Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.
Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes.
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D004557:Electric Organ / Q000378:metabolism*; D005399:Fishes; D011950:Receptors, Cholinergic / Q000302:isolation & purification* / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism / Q000648:ultrastructure*
D002038:Bungarotoxins; D011950:Receptors, Cholinergic