Separation of protease activity from acetylcholinesterase of the electric EEL

We examined the protease activity reported to be associated with acetylcholinesterase (AChE) by extensive purification of the electric eel enzyme. Upon edrophonium-Sepharose chromatography of a commercial preparation, a majority of the protease activity was recovered in the effluent with no AChE activity, while a marginal activity was detected in the AChE fraction eluted with edrophonium chloride. Further chromatography of the edrophonium eluate on hydroxyapatite gave partially overlapping peaks of protease and AChE activities. Finally, the protease activity was mostly removed from the AChE fraction by passing through an ovoinhibitor-agarose column. The protease activity in the edrophonium eluate was inhibited by various serine protease inhibitors, but not by AChE inhibitors. These results suggest that the AChE and protease activities are physically separable, and thus that the protease activity, so far reported as intrinsic to AChE, is probably due to contaminants.