24] Respiratory control and oxidative phosphorylation measurements in mitochondria

Abstract

The main function of mitochondria is to convert the free energy of oxidation that is present in oxidizable substrates into the free energy of hydrolysis of adenosine triphosphate (ATP). Therefore, an important criterion for the functioning of mitochondria is the efficiency with which this conversion is catalyzed. This efficiency is usually estimated in either of two ways: from the number of moles of ATP formed per mole of substrate oxidized (the P/O ratio) or from the dependence of the rate of substrate oxidation on the presence of adenosine diphosphate (ADP) and phosphate. Both oxidation and phosphorylation reactions in mitochondria are linked to the formation of an electrochemical gradient of H + ions across the membrane. This gradient may be the intermediate between the two reactions. In view of the close connection between movement of H + ions across the membrane and oxidative phosphorylation, methods have been developed to quantitate the relationship between these processes. The most direct of these is the oxygen pulse method. In this method, the mitochondria are incubated in the presence of a respiratory substrate under anaerobic conditions in the presence of valinomycin and K + . The oxygen pulse experiments provide useful information in the case of intact bacteria, in which direct measurement of ATP synthesis can be carried out only on the endogenous adenine nucleotides.