BRAIN ACETYLCHOLINE STORAGE SYSTEM

这个项目将描述乙酰胆碱的生物化学特性。 (ACh)哺乳动物脑突触小泡的储存。牛脑 将作为等渗分离的起始原料 纯化的混合型神经递质突触小泡 常规用于提纯的生化分离技术 鱼雷电器官突触小泡。在鱼雷小泡里 已知ACH存储系统由ATPase组成， S乙酰胆碱转运体和抑制物受体 2-(4-苯基哌啶)环己醇(维沙米考，前身为AH5183)。 纯化囊泡中(~3H)维沙米考与脑受体的结合 将被表征，包括确定受体是否可以 以一种神秘的形式存在，就像在鱼雷泡中一样。测量结果 将使用真空辅助过滤技术获得。 放射性标记ACh在纯化脑泡中的主动转运 也将通过滤波法进行研究。两者之间的联系 (~3H)维沙霉素结合和(~(14)C)ACh主动转运抑制 相同的样本将被表征，看看它是否像 就像鱼雷的小泡一样。脑泡的维沙米考受体 会被洗涤剂溶解和提纯到均质 确定其组成与鱼雷的相似程度 受体。最后，脑泡中的ACh转运蛋白将是 光亲和-标记放射性ACh类似物和 用凝胶电泳法和荧光法鉴定标记的亚基。 该项目的总体目标是确定 脑内ACh存储系统与脑内ACh存储系统的相似性 电子琴。其长期目标是理解 ACh运输和储存的生物化学，并确定 哺乳动物ACh转运与ACh释放的关系 胆碱能神经末梢。这包括对所有 系统中的监管功能，以便它们可能 在药理上或其他方面被操纵以增加ACh的储存 进入和释放出受损的胆碱能终末。因为有很多人 疾病或中毒状态选择性地影响胆碱能 人类的神经系统，比如说阿尔茨海默氏症，这个 这种能力可能会在临床上带来显著的好处。

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.