Kinetic analysis of ATP hydrolysis by complex V in four murine tissues: Towards an assay suitable for clinical diagnosis

PLoS One. 2019 Aug 28;14(8):e0221886. doi: 10.1371/journal.pone.0221886. eCollection 2019.


Background: ATP synthase, the mitochondrial complex V, plays a major role in bioenergetics and its defects lead to severe diseases. Lack of a consensual protocol for the assay of complex V activity probably explains the under-representation of complex V defect among mitochondrial diseases. The aim of this work was to elaborate a fast, simple and reliable method to check the maximal complex V capacity in samples relevant to clinical diagnosis.

Methods: Using homogenates from four different murine organs, we tested the use of dodecylmaltoside, stability of the activity, linearity with protein amount, sensitivity to oligomycin and to exogenous inhibitory factor 1 (IF1), influence of freezing, and impact of mitochondrial purification.

Results: We obtained organ-dependent, reproducible and stable complex V specific activities, similar with fresh and frozen organs. Similar inhibition by oligomycin and exogenous IF1 demonstrated tight coupling between F1 and F0 domains. The Michaelis constant for MgATP had close values for all organs, in the 150-220 μM range. Complex V catalytic turnover rate, as measured in preparations solubilized in detergent using immunotitration and activity measurements, was more than three times higher in extracts from brain or muscle than in extracts from heart or liver. This tissue specificity suggested post-translational modifications. Concomitant measurement of respiratory activities showed only slightly different complex II/complex V ratio in the four organs. In contrast, complex I/complex V ratio differed in brain as compared to the three other organs because of a high complex I activity in brain. Mitochondria purification preserved these ratios, except for brain where selective degradation of complex I occurred. Therefore, mitochondrial purification could introduce a biased enzymatic evaluation.

Conclusion: Altogether, this work demonstrates that a reliable assay of complex V activity is perfectly possible with very small samples from frozen biopsies, which was confirmed using control and deficient human muscles.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Animals
  • Biological Assay / methods*
  • Glucosides / pharmacology
  • Humans
  • Hydrolysis
  • Kinetics
  • Male
  • Mice, Inbred C57BL
  • Mitochondria, Heart / drug effects
  • Mitochondria, Heart / metabolism
  • Mitochondrial Proton-Translocating ATPases / metabolism*
  • Organ Specificity* / drug effects
  • Proton-Translocating ATPases / metabolism
  • Solubility
  • Time Factors


  • Glucosides
  • dodecyl maltoside
  • Adenosine Triphosphate
  • Mitochondrial Proton-Translocating ATPases
  • Proton-Translocating ATPases
  • oligomycin sensitivity-conferring protein

Grant support

The authors are grateful for the support of the Fondation pour la Recherche Médicale (FRM) (grant DPM20121125550 to AL and FH), the AFM Telethon (grant 16416 to AL), and the French Infrastructure for Integrated Structural Biology (FRISBI, (to FH).