title: An evaluation of the measurement of the activities of complexes I-IV in the respiratory chain of human skeletal muscle mitochondria.
abstract: The measurement of individual respiratory chain complexes is an important component of the investigation of diseases due to mitochondrial dysfunction. We have evaluated assays which measure complexes I to IV in human skeletal muscle mitochondria and in addition optimized these assays to provide sensitive and reliable diagnostic techniques, particularly in situations where a partial interruption at a single complex needs to identified. Using several established methods of membrane disruption we have found that optimal activities of complexes I and II are obtained by freeze-thawing the mitochondria in hypotonic potassium phosphate buffer, whereas complex III and IV activities are markedly increased by the addition of the detergent n-dodecyl-beta-D-maltoside. Complex I activity is measured in the presence of 2.5 mg.ml-1 bovine serum albumin, which increases rotenone sensitivity, and we have shown that NADH-cytochrome b5 reductase makes an important contribution to the rotenone-insensitive NADH-ubiquinone oxidoreductase activity. Complex II activity is measured after preincubation of the mitochondrial fraction with succinate to fully activate the complex. Complex I and III activities are dependent upon the length of the isoprenoid chain of the ubiquinone and ubiquinol, respectively. These assays have been used to establish a control range.
authors.affiliation: Division of Clinical Neuroscience, University of Newcastle upon Tyne, England.
authors.forename: M A
authors.initials: MA
authors.lastname: Birch-Machin
authors.forename: H L
authors.initials: HL
authors.lastname: Briggs
authors.forename: A A
authors.initials: AA
authors.lastname: Saborido
authors.forename: L A
authors.initials: LA
authors.lastname: Bindoff
authors.forename: D M
authors.initials: DM
authors.lastname: Turnbull
mesh_terms: D015786:Cytochromes b5 / Q000032:analysis / Q000502:physiology; D004579:Electron Transport / Q000502:physiology*; D042967:Electron Transport Complex I; D042963:Electron Transport Complex II; D014450:Electron Transport Complex III / Q000032:analysis* / Q000502:physiology; D005960:Glucosides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008931:Mitochondria, Muscle / Q000201:enzymology / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000032:analysis* / Q000502:physiology; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000032:analysis* / Q000502:physiology; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis / Q000502:physiology; D010088:Oxidoreductases / Q000032:analysis* / Q000502:physiology; D013053:Spectrophotometry; D013385:Succinate Dehydrogenase / Q000032:analysis* / Q000502:physiology
chemical_list: D005960:Glucosides; D009097:Multienzyme Complexes; C040358:dodecyl maltoside; D015786:Cytochromes b5; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D016660:NAD(P)H Dehydrogenase (Quinone); D042967:Electron Transport Complex I; D014450:Electron Transport Complex III