An acetylcholinesterase has been purified from Torpedo californica by mild proteolysis of electroplax membranes with trypsin (5 µg/ml for 5 min) to solubilize the enzyme, followed by affinity chromatography of the soluble enzyme. The procedure yields an apparently homogeneous enzyme whose molecular weight (approximately 335,000), frictional coefficient (1.65), and amino acid composition all distinguished the Torpedo acetylcholinesterase from that which has been prepared by lytic procedures from Electrophorus. The enzyme is composed of four similar, if not identical, subunits, each of which possesses a catalytic and inhibitor binding site. Torpedo acetylcholinesterase contains 7.9% carbohydrate present as hexoses, hexosamines, and sialic acid, and at least some of these residues are exposed on the outer surface of the molecule. Concanavalin A, a plant lectin with specificity toward mannose residues at nonreducing positions, forms a sedimentable complex with the purified acetylcholinesterase which can be partially reversed by α-methyl-D-mannoside. Addition of concanavalin A to electroplax membranes markedly inhibits trypsin-induced acetylcholinesterase release from the membrane surface.
ACKNOWLEDGMENTS We wish to thank Drs. Susan Taylor and Johannes Everse of the Department of Chemistry, University of California, San Diego, for their assistance with the amino acid and sedimentation analyses.
- Copyright ©, 1974, by Academic Press, Inc.