TRANSPORT ENZYMES IN SYNAPTIC VESICLES

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The project would characterize the two ATPase activities of purified mature cholinergic synaptic vesicles (VP1) from Torpedo californica to determine whether one is a transport ATPase stimulated by ionophores or acetylcholine and another is a myosin-like ATPase. Myosin-like AtPase would be removed, if possible, to allow uncomplicated study of transport ATPase activity. Determination of which metal ions support each type of ATPase activity would be made to further distinguish them. Alternative high energy substrates would be tested for hydrolysis by VP1 vesicles which is stimulated by exogenous acetylcholine in a broader search for a vesicle acetylcholine transport enzyme. Immature recycled synaptic vesicles (VP2) which are actively engaged in acetylcholine uptake would be isolated from the electric organ and similar studies conducted on them. Cytoplasm would be tested for effects on VP1 and VP2 enzymes exhibiting transport properties, and any effector molecules preliminarily characterized. Wheter activation of VP1 and VP2 transport system acetylcholine was due to direct interaction of acetylcholine with the energy transducing system or indirect interaction via an intermediary pumped ion gradient would be determined with ionophores, uncouplers and vesicle ghosts. Right-side-out sealed ghosts would be pruduced to allow control of vesicle interior conditions and further sidedness studies. Identification of VP1 and VP2 transport polypeptides would be attempted by SDS gel electrophoresis of vesicles labeled specifically with radioactive substrate, inhibitor of affinity label. Possible maturation or regulation of acetylcholine transport system would be studied by comparing the structure and kinetics of the system in VP1 and VP2 vesicles.