Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation

A protein which inhibits mitochondrial ATPase has been isolated from bovine heart mitochondria in pure form and its properties have been characterized. 2. To obtain inhibition it was necessary to incubate the protein with ATPase in the presence of Mg++ and ATP. The specificity of this requirement for cation and nucleotide was similar to that reported previously for ATPase activity. About 4 times as much inhibitor was required to inhibit soluble ATPase as compared to particle-bound ATPase. 3. Soluble ATPase purified from a mitochondrial extract prepared with a Nossal shaker as described previously contained considerable amounts of ATPase inhibitor. A new simple procedure for the purification of ATPase was developed starting with an extract obtained from mitochondria by sonic oscillation. A second, more complex procedure was developed starting with an extract made from submitochondrial particles that had been depleted of inhibitor. The first preparation still contained inhibitor, although less than previously described preparations. The second preparation was virtually free of inhibitor. 4. Exposure of soluble ATPase to trypsin or chymotrypsin resulted in the removal of residual inhibitor and in a slight increase in ATPase activity. This treatment abolished coupling activity although the ATPase would still bind to the particles and its activity was partially sensitive to rutamycin.