Mitochondrial ATP synthesis in permeabilized cells: assessment of the ATP/O values in situ.

We have developed a method to assess the actual mitochondrial ATP synthesis in cells (fibroblasts and Ehrlich ascites tumor cells). This method relies on gentle permeabilization of the cells, inhibition of nonmitochondrial ATP synthesis systems (glycolysis and adenylate kinase), and the inhibition of all ATPase activities (ATP hydrolysis) by decreasing the cytosolic magnesium concentration with EDTA. We have simultaneously measured the rate of respiration, and the ATP/O values obtained with this method are similar to those obtained with isolated mitochondria using the same respiratory substrates. This method is highly appropriate for dealing with small amounts of tissue such as human biopsies.