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，旨在本地化三个 细胞内膜系统内的特异性大分子， 细胞体和轴突。这些大分子的定位将 阐明参与特定的体膜系统， 用于血浆不同靶区的蛋白质的包装 膜或细胞外间隙和轴突膜的参与 轴突运输系统。这三个目标大分子将被 研究的是：1）Na+ +K+ ATP酶; 2）电压依赖性钠 通道;和3）乙酰胆碱酯酶（AChE）。这些大分子（它们的 个体组成亚基或不同的分子形式）将被 借助光可视化的特异性抗体探针定位 和电子显微镜使用金，铁蛋白，过氧化物酶，或荧光 抗体缀合物。所研究的膜系统都是 细胞体和轴突。在细胞体中，那些参与 膜蛋白或分泌产物的生物合成和包装; 例如，在一个实施例中，粗面内质网（RER）、滑面内质网 内质网（SER）和高尔基体。在轴突中，至少有 三种形态和功能不同的膜系统。之一 这些明显参与了顺行性快速轴突运输，另一个 在逆行运输和第三个似乎并不迅速 其功能或与其他两个的关系是 未知这些计划中的研究是关于 所有这三种大分子的能力， 10种Na+ +K+特异性抗体的制备和表征 大鼠和鳗鱼的ATP酶及其α和β亚基（Electrophorus electricus）组织，2）来自鳗鱼的Na+通道，以及3）两种主要的 分子形式的乙酰胆碱酯酶。我们已经证明了抗体 这些大分子中的每一种都适用于高分辨率 免疫电子显微镜研究、免疫荧光研究，或 生物化学探针与技术，包括蛋白质印迹，酶 连接的免疫吸附测定或放射免疫测定。物种和组织 抗体的特异性已被检测。所有人都有能力 识别每种抗原的神经元形式， 标签能力已经得到确认。