Expression of recombinant human acetylcholinesterase and its application in screening its inhibitors.
This study is designed to obtain recombinant human acetylcholinesterase (rhAChE) and apply it in screening acetylcholinesterase inhibitors. The rhAChE was overexpressed in HEK293 cells transfected by plasmid of pCMV-AChE with the cationic liposome and rhAChE was found to be secreted into cell culture medium. AChE activity was assayed according to modified Ellman method to obtain kinetic parameters. IC so50 values for donepezil compounds of rhAChE were calculated to determine their activities of inhibition. The results showed that Km value was 151.9 micromol.L-1 donepezil inhibited rhAChE in a mixed competitive-noncompetitive way (Ki= 16.03 nmol.L-1, Ki = 18.36 nmol.L-1) and that most new compounds tested exhibited high activities of inhibition on rhAChE. The study suggests that rhAChE is available to be applied in screening AChE inhibitors in vitro.
Xiang-Jun
XJ
Wang
Huai-Xiu
HX
Wu
Shan-Shan
SS
Ye
Lan-Ying
LY
Pan
Yong-Chang
YC
Qian
D000110:Acetylcholinesterase / Q000235:genetics / Q000378:metabolism*; D002800:Cholinesterase Inhibitors / Q000032:analysis* / Q000494:pharmacology; D000077265:Donepezil; D057809:HEK293 Cells; D006801:Humans; D007189:Indans / Q000032:analysis* / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007700:Kinetics; D010880:Piperidines / Q000032:analysis* / Q000494:pharmacology; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002800:Cholinesterase Inhibitors; D007189:Indans; D010880:Piperidines; D011994:Recombinant Proteins; D000077265:Donepezil; D000110:Acetylcholinesterase