title: Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratoire de Résonance Magnétique des Systèmes Biologiques, UMR 5536 CNRS-Université Victor Segalen Bordeaux 2, Bordeaux, France. gilles.gouspillou@gmail.com
authors.forename: Gilles
authors.initials: G
authors.lastname: Gouspillou
authors.forename: Richard
authors.initials: R
authors.lastname: Rouland
authors.forename: Guillaume
authors.initials: G
authors.lastname: Calmettes
authors.forename: Véronique
authors.initials: V
authors.lastname: Deschodt-Arsac
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Franconi
authors.forename: Isabelle
authors.initials: I
authors.lastname: Bourdel-Marchasson
authors.forename: Philippe
authors.initials: P
authors.lastname: Diolez
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism; D008931:Mitochondria, Muscle / Q000378:metabolism; D010084:Oxidation-Reduction; D010085:Oxidative Phosphorylation; D051381:Rats; D017208:Rats, Wistar
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate