STRUCTURAL DISTINCTIONS AMONG ACETYLCHOLINESTERASES

乙酰胆碱酯酶之间的结构差异

• 批准号: 3396994
• 负责人: TERRONE L ROSENBERRY
• 金额: $ 29.58万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3396994
• 关键词: Drosophilidae; Torpedo; acetylcholinesterase; alternatives to animals in research; animal tissue; basement membrane; chemical structure function; chromatography; conformation; fish electric organ; gel electrophoresis; genetically modified animals; human tissue; inositol phosphates; laboratory rat; membrane activity; membrane proteins; neurochemistry; nucleic acid sequence; phospholipids; phosphorylation; protein sequence; protein structure function; saltwater environment; synapses

Long-term objectives are to characterize the biochemical features of distinct acetyl-cholinesterase (AChE) forms in synapses: their structures, processes of assembly and localization, and functions in synaptic transmission. The current program focuses on the structures that attach AChEs to cell membranes. Previous work has demonstrated three distinct classes of attachment structures, each rather unusual for membrane proteins; 1) Dodecameric (A12) AChE is localized in skeletal neuromuscular junctions by collagen-like subunits. 2) Dimeric (G2) AChE in mammalian erythrocytes, nerve endings in torpedo electric organ, and insect heads in anchored by a glycoinositol phospholipid covalently linked to the C-terminus. 3) Tetrameric (G4) AChE in mammalian brain appears to bind to membranes through a small hydrophobic noncatalytic subunit. The proposed Specific Aims involve the latter two AChE classes. 1. A 20-kDa subunit in G4 bovine brain AChE has been identified that appears responsible for membrane interaction, and its structure will be determined to establish whether it is a peptide a lipid, or both. The subunit amino acid sequence will be pursued, and antisera against this subunit will be obtained 2. Additional structural features of the glycoinositol phospholipid anchor of human erythrocyte AChE will be defined. These include the hexose and hexose phosphate components and the positions of hexose and inositol linkage. Peptides that include the C- terminus of this AChE will sequenced. 3. Palmitoylation of inositol renders the anchor of human erythrocyte AChE resistant to a characteristic phospholipase C. Evidence of this modification will be sought in a variety of cell lines by examining both endogenous G2 AChE and transfected Drosophila G2 AChE. Free glycoinositol phospholipid precursors of AChE anchors will be investigated. 4. A nucleotide sequence corresponding to a stable transmembrane peptide will be substituted for the 3' coding region that directs the glycoinositol phospholipid anchor addition in Drosophila AChE. Our long-term goal in this aim is to produce a transgenic fly in which AChE is anchored only by peptide and assess the effects of this changes on fly development and AChE location. Because 30- 40 membrane proteins currently are known to be anchored by glycoinositol phospholipids, further information about these anchors is vital to a better understanding of membrane protein function.

长期目标是表征 突触中不同的乙酰胆碱酯酶（ACHE）形式：它们 结构，组装和本地化的过程以及功能 突触传输。当前的计划着重于结构 那将酸痛连接到细胞膜上。以前的工作已经证明了三个 不同类别的依恋结构，每种结构都不寻常 膜蛋白； 1）十二焦点（A12）ACHE位于骨骼中 胶原蛋白样亚基的神经肌肉连接。 2）二聚体（G2）疼痛 在哺乳动物的红细胞，鱼雷电动器官中的神经末端，以及 昆虫的头部由乙二醇磷脂锚定为共价 与C端相关。 3）哺乳动物大脑中的四聚体（G4）ACHE 似乎通过小疏水非催化与膜结合 亚基。拟议的特定目的涉及后两个ACHE类。 1。在G4牛脑疼痛中有一个20 kDa亚基 似乎负责膜相互作用，其结构将是 确定它是脂质是脂质还是两者兼而有之。这 将追求亚基氨基酸序列，并针对此 亚基将获得2。 人红细胞ACHE的甘氨酸肌醇磷脂锚将是 定义。这些包括己糖和己糖磷酸成分以及 己糖和肌醇链接的位置。包括c-的肽 该疼痛的末端将测序。 3。肌醇的棕榈酰化 使人类红细胞疼痛的锚点具有抗性 特征性磷脂酶C.这种修饰的证据将是 通过检查两个内源性G2疼痛，在各种细胞系中寻求 并转染果蝇G2酸痛。游离糖肌醇磷脂 将研究ACHE锚的前体。 4。核苷酸序列 对应于稳定的跨膜肽的对应 指导甘油辛醇磷脂锚的3'编码区域 在果蝇中的添加。我们的长期目标是生产 一种转基因苍蝇，其中ACHE仅通过肽锚定并评估 这种变化对苍蝇发育和疼痛位置的影响。因为30- 目前已知40种膜蛋白被糖溶质醇锚定 磷脂，有关这些锚的更多信息对于A至关重要 更好地了解膜蛋白功能。