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.

该提案详细介绍了一项研究计划，旨在将三个 细胞内膜系统中的特定大分子 细胞体和轴突。这些大分子的本地化将 阐明特定的胞体膜系统参与 去往血浆不同靶区的蛋白质包装 膜或细胞外间隙与轴突膜的受累 轴突运输系统。这三个目标大分子是 研究有：1)Na++K+-ATPase；2)电压依赖性钠 3)乙酰胆碱酯酶(AChE)。这些大分子(他们的 个人组成亚单位或不同的分子形式)将是 借助光显示的特异性抗体探针进行定位 使用金、铁蛋白、过氧化物酶或荧光的电子显微镜 抗体偶联。正在研究的膜系统都是 细胞体和轴突。在细胞体中，那些参与 膜蛋白或分泌物的生物合成和包装； 例如，粗面内质网(RER)、光滑内质网 网(SER)和高尔基体。在轴突中，至少有 三个在形态和功能上截然不同的膜系统。其中之一 这些明显参与了顺行快速轴突运输，另一种 在逆行运输中，第三个看起来不是很快 其功能或与其他两个人的关系是 未知。这些计划对生物合成和运输的调查 所有这三个大分子现在都能通过最近的 抗10-Na++K+特异性抗体的制备及鉴定 大鼠和鳗鱼的ATPase及其α、β亚基 电)组织，2)鳗鱼的Na+通道，以及3)两个主要的 来自鱼雷的乙酰胆碱酯酶的分子形式。我们已经证实了抗体 适用于高分辨率的每一种大分子 免疫电子显微镜研究、免疫荧光研究或AS 带有蛋白质印迹、酶等技术的生化探针 联用免疫吸附测定法或放射免疫测定法。物种和组织 对抗体的特异性进行了检测。所有人都有能力 识别每个抗原的神经元形式，在大多数情况下是胞浆 贴标能力已经得到确认。