Characterization of acetylcholinesterase in Caco-2 cells.
Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-beta-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.
Plageman
Lauren R
LR
Division of Pharmaceutical Sciences, College of Pharmacy, University of Cincinnati, 3223 Eden Avenue, Cincinnati, OH 45267, USA.
Pauletti
Giovanni M
GM
Skau
Kenneth A
KA
D000109:Acetylcholine / Q000378:metabolism*; D000110:Acetylcholinesterase / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology*; D018938:Caco-2 Cells / Q000201:enzymology*; D002454:Cell Differentiation / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology; D004298:Dopamine / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D012995:Solubility; D013347:Subcellular Fractions; D013379:Substrate Specificity
D000110:Acetylcholinesterase; D000109:Acetylcholine; D004298:Dopamine