Acetylcholinesterase from the horn fly (Diptera: Muscidae) II: Biochemical and molecular properties

Purified acetylcholinesterase (AChE) of the horn fly was characterized to elucidate the enzymological, inhibitory, and molecular properties of the enzyme. Maximum activity of the AChE against the substrate acetylthiocholine (ATCh) occurred when reactions were conducted at 37°C and pH 7.5. Km and Vmax values were (9.2 ± 0.35) × 10−6 M and 239.8 ± 10.8 units/mg, respectively, for ATCh and (1.5 ± 0.07) × 10−5 M and 138.5 ± 5.5 units/mg, respectively, for butyrylthiocholine (BTCh). The activity of AChE decreased when concentrations of ATCh or BTCh were higher than 1 mM. Studies of the interaction of AChE with different inhibitors revealed pl50 values of 8.88 for eserine, 6.90 for BW284C51, and 4.97 for ethopropazine. Bimolecular reaction constants (kis) for the organophosphorus (OP) anticholinesterases were (2.74 ± 0.14) × 106 M−1 min−1 for coroxon, (7.20 ± 0.28) × 105 M−1 min−1 for paraoxon, and (2.33 ± 0.12) × 105 M−1 min−1 for stirofos. Two major forms of native AChE molecules were found on non-denaturing polyacrylamide gel electrophoresis (PAGE) with Triton X-100, corresponding to bands AChE-2 and AChE-4 found on PAGE without Triton X-100. AChE-2 had an estimated molecular weight of 603,000 and was amphiphilic. AChE-4 had a molecular weight of 147,000 and was hydrophilic. Results of PAGE analyses indicated that the purified enzyme had two bands, one of about 123 kDa and the other greater than 320 kDa, prior to disulfide reduction and only one band at about 54 kDa after reduction on SDS-PAGE. © 1994 Wiley-Liss, Inc.1