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MONOCLONAL ANTIBODIES AS PROBES OF CYTOCHROME OXIDASE

单克隆抗体作为细胞色素氧化酶的探针

基本信息

批准号：
3280901
负责人：
SAMUEL H CHAN
金额：
$ 11.92万
依托单位：
SYRACUSE UNIVERSITY AT SYRACUSE
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-09-05 至 1989-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3280901
关键词：
antigens bioenergetics chemical structure function cytochrome oxidase electron transport enzyme linked immunosorbent assay enzyme reconstitution enzyme structure gel electrophoresis genetic manipulation membrane model monoclonal antibody

项目摘要

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!

细胞色素氧化酶是细胞中的最终电子传递酶，真核生物的线粒体内膜，以某种形式被所有需氧生物。因为它能立刻分解氧气在能量转换的过程中，它对生命的可能性至关重要。地球然而，很少有人知道的实际构象的流动性，酶在电子传递中发挥作用，能量转换拟议中的研究将联合收割机与新出现的单克隆抗体生产技术与线粒体生物化学领域相关。我们将准备针对酶及其亚基的单克隆抗体。然后我们将筛选对于决定簇识别敏感的克隆，活性状态（使用新开发的ELISA（酶联免疫吸附剂抗原测定）或识别与其结合的决定簇影响氧化还原功能（通过测定细胞色素c氧化酶活性，已与克隆抗体预孵育的酶）。决定因素它们显示出有趣的结合活性关系（以及抗原性对去污剂变性不敏感）将定位于用蛋白质印迹技术分析酶的亚基。一二维SDS-PAGE将解析亚基并显示肽由有限的蛋白质水解引起。这些肽的蛋白质印迹可以揭示了亚基的相关部分，用于确定初级结构通过使用这些技术在体内或体内探测酶，分离的（溶解的或人工重构的）、静止的或活性的，有或没有配体，以及有或没有亚基，似乎与能量转换最直接相关，我们将获得一系列地形变异的结构-功能图氧化酶的，最终，导致全面概述这种酶的结构和功能之间的动态相互作用，对氧化磷酸化如此重要--对生命本身！