Three spectrophotometric assays for the measurement of the five respiratory chain complexes in minuscule biological samples

被引:82
作者
Benit, Paule
Goncalves, Sergio
Dassa, Emmanuel Philippe
Briere, Jean-Jacques
Martin, Gail
Rustin, Pierre
机构
[1] Inserm U676, F-75019 Paris, France
[2] Univ Paris 07, Fac Med Denis Diderot, IFR02, Paris, France
[3] Univ Calif San Francisco, Dept Anat, San Francisco, CA 94143 USA
[4] Univ Calif San Francisco, Dept Dermatol, San Francisco, CA 94143 USA
关键词
respiratory chain; spectrophotornetry; mitochondrial deficiency; Aif;
D O I
10.1016/j.cca.2006.05.034
中图分类号
R446 [实验室诊断]; R-33 [实验医学、医学实验];
学科分类号
1001 ;
摘要
Background: The measurement of the activities of the five complexes comprising the respiratory chain has proven to be a major challenge when a limiting amount of biological material is available. Here we report a set of three convenient assays that allows this measurement under such circumstances. Methods: One assay relies on the sequential addition of reagents to measure first complex IV activity, followed by complex II+III, and then glycerol-3-phosphate dehydrogenase + complex III activities and finally isolated complex III activity. A second assay measures the activity of complex II followed by glycerol-3-phosphate dehydrogenase and isocitrate dehydrogenase. A third assay measures rotenone-sensitive complex. I activity and subsequently oligomycin-sensitive complex V activity. Results: These assays have been successfully used on extracts of small numbers of human cells displaying various defects in the respiratory chain, and on frozen tissue homogenates of retina and very early mouse embryos. Conclusions: The strength of this set of assays lies both in its rapid and simple execution and its capacity for immediate detection of partial defects, because each activity can be compared with one or two other activities measured in the same sample. (c) 2006 Elsevier B.V. All rights reserved.
引用
收藏
页码:81 / 86
页数:6
相关论文
共 28 条
[1]   Large-scale deletion and point mutations of the nuclear NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency [J].
Bénit, P ;
Chretien, D ;
Kadhom, N ;
de Lonlay-Debeney, P ;
Cormier-Daire, V ;
Cabral, A ;
Peudenier, S ;
Rustin, P ;
Munnich, A ;
Rötig, A .
AMERICAN JOURNAL OF HUMAN GENETICS, 2001, 68 (06) :1344-1352
[2]   Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues [J].
Birch-Machin, MA ;
Turnbull, DM .
METHODS IN CELL BIOLOGY, VOL 65: MITOCHONDRIA, 2001, 65 :97-117
[3]   MUTATION OF A NUCLEAR SUCCINATE-DEHYDROGENASE GENE RESULTS IN MITOCHONDRIAL RESPIRATORY-CHAIN DEFICIENCY [J].
BOURGERON, T ;
RUSTIN, P ;
CHRETIEN, D ;
BIRCHMACHIN, M ;
BOURGEOIS, M ;
VIEGASPEQUIGNOT, E ;
MUNNICH, A ;
ROTIG, A .
NATURE GENETICS, 1995, 11 (02) :144-149
[4]  
BOURGERON T, 1993, J BIOL CHEM, V268, P19369
[5]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[6]  
BROWN D, 2006, P NTAL ACAD SCI, P103
[7]   REFERENCE CHARTS FOR RESPIRATORY-CHAIN ACTIVITIES IN HUMAN TISSUES [J].
CHRETIEN, D ;
RUSTIN, P ;
BOURGERON, T ;
ROTIG, A ;
SAUDUBRAY, JM ;
MUNNICH, A .
CLINICA CHIMICA ACTA, 1994, 228 (01) :53-70
[8]   Revisiting pitfalls, problems and tentative solutions for assaying mitochondrial respiratory chain complex III in human samples [J].
Chretien, D ;
Slama, A ;
Brière, JJ ;
Munnich, A ;
Rötig, A ;
Rustin, P .
CURRENT MEDICINAL CHEMISTRY, 2004, 11 (02) :233-239
[9]   Biochemical parameters for the diagnosis of mitochondrial respiratory chain deficiency in humans, and their lack of age-related changes [J].
Chretien, D ;
Gallego, J ;
Barrientos, A ;
Casademont, J ;
Cardellach, F ;
Munnich, A ;
Rötig, A ;
Rustin, P .
BIOCHEMICAL JOURNAL, 1998, 329 :249-254
[10]   Assay of mitochondrial respiratory chain complex I in human lymphocytes and cultured skin fibroblasts [J].
Chretien, D ;
Bénit, P ;
Chol, M ;
Lebon, S ;
Rötig, A ;
Munnich, A ;
Rustin, P .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2003, 301 (01) :222-224