MONOCLONAL ANTIBODIES AS PROBES OF CYTOCHROME OXIDASE

细胞色素氧化酶，细胞中最重要的电子传递酶

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!