Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants

Yuxia Wang, Lawrence M. Schopfer, Ellen G. Duysen, Florian Nachon, Patrick Masson, Oksana Lockridge

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)131-138
Number of pages8
JournalAnalytical Biochemistry
Volume329
Issue number1
DOIs
StatePublished - Jun 1 2004

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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