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

Gouspillou G, Rouland R, Calmettes G, Deschodt-Arsac V, Franconi JM, Bourdel-Marchasson I, Diolez P - PLoS ONE (2011)

Bottom Line: The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria.This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations.Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Résonance Magnétique des Systèmes Biologiques, UMR 5536 CNRS-Université Victor Segalen Bordeaux 2, Bordeaux, France. gilles.gouspillou@gmail.com

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.

Methodology/principal findings: 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/significance: 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.

Show MeSH

Related in: MedlinePlus

Changes in the P/O ratio as a function of ADP concentration in liver and muscle mitochondria.P/O ratio was determined by calculating the phosphorylation to oxidation rates ratio. Data for liver (n = 4) and muscle (n = 5) are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3111431&req=5

pone-0020709-g004: Changes in the P/O ratio as a function of ADP concentration in liver and muscle mitochondria.P/O ratio was determined by calculating the phosphorylation to oxidation rates ratio. Data for liver (n = 4) and muscle (n = 5) are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle.

Mentions: The coupling efficiency of oxidative phosphorylation can be defined as the amount of ATP molecules that mitochondria can synthesize for each atom of oxygen consumed. This coupling efficiency is central to the physiology of energy metabolism and can be assessed by the determination of the well-known P/O ratio [27], [30]. Since both oxidation and phosphorylation rates are determined simultaneously using the proposed method, this crucial parameter is directly accessible. P/O ratio was therefore calculated for every ADP concentration (figure 4). Maximal P/O ratio, obtained under the highest ADP concentrations (from 200 to 900 µM), was similar between liver and muscle mitochondria. However and most interestingly, under low ADP concentrations (from approximately 7 to 150 µM), the P/O ratio was significantly higher in muscle mitochondria. According to this result, under low ADP concentrations, muscle mitochondria synthesize a higher amount of ATP for the same amount of oxygen as compared to liver mitochondria. In this way muscle mitochondria appear optimized toward ATP production at a high yield even at low ADP concentrations when compared to liver mitochondria.


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

Gouspillou G, Rouland R, Calmettes G, Deschodt-Arsac V, Franconi JM, Bourdel-Marchasson I, Diolez P - PLoS ONE (2011)

Changes in the P/O ratio as a function of ADP concentration in liver and muscle mitochondria.P/O ratio was determined by calculating the phosphorylation to oxidation rates ratio. Data for liver (n = 4) and muscle (n = 5) are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111431&req=5

pone-0020709-g004: Changes in the P/O ratio as a function of ADP concentration in liver and muscle mitochondria.P/O ratio was determined by calculating the phosphorylation to oxidation rates ratio. Data for liver (n = 4) and muscle (n = 5) are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle.
Mentions: The coupling efficiency of oxidative phosphorylation can be defined as the amount of ATP molecules that mitochondria can synthesize for each atom of oxygen consumed. This coupling efficiency is central to the physiology of energy metabolism and can be assessed by the determination of the well-known P/O ratio [27], [30]. Since both oxidation and phosphorylation rates are determined simultaneously using the proposed method, this crucial parameter is directly accessible. P/O ratio was therefore calculated for every ADP concentration (figure 4). Maximal P/O ratio, obtained under the highest ADP concentrations (from 200 to 900 µM), was similar between liver and muscle mitochondria. However and most interestingly, under low ADP concentrations (from approximately 7 to 150 µM), the P/O ratio was significantly higher in muscle mitochondria. According to this result, under low ADP concentrations, muscle mitochondria synthesize a higher amount of ATP for the same amount of oxygen as compared to liver mitochondria. In this way muscle mitochondria appear optimized toward ATP production at a high yield even at low ADP concentrations when compared to liver mitochondria.

Bottom Line: The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria.This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations.Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Résonance Magnétique des Systèmes Biologiques, UMR 5536 CNRS-Université Victor Segalen Bordeaux 2, Bordeaux, France. gilles.gouspillou@gmail.com

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.

Methodology/principal findings: 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/significance: 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.

Show MeSH
Related in: MedlinePlus