Postsynaptic membranes in the electric tissue of Narcine: III. Isolation and characterization.
Postsnyaptic membranes in homogenates of the electric tissue of Narcine were identified by labelling nicotinic acetylcholine receptors in the membranes with radioactive alpha-bungarotoxin. Various media and centrifugation conditions were examined in an attempt to obtain highly purified postsynaptic membranes. The main criterion for purification was approach towards the specific activity of the pure receptor protein, 9--10 nmol toxin-sites/mg protein. Isolation of tissue microsomes with Tris buffer, EDTA and the protease inhibitor phenylmethylsulfonylfluoride (PMSF), conditions which preserve the receptor molecules optimally, yielded about 50% of the tissue toxin-sites, 5% of the protein, 4% of the ATPase and less than 2% of the acetylcholinesterase (AChE). Further separation of vesiculated membranes in continuous density gradients of sucrose showed that the major contaminants of postsynaptic membrane vesicles were damaged mitochondria and tubular vesicles of dorsal electroplaque membranes rich in ATPase. Mitochondria were effectively removed from homogenates by 'differential' centrifugation, and ATPase-rich vesicles could be largely removed by causing their agglutination with calcium ions, or by controlled proteolysis in the absence of PMSF. Partially purified postsynaptic membranes were obtained having about 7 nmol toxin-sites/mg membrane protein. Further purification appears possible by affinity techniques.
D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D004557:Electric Organ / Q000648:ultrastructure*; D005399:Fishes / Q000033:anatomy & histology*; D008055:Lipids / Q000032:analysis; D011978:Receptors, Nicotinic / Q000032:analysis; D013570:Synaptic Membranes / Q000032:analysis* / Q000648:ultrastructure
D008055:Lipids; D011978:Receptors, Nicotinic