A rapid percoll gradient procedure for the preparation of acetylcholine receptor-rich vesicles from Torpedo marmorata electric organ

Abstract

A rapid method for the isolation of acetylcholine receptor-rich membranes from Torpedo marmorata electric organ, using a Percoll density gradient, is presented. The preparation of purified membranes appeared on electron microscope examination as a homogeneous population of sealed vesicles, covered with the characteristic rosettes identified as acetylcholine receptor clusters. Biochemical characterization revealed an α-bungarotoxin specific binding activity of 1.6–2.1 nmol/mg of protein, low acetylcholinesterase specific activity, a protein:lipid ratio (w/w) of 2.1 with high cholesterol content. Polyacrylamide gel electrophoresis under denaturing conditions showed the polypeptide bands characteristic of the receptor (α, β, γ and δ), together with 43 kDa and ∼100 kDa proteins (already described as ν and ϵ). The method is simple and rapid, and maintains constant osmotic conditions throughout. It thus represents an improvement over previous methods, and will be useful for routine preparation and specially for studying post-synaptic membrane components interactions.