SYNAPTIC VESICLE WITH GLUTAMATE UPTAKE SYSTEM

具有谷氨酸摄取系统的突触小泡

• 批准号: 3412993
• 负责人: TETSUFUMI UEDA
• 金额: $ 12.12万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3412993
• 关键词: SDS polyacrylamide gel electrophoresis; adenosine triphosphate; adenosinetriphosphatase; affinity chromatography; autoradiography; central nervous system; chloride channels; density gradient ultracentrifugation; gel electrophoresis; glutamate receptor; glutamates; high performance liquid chromatography; laboratory mouse; membrane potentials; membrane transport proteins; molecular weight; neural information processing; neural transmission; neurotransmitters; phosphorylation; radioassay; synaptic vesicles

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.

这项研究的目的是扩大对 突触小泡中 ATP 依赖性谷氨酸摄取系统， 特别是要调查其监管。 有令人信服的 有证据表明谷氨酸是主要的兴奋剂 中枢神经系统中的神经递质。 ATP依赖性 囊泡摄取对谷氨酸具有高度特异性，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，我们获得了水泡的证据 谷氨酸的吸收受到蛋白质物质的抑制。 我 建议 (a) 通过经典和 高压（HPLC）液相色谱法； (b) 确定 SDS-聚丙烯酰胺凝胶的理化性质 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 酶的影响来抑制抑制， 膜电位、氯离子流入和谷氨酸流出； (四) 检查蛋白质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 HPLC显示保留抑制活性的片段；和 (f) 识别与抑制剂相互作用的蛋白质，通过 抑制剂结合的亲和层析。 这项研究将 不仅增强了对囊泡谷氨酸吸收的理解 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 涉及谷氨酸传输。