Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.

<h4>Background</h4>Mitochondrial dysfunctions appear strongly implicated in a wide range of pathologies. Therefore, there is a growing need in the determination of the normal and pathological integrated response of oxidative phosphorylation to cellular ATP demand. The present study inten...

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Autores principales:	Gilles Gouspillou, Richard Rouland, Guillaume Calmettes, Véronique Deschodt-Arsac, Jean-Michel Franconi, Isabelle Bourdel-Marchasson, Philippe Diolez
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/3f4a36c8711f4b90b3b248039ef4a77a
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spelling	oai:doaj.org-article:3f4a36c8711f4b90b3b248039ef4a77a2021-11-18T06:52:21ZAccurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.1932-620310.1371/journal.pone.0020709https://doaj.org/article/3f4a36c8711f4b90b3b248039ef4a77a2011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21694779/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Mitochondrial dysfunctions appear strongly implicated in a wide range of pathologies. Therefore, there is a growing need in the determination of the normal and pathological integrated response of oxidative phosphorylation to cellular ATP demand. The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria.<h4>Methodology/principal findings</h4>The proposed method is based on the simultaneous monitoring of substrate oxidation (determined polarographically) and phosphorylation (determined using the glucose-hexokinase glucose-6-phosphate dehydrogenase-NADP(+) enzymatic system) rates, coupled to the determination of actual ADP and ATP concentrations by bioluminescent assay. This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations. We demonstrate how the application of this method allows an accurate determination of mitochondrial affinity for ADP from both oxidation (K(mVox)) and phosphorylation (K(mVp)) rates. We also demonstrate that determination of K(mVox) leads to an important overestimation of the mitochondrial affinity for ADP, indicating that mitochondrial affinity for ADP should be determined using phosphorylation rate. Finally, we show how this method allows the direct and precise determination of the mitochondrial coupling efficiency. Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method.<h4>Conclusions/significance</h4>Because the proposed method allows the accurate determination of mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria, it also opens the route to a better understanding of functional consequences of mitochondrial adaptations/dysfunctions arising in various physiological/pathophysiological conditions.Gilles GouspillouRichard RoulandGuillaume CalmettesVéronique Deschodt-ArsacJean-Michel FranconiIsabelle Bourdel-MarchassonPhilippe DiolezPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 6, p e20709 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Gilles Gouspillou Richard Rouland Guillaume Calmettes Véronique Deschodt-Arsac Jean-Michel Franconi Isabelle Bourdel-Marchasson Philippe Diolez Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.
description	<h4>Background</h4>Mitochondrial dysfunctions appear strongly implicated in a wide range of pathologies. Therefore, there is a growing need in the determination of the normal and pathological integrated response of oxidative phosphorylation to cellular ATP demand. The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria.<h4>Methodology/principal findings</h4>The proposed method is based on the simultaneous monitoring of substrate oxidation (determined polarographically) and phosphorylation (determined using the glucose-hexokinase glucose-6-phosphate dehydrogenase-NADP(+) enzymatic system) rates, coupled to the determination of actual ADP and ATP concentrations by bioluminescent assay. This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations. We demonstrate how the application of this method allows an accurate determination of mitochondrial affinity for ADP from both oxidation (K(mVox)) and phosphorylation (K(mVp)) rates. We also demonstrate that determination of K(mVox) leads to an important overestimation of the mitochondrial affinity for ADP, indicating that mitochondrial affinity for ADP should be determined using phosphorylation rate. Finally, we show how this method allows the direct and precise determination of the mitochondrial coupling efficiency. Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method.<h4>Conclusions/significance</h4>Because the proposed method allows the accurate determination of mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria, it also opens the route to a better understanding of functional consequences of mitochondrial adaptations/dysfunctions arising in various physiological/pathophysiological conditions.
format	article
author	Gilles Gouspillou Richard Rouland Guillaume Calmettes Véronique Deschodt-Arsac Jean-Michel Franconi Isabelle Bourdel-Marchasson Philippe Diolez
author_facet	Gilles Gouspillou Richard Rouland Guillaume Calmettes Véronique Deschodt-Arsac Jean-Michel Franconi Isabelle Bourdel-Marchasson Philippe Diolez
author_sort	Gilles Gouspillou
title	Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.
title_short	Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.
title_full	Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.
title_fullStr	Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.
title_full_unstemmed	Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.
title_sort	accurate determination of the oxidative phosphorylation affinity for adp in isolated mitochondria.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/3f4a36c8711f4b90b3b248039ef4a77a
work_keys_str_mv	AT gillesgouspillou accuratedeterminationoftheoxidativephosphorylationaffinityforadpinisolatedmitochondria AT richardrouland accuratedeterminationoftheoxidativephosphorylationaffinityforadpinisolatedmitochondria AT guillaumecalmettes accuratedeterminationoftheoxidativephosphorylationaffinityforadpinisolatedmitochondria AT veroniquedeschodtarsac accuratedeterminationoftheoxidativephosphorylationaffinityforadpinisolatedmitochondria AT jeanmichelfranconi accuratedeterminationoftheoxidativephosphorylationaffinityforadpinisolatedmitochondria AT isabellebourdelmarchasson accuratedeterminationoftheoxidativephosphorylationaffinityforadpinisolatedmitochondria AT philippediolez accuratedeterminationoftheoxidativephosphorylationaffinityforadpinisolatedmitochondria
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Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.

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