TRANSPORT ENZYMES IN SYNAPTIC VESICLES

这个项目将阐明生物化学机制负责 神经递质乙酰胆碱（ACh）通过突触囊泡的储存 神经末梢 指导性假设是新陈代谢的再循环 主动VP2囊泡进行大部分ACh主动转运。 一 一种从加州电鳐电器官中纯化VP2囊泡的方法 将被开发。 VP2的总体物理和化学性质将 与研究充分的静息VP1囊泡进行比较， 乙酰胆碱储存过程的机制方面将重新调查， VP2囊泡。 含有ACh和药物的VP2囊泡多肽 ACh转运系统的抑制结合位点将通过 放射性亲和标记实验。 质子泵ATP酶， 驱动乙酰胆碱储存，药物受体和乙酰胆碱转运蛋白将是 以天然去污剂溶解形式纯化。 分离的蛋白质将 被重构到脂质体中，并且每个WLL的离子机制是 表征了 将比较每种蛋白质的生化特性， 从VP1和VP2囊泡中分离的形式， ACh储存系统的局部调节。 预计该项目将为以下方面提供必要的背景资料： 哺乳动物脑和其他器官中乙酰胆碱储存的生物化学研究。 这 应允许开发药理学或其他干预策略 这将影响刺激ACh储存和释放， 或中毒的中枢或外周胆碱能神经末梢。 一 长期目标是找到大范围使用的改进策略 许多神经功能障碍，如阿尔茨海默氏病，这是 与胆碱能神经末梢相连

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.