TRANSPORT ENZYMES IN SYNAPTIC VESICLES

这一项目将阐明导致生物化学机制 神经递质乙酰胆碱(ACh)的突触小泡储存 神经末梢。指导性假设是新陈代谢循环 激活的VP2囊泡执行ACh的大部分主动转运。一个 一种从鱼雷加州电器官中提纯VP2囊泡的方法 将会被开发出来。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，以及 将在中重新研究ACH存储过程的机械方面 VP2囊泡。含ACh和药物的VP2囊泡多肽 ACh运输系统的抑制结合位点将通过以下方式确定 放射性亲和标记实验。质子泵ATPase 推动ACh储存、药物受体和ACh转运蛋白 纯净的天然洗涤剂溶解形式。分离出的蛋白质将 被重组为脂质体，其离子机制将是 特色化的。每种蛋白质的生化性质将被比较 从VP1和VP2囊泡中分离出来的形式，以努力鉴定和 在ACH存储系统中进行本地化监管。 预计该项目将为以下项目提供必要的背景 哺乳动物脑和其他器官ACh储存的生化研究。这 应允许开发药理学或其他干预策略 这会刺激ACh在病人体内的储存和释放 或中枢性或外周性胆碱能神经末梢。一个 长期目标是在更大范围内找到改进的使用策略 神经功能障碍的数量，如阿尔茨海默氏症，已经被 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.