MONOCLONAL ANTIBODIES AS PROBES OF CYTOCHROME OXIDASE

细胞色素氧化酶，细胞中的终极电子传递酶 真核生物的线粒体内膜，以某种形式由 都是好氧生物。因为它能立刻排出氧气，是一处 对于生命存在的可能性，它是至关重要的 地球。然而，人们对分子的实际构象迁移率知之甚少 TOTO中的酶，因为它在电子传递和 能量转导。拟议的研究将结合新出现的 用传统工艺生产单抗的技术 与线粒体生物化学领域相关。我们会做好准备 抗酶及其亚基的单抗。然后我们会放映 对于决定因素识别敏感的克隆 活性状态(使用新开发的酶联免疫吸附剂 检测)抗原检测)或识别与其结合的决定因素 影响氧化还原功能(通过分析细胞色素C氧化酶活性) 已与克隆抗体预先孵育的酶)。决定因素 它们显示了有趣的结合-活性关系(以及其 抗原性对洗涤剂变性不敏感)将定位于 用Western blotting技术测定该酶的亚基。一个 二维SDS-PAGE将解析亚基并展示多肽 由有限的蛋白质分解引起的。这些多肽的蛋白质印迹图可以 揭示亚基的相关部分，以确定初级 结构。通过使用这些技术来探测体内或 隔离(溶解的或人工重组的)，休眠或 活性，有或没有配体，以及有或没有亚基 似乎与能量传递有最直接的联系，我们将 获得一系列地形变异性的结构-功能图 最终，导致了对酶的全面概述 这种酶的结构和功能之间的动态相互作用 对氧化磷酸化至关重要--对生命本身来说！

Cytochrome oxidase, the ultimate electron transport enzyme in the mitochondrial inner membrane of eukaryotes, is possessed in some form by all aerobic organisms. Because it at once detoxifies oxygen and is a site of energy transduction, it is essential to the very possibility of life on earth. Yet little is known of the actual conformational mobility of the enzyme in toto as it performs its functions on electron transport and energy transduction. The proposed study will combine the newly emerging technology of monoclonal antibody production with techniques traditionally associated with the field of mitochondrial biochemistry. We will prepare monoclonal antibodies to the enzyme and its subunits. We will then screen for clones for which the recognition of determinants is sensitive to activity state (using a newly developed ELISA (enzyme linked immuno-sorbant assay) antigen assay) or which recognize determinants to which binding affects redox function (by assaying the cytochrome c oxidase activity of enzyme which has been preincubated with the cloned antibody). Determinants which show interesting binding-activity relationships (and whose antigenicity is insensitive to detergent denaturation) will be localized on the subunits of the enzyme using the technique of Western blotting. A two-dimensional SDS-PAGE will resolve the subunits and display the peptides resulting from limited proteolysis. Western blots of these peptides can reveal the relevant portions of the subunit for determination of primary structure. By using these techniques to probe the enzyme in vivo or isolated (either solubilized or artificially reconstituted), resting or active, with and without ligands, and with or without the subunit which appears to be most directly associated with energy transduction, we will obtain a series of structure-function maps of the topographical variability of the oxidase, ultimately, leading to a comprehensive overview of the dynamic interaction between structure and function of this enzyme which is so vital to oxidative phosphorylation-to life itself!