title: Partial purification of presynaptic plasma membrane by immunoadsorption.
abstract: During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or "active zones." In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased approximately 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis*; D004557:Electric Organ; D007106:Immune Sera; D007163:Immunosorbent Techniques; D007455:Iodine / Q000378:metabolism; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000378:metabolism; D013570:Synaptic Membranes / Q000032:analysis*; D013572:Synaptic Vesicles / Q000276:immunology; D013574:Synaptosomes / Q000032:analysis*; D014101:Torpedo
chemical_list: D007106:Immune Sera; D011950:Receptors, Cholinergic; D007455:Iodine; D000109:Acetylcholine