Figure 1.
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.
Figure 2.
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.
Figure 3.
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.
Figure 4.
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.