Mitochondrial oxydative phosphorylation biochemistry (enzymology/polarography)
Within the Mitochondrial Bioenergetic, Structure and Plasticity Plateform, the mitochondrion is approached in its energetic, metabolic and dynamic function. Energetic properties and cell metabolism are analysis using dynamic approaches (oxygraphy), measurement of maximal activity of complexes (enzymology) or structural analysis of the respiratory chain (Western Blot, Blue-Native Page 1D or 2D BN/BN)
The respiratory chain : The 4 complexes transfer the electrons provide from the reduced equivalents (NADH, H+, FADH2) to the final acceptor oxygen. During this transfer a protomotive force is generated (H+ translocation), and used by the ATP synthase for the phosphorylation of ADP in ATP: this process is called oxidative phosphorylation.
Mitochondrial oxidative phosphorylation biochemistry
Oxygraphy allows functional analyses of the respiratory chain on isolated mitochondria, cells or tissue. The analyses carried out relate to the measurement of respiration related to the different complexes, the measurement of the cellular oxygen uptake, the analysis of the mitochondrial membrane potential, or the efficiency of ATP synthesis.
The laboratory is equipped with a high-precision oxygraph allowing the analysis of oxygen concentration and flow. These ones are measured thanks to a Clark electrode, placed in a thermostated chamber. It is currently coupled with aTPP+ electrode, but other electrodes can also be added, in particular calcium or NO ones.
Left : the High-resolution oxygraph.Right :Oxygraphic traces of the respiration linked to the different complexes (A, complex 1,compared to B, complex II) in permeabilized fibroblasts. Red line : oxygen flux, blue line : oxygen concentration.
The analysis of the maximal activity of each complex of the respiratory chain (complexes I to IV), of ATPsynthase and the enzymes of the Krebs cycle or beta-oxidation, is carried out by spectrophotometry, on isolated mitochondria, cells, or tissue homogenates.
The laboratory disposes of a thermostated spectrophotometer with double wavelengths (SAFAS, UVmc2), and a microplate reader allowing the detection of luminescence, fluorescence and absorptance (SAFAS, Xenius).