Isolation of acetylcholinesterase from apoptotic human lung fibroblast cells by antibody affinity chromatography.
Acetylcholinesterase (AChE; EC3.1.1.7) is well known for its role in the hydrolysis of acetylcholine at cholinergic synapses to terminate neurotransmission. In addition to its synaptic presence, AChE has been found to be in non-cholinergic cells such as hematopoietic and osteogenic cells. We have recently reported that AChE is expressed in various cells undergoing apoptosis. To characterize AChE in apoptotic cells and to investigate the role of AChE expression in apoptosis, we devised a method to purify AChE expressed in apoptotic human lung fibroblast cell line HLF. The isolation of this enzyme is mainly based on inhibitor ligand affinity chromatography using immobilized tacrine. However, this method is only effective in isolating active AChE. Here we employed antibody-based chromatography and found that both active and inactive AChE were present in apoptotic HLF cells. Active AChE was predominantly observed in the nuclei of apoptotic cells, while inactive AChE was mainly present in the cytoplasm. Therefore, our method provides an opportunity to investigate further the role of AChE, especially inactive AChE, in apoptosis.
Jin
Qi-Huang
QH
Chinese Academy of Sciences, Shanghai.
Shi
Yu-Fang
YF
He
Heng-Yi
HY
Ng
Kelvin K W
KK
Jiang
Hua
H
Yang
Lei
L
Jiang
Zi-Qing
ZQ
Zhang
Xue-Jun
XJ
D000110:Acetylcholinesterase / Q000276:immunology / Q000302:isolation & purification*; D000122:Acetylthiocholine / Q000378:metabolism; D000818:Animals; D000918:Antibody Specificity; D017209:Apoptosis; D015153:Blotting, Western; D002478:Cells, Cultured / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods*; D003593:Cytoplasm / Q000201:enzymology; D004524:Eels; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D020287:In Situ Nick-End Labeling; D008168:Lung / Q000166:cytology* / Q000201:enzymology; D011817:Rabbits; D013053:Spectrophotometry
D000122:Acetylthiocholine; D000110:Acetylcholinesterase