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.
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