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Gene structure and Function of H^+-ATPase.

H^-ATP酶的基因结构和功能。

基本信息

批准号：
60440032
负责人：
KAGAWA Yasuo
金额：
$ 9.73万
依托单位：
Jichi Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1988
项目状态：
已结题

项目摘要

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

为了研究ATP合成酶分子上ATP合成与质子转运偶联的分子机制，来自嗜热细菌的ATP合成酶具有很大的分析优势，因为该蛋白质非常稳定并且对各种实验处理具有抗性。其次，对于在细胞水平上调控基因表达的研究，人培养细胞是很好的目标，因为已经建立了许多细胞系，并且已经报道了一些线粒体疾病。因此，我们已经采取的方法来了解ATP合成的分子机制和细胞对ATP形成的反应，分别。(A)ATP合成酶的分子机制。1)从嗜热菌PS3中克隆了ATP合酶操纵子的基因并进行了测序。2)α、β和γ亚基在E.通过引入与强大肠杆菌连接的嗜热基因， ...更多信息 . coli启动子。在纯化过程中，在不使亚基变性的情况下纯化各个亚基。3)过表达的亚基可以被重组。上述温和的纯化程序能够重建α和β亚基。4)通过定点突变的方法，将亚基中的某些氨基酸人工替换为其它氨基酸。突变亚基的组合提供了一些关于分子机制的信息。5)过表达的亚基用于一些物理化学方法，包括中子散射和蛋白质结晶。(B)人类ATP合成酶的细胞生物学。1)对编码ATP合成酶β亚基等7个线粒体蛋白的cDNA进行了克隆和测序。2)还克隆和测序了人ATP合酶β亚基和人丙酮酸脱氢酶α亚基的基因组基因。3)通过启动子活性分析5 '上游区。这类实验揭示了一种新的增强子的存在，增强转录。此外，增强子是一些线粒体蛋白的一些基因所共有的。4)通过北方印迹法检测不同细胞条件下基因表达的调控。5)氧化磷酸化缺陷细胞系是从线粒体疾病患者中建立的。少