PACKAGING AND AXONAL TRANSPORT OF THREE MACROMOLECULES

三种大分子的包装和轴突运输

• 批准号: 2266074
• 负责人: Mark H Ellisman
• 金额: $ 19.57万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2266074
• 关键词: Torpedo; acetylcholinesterase; alternatives to animals in research; axon; cell membrane; electron microscopy; endoplasmic reticulum; enzyme linked immunosorbent assay; extracellular matrix; laboratory rat; macromolecule; membrane structure; neuroanatomy; neuronal transport; neurons; potassium channel; protein biosynthesis; protein structure function; protein transport; radioimmunoassay; sodium channel; sodium potassium exchanging ATPase; soma; voltage gated channel

This proposal details a research program designed to localize three specific macromolecules within intracellular membrane systems of both the cell body and the axon. Localization of these macromolecules will elucidate the involvement of specific somal membrane systems in the packaging of proteins destined for different target areas of the plasma membrane or extracellular space and the involvement of axonal membrane systems in axonal transport. The three target macromolecules to be studied are; 1) the Na+ +K+ ATPase; 2) the voltage dependent SODIUM CHANNEL; and 3) ACETYLCHOLINESTERASE (AChE). These macromolecules (their individuals constituent subunits or distinct molecular forms) will be localized with the aid of specific antibody probes visualized by light and electron microscopy using gold, ferritin, peroxidase, or fluorescent antibody conjugates. The membrane systems under study are both those of the cell body and of the axon. In the cell body, those involved in the biosynthesis and packaging of membrane proteins or secretion products; e.g., the rough endoplasmic reticulum (RER), the smooth endoplasmic reticulum (SER), and the Golgi apparatus. In the axon, there are at least three morphologically and functionally distinct membrane systems. One of these is clearly involved in anterograde rapid axonal transport, another in retrograde transport and the third which does not appear to be rapidly transported and whose function or relationship to the other two is unknown. These planned investigations into the biosynthesis and transport of all three of these macromolecules are now enabled by the recent production and characterization of specific antibodies to 10 the Na+ +K+ ATPase and its alpha and beta subunits from rat and eel (Electrophorus electricus) tissues, 2) the Na+ channel from eel, and 3) two of the major molecular forms of AChE from Torpedo. We have already proven antibodies to each of these macromolecules suitable for use in high resolution immunoelectron microscopic studies, immunofluorescent studies, or as biochemical probes with techniques including Western blotting, enzyme linked immunoadsorbent assays or radioimmunoassays. Species and tissue specificity of antibodies have been examined. All are capable of recognizing neuronal forms of each antigen and in most cases cytoplasmic labeling capacity has been already confirmed.

该建议详细介绍了一项旨在本地化三个的研究计划 两者的细胞内膜系统中的特定大分子 细胞体和轴突。这些大分子的定位将 阐明特定的索马膜系统参与 蛋白质的包装，注定要针对等离子体的不同靶区域 膜或细胞外空间以及轴突膜的参与 轴突运输的系统。三个目标大分子为 研究是； 1）Na++ K+ ATPase； 2）依赖电压的钠 渠道; 3）乙酰胆碱酯酶（ACHE）。这些大分子（它们的 个体组成的亚基或不同的分子形式）将是 借助光可视化的特定抗体探针局部 使用金，铁蛋白，过氧化物酶或荧光的电子显微镜 抗体结合物。所研究的膜系统都是 细胞体和轴突。在细胞体中，参与的人 膜蛋白或分泌产物的生物合成和包装； 例如，粗糙的内质网（RER），光滑的内质 网状（SER）和高尔基体。在轴突中，至少有 三个形态和功能上不同的膜系统。之一 这些显然参与了顺行快速轴突运输，另一种 在逆行运输中，第三次似乎不迅速 运输以及与其他两个的功能或关系是 未知。这些计划对生物合成和运输的调查 现在，所有这三个大分子都在最近的 对10个Na++ K+的特定抗体的生产和表征 ATPase及其大鼠和鳗鱼的Alpha和β亚基（电子 电动组织，2）鳗鱼的Na+通道，3）两个主要 鱼雷的分子疼痛形式。我们已经证明了抗体 这些大分子适合于高分辨率 免疫电子显微镜研究，免疫荧光研究或AS 具有包括蛋白质印迹，酶在内的技术的生化探针 链接的免疫吸附测定法或放射免疫测定。物种和组织 已经检查了抗体的特异性。所有人都有能力 识别每种抗原的神经元形式，在大多数情况下，细胞质形式 标签能力已经被确认。