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, the electric organ of the marine ray was utilized. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an [rabbit] 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 [from rabbit], the bead-bound synaptosomes were lysed by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with I before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an .apprx. 5-fold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased .apprx. 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic visicle membrane since 2-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numberous nonvesicle components. Antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.