This project will elucidate the biochemical mechanisms responsible for storage of the neurotransmitter acetylcholine (ACh) by synaptic vesicles of nerve terminals. The guiding hypothesis is that recycling metabolically active VP2 vesicles carry out the majority of ACh active transport. A method for purifying VP2 vesicles from Torpedo californica electric organ will be developed. The gross physical and chemical properties of VP2 will be compared with those of the well-studied resting VP1 vesicles, and mechanistic aspects of the ACh storage process will be reinvestigated in VP2 vesicles. The VP2 vesicle polypeptides containing the ACh and drug inhibition binding sites of the ACh transport system will be identified by radioactive affinity labeling experiments. The proton pump ATPase which drives ACh storage, drug receptor and ACh transporter proteins will be purified in native detergent solubilized form. The isolated proteins will be reconstituted into liposomes and the ionic mechanism for each wll be characterized. Biochemical properties of each protein will be compared for the forms isolated from VP1 and VP2 vesicles in an effort to identify and localize regulation in the ACh storage system. It is expected that the project will develop the necessary background for biochemical study of ACh storage in mammalian brain and other organs. This should allow development of pharmacologic or other intervention strategies which would effect stimulation of ACh storage in and release from diseased or intoxicated central or peripheral cholinergic nerve terminals. A long-term goal is to find ameliorative strategies of use in the large number of neural dysfunctions, such as Alzheimer's disease, which have been linked to the cholinergic nerve terminal.
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