Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase.
Acetylcholinesterase is an enzyme whose best-known function is to hydrolyze the neurotransmitter acetylcholine. Acetylcholinesterase is expressed in several noncholinergic tissues. Accordingly, we report for the first time the identification of acetylcholinesterase in human umbilical cord vein endothelial cells. Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubilization of human endothelial cell membranes with Triton X-100. These extracts were analyzed under polyacrylamide gel electrophoresis in the presence of Triton X-100 and under nondenaturing conditions, followed by specific staining for cholinesterase or acetylcholinesterase activity. The gels revealed one enzymatically active acetylcholinesterase band in the extracts that disappeared when staining was performed in the presence of eserine (an acetylcholinesterase inhibitor). Performing western blotting with the C-terminal anti-acetylcholinesterase IgG, we identified a single protein band of approximately 70 kDa, the molecular mass characteristic of the human monomeric form of acetylcholinesterase. The western blotting with the N-terminal anti-acetylcholinesterase IgG antibody revealed a double band around 66-70 kDa. Using the Ellman's method to measure the cholinesterase activity in human umbilical vein endothelial cells, regarding its substrate specificity, we confirmed the existence of an acetylcholinesterase enzyme. Our studies revealed a predominance of acetylcholinesterase over other cholinesterases in human endothelial cells. In conclusion, we have demonstrated the existence of a membrane-bound acetylcholinesterase in human endothelial cells. In future studies, we will investigate the role of this protein in the endothelial vascular system.
Carvalho
Filomena A
FA
Instituto de Biopatologia Química, Faculdade de Medicina de Lisboa/Unidade de Biopatologia Vascular, Instituto de Medicina Molecular, Lisbon, Portugal. filomenacarvalho@fm.ul.pt
Graça
Luís M
LM
Martins-Silva
João
J
Saldanha
Carlota
C
D000107:Acetylation; D000110:Acetylcholinesterase / Q000378:metabolism*; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D008078:Cholesterol, LDL / Q000737:chemistry / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000166:cytology* / Q000201:enzymology*; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D014471:Umbilical Veins / Q000201:enzymology*
D008078:Cholesterol, LDL; D002800:Cholinesterase Inhibitors; D000110:Acetylcholinesterase