ACETYLCHOLINESTERASES FROM ELAPIDAE SNAKE-VENOMS - BIOCHEMICAL, IMMUNOLOGICAL AND ENZYMATIC CHARACTERIZATION

We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Vipe ridae and Crotalidae). We found high acetylcholinesterase (AChE) activ ity in all venoms from Elapidae, except in those from the Dendroaspis genus, AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom, We could not detect ace tylcholinesterase activity in any batch of venom from Viperidae or Cro talidae. Titration of active sites with an organophosphorous agent (MP T) revealed that the AChE of all venoms have similar turnovers (6000 t o 8000 s(-1)) which are clearly higher than those of Torpedo and mamma lian enzymes but lower than that of Electrophorus. AChEs from the veno m of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus g enera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hyd rolyzing acetylthiocholine faster than propionylthiocholine and butyry lthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fa sciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from ot her venoms. Polyspecific mAbs were used to demonstrate that venoms fro m Dendroaspis, which contain the AChE inhibitor fasciculin but lack AC hE activity, were also devoid of immunoreactive AChE protein, AChE inh ibitors acting at the active site (edrophonium, tacrine) and at the pe ripheral site (propidium, fasciculin), as well as bis-quaternary ligan ds (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of react ivity with regard to the different inhibitors, with the exception of f asciculin. AChEs from Naja and Haemacatus venoms were relatively insen sitive to fasciculin inhibition (IC(50)much greater than 10(-6) M), wh ile Bungarus (IC(50)approximate to 10(-8) M) and especially Ophiophagu s (IC50<10(-10) M) AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal ant ibody (Elec-410) previously described as a specific ligand for the Ele ctrophorus electricus peripheral site. Taken together, these results s how that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs s how strong immunological similarities and possess very similar enzymat ic properties. However, they present quite different sensitivity to pe ripheral site inhibitors, fasciculin and the monoclonal antibody Elec- 410.