Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus

Rhipicephalus (Boophilus) microplus cDNAs BmAChE1 BmAChE2 and BmAChE3 were previously identified as presumptively encoding acetylchohnesterases (AChEs) but biochemical identity was confirmed only for recombinant BmAChE3 In the present study four recombinant BmAChE1 constructs and single recombinant constructs of BmAChE2 and BmAChE3 were expressed in baculovirus Biochemical characterization of the recombinant proteins supports classification of rBmAChEl rBmAChE2 and rBmAChE3 as AChEs (E C 3 11 7) as evidenced by (i) substrate preference for acetylthiocholine (II) inhibition by eserine BW284c51 and the organophosphates (OPs) malaoxon and paraoxon (in) insensitivity to iso-OMPA and (iv) rapid hydrolysis of acetyl-beta-methyl-thiocholine Unlike reports for insect AChEs we did not observe substrate inhibition of activity at acetylthlocholine concentrations as high as 40 mM however product inhibition was apparent at 10-100 mu M choline in agreement with properties reported for the catalytic domain of Anopheles gamMae acetylcholinesterase-1 Substrate affinity and V-max values were highest for rBmAChE1 proteins and one rBmAChE1 enzyme (Tx11 derived from the OP-resistant strain Tuxpan) was insensitive to paraoxon and exhibited a greatly reduced Vmax near that of rBmAChE2 To date recombinant BmAChEl and BmAChE3 enzymes with reduced sensitivity to OP-inhibition have been cloned and expressed from OP-resistant strains The presence of at least three genes expressing AChEs in R. (B) microplus at least two of which contain mutations expressed as OP-insensitive enzymes strongly suggests that phenotypic resistance to OPs may be complex and multigenic in character Published by Elsevier B V