellulaire chezles eucaryotes aérobes sous forme d’ATP par le processus des oxydations phosphorylantes.Chez la plupart des espèces, cette enzyme possède une origine génétique double, nucléaire etmitochondriale. Dans la première partie de ce travail, je décris la construction de modèles delevure de mutations du gène mitochondrial ATP6 de l’ATP synthase découvertes chez despatients atteints de maladies neurologiques (9185T>C and 9191T>C) ou dans des tumeurs(8716A>G, 8914C>A, 8932C>T, 8953A>G and 9131T>C). Le gène ATP6 code une sousunitéessentielle (a/6) du domaine FO de l’ATP synthase. J’ai trouvé que la mutation 9185T>Cn’affecte pas l’assemblage de l’ATP synthase, mais conduit à une diminution de la vitesse desynthèse d’ATP d’environ 30%. La mutation 9191T>C empêche presque entièrementl’incorporation de la sous-unité a/6 dans l’ATP synthase. Les cinq mutations identifiées dansles tumeurs ont un effet modeste à nul, indiquant que ces mutations ne favorisent pas latumorigenèse en affectant le processus énergétique mitochondrial, comme évoquéprécédemment. J’ai ensuite étudié la régulation de la synthèse des sous-unités a/6 et 9 dans lesmitochondries de levures. La sous-unité 9 est présente sous la forme d’un anneau de 10 copiesqui interagit avec la sous-unité 6. Durant la catalyse, la rotation de cet anneau provoque deschangements conformationnels favorisant la synthèse d’ATP dans le secteur F1 de l’ATPsynthase. Je montre que la synthèse de ces protéines est couplée à leur assemblage, demanière à ce qu’elles soient produites dans une stoechiométrie adéquate et pour éviterl’accumulation d’intermédiaires d’ATP synthase potentiellement délétères / Mitochondrial F1FO-ATP synthase produces most of the cellular energy in aerobiceukaryotes under the form of ATP in the process of oxidative phosphorylation. This enzymehas in most species a double genetic origin, nuclear and mitochondrial. In the first part of thiswork, I describe the construction of yeast models of ATP synthase mutations in themitochondrial ATP6 gene, that have been found in patients presenting with neurologicaldisorders (9185T>C and 9191T>C) and in tumors (8716A>G, 8914C>A, 8932C>T,8953A>G and 9131T>C). The ATP6 gene encodes an essential subunit (called a/6) of theATP synthase proton-translocating domain (FO). The 9185T>C mutation had no effect on theassembly of ATP synthase, but reduces the rate of ATP synthesis by 30%. The 9191T>Cmutation almost completely prevented incorporation of the subunit a/6 into the ATP synthase.The five mutations found in tumors had modest, if at all, effect, indicating that thesemutations probably do not favor tumorigenesis, as was hypothesized. In the second part of mythesis, I studied the regulation of synthesis of subunits a/6 and 9 in yeast mitochondria. Thesubunit 9 is present in 10 copies forming a ring that interacts with subunit 6. Protonmovements through the FO induce the rotation of the subunit 9-ring, which results inconformational changes that promote ATP synthesis in the catalytic sector (F1) of ATPsynthase. I discovered mechanisms that enable the coupling of the synthesis of these proteinsto their assembly, as a means to ensure the production of subunits 6 and 9 in the rightstoichiometry and to avoid accumulation of potentially harmful assembly intermediates of theATP synthase.

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No description available.