BINDING OF I-125 FASCICULIN TO RAT-BRAIN ACETYLCHOLINESTERASE - THE COMPLEX STILL BINDS DIISOPROPYL FLUOROPHOSPHATE

Iodination of fasciculin 3 (FAS3) from Dendroaspis viridis venom provi ded us with a fully active specific probe of fasciculin binding sites on rat brain acetylcholinesterase (AChE). Binding and inhibition are c oncomitant, as association and inhibition rate constants k1 and k(i) a re identical. The I-125-FAS3.AChE complex dissociates very slowly (t1/ 2 = 48 h) and is characterized by a dissociation constant, K(d), of 0. 4 pM. All the specific binding of I-125-FAS3 to AChE is prevented by F AS3 as well as by the two other fasciculins, FAS1 and FAS2, from D. an gusticeps venom (K(d) = 0.4, 14, and 25 pM, respectively). It is also prevented by propidium iodide, BW284C51, and d-tubocurarine, which bin d to peripheral anionic sites of AChE, by Ca2+ and Mg2+, known to enha nce AChE activity through an allosteric phenomenon and by acetylthioch oline concentrations which lead to excess substrate inhibition of the enzyme. Diisopropyl fluorophosphate and paraoxon, which inhibit AChE b y phosphorylating the catalytic serine, have no effect on either the b inding rate or the number of binding sites of I-125-FAS3. O-Ethyl-S2-d iisopropylaminoethyl methylphosphonothionate, however, which binds irr eversibly to the AChE catalytic site but reversibly to a peripheral si te, induces a 130% increase in the binding rate of I-125-FAS3, without changing the total number of I-125-FAS3 binding sites. Our results de monstrate that fasciculins bind on a peripheral site of AChE, distinct from the catalytic site and, at least partly, common with the sites o n which some cationic inhibitors and the substrate in excess bind. Sin ce phosphorylation of the catalytic serine (esteratic subsite) by [1,3 -H-3]diisopropyl fluorophosphate can still occur on the FAS3.AChE comp lex, the structural modification induced by fasciculins may affect the anionic subsite of AChE catalytic site.