title: Small-scale immunopurification of cytochrome c oxidase for a high-throughput multiplexing analysis of enzyme activity and amount.
abstract: 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.
authors.affiliation: Mitosciences Inc., Eugene, OR 97403-2095, USA.
authors.forename: James
authors.initials: J
authors.lastname: Murray
authors.forename: Birgit
authors.initials: B
authors.lastname: Schilling
authors.forename: Richard H
authors.initials: RH
authors.lastname: Row
authors.forename: Chris B
authors.initials: CB
authors.lastname: Yoo
authors.forename: Bradford W
authors.initials: BW
authors.lastname: Gibson
authors.forename: Michael F
authors.initials: MF
authors.lastname: Marusich
authors.forename: Roderick A
authors.initials: RA
authors.lastname: Capaldi
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D021122:Protein Subunits; D016047:Zalcitabine; D003576:Electron Transport Complex IV