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1. 1. The activation by phospholipids of particulate mitochondrial ATPase from rat liver was studied on a preparation extracted from submitochondrial particles with cholate. 2. 2. Lysophosphatidylcholine and the highly acidic phospholipids diphosphatidylglycerol, phosphatidylinositol and phosphatidylserine stimulated the ATPase at low concentration (50–60 μg/mg protein); phosphatidylethanolamine was less effective whereas phosphatidylcholine exhibited the lowest activity. 3. 3. The saturation curves of phospholipids acting at low concentration showed in most cases a deviation from normal Michaelis-Menten kinetics. This devation was absent or less manifest with phosphatidylethanolamine and phosphatidylcholine. Competition between phospholipids and oligomycin was seen with acidic phospholipids or mixtures of neutral and acidic phospholipids. 4. 4. Similarly to oligomycin, dicyclohexylcarbodiimide, tributyltin and chlorpromazine competitively inhibited the activation produced by phosphatidylserine, phosphatidylinositol and diphosphatidylglycerol. 5. 5. It is concluded that both the hydrophobic-hydrophilic balance and the negative charge of phospholipids are important in the interaction between phospholipids and protein at the level of particulate mitochondrial ATPase. The competition between phospholipids and inhibitors of mitochondrial ATPase suggests that the activation induced by phospholipids is involved in the inhibitory effect.

Mixed phospholipids, purified phosphatidylcholine and phosphatidylserine restored the ATPase activity of submitochondrial particles pretreated with dicyclohexylcarbodiimide (DCCD). Pretreatment of mixed phospholipids with DCCD resulted in preparations which were able to transfer the inhibitor to submitochondrial particles. Phosphatidylcholine alone was fully effective. Phosphatidylserine, which apparently forms a stable bond with DCCD, was inactive. These findings suggest different functions of individual phospholipids in the DCCD-induced inhibition. The observation that phosphatidylserine displays unusual high activity in the restoration of ATPase activity in phospholipid-depleted submitochondrial particles indicates that it plays an important role in the activation of mitochondrial ATPase and deserves attention as costituent of the final receptor site for DCCD.

1. 1. The stimulation of mitochondrial particulate ATPase by natural phosphatidylcholines was studied in a phospholipid-free preparation from bovine heart obtained by extraction with cholate. The low stimulatory activity of these phospholipids was increased by: (a) introduction of negatively charged amphiphatic substances into the zwitterionic liposomes; (b) addition of Cl − to the incubation medium. 2. 2. Liposomes of acidic phospholipids or phosphatidylcholine containing anionic amphipaths prevented the ATP plus Mg 2+ -induced decrease of ATPase activity in Mg-ATP particles which have a high content of ATPase inhibitor. 3. 3. Acidic phospholipids, in contrast to phosphatidylcholine, prevented the inhibition of soluble and particulate ATPase by purified ATPase inhibitor. 4. 4. It is concluded that the structure of the lipid-water interface is of primary importance in the stimulation produced by liposomes of diacylphospholipids on particulate mitochondrial ATPase. This effect is associated with the removal of inhibition induced by endogenous ATPase inhibitor on phospholipid-depleted preparations.