Assembly & Conductance of Fo Sector of E coli H+-ATPase

描述：（申请人的描述）F-型ATP酶合成大部分的 生物系统中的ATP这些酶将储存在细胞中的能量相互转化， 跨膜质子梯度与细胞ATP水平。三个具体目标 在本提案中，我们将讨论有关F0的结构和功能的问题， 扇区，可以存在于不同的物理和功能状态，取决于 对组装状态、基因表达的变化和/或对代谢的反应的影响 情节 第一个目的是检验假设， 亚基改变酶的催化和能量偶联特性。的 C化学计量将被操纵， uncE（c亚基）或通过在不同的生长环境中生长野生型细胞代谢 碳源，在不同pH下，或在有氧与厌氧条件下。 将对分离的膜进行膜结合ATP酶和ATP合酶测定 活动，以及不同F0部门的相对化学计量 将通过免疫沉淀和抗c免疫印迹进行定量 抗体的 第二个目的是检验这样一个假设，即碳化学计量的变化可以 改变纯化的F0扇区的质子电导。F0含有不同的 大量的c亚基将被纯化并重组到脂质体中， 单通道F0质子通量将在形成后测量。 钾/缬氨霉素诱导的电梯度。这些目标的另一个目标是 研究的目的是修改F0纯化程序，以尽量减少或消除 污染离子通道活动。 第三个目的是检查F0的组装，首先通过表征 在不存在F1亚基的情况下合成和组装的F0的性质， 然后通过确定哪些F1亚基与未成熟的F0相互作用， 产生成熟的质子传导F0扇区。通过结合标准膜 用改进的脂质体质子通量测定法进行荧光猝灭测定， 这些研究将测试不同的F1亚基协助 新合成的FO的插入、组装或稳定性。

DESCRIPTION: (Applicant's Description) F-type ATPases synthesize most of the ATP in biological systems. These enzymes interconvert the energy stored in a transmembrane proton gradient with cellular ATP levels. All three specific aims in this proposal address questions about the structure and function of the F0 sector, which can exist in different physical and functional states, depending on assembly state, changes in gene expression, and/or response to metabolic circumstances. The first aim is to test the hypothesis that changes in stoichiometry of the C subunit changes the catalytic and energy-coupling properties of the enzyme. The C stoichiometry will be manipulated either genetically by altering expression of uncE (c subunit) or metabolically by growing wild type cells on different carbon sources, at different pH's, or under aerobic vs. anaerobic conditions. Isolated membranes will be assayed for membrane-bound ATPase and ATP synthase activities, and the relativtive stoichiometries of the different F0 sectors will be quantified by immunoprecipitation and immunoblotting with anti-c antibodies. The second aim is to test the hypothesis that a change in c stoichiometry can change the proton conductance of purified F0 sectors. F0 containing different numbers of c subunit will be purified and reconstituted into liposomes, and the single-channel F0 proton flux will by measured after the formation of a potassium/valinomycin-induced electrical gradient. An additional goal of these studies is to modify the F0 purification procedure to minimize or eliminate contaminating ion-channel activities. The third aim is to examine the assembly of the F0, first by characterizing the nature of the F0 synthesized and assembled in the absence of the F1 subunits, and then by determining which F1 subunits interact with that immature F0 to produce the mature, proton-conducting F0 sector. By combining standard membrane fluorescence quenching assays with an improved liposome proton-flux assay, these studies will test the ability of different F1 subunits to assist the insertion, assembly, or stability of the newly-synthesized F0.