Gene structure and Function of H^+-ATPase.

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For investigating the molecular mechanism of ATP synthesis on an ATP synthase molecule coupled with the proton translocation, ATP synthase from a thermophilic bacterium has a great advantage to be analyzed, because the protein is extremely stable and resistant against various kinds of experimental treatments. Next, for the studies on the regulation of the gene expression in the cellular level, the human cultured cells are good targets because a lot of cell-lines have been established and because some mitochondrial diseases have been reported. Therefore, we have taken approaches to understand the molecular mechanisms for ATP synthesis and the cellular response against ATP formation, respectively.(A) For molecular mechanism of ATP synthase. 1) The gene for the ATP synthase operon was cloned and sequenced from a thermophilic bacterium PS3. 2) The alpha ,bata and gamma subunits could be over-expressed in E. coli by introducing the thermophilic genes which had been connected with a strong E … More . coli promoter. The individual aubunits were purified without denaturating the subunits during the purification procedure. 3) The over-expressed subunit could be reconstituted. The above mentioned mild purification procedure enables the reconstitution of the alpha and bata subunits. 4) By site-directed mutagenesis method, some amino acid in the subunits were substituted with the other amino acids artificially. The combination of the mutated subunits gave several information on the molecular mechanisms. 5) The over-expressed subunits were used for some physico-chemical approaches including neutron scattering, and crystallization of the proteins.(B) The cell biology of the human ATP synthase. 1) The human cDNAs encoding 7 mitochondrial proteins including ATP synthase bata-subunit were cloned sequenced. 2) The genomic genes for the human ATP synthase bata-subunit and the human pyruvate dehydrogenase alpha-subunit were also cloned and sequenced. 3) The 5' upstream region were analyzed by the promoter activity. These kinds experiments revealed the presence of a novel enhancer which enhances the transcription. In addition, the enhancer were common to some genes for some mitochondrial proteins. 4) The regulation of the gene expression were examined by Northern blotting in the various kind of cellular conditions. 5) The oxidative phosphorylation-deficient cell lines were established from patients with mitochondrial disease. Less