Assessment of Mitochondrial Respiratory Chain Enzymatic Activities on Tissues and Cultured Cells

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2012 Jun 2

PMID 22653162

Citations 439

Authors

Marco Spinazzi

Alberto Casarin

Vanessa Pertegato

Leonardo Salviati

Corrado Angelini

Affiliations

Soon will be listed here.

Abstract

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.

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References

Gellerich F, Mayr J, Reuter S, Sperl W, Zierz S . The problem of interlab variation in methods for mitochondrial disease diagnosis: enzymatic measurement of respiratory chain complexes. Mitochondrion. 2005; 4(5-6):427-39. DOI: 10.1016/j.mito.2004.07.007. View

Rowley N, Prip-Buus C, Westermann B, Brown C, Schwarz E, Barrell B . Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding. Cell. 1994; 77(2):249-59. DOI: 10.1016/0092-8674(94)90317-4. View

Kirby D, Thorburn D, Turnbull D, Taylor R . Biochemical assays of respiratory chain complex activity. Methods Cell Biol. 2007; 80:93-119. DOI: 10.1016/S0091-679X(06)80004-X. View

Moghaddas S, Distler A, Hoppel C, Lesnefsky E . Quinol type compound in cytochrome c preparations leads to non-enzymatic reduction of cytochrome c during the measurement of complex III activity. Mitochondrion. 2008; 8(2):155-63. DOI: 10.1016/j.mito.2007.12.001. View

Hauptmann S, Scherping I, Drose S, Brandt U, Schulz K, Jendrach M . Mitochondrial dysfunction: an early event in Alzheimer pathology accumulates with age in AD transgenic mice. Neurobiol Aging. 2008; 30(10):1574-86. DOI: 10.1016/j.neurobiolaging.2007.12.005. View

Kuznetsov A, Veksler V, Gellerich F, Saks V, Margreiter R, Kunz W . Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc. 2008; 3(6):965-76. DOI: 10.1038/nprot.2008.61. View

Janssen A, Trijbels F, Sengers R, Smeitink J, van den Heuvel L, Wintjes L . Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts. Clin Chem. 2007; 53(4):729-34. DOI: 10.1373/clinchem.2006.078873. View

Lebrecht D, Setzer B, Ketelsen U, Haberstroh J, Walker U . Time-dependent and tissue-specific accumulation of mtDNA and respiratory chain defects in chronic doxorubicin cardiomyopathy. Circulation. 2003; 108(19):2423-9. DOI: 10.1161/01.CIR.0000093196.59829.DF. View

Trounce I, Kim Y, Jun A, Wallace D . Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol. 1996; 264:484-509. DOI: 10.1016/s0076-6879(96)64044-0. View

10.

Barrientos A, Fontanesi F, Diaz F . Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography and spectrophotometric enzyme assays. Curr Protoc Hum Genet. 2009; Chapter 19:Unit19.3. PMC: 2771113. DOI: 10.1002/0471142905.hg1903s63. View

11.

Janssen A, Trijbels F, Sengers R, Wintjes L, Ruitenbeek W, Smeitink J . Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology. Clin Chem. 2006; 52(5):860-71. DOI: 10.1373/clinchem.2005.062414. View

12.

Zheng X, Shoffner J, Voljavec A, Wallace D . Evaluation of procedures for assaying oxidative phosphorylation enzyme activities in mitochondrial myopathy muscle biopsies. Biochim Biophys Acta. 1990; 1019(1):1-10. DOI: 10.1016/0005-2728(90)90118-n. View

13.

Grad L, Sayles L, Lemire B . Isolation and functional analysis of mitochondria from the nematode Caenorhabditis elegans. Methods Mol Biol. 2008; 372:51-66. DOI: 10.1007/978-1-59745-365-3_4. View

14.

Jonckheere A, Smeitink J, Rodenburg R . Mitochondrial ATP synthase: architecture, function and pathology. J Inherit Metab Dis. 2011; 35(2):211-25. PMC: 3278611. DOI: 10.1007/s10545-011-9382-9. View

15.

Fischer J, Ruitenbeek W, STADHOUDERS A, Trijbels J, Sengers R, Janssen A . Investigation of mitochondrial metabolism in small human skeletal muscle biopsy specimens. Improvement of preparation procedure. Clin Chim Acta. 1985; 145(1):89-99. DOI: 10.1016/0009-8981(85)90022-1. View

16.

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

17.

Winklhofer K, Haass C . Mitochondrial dysfunction in Parkinson's disease. Biochim Biophys Acta. 2009; 1802(1):29-44. DOI: 10.1016/j.bbadis.2009.08.013. View

18.

Angelini C, Bresolin N, Pegolo G, Bet L, Rinaldo P, Trevisan C . Childhood encephalomyopathy with cytochrome c oxidase deficiency, ataxia, muscle wasting, and mental impairment. Neurology. 1986; 36(8):1048-52. DOI: 10.1212/wnl.36.8.1048. View

19.

Chandra D, Singh K . Genetic insights into OXPHOS defect and its role in cancer. Biochim Biophys Acta. 2010; 1807(6):620-5. PMC: 4681500. DOI: 10.1016/j.bbabio.2010.10.023. View

20.

Chretien D, Bourgeron T, Rotig A, Munnich A, Rustin P . The measurement of the rotenone-sensitive NADH cytochrome c reductase activity in mitochondria isolated from minute amount of human skeletal muscle. Biochem Biophys Res Commun. 1990; 173(1):26-33. DOI: 10.1016/s0006-291x(05)81016-2. View