Interaction of a benzomorphan opiate with acetylcholinesterase and the nicotinic acetylcholine receptor.

The benzomorphan opiate, (-)N-allynormetazocine [(-)ANMC, (-)SKF10047], has been shown previously to bind two distinct sites on acetylcholine receptor (AChR)-rich membranes from Torpedo electroplaque. The low affinity site seems to correspond to the site for noncompetitive blockers on the AChR. The high affinity site, which can be photoaffinity labeled using UV irradiation, was distinct from this site. We show here, using a variety of techniques, that the high affinity binding site for (-)ANMC is on the acetylcholinesterase (AChE) associated with these membranes. The Triton X-100-solubilized peptide photolabeled with (-)[3H]ANMC co-migrates with acetylcholinesterase activity on velocity sucrose gradient centrifugation and fast protein liquid chromatography. In addition, the labeled peptide cannot be precipitated with monoclonal or polyclonal antibodies raised against the nicotinic AChR but can be precipitated with anti-AChE antibodies. Localization of the binding site on AChE was confirmed by photolabeling of and reversible binding to the 11 S AChE purified from Torpedo californica. The binding and photolabeling had characteristics and affinity similar to those for the high affinity binding site in Torpedo electroplaque membranes. Competition studies with specific AChE inhibitors suggest that the binding site may be the catalytic site of the enzyme, which exists on the 66-kDa globular protein. The effect of (-) and (+)ANMC on AChE activity was also investigated. ANMC inhibited AChE activity at micromolar concentrations in a stereoselective fashion, with the (-)isomer exhibiting a 2-fold higher affinity than the (+) isomer. The inhibition was consistent with a competitive blockade of AChE activity.