Large-scale Purification of Membranes from Torpedo Presynaptic Plasma marmorata Electric Organ

The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of >100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy. Synaptic transmission occurs in organized areas where specialized domains of the nerve ending plasma membrane, the so-called "active zones" (6), are facing specialized domains of the postsynaptic cell membrane. A detailed knowledge of the protein composition of the presynaptic plasma membrane (PSPM) ~ is essential to understand the events leading to transmitter release. Torpedo marmorata electric organs possess a very abundant and homogeneous cholinergic innervation (1 I). The nerve electroplaque junction has proven to be a useful model for biochemical studies on peripheral cholinergic transmission (for reviews see references 12, 16, 33, and 36). In addition to synaptic vesicles (13, 35), the presynaptic organelles where acetylcholine (ACh) is stored, nerve terminals (synaptosomes) have been isolated and purified from Torpedo electric organ (15, 23). PSPM fractions were obtained after subfractionation of these highly purified synaptosomes (24, 32). This two-step procedure permitted us to overcome a major difficulty, i.e., the lack of specific markers for the PSPM, but only small quantities of PSPM proteins could be obtained. The aim of the present work was to develop a large-scale procedure permitting the purification of PSPM directly from Torpedo electric organ. This was possible since several proAbbreviations used in this paper: ACh, acetylcholine; ACHE, acetylcholinesterase; AChR, acetylcholine receptor; ELISA, enzymelinked immunosorbent assay; GCV, Glycera convoluta venom; PSPM, presynaptic plasma membrane. THE JOURNAL OF CELt BIOLOGY. VOtUME 101 NOVEMBER 1985 1757-1762 © The Rockefeller University Press • 0021-9525/85/11/1757/06 $1.00 teins were recently shown to be specific for, or at least highly concentrated in, the PSPM. First, a high acetylcholinesterase (ACHE) activity was found to be associated with this plasma membrane (24, 32). This AChE activity corresponds to the hydrophobic dimeric form of the enzyme (18, 22). Second, a presynaptic neurotoxin extracted from the venom glands of the polychaete annelid Glycera convoluta (GCV), which triggers a massive quantal ACh release in neuromuscular junctions and Torpedo electric organ (19), was found to bind specifically to a protein of the PSPM (26). Finally, two monoclonal antibodies to PSPM antigens were obtained: one (C18) is directed against a 67-kD ectocellularly exposed protein (21); the second (H6-1) is characterized in the present report. Using these protein markers, we were able to isolate large amounts of PSPM by fractionating in a single run up to 500 g of frozen electric organ. The purity of the PSPM fraction was similar to that of the fraction purified from isolated synaptosomes. This large-scale procedure will enable us to attempt the purification of presynaptic membrane proteins involved in synaptic activity. MATERIALS AND METHODS Preparation of the PSPM Fraction: Torpedo marmorata were obtained alive from the Marine Station of Arcachon (France). Electric organs were dissected out and cut into 0.5-g pieces after removal of the skin and large nerve trunks. Pieces were washed in 300 mM NaC1, I mM EDTA, l0 mM Tris buffer, pH 8.0 (~25 g tissue per 100 ml solution) for 2 h in a cold room. They were then blotted dry and frozen in 50-g batches at -70"C. The frozen tissue 1757 on A ril 3, 2017 D ow nladed fom Published November 1, 1985