Studies on the activation of purified mitochondrial ATPase by phospholipids

1. An ATPase which is activated by phospholipids and inhibited by oligomycin, has been purified from beef heart submitochondrial particles using affinity chromatography. Phospholipid and detergent are removed by washing the enzyme with a solution of serum albumin while it is attached to the biospecific adsorbent.

2. The ATPase is activated up to 18-fold by lysolecithin and to a smaller extent by cardiolipin, phosphatidylinositol and phosphatidylethanolamine. The amount required of each of these phospholipids to give half-maximal activation is apparently inversely related to the number of fatty acid chains in the lipid. Lecithin, which is a poor activator of the ATPase, competitively inhibits the activation by cardiolipin.

3. The activation of the ATPase consists of an increase in both the maximal velocity of the reaction and the affinity for substrate ATP. The pH optimum of the reaction is not influenced by the charge of the lipid.

4. Arrhenius plots of ATPase activated with lysolecithin show a transition to a higher activation energy at temperatures below 19 °C. The sensitivity of the lysolecithin-activated enzyme to oligomycin is markedly reduced below the same temperature. With cardiolipin the transition is observed at 13 °C.

5. ADP, Mg²⁺ and to a smaller extent ATP, Mg²⁺ enhance the activation of ATPase by suboptimal amounts of phospholipid.