Purification of acetylcholinesterase by affinity chromatography from torpedo japonica

Abstract

Acetylcholinesterase (EC 3.1.1.7. AChE) hydrolyzes acetylcholine from the nerve terminal. It has been suggested that native acetylcholinesterase is associated with the basal lamina (basement membrane) that runs through the synaptic cleft. Three native forms of acetylcholinesterase with sedimentation coefficients of 9S(A form), 14.2S(C form) and 18.4S(D form) are persent in the electric organ of eel Electrophorus electricus. Acetylcholinesterase from Torpedo marmorata also exhibits three molecular forms with sedimentation coefficients of 7.9S, 13.4S and 17.4S. These forms were assumed to be homologous to the A, C and D Electrophorus electricus forms. In the present study, 9-(5-carboxypentylamino)-acridine was synthesized and used as a ligand for affinity chromatography of acetylcholinesterase from Torpedo japonica. When the affinity-purified acetylcholinesterase was subjected to a sucrosegradient sedimentation, three peaks with the enzyme activity were observed. Two of these sedimented faster than the marker protein (catalase) and one sedimented much slower than the marker protein. Although precise sedimentation coefficients were not determined, it appears that three peaks correspond to A(7.9S), C(13.4S) and D (17.4S) forms of Torpedo marmorata. The 11S form was not observed. A preliminary study on the enzyme aggregation at low ionic strength was made.