Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells.
The assessment of mitochondrial respiratory chain (RC) enzymatic activities is essential for investigating mitochondrial function in several situations, including mitochondrial disorders, diabetes, cancer, aging and neurodegeneration, as well as for many toxicological assays. Muscle is the most commonly analyzed tissue because of its high metabolic rates and accessibility, although other tissues and cultured cell lines can be used. We describe a step-by-step protocol for a simple and reliable assessment of the RC enzymatic function (complexes I-IV) for minute quantities of muscle, cultured cells and isolated mitochondria from a variety of species and tissues, by using a single-wavelength spectrophotometer. An efficient tissue disruption and the choice for each assay of specific buffers, substrates, adjuvants and detergents in a narrow concentration range allow maximal sensitivity, specificity and linearity of the kinetics. This protocol can be completed in 3 h.
Neuromuscular Laboratory, Department of Neurosciences, University of Padova, Padova, Italy. maspinazzi@gmail.com
Marco
M
Spinazzi
Alberto
A
Casarin
Vanessa
V
Pertegato
Leonardo
L
Salviati
Corrado
C
Angelini
D000818:Animals; D002478:Cells, Cultured; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000378:metabolism; D042963:Electron Transport Complex II / Q000378:metabolism; D014450:Electron Transport Complex III / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D013053:Spectrophotometry / Q000295:instrumentation / Q000379:methods
D042963:Electron Transport Complex II; D003576:Electron Transport Complex IV; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III