Ischemia-induced inhibition of mitochondrial complex I in rat brain: effect of permeabilization method and electron...

In this study we have examined the effect of global brain ischemia/reperfusion on biochemical properties of the mitochondrial respiratory complex I (CI) in rat hippocampus and cortex. Since the inner mitochondrial membrane forms the permeability barrier for NADH, the methodology of enzymatic activity determinations employs membrane permeabilization methods. This action affects the basic character of electrostatic and hydrophobic interactions inside the membrane and might influence functional properties of membrane embedded proteins. Therefore we have performed the comparative analysis of two permeabilization methods (sonication, detergent) and their impact on CI enzymatic activities under global brain ischemic-reperfusion conditions. We have observed that ischemia led to significant decrease of CI activities using both permeabilization methods in both brain areas. However, significant differencies in enzymatic activities were registered during reperfusion intervals according to used permeabilization method. We have also tested the effect of electron acceptors (decylubiquinone, potassium ferricyanide, nitrotetrazolium blue) on CI activities during I/R. Based on our results we assume that the critical site where ischemia affects CI activities is electron transfer to electron acceptor. Further, the observed mitochondrial dysfunction was analyzed by means of one and 2-dimensional BN PAGE/SDS PAGE with the focus on 3-nitrotyrosine immunodetection as a marker of oxidative damage to proteins. Add to this, initialization of p53 mitochondrial apoptosis through p53, Bax, Bcl-X(L) proteins and a possible involvement of GRIM-19, the CI structural subunit, in apoptotic processes were also studied.