Abstract
Iodination of fasciculin 3 (FAS3) from Dendroaspis viridis venom provided us with a fully active specific probe of fasciculin binding sites on rat brain acetylcholinesterase (AChE). Binding and inhibition are concomitant, as association and inhibition rate constants k1 and ki are identical. The 125I-FAS3.AChE complex dissociates very slowly (t 1/2 = 48 h) and is characterized by a dissociation constant, Kd, of 0.4 pM. All the specific binding of 125I-FAS3 to AChE is prevented by FAS3 as from D. angusticeps venom (Kd = 0.4, 14, and 25 pM, respectively). It is also prevented by propidium iodide, BW284C51, and d-tubocurarine, which bind to peripheral anionic sites of AChE, by Ca2+ and Mg2+, known to enhance AChE activity through an allosteric phenomenon and by acetylthiocholine concentrations which lead to excess substrate inhibition of the enzyme. Diisopropyl fluorphosphate and paroxon, which inhibit AChE by phosphorylating the catalytic serine, have no effect on either the binding rate or the number of binding sites of 125I-FAS3. O-Ethyl-S2-diisopropylaminoethyl methylphosphonothionate, however, which binds irreversibly to the AChE catalytic site but reversibly to a peripheral site, induces a 130% increase in the binding rate of 125I-FAS3, without changing the total number of 125I-FAS3 binding sites. Our results demonstrate that fasciculins bind on a peripheral site of AChE, distinct from the catalytic site and, at least partly, common with the sites on which some cationic inhibitors and the substrate in excess bind. Since phosphorylation of the catalytic serine (esteratic subsite) by [1,3-3H]diisopropyl fluorophosphate can still occur on the FAS3.AChE complex, the structural modification induced by fasciculins may affect the anionic subsite of AChE catalytic site.
Highlights
Few natural inhibitors of AChE are known by [1,3-3H]diisopropyl fluorophosphate can still occur on the fasciculin 3 (FAS3).AChE complex, the structuralmodification induced by fasciculins may affect the anioniscubsite of AChE catalytic site
Successive steps of ion-exchangechromatographyandreverse-phase HPLC allowed FAS3 to be purified to homogeneity (Fig. 1,B-E)
When compared FAS3, we studied the rate of inhibition of AChE activity by with FAS3, FASB has only 3 Tyr residues at positions 4,23, FAS3 for concentrations ranging from 0.1 to 1.5 nM
Summary
(Received for publication, October 20, 1992, and in revised form, February 22, 1993). T h e AChE catalytic site was already known t o be composed well as by the two other fasciculins,FASl and FASB, of an esteratic and an anionic subsite (aocretyl- and cholinefrom D. angusticeps venom (Kd= 0.4, 14,and 25 p ~ ,binding sites, respectively). Diisopropyl fluorophosphate and paraoxon, which in- chemical studies and proposed to actalalossteric sites Few natural inhibitors of AChE are known by [1,3-3H]diisopropyl fluorophosphate can still occur on the FAS3.AChE complex, the structuralmodification induced by fasciculins may affect the anioniscubsite of AChE catalytic site. As AChE inhibitors could be used as therapeutic agents against some cholinergic deficiencies, the detailed knowledge of the molecular mechanism of action of proteinic and peripheral-specific AChE ligands such as fasciculins is of particular interest
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