Dataset Information

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

ABSTRACT:

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 ?M 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.

SUBMITTER: Haraux F

PROVIDER: S-EPMC6713359 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

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

Haraux Francis F Lombès Anne A

PloS one 20190828 8

<h4>Background</h4>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.<h4>Methods</h4>Using homogenates from four dif ...[more]

PMID: 31461494

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Project description:P-Glycoprotein, a member of the ATP-binding cassette (ABC) superfamily, is a multidrug transporter responsible for cellular efflux of hundreds of structurally unrelated compounds, including natural products, many clinically used drugs and anti-cancer agents. Expression of P-glycoprotein has been linked to multidrug resistance in human cancers. ABC transporters are driven by ATP hydrolysis at their two cytoplasmic nucleotide-binding domains, which interact to form a closed ATP-bound sandwich dimer. Intimate knowledge of the catalytic cycle of these proteins is clearly essential for understanding their mechanism of action. P-Glycoprotein has been proposed to hydrolyse ATP by an alternating mechanism, for which there is substantial experimental evidence, including inhibition of catalytic activity by trapping of ortho-vanadate at one nucleotide-binding domain, and the observation of an asymmetric occluded state. Despite many studies of P-glycoprotein ATPase activity over the past 20 years, no comprehensive kinetic analysis has yet been carried out, and some puzzling features of its behaviour remain unexplained. In this work, we have built several progressively more complex kinetic models, and then carried out simulations and detailed analysis, to test the validity of the proposed reaction pathway employed by P-glycoprotein for ATP hydrolysis. To establish kinetic parameters for the catalytic cycle, we made use of the large amount of published data on ATP hydrolysis by hamster P-glycoprotein, both purified and in membrane vesicles. The proposed kinetic scheme(s) include a high affinity priming reaction for binding of the first ATP molecule, and an independent pathway for ADP binding outside the main catalytic cycle. They can reproduce to varying degrees the observed behavior of the protein's ATPase activity and its inhibition by ortho-vanadate. The results provide new insights into the mode of action of P-glycoprotein, and some hypotheses about the nature of the occluded nucleotide-bound state.

Dataset Information

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

Background

Methods

Results

Conclusion

Publications

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

Similar Datasets

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