Monoclonal Antibodies of Mammalian Cytochrome Oxidase and its Protein Subunits

SUMMARY Ba1b/c mice were immunized with cytochrome oxidase holoenzyme and individual subunit-precipitated nitrocellulose paper strips, and their spleen cells were fused with myeloma cells. After selection of hybrids, the cultures were initially screened for whole oxidase and subunit antibody titers using microplate ELISA procedures. The positive clones were expanded and screened again. Identification and confirmation of subunit specific monoclonal antibodies were carried out with Western blot techniques. To date, we have been able to obtain about 25 different hybridoma cell lines that produce antibodies directed against the whole oxidase, subunits I through VII individually and also lines that react with more than one subunit (e.g., VIa, VIb, and VIIa,b,c on highly resolved gels). Many antibodies exhibit inhibition of electron transport activity of both solubilized oxidase and oxidase vesicles. However, some lines inhibit the soluble oxidase to a greater extent than that of vesicular oxidase while others show the opposite, indicating different epitope and/or subunit locations in the vesicular oxidase. In addition, some antibodies improve the respiratory control index of the vesicular oxidase, possibly due to a decrease of proton leaks and more tightly-coupled vesicles in the presence of the antibod:ies. By using these specific monoclonal antibodies together with other experimental results, a more complete picture on the structure and function of this complex membrane-bound enzyme and its protein subunits is emerging.