Isolation of a synaptic membrane fraction enriched in cholinergic receptors by controlled phospholipase A2 hydrolysis of synaptic membranes.
A procedure is described for the isolation of synaptic membrane fragments that retain such functionally important proteins as acetylcholine receptors, acetylcholinesterase, 3',5'-cyclic nucleotide phosphodiesterase, and (Na+ + K+)-ATPase. The method is based on the observation, made in brain slices, that junctional membranes are more resistant to phospholipase A2 attack than mitochondrial or plasma membranes. Hydrolysis by phospholipase A2 was controlled by addition of fatty acid-free bovine serum albumin. The membrane fraction obtained represents approximately a 15-fold enrichment of the postsynaptic marker proteins muscarinic and nicotinic acetylcholine receptor and 3',5'-cyclic nucleotide phosphodiesterase over an ordinary synaptic plasma membrane preparation, and is devoid of mitochondrial and microsomal contaminations. The membranes appear on the electron micrographs as rigid fragments (average length 2500-4000A), which do not form vesicles.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods; D008297:Male; D010740:Phospholipases; D051381:Rats; D011950:Receptors, Cholinergic; D013570:Synaptic Membranes / Q000378:metabolism / Q000648:ultrastructure*
D011950:Receptors, Cholinergic; D010740:Phospholipases