Hooper_1982_J.Physiol.Paris_78_443

Exocytosis at the nerve terminal will only be fully understood when purified synaptic vesicles can be made to fuse with presynaptic plasma membrane in vitro. While the purification of synaptic vesicles from electric organ is now straightforward, isolation of the presynaptic plasma membrane presents a greater difficulty because of lack of specific markers. We have utilized pure synaptic vesicles in a novel way to overcome this difficulty. Antibodies raised to pure synaptic vesicles can be used to detect the presence of vesicle antigens. Thus, for example, we can show that synaptosome preparations isolated from electric organ by conventional procedures have about 5% of their protein in synaptic vesicles. The synaptic vesicle antigens and choline acetyltransferase both disappear from the synaptosome fraction after denervation of the electric organ, verifying that they are nerve terminal specific. Some of the synaptic vesicle antigens can be detected on the outside of the intact synaptosomes by binding rabbit anti-synaptic vesicle antibodies. Such antibody-coated synaptosomes will absorb specifically to goat anti-rabbit IgG attached to polyacrylamide beads. Lysis of such bead-bound synaptosomes leaves a plasma membrane fraction firmly attached to the beads. By raising antibodies to this membrane fraction an antiserum has been generated that binds to antigenic determinants in the electric organ. Some of these antibodies bind to the frog neuromuscular junction. The antigens recognized by this antiserum are, like synaptic vesicle antigens, lost on denervation. Unlike synaptic vesicle antigens, however, the antigens recognized by the anti-plasma membrane serum are present on the outside of resting frog nerve terminals.