Small-scale immunopurification of cytochrome c oxidase for a high-throughput multiplexing analysis of enzyme activity and amount.
COX (cytochrome c oxidase) deficiency is one of the main causes of genetic mitochondrial disease and presents with multiple phenotypes, depending on whether the causative mutation exists in a mitochondrial or nuclear gene and on whether it involves an altered catalytic or structural component or an assembly factor for this membrane-embedded 13-subunit enzyme complex. COX deficiency is routinely observed in AD (Alzheimer's disease), although there is continuing debate about whether this is a causative or a secondary consequence of the condition. Altered levels of COX and reduced oxidative phosphorylation capacity have been reported in other common diseases, including cancer, and are seen as unwanted side effects in a number of drug treatments, particularly with antiretroviral and antibiotic treatments. Here, we introduce a simple, rapid, high-throughput 96-well plate protocol that uses a multiplex approach to determine the amount and activity of COX, which should find widespread use in evaluating the above diseases and in drug safety studies. Importantly, the method uses very small amounts of cell material or tissue and does not require the isolation of mitochondria. We show the utility of this approach by example of the analysis of fibroblasts from patients with COX activity deficiency and the effect of the antiretroviral drug ddC (2',3'-dideoxycytidine) on the biogenesis of the enzyme.
Murray
James
J
Mitosciences Inc., Eugene, OR 97403-2095, USA.
Schilling
Birgit
B
Row
Richard H
RH
Yoo
Chris B
CB
Gibson
Bradford W
BW
Marusich
Michael F
MF
Capaldi
Roderick A
RA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D002457:Cell Extracts; D030562:Databases, Protein; D003576:Electron Transport Complex IV / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D028361:Mitochondrial Diseases / Q000201:enzymology / Q000235:genetics; D009206:Myocardium / Q000378:metabolism; D009928:Organ Specificity; D021122:Protein Subunits / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D016047:Zalcitabine / Q000633:toxicity
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D021122:Protein Subunits; D016047:Zalcitabine; D003576:Electron Transport Complex IV