SYNAPTIC VESICLE WITH GLUTAMATE UPTAKE SYSTEM

这项研究的目的是扩大对 突触小泡中依赖于ATP的谷氨酸摄取系统， 特别是调查其监管情况。有令人信服的 谷氨酸是一种主要的兴奋性物质的证据 中枢神经系统中的神经递质。依赖三磷酸腺苷 囊泡摄取是谷氨酸高度特异的，并被认为 在确定神经递质的作用中发挥重要作用 谷氨酸。最近，我们获得了水泡病毒的证据 谷氨酸摄取被一种蛋白质类物质抑制。我 建议(A)用经典的和分离的方法提纯抑制物质 高压(高效液相色谱)；(B)测定 十二烷基硫酸钠-聚丙烯酰胺凝胶的物化性质 电泳、凝胶过滤、蔗糖密度梯度 离心和氨基酸分析；(C)确定机理 通过检测它对质子泵ATPase的影响来进行抑制， 膜电位、氯离子内流和谷氨酸外流；(D) 检测蛋白质磷酸化在抑制中的作用 十二烷基硫酸钠凝胶电泳法和放射自显影技术；(E)鉴定多肽 经高效液相色谱检测保持抑制活性的片段；和(F) 通过以下方式确定与抑制剂相互作用的蛋白质(S) 抑制剂偶联亲和层析。这项研究将 不仅提高了对囊泡谷氨酸摄取的理解 系统，但也可能最终提供新的见解 神经病理生理学异常类型的探讨 谷氨酸的传递也有牵连。

The objective of this research is to broaden understanding of the ATP-dependent glutamate uptake system in the synaptic vesicle, particularly to investigate its regulation. There is compelling evidence that glutamate serves as a major excitatory neurotransmitter in the central nervous system. The ATP-dependent vesicular uptake is highly specific for glutamate and considered to play an important role in determining the neurotransmitter role of glutamate. Recently, we have obtained evidence that vesicular glutamate uptake is inhibited by a proteinaceous substance. I propose to (a) purify the inhibitory substance by classical and high pressure (HPLC) liquid chromatography; (b) determine physicochemical properties by SDS-polyacrylamide gel electrophoresis, gel filtration, sucrose density gradient centrifugation and amino acid analysis; (c) determine the mechanism of inhibition by examining its effects on a proton-pump ATPase, membrane potential, chloride influx, and glutamate efflux; (d) examine the role of protein phosphorylation in the inhibition by SDS-gel electrophoresis and autoradiography; (e) identify a peptide fragment which retains the inhibitory activity by HPLC; and (f) identify the protein(s) with which the inhibitor interacts, by inhibitor-conjugated affinity chromatography. This research will not only enhance understanding of the vesicular glutamate uptake system, but also may ultimately provide new insights into the investigation of those types of neuropathophysiology where abnormal glutamate transmission is implicated.