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

Experimental determination of oxidation rate, phosphorylation rate and ADP/ATP concentrations.Our experimental set-up was composed of an oxygraph, a spectrophotometer and a luminometer. An optic fiber, connected to the spectrophotometer, was inserted in the oxygraphic vessel (picture on the top-left hand corner). Mitochondrial oxidation rate was determined using the Clark electrode of the oxygraph. Phosphorylation rate was assessed, with the help of the optic fiber, by the continuous monitoring of NADPH production in the oxygraphic vessel. Samplings were performed at the onset and the end of the recording to assess both ADP and ATP concentrations using a bioluminescence-based assay with the help of a luminometer (picture on the top-right hand corner). For clarity, all parameters that were measured during each experiment are highlighted by colored circles. HK: hexokinase, G6PDH: glucose 6 phosphate dehydrogenase, G6P: glucose 6 phosphate, OM: outer membrane, IM: inner membrane.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3111431&req=5

pone-0020709-g001: Experimental determination of oxidation rate, phosphorylation rate and ADP/ATP concentrations.Our experimental set-up was composed of an oxygraph, a spectrophotometer and a luminometer. An optic fiber, connected to the spectrophotometer, was inserted in the oxygraphic vessel (picture on the top-left hand corner). Mitochondrial oxidation rate was determined using the Clark electrode of the oxygraph. Phosphorylation rate was assessed, with the help of the optic fiber, by the continuous monitoring of NADPH production in the oxygraphic vessel. Samplings were performed at the onset and the end of the recording to assess both ADP and ATP concentrations using a bioluminescence-based assay with the help of a luminometer (picture on the top-right hand corner). For clarity, all parameters that were measured during each experiment are highlighted by colored circles. HK: hexokinase, G6PDH: glucose 6 phosphate dehydrogenase, G6P: glucose 6 phosphate, OM: outer membrane, IM: inner membrane.

Mentions: Phosphorylation rates were determined using a 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) [23] (figure 1). Chemicals composing this enzymatic system were in excess in order to avoid any limitation of mitochondrial activity. By this system, the ADP phosphorylated by mitochondria into ATP is regenerated by the phosphorylation of glucose into glucose-6-phosphate (G6P) catalyzed by Hexokinase (HK) (eq. 1). This first reaction allows the establishment of a constant ATP turnover ensuring the study of oxidative phosphorylation during steady states. Resulting G6P is then oxidized by Glucose-6-phosphotate dehydrogenase (G6PDH) to form 6-phosphogluconate, using NADP+ as electron acceptor (eq. 2). ATP(1)(2)


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)

Experimental determination of oxidation rate, phosphorylation rate and ADP/ATP concentrations.Our experimental set-up was composed of an oxygraph, a spectrophotometer and a luminometer. An optic fiber, connected to the spectrophotometer, was inserted in the oxygraphic vessel (picture on the top-left hand corner). Mitochondrial oxidation rate was determined using the Clark electrode of the oxygraph. Phosphorylation rate was assessed, with the help of the optic fiber, by the continuous monitoring of NADPH production in the oxygraphic vessel. Samplings were performed at the onset and the end of the recording to assess both ADP and ATP concentrations using a bioluminescence-based assay with the help of a luminometer (picture on the top-right hand corner). For clarity, all parameters that were measured during each experiment are highlighted by colored circles. HK: hexokinase, G6PDH: glucose 6 phosphate dehydrogenase, G6P: glucose 6 phosphate, OM: outer membrane, IM: inner membrane.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020709-g001: Experimental determination of oxidation rate, phosphorylation rate and ADP/ATP concentrations.Our experimental set-up was composed of an oxygraph, a spectrophotometer and a luminometer. An optic fiber, connected to the spectrophotometer, was inserted in the oxygraphic vessel (picture on the top-left hand corner). Mitochondrial oxidation rate was determined using the Clark electrode of the oxygraph. Phosphorylation rate was assessed, with the help of the optic fiber, by the continuous monitoring of NADPH production in the oxygraphic vessel. Samplings were performed at the onset and the end of the recording to assess both ADP and ATP concentrations using a bioluminescence-based assay with the help of a luminometer (picture on the top-right hand corner). For clarity, all parameters that were measured during each experiment are highlighted by colored circles. HK: hexokinase, G6PDH: glucose 6 phosphate dehydrogenase, G6P: glucose 6 phosphate, OM: outer membrane, IM: inner membrane.
Mentions: Phosphorylation rates were determined using a 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) [23] (figure 1). Chemicals composing this enzymatic system were in excess in order to avoid any limitation of mitochondrial activity. By this system, the ADP phosphorylated by mitochondria into ATP is regenerated by the phosphorylation of glucose into glucose-6-phosphate (G6P) catalyzed by Hexokinase (HK) (eq. 1). This first reaction allows the establishment of a constant ATP turnover ensuring the study of oxidative phosphorylation during steady states. Resulting G6P is then oxidized by Glucose-6-phosphotate dehydrogenase (G6PDH) to form 6-phosphogluconate, using NADP+ as electron acceptor (eq. 2). ATP(1)(2)

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