BRAIN ACETKYLCHOLINE STORAGE SYSTEM

这个项目将表征乙酰胆碱的生化方面 (ACh)通过哺乳动物脑突触囊泡储存。 牛脑 将用作等渗分离高度 纯化的混合神经递质型突触囊泡， 常规用于纯化的生化分离技术 电鳐电器官的突触囊泡 在鱼雷囊泡中 已知ACh贮存系统由ATP酶组成， 乙酰胆碱转运蛋白和抑制性化合物受体 2-（4-苯基哌啶子基）环己醇（vesamicol，原名AH 5183）。 在纯化的囊泡中（3 H）囊泡霉素与脑受体的结合 将被表征，包括确定受体是否可以 以一种隐蔽的形式存在，如鱼雷囊泡。 测量 将使用真空辅助过滤技术获得。 放射性标记乙酰胆碱在纯化脑泡中的主动转运 也将通过过滤器方法进行研究。 之间的联系 （3 H）vesamicol结合和（14 C）ACh主动转运抑制， 同样的样品将被表征， 就像鱼雷囊泡一样 脑泡的囊泡霉素受体 将被洗涤剂溶解并纯化至均匀， 确定它的成分与鱼雷的成分有多相似 受体的 最后，脑囊泡中的ACh转运蛋白将被 用放射性ACh类似物进行光亲和标记， 通过凝胶电泳和荧光照相鉴定标记的亚基。 本项目的总体目标是确定 脑中ACh储存系统与脑中ACh储存系统的相似性。 电子琴 长期目标是了解 乙酰胆碱运输和储存的生物化学，并确定 哺乳动物乙酰胆碱转运与释放的关系 胆碱能神经末梢 这包括了解所有 系统中的监管功能， 乙酰胆碱储存量增加 并从受损的胆碱能末梢释放。 由于许多 疾病或中毒状态选择性地影响胆碱能 人类的神经系统，例如，阿尔茨海默氏病， 能力可能具有显著的临床益处。

This project will characterize biochemical aspects of acetylcholine (ACh) storage by mammalian brain synaptic vesicles. Bovine brain will be used as starting material for isosmotic isolation of highly purified synaptic vesicles of mixed neurotransmitter type using biochemical separation techniques routinely used for purification of Torpedo electric organ synaptic vesicles. In Torpedo vesicles it is known that the ACh storage system is composed of an ATPase, s transporter for ACh and a receptor for the inhibitory compound 2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183). Binding of (3H)vesamicol to the brain receptor in purified vesicles will be characterized, including determining whether receptor can exist in a cryptic form as in Torpedo vesicles. The measurements will be obtained using a vacuum assisted filtration technique. Radiolabeled ACh active transport by the purified brain vesicles will be studied also by the filter method. The linkage between (3H)vesamicol binding and (14C)ACh active transport inhibition in the same samples will be characterized to see if it is as complex as in Torpedo vesicles. The vesamicol receptor of brain vesicles will be detergent solubilized and purified to homogeneity to determine how similar its composition is to that of the Torpedo receptor. Finally, the ACh transporter in brain vesicles will be photoaffinity-labeled with a radioactive ACh analogue and the labeled subunit identified by gel electrophoresis and fluorography. The overall aim of this project is to determine the extent of similarity of the ACh storage system in brain to that of the electric organ. The long term aim is to understand the biochemistry of ACh transport and storage, and to determine the relationship of ACh transport to ACh release in the mammalian cholinergic nerve terminal. This includes an understanding of all the regulatory features in the system so that they might be pharmacologically or otherwise manipulated to increase ACh storage in and release from compromised cholinergic terminals. Since many disease or toxic states impact selectively on the cholinergic nervous system of humans, for example, Alzheimer's disease, this capability could be of significant clinical benefit.