Rhipicephalus (Boophilus) microplus cDNAs, BmAChE1, BmAChE2, and BmAChE3, were previously identified as presumptively encoding acetylcholinesterases (
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 rBmAChE1, rBmAChE2, and rBmAChE3 as
AChEs (E.C.3.1.1.7), as evidenced by (i) substrate preference for
acetylthiocholine, (ii) inhibition by
eserine,
BW284c51, and the
organophosphates (OPs)
malaoxon and
paraoxon, (iii) 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
acetylthiocholine concentrations as high as 40 mM, however, product inhibition was apparent
at 10-100 microM
choline in agreement with properties reported for the catalytic domain of Anopheles gambiae 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 V(max) near that of rBmAChE2. To date, recombinant BmAChE1 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.