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- Title
Expi 293 CELLS EXPRESSING AN ENDOGENOUS WILD-TYPE BUTYRYLCHOLINESTERASE, AND A VARIETY OF ESTERASES THAT SELF-REACTIVATES AFTER PHOSPHYLATION BY ALL TYPES OF ORGANOPHOSPHORUS AGENTS.
- Authors
Zueva, Irina; Lockridge, Oksana; Schopfer, Lawrence M.; Masson, Patrick
- Abstract
A human embryonic kidney cell line (Expi293), adapted for suspension growth in serum-free medium, secretes a tetrameric butyrylcholinesterase (BChE). Expression levels are very low, but are increased 10-fold upon treatment with polyethylenimine. DNA sequencing shows that this enzyme is wild-type BCHE. This endogenous BChE displays catalytic properties very close to that of natural huBChE with butyrylthiocholine and N-methylindoxyl acetate as substrates [1]. Several endogenous co-secreted esterases self-reactivate after inhibition by echothiophate, paraoxon, cresyl saligenin phosphate (CBDP), racemic coumarin(CM)-soman, CM-tabun and CM-VX. Overall reactivation rate constants, kr, of diethylphosphorylated enzymes after inhibition by echothiophate and paraoxon are 0.171 min-1 and 0.059 min-1, respectively, suggesting multiple OP-hydrolyzing enzymes. After phosphonylation by CM-soman, CM-tabun and CM-VX, kr values range from 0.0375 min-1 to 0.0078 min-1. kr of CBDP-inhibited enzyme is 0.028 min-1. Interestingly, an apparent aging rate is observed after phosphylation. The aging rate of the soman-phosphonylated enzyme(s) is approximately 2-fold slower than for wtBChE (half-time =16 min against 9 min for wtBChE [2]). The half-time for aging after inhibition by CBDP is 31 min whereas aging of wtBChE-CBDP is almost instantaneous [3]. Diethylphosphorylated enzyme(s) inhibited by paraoxon and echiothiophate age(s) with apparent ka =0.162 min-1 and 0.057 min-1, respectively. This difference also supports the multiple enzyme hypothesis. Further studies are in progress to indentify the different OP-reacting enzymes produced by this Expi293 cell line.
- Subjects
BUTYRYLCHOLINESTERASE; ESTERASES; ORGANOPHOSPHORUS compounds; SERUM-free culture media; NUCLEOTIDE sequencing
- Publication
Military Medical Science Letters / Vojenské zdravotnické Listy, 2018, Vol 87, p96
- ISSN
0372-7025
- Publication type
Article