A microtiter plate assay for cytochrome c oxidase in permeabilized whole cells.
Defects of the mitochondrial respiratory chain often involve dysfunction of cytochrome c oxidase, the terminal component of this chain. We have developed a microassay, suitable for various cell types, which can be carried out in microwell plates. This assay has advantages over the traditional spectrophotometric method as only a small number of cells are used, without the need for mitochondrial fractionation. This makes the assay faster and use of the microtiter plate allows many samples to be screened simultaneously. We demonstrate that lowered levels of cytochrome c oxidase activity can be detected in patients' blood samples, avoiding the necessity for a more invasive tissue biopsy. This cytochrome c oxidase assay is also suitable for use as an intramitochondrial marker for monitoring the effect of mitochondrial translation inhibitors or effectors which modulate mitochondrial biogenesis and could be used for assessing the effect of mitochondrial DNA mutations.
Chrzanowska-Lightowlers
Z M
ZM
Division of Clinical Neuroscience, University of Newcastle upon Tyne, England.
Turnbull
D M
DM
Lightowlers
R N
RN
D015100:3,3'-Diaminobenzidine / Q000494:pharmacology; D002460:Cell Line; D002461:Cell Line, Transformed; D002463:Cell Membrane Permeability; D003574:Cytochrome c Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D008099:Liver; D008832:Microchemistry / Q000379:methods; D011190:Potassium Cyanide / Q000494:pharmacology; D012503:Saponins; D013053:Spectrophotometry / Q000379:methods; D013385:Succinate Dehydrogenase / Q000032:analysis; D014407:Tumor Cells, Cultured
D003574:Cytochrome c Group; D012503:Saponins; D015100:3,3'-Diaminobenzidine; D013385:Succinate Dehydrogenase; D003576:Electron Transport Complex IV; D011190:Potassium Cyanide