SYNAPTIC VESICLE WITH GLUTAMATE UPTAKE SYSTEM

这项研究的目的是扩大对 突触囊泡中ATP依赖的谷氨酸摄取系统， 特别是研究它的规则。 有令人信服 证明谷氨酸是一种主要的兴奋性物质 中枢神经系统中的神经递质。 atp依赖 囊泡摄取对谷氨酸具有高度特异性， 在决定神经递质的作用方面发挥重要作用 谷氨酸盐 最近，我们获得的证据表明， 谷氨酸摄取被蛋白质物质抑制。 我 建议（a）通过经典的和 高压（HPLC）液相色谱法;（B）测定 SDS-聚丙烯酰胺凝胶理化性质 电泳，凝胶过滤，蔗糖密度梯度 离心和氨基酸分析;（c）确定机制 通过检查其对质子泵ATP酶的影响， 膜电位、氯离子流入和谷氨酸流出;（d） 研究蛋白磷酸化在抑制中的作用， SDS-凝胶电泳和放射自显影;（e）鉴定肽 通过HPLC保留抑制活性的片段;和（f） 鉴定与抑制剂相互作用的蛋白质， 标记物偶联亲和色谱法。 这项研究将 不仅增强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， 研究那些异常的神经病理生理学类型 谷氨酸传输也有牵连。

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.