Cholinergic Synaptic Vesicles Contain a V‐Type and a P‐Type ATPase

Fifty to eighty‐five percent of the ATPase activity in different preparations of cholinergic synaptic vesicles isolated from Torpedo electric organ was half‐inhibited by 7 μ M vanadate. This activity is due to a recently purified phos‐phointermediate, or P‐type, ATPase. Acetylcholine (ACh) active transport by the vesicles was stimulated about 35% by vanadate, demonstrating that the P‐type enzyme is not the proton pump responsible for ACh active transport. Nearly all of the vesicle ATPase activity was inhibited by N ‐ethyl‐maleimide. The P‐type ATPase could be protected from N ‐ethylmaleimide inactivation by vanadate, and subsequently reactivated by complexation of vanadate with deferoxamine. The inactivation‐protection pattern suggests the presence of a vanadate‐insensitive, A‐ethylmaleimide‐sensitive ATPase consistent with a vacuolar, or V‐type, activity expected to drive ACh active transport. ACh active transport was half‐inhibited by 5 μ M N ‐ethylmaleimide, even in the presence of vanadate. The presence of a V‐type ATPase was confirmed by Western blots using antisera raised against three separate subunits of chromaffin granule vacuolar ATPase I. Both ATPase activities, the P‐type polypeptides, and the 38‐kilo‐dalton polypeptide of the V‐type ATPase precisely copurify with the synaptic vesicles. Solubilization of synaptic vesicles in octaethyleneglycol dodecyl ether detergent results in several‐fold stimulation of the P‐type activity and inactivation of the V‐type activity, thus explaining why the V‐type activity was not detected previously during purification of the P‐type ATPase. It is concluded that cholinergic vesicles contain a P‐type ATPase of unknown function and a V‐type ATPase which is the proton pump