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

描述：(申请者描述)F型ATPase合成了大部分 生物系统中的三磷酸腺苷。这些酶将储存在体内的能量相互转化 跨膜质子梯度与细胞三磷酸腺苷水平。所有三个具体目标 在本提案中，解决了有关F0的结构和功能的问题 扇区，它可以存在于不同的物理和功能状态，具体取决于 在组装状态，基因表达的变化，和/或对代谢的反应 情况。 第一个目标是检验C的化学计量比变化的假设 亚基改变了酶的催化和能量偶联性质。这个 化学计量学将通过改变表达从基因上进行操纵 或通过在不同的细胞上生长野生型细胞来代谢 碳源，在不同的pH值下，或在好氧和厌氧条件下。 将对分离的膜进行膜结合ATPase和ATP合成酶的检测 活度，以及不同F0部门的相对化学计量 将通过免疫沉淀和抗c抗体免疫印迹来定量。 抗体。 第二个目的是检验这样一个假设，即c化学计量比的变化可以 改变纯化的F0扇区的质子电导。F0包含不同的 多个c亚基将被提纯并重组为脂质体，而 单通道F0质子通量将在形成后被测量 钾/呋喃西林诱导的电梯度。这些项目的另一个目标 研究是修改F0的提纯程序，以最大限度地减少或消除 污染离子通道活动。 第三个目标是检查F0的组装，首先通过表征 在没有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 生产成熟的质子传导F0扇区。通过结合标准膜 用改进的脂质体质子流量法进行荧光猝灭分析， 这些研究将测试不同F1亚基协助 新合成的F0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.