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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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Dependence of oxidation and phosphorylation rates on ADP concentration in liver and muscle mitochondria.Oxidation and phosphorylation rates were recorded simultaneously in liver (n = 4) and muscle (n = 5) mitochondria oxidizing glutamate+malate+succinate as substrates. True steady state of oxidation and phosphorylation rates were obtained using 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). Data presented in panels A and B correspond to absolute oxidation and phosphorylation rates, respectively. Maximal oxidation and phosphorylation rates obtained for muscle and liver mitochondria were 408.0±42.5 vs. 85.2±5.5 nmolO2.min−1.mg−1 and 1672.8±134.1 vs. 361.3±33.41 nmolATP.min−1.mg−1, respectively. Panels C and D show normalized oxidation and phosphorylation rates, respectively. Data were fitted using the Michaelis-Menten equation presented in the materials and methods section. Data are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle, # p<0.01 vs. KmVox.
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pone-0020709-g003: Dependence of oxidation and phosphorylation rates on ADP concentration in liver and muscle mitochondria.Oxidation and phosphorylation rates were recorded simultaneously in liver (n = 4) and muscle (n = 5) mitochondria oxidizing glutamate+malate+succinate as substrates. True steady state of oxidation and phosphorylation rates were obtained using 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). Data presented in panels A and B correspond to absolute oxidation and phosphorylation rates, respectively. Maximal oxidation and phosphorylation rates obtained for muscle and liver mitochondria were 408.0±42.5 vs. 85.2±5.5 nmolO2.min−1.mg−1 and 1672.8±134.1 vs. 361.3±33.41 nmolATP.min−1.mg−1, respectively. Panels C and D show normalized oxidation and phosphorylation rates, respectively. Data were fitted using the Michaelis-Menten equation presented in the materials and methods section. Data are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle, # p<0.01 vs. KmVox.

Mentions: In the present study, both oxidation and phosphorylation rates were therefore determined as described above during steady states under measured ADP concentrations ranging from 7 to 900 µM. The dependence of oxidation and phosphorylation rates on ADP concentration for liver and muscle mitochondria are shown in figure 3. Maximal oxidation and phosphorylation rates were more than four times higher in muscle as compared to liver mitochondria (figure 3 A and B). These results are in accordance with previously shown higher state III oxidation rate in muscle mitochondria [24]. In addition, it is interesting to note that even for the very low ADP concentrations used in the present study, both oxidation and phosphorylation rates were significantly much higher in muscle mitochondria (figure 3A and B). To our knowledge, these oxidation and phosphorylation rates kinetics allowed for the first time the accurate determination of KmVox and KmVp. As shown in figure 3A and B, mitochondria isolated from skeletal muscle were characterized by significantly lower KmVox and KmVp as compared to liver mitochondria, clearly indicating a much higher affinity for ADP in muscle 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)

Dependence of oxidation and phosphorylation rates on ADP concentration in liver and muscle mitochondria.Oxidation and phosphorylation rates were recorded simultaneously in liver (n = 4) and muscle (n = 5) mitochondria oxidizing glutamate+malate+succinate as substrates. True steady state of oxidation and phosphorylation rates were obtained using 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). Data presented in panels A and B correspond to absolute oxidation and phosphorylation rates, respectively. Maximal oxidation and phosphorylation rates obtained for muscle and liver mitochondria were 408.0±42.5 vs. 85.2±5.5 nmolO2.min−1.mg−1 and 1672.8±134.1 vs. 361.3±33.41 nmolATP.min−1.mg−1, respectively. Panels C and D show normalized oxidation and phosphorylation rates, respectively. Data were fitted using the Michaelis-Menten equation presented in the materials and methods section. Data are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle, # p<0.01 vs. KmVox.
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Related In: Results  -  Collection

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pone-0020709-g003: Dependence of oxidation and phosphorylation rates on ADP concentration in liver and muscle mitochondria.Oxidation and phosphorylation rates were recorded simultaneously in liver (n = 4) and muscle (n = 5) mitochondria oxidizing glutamate+malate+succinate as substrates. True steady state of oxidation and phosphorylation rates were obtained using 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). Data presented in panels A and B correspond to absolute oxidation and phosphorylation rates, respectively. Maximal oxidation and phosphorylation rates obtained for muscle and liver mitochondria were 408.0±42.5 vs. 85.2±5.5 nmolO2.min−1.mg−1 and 1672.8±134.1 vs. 361.3±33.41 nmolATP.min−1.mg−1, respectively. Panels C and D show normalized oxidation and phosphorylation rates, respectively. Data were fitted using the Michaelis-Menten equation presented in the materials and methods section. Data are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle, # p<0.01 vs. KmVox.
Mentions: In the present study, both oxidation and phosphorylation rates were therefore determined as described above during steady states under measured ADP concentrations ranging from 7 to 900 µM. The dependence of oxidation and phosphorylation rates on ADP concentration for liver and muscle mitochondria are shown in figure 3. Maximal oxidation and phosphorylation rates were more than four times higher in muscle as compared to liver mitochondria (figure 3 A and B). These results are in accordance with previously shown higher state III oxidation rate in muscle mitochondria [24]. In addition, it is interesting to note that even for the very low ADP concentrations used in the present study, both oxidation and phosphorylation rates were significantly much higher in muscle mitochondria (figure 3A and B). To our knowledge, these oxidation and phosphorylation rates kinetics allowed for the first time the accurate determination of KmVox and KmVp. As shown in figure 3A and B, mitochondria isolated from skeletal muscle were characterized by significantly lower KmVox and KmVp as compared to liver mitochondria, clearly indicating a much higher affinity for ADP in muscle 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