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The role of butyrylcholmesterase (BuChE) and acetylcholmesterase (AChE) in regulating acetylcholine (ACh) lifetime was investigated by use of selective cholinesterase (ChE) inhibitors. Addition of 1 PM tetraisopropylpyrophosphoramlde (ISO-OMPA) led to a 98% inlubltion of BuChE activity with httle or no effect on AChE activity This inhIbItion wa accompamed by a 26% increase m the amplitude and a 43% prolongation in the half-relawatlon time of contractions ehclted by electnc field stimulation (EFS) Coapplicatlon of BW 284C51 (a selective AChE inhibltor) and 1 PM ISO-OMPA resulted m increases of 2-fold in the amplitude and lo-fold m the half-relaxation time of EFS-Induced contractions. These aIteratIons were accompamed by small but sustained baseline contractures that were antagonized completely by incubation with exogenous BuChE (2.5 U/ml). The results suggest that BuChE serves to coregulate the hfetime of ACh m camne tracheal smooth muscle. Pseudocholmesterase;Acetylcholinesterase.Tetralsopropylpyrophosphoramlde. BW 284C51

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The binding activity of a rabbit polyclonal antiserum raised against a 51‐residue peptide (P51) homologous to human VAMP2 (residues 44–94) was examined. Human VAMP2 is an 18‐kDa protein located on the external membrane of small synaptic vesicles and is targeted by four of the seven botulinum neurotoxin (BoNT) serotypes (B, D, F and G). The antiserum, designated anti‐P51, recognized P51 but exhibited little cross‐reactivity with the two cleavage products that result from BoNT/B‐mediated proteolysis of P51. The larger of these fragments, designated as P33 (residues 44–76), exhibited a weak but measurable interaction with the antiserum. The smaller cleavage product, designated as P18 (residues 77–94), was not recognized by the antiserum. Anti‐P51 was used to monitor BoNT/B light chain (LC)‐mediated cleavage of P51 using an indirect ELISA. The serine protease inhibitor phenylmethylsulfonyl fluoride did not inhibit BoNT/B activity, but the zinc chelator N,N,N′,N′‐tetrakis (2‐pyridylmethyl)ethylenediamine (TPEN) and the elastase inhibitor 7‐ N ‐phenylcarbamoylamino‐4‐chloro‐3‐propyloxyisocoumarin (ICD 1578) produced complete blockade of BoNT/B LC action. Under ideal conditions, it will be possible to evaluate up to seven candidate anti‐BoNT/B drugs in triplicate at four concentrations using a single 96‐well microtiter plate. These findings indicate that the ELISA will be suitable for rapid screening of BoNT/B inhibitors.