Complex I spectrophotometric assay in cultured cells: Detailed analysis of key factors

Complex I spectrophotometric assay in cultured cells: Detailed analysis of key factors

Author Oliveira, Katia Klug Autor UNIFESP Google Scholar
Kiyomoto, Beatriz Hitomi Autor UNIFESP Google Scholar
Rodrigues, Andresa De Santi Autor UNIFESP Google Scholar
Tengan, Celia Harumi Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract The diagnosis of mitochondrial encephalomyopathies caused by complex I (C-I) deficiency relies mainly on the spectrophotometric C-I assay. Considered difficult, this assay lacks reliability and has high nonspecific activity. We studied the key factors of this assay in cultured cells (cybrid and fibroblast): ubiquinone analogues, rotenone inhibition to determine specific activity, and mode of permeabilization of mitochondria] membranes. We showed that ubiquinone 1 allows a more stable and reliable assay, better results were obtained with rotenone inhibition done in the same assay, and mitochondria] permeability was improved just by freeze/thawing the sample prior to the assay. (C) 2012 Elsevier Inc. All rights reserved.
Keywords Respiratory chain
Mitochondrial diseases
Complex I
NADH:ubiquinone reductase
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Date 2013-04-01
Published in Analytical Biochemistry. San Diego: Academic Press Inc Elsevier Science, v. 435, n. 1, p. 57-59, 2013.
ISSN 0003-2697 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 57-59
Access rights Closed access
Type Article
Web of Science ID WOS:000315477600009

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