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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.

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Related in: MedlinePlus

Typical recording of oxidation rate, phosphorylation rate and ADP/ATP concentrations.Oxidation and phosphorylation rates were recorded simultaneously in liver and muscle mitochondria oxidizing glutamate+malate+succinate as substrates. Mitochondrial protein concentration in the oxygraphic vessel was 25 µg.ml−1. Steady states of oxygen consumption and phosphorylation rates were obtained using the coupled enzymatic system composed of Glucose (5 mM) - Hexokinase (2.5 U.ml−1, Sigma-Aldrich, H4502) - Glucose-6-phosphotate dehydrogenase (2.5 U.ml−1, Sigma-Aldrich, G6378) - NADP+ (1.6 mM). Dashed arrows correspond to the sampling of measurement medium taken from the oxygraphic vessel during each recording for determination of ADP and ATP concentrations.
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pone-0020709-g002: Typical recording of oxidation rate, phosphorylation rate and ADP/ATP concentrations.Oxidation and phosphorylation rates were recorded simultaneously in liver and muscle mitochondria oxidizing glutamate+malate+succinate as substrates. Mitochondrial protein concentration in the oxygraphic vessel was 25 µg.ml−1. Steady states of oxygen consumption and phosphorylation rates were obtained using the coupled enzymatic system composed of Glucose (5 mM) - Hexokinase (2.5 U.ml−1, Sigma-Aldrich, H4502) - Glucose-6-phosphotate dehydrogenase (2.5 U.ml−1, Sigma-Aldrich, G6378) - NADP+ (1.6 mM). Dashed arrows correspond to the sampling of measurement medium taken from the oxygraphic vessel during each recording for determination of ADP and ATP concentrations.

Mentions: Figure 2 shows a combined typical recording obtained using Glutamate+Malate+Succinate substrates in order to reconstitute the tricarboxylic acid cycle function and consequently to approach physiological conditions [29]. Mitochondria were added at the onset of the recording and quickly reached state 4 oxidation rate. In this example, the addition of 50 µM ADP triggered phosphorylation, leading to a corresponding increase in oxidation rate. Figure 2 shows that both ADP-induced oxidation and phosphorylation rates were constant during the duration of the recording (approximately 6 min). In order to determine ADP and ATP concentrations, two samplings were performed at the beginning and at the end of recording. As it can be seen in figure 2, the stability of these two concentrations demonstrates the achievement of true steady states during the time course of the experiment.


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)

Typical recording of oxidation rate, phosphorylation rate and ADP/ATP concentrations.Oxidation and phosphorylation rates were recorded simultaneously in liver and muscle mitochondria oxidizing glutamate+malate+succinate as substrates. Mitochondrial protein concentration in the oxygraphic vessel was 25 µg.ml−1. Steady states of oxygen consumption and phosphorylation rates were obtained using the coupled enzymatic system composed of Glucose (5 mM) - Hexokinase (2.5 U.ml−1, Sigma-Aldrich, H4502) - Glucose-6-phosphotate dehydrogenase (2.5 U.ml−1, Sigma-Aldrich, G6378) - NADP+ (1.6 mM). Dashed arrows correspond to the sampling of measurement medium taken from the oxygraphic vessel during each recording for determination of ADP and ATP concentrations.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020709-g002: Typical recording of oxidation rate, phosphorylation rate and ADP/ATP concentrations.Oxidation and phosphorylation rates were recorded simultaneously in liver and muscle mitochondria oxidizing glutamate+malate+succinate as substrates. Mitochondrial protein concentration in the oxygraphic vessel was 25 µg.ml−1. Steady states of oxygen consumption and phosphorylation rates were obtained using the coupled enzymatic system composed of Glucose (5 mM) - Hexokinase (2.5 U.ml−1, Sigma-Aldrich, H4502) - Glucose-6-phosphotate dehydrogenase (2.5 U.ml−1, Sigma-Aldrich, G6378) - NADP+ (1.6 mM). Dashed arrows correspond to the sampling of measurement medium taken from the oxygraphic vessel during each recording for determination of ADP and ATP concentrations.
Mentions: Figure 2 shows a combined typical recording obtained using Glutamate+Malate+Succinate substrates in order to reconstitute the tricarboxylic acid cycle function and consequently to approach physiological conditions [29]. Mitochondria were added at the onset of the recording and quickly reached state 4 oxidation rate. In this example, the addition of 50 µM ADP triggered phosphorylation, leading to a corresponding increase in oxidation rate. Figure 2 shows that both ADP-induced oxidation and phosphorylation rates were constant during the duration of the recording (approximately 6 min). In order to determine ADP and ATP concentrations, two samplings were performed at the beginning and at the end of recording. As it can be seen in figure 2, the stability of these two concentrations demonstrates the achievement of true steady states during the time course of the experiment.

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