Purification by affinity chromatography of acetylcholinesterase from electric organ tissue of the electric eel subsequent to tryptic treatment

Abstract

1. 1.|A Sepharose-acetylcholinesterase inhibitor conjugate was prepared by synthesis of the inhibitor [ N-( ε-aminocaproyl)- p-aminophenyl]trimethylammonium bromide hydrobromide and its covalent linkage to the CNBr-activated resin. 2. 2.|The Sepharose-inhibitor conjugate was employed for purification of acetyl-cholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from electric organ tissue by affinity chromatography. Thus the enzyme was selectively adsorbed on the resin and specifically eluted with the soluble acetylcholinesterase inhibitor, decamethonium bromide. 3. 3.|The Sepharose-inhibitor conjugate adsorbed the different molecular species of acetylcholinesterase (differing in sedimentation coefficient) present in electric organ tissue. Since two of these species aggregate at low ionic strength, the partially purified enzyme also displayed this property. 4. 4.|Highly purified acetylcholinesterase was obtained if affinity chromatography was preceded by controlled tryptic digestion or prolonged autolysis causing conversion of the enzyme to an 11-S form which does not aggregate at low ionic strength. 5. 5.|Purified acetylcholinesterase was obtained in an overall yield of 40%. It was essentially homogeneous on acrylamide-gel electrophoresis, and its sedimentation coefficient (approx. 11 S), specific activity and amino acid composition resembled those previously reported for purified acetylcholinesterase. 6. 6.|Active site titration of the purified enzyme yielded an equivalent weight of 107 000 per active site. Acrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed two major polypeptide components of molecular weights approx. 88 000 and 64 000. 7. 7.|The properties of the purified acetylcholinesterase are compared with those of the purified preparations previously reported, and its relationship to the molecular species present in intact electric organ tissue is discussed.