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 microM vanadate. This activity is due to a recently purified phosphointermediate, 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-ethylmaleimide. 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, N-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 microM 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-kilodalton 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.
Yamagata
S K
SK
Department of Chemistry, IES, University of California, Santa Barbara 93106.
Parsons
S M
SM
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000145:classification*; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D010042:Ouabain / Q000494:pharmacology; D010275:Parasympathetic Nervous System / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013572:Synaptic Vesicles / Q000187:drug effects / Q000201:enzymology*; D014101:Torpedo; D014638:Vanadates / Q000494:pharmacology
D014638:Vanadates; D010042:Ouabain; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D005033:Ethylmaleimide