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Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents

Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents

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KIENHÖFER, Joachim, 2009. Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents

@phdthesis{Kienhofer2009Assoc-8512, title={Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents}, year={2009}, author={Kienhöfer, Joachim}, address={Konstanz}, school={Universität Konstanz} }

2011-03-24T17:44:17Z application/pdf Kienhöfer, Joachim eng Kienhöfer, Joachim Mitochondrial DNA (mtDNA) is organized in protein-DNA macro-complexes, called nucleoids. Average nucleoids contain 2-8 mtDNA molecules, which are organized by the histone like mitochondrial transcription factor A. Besides well-characterized constituents, such as single-stranded binding protein or polymerase γ, various other proteins with illdefined functions have been identified.<br />We report for the first time that mammalian nucleoids contain essential enzymes of an integral antioxidant system. Intact nucleoids were isolated with sucrose density gradients from several tissues of different species as well as from several cell lines. Manganese superoxide dismutase (SOD2) was detected by Western blot in the nucleoid containing fractions. Since the mitochondrial matrix protein fumarate hydratase as well as malate dehydrogenase does not co-purify with the nucleoids it is unlikely that association of SOD2 with mtDNA is due to contamination of the nucleoid fraction. DNA, mitochondrial glutathione peroxidase, and polymerase γ were co-immunoprecipitated with SOD2 from nucleoid fractions, which suggests that an antioxidant system composed of SOD2 and glutathione peroxidase are integral constituents of nucleoids.<br />Using a sandwich filter-binding assay, direct association of SOD2 by salt-sensitive ionic forces with a chemically synthesized mtDNA fragment was demonstrated. Increasing salt concentrations during nucleoid isolation on sucrose density gradients disrupted the association of SOD2 with mitochondrial nucleoids and confirmed the involvement of ionic forces for the binding. Our biochemical data reveal that nucleoids contain an integral antioxidant system that may protect mtDNA from superoxide-based oxidative damage.<br />More important for the generation of oxidative DNA damage seems to be peroxynitrite which is generated by equimolar fluxes of superoxide and nitrogen monoxide. With a modified protocol for the fluorimetric analysis of DNA unwinding-assay significantly higher amounts of the highly mutagenic 8-oxo-7,8-dihydroguanine was found to be generated by peroxynitrite than by superoxide or hydrogen peroxide in a mtDNA model. Manganese superoxide dismutase was able to protect DNA from oxidative damage generated by equimolar fluxes of superoxide and nitrogen monoxide (resulting in peroxynitrite formation) in this model. Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents 2009 deposit-license 2011-03-24T17:44:17Z Assoziation mitochondrialer antioxidativer Enzyme mit der mitochondrialen DNA als ein integraler Bestandteil des Nukleoid-Komplex

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

Dissertation_Kienhoefer.pdf 128

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