Specific Labelling of the Curare Binding Sites of Acetylcholinesterase and Some Properties of the Modified Enzyme

Abstract

Electric eel acetylcholinesterase was found to selectively react covalently with two molecules of labelled N,N-dimethyl-2-phenylaziridinium chloride (14C-DPA) per unit of 65 000 daltons. One molecule of bound DPA is selectively labilized at pH 9.5. Both alkylated enzyme species (E.2DPA and E.DPA) are inactive toward acetylcholine as substrate, but considerably more reactive toward indophenylacetate (IPA) as substrate. The IPA activities of both the E.2DPA and E.DPA enzymes are unaffected by decamethonium bromide (C10), whereas only the latter suffers inhibition by d-tubocurarine. It is concluded that the enzyme moiety where quaternary effectors interact possesses two topographically distinct classes of sites: one class is essential for C10 inhibition of the esteratic activity, and the other would serve as an allosteric binding site for curare. This latter site may play the role of receptor on excitable membranes.