TY - JOUR
T1 - Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants
AU - Wang, Yuxia
AU - Schopfer, Lawrence M.
AU - Duysen, Ellen G.
AU - Nachon, Florian
AU - Masson, Patrick
AU - Lockridge, Oksana
N1 - Funding Information:
This study was supported by U.S. Army Medical Research and Materiel Command Grant DAMD17-01-2-0036 (to O.L.), UNMC Eppley Cancer Center Support Grant P30CA36727-19, and Contract 00-2-032-0-00 from the Office of the Defense Cooperation Attache, Republique Francaise (to P.M.). The information does not necessarily reflect the position or the policy of the United States or French governments, and no official endorsement should be inferred.
PY - 2004/6/1
Y1 - 2004/6/1
N2 - Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5μg/ml human G117H BChE, 2μg/ml wild-type mouse BChE, and 0.06μg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.
AB - Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5μg/ml human G117H BChE, 2μg/ml wild-type mouse BChE, and 0.06μg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.
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U2 - 10.1016/j.ab.2004.02.038
DO - 10.1016/j.ab.2004.02.038
M3 - Article
C2 - 15136175
AN - SCOPUS:2342529041
SN - 0003-2697
VL - 329
SP - 131
EP - 138
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -