Acetylcholinesterase (AChE) expression is regulated in cell types at the transcriptional and translational levels. In this study, we characterized and compared AChE catalytic activity, mRNA, protein expression, and protein localization in a variety of neuronal (SH-SY5Y neuroblastoma and primary cerebellar granule neurons (CGN)) and non-neuronal (LLC-MK2, HeLa, THP-1, and primary astrocytes) cell types. All cell lines expressed AChE catalytic activity; however the levels of AChE-specific activity were higher in neuronal cells than in the non-neuronal cell types. CGN expressed significantly more AChE activity than SH-SY5Y cells. All cell lines analyzed expressed AChE protein at equivalent levels, as well as mRNA splice variants. Localization of AChE was characterized by immunofluorescence and confocal microscopy. SH-SY5Y, CGN, and nerve-growth factor-differentiated PC-12 cells exhibited a pattern of AChE localization characterized as diffuse in the cytoplasm and punctate staining along neurites and on the plasma membrane. The localization in HeLa, LLC-MK2, fibroblasts, and undifferentiated PC-12 cells was significantly different than in neuronal cells-AChE was intensely localized in the perinuclear region, without staining near or on the plasma membrane. Based on the evidence presented here, we hypothesize that the presence of AChE protein doesn't correlate with catalytic activity, and the diffuse cytoplasmic and plasma membrane localization of AChE is a property of neuronal cell types. Keywords acetylcholinesterase; confocal microscopy; cerebellar granule neuron; neuroblastoma; SH-SY5Y; HeLa; PC-12 Acetylcholinesterase (AChE) belongs to the serine hydrolase family that contains the α/β hydrolase fold as a common structural element [Ollis et al., 1992]. The catalytic active site of AChE is made up of a precise arrangement of active site functional groups that catalyze the hydrolytic breakdown of esters that bear quaternary ammonium groups. The biological importance of AChE-catalyzed hydrolysis is manifested in the rapid termination of the neuronal impulse that occurs when acetylcholine (ACh) is released into the synaptic cleft. AChE rapidly hydrolyzes ACh into acetic acid and choline at extremely fast turnover rates [Taylor and Radic, 1994].

Two types of polyclonal antibodies were generated from (a) a decapeptide sequence that includes the active site serine of acetylcholinesterase (anti-AChE 10S ) and (b) the identical decapeptide sequence phosphorylated at the active site serine of acetylcholinesterase (antiAChE 10SP ). The anti-AChE 10S antiserum was found to specifically recognize native, control, and vehicletreated recombinant mouse AChE (rMoAChE) but did not recognize rMoAChE that was phosphorylated by the four organophosphate (OP) compounds tested. Conversely the anti-AChE 10SP antiserum recognized phosphoserine rMoAChE that resulted from reaction with phosphorous oxychloride (POCl 3 ) but did not recognize native or vehicle-treated rMoAChE. Anti-AChE 10SP also did not recognize OP-AChE conjugates that resulted from the reaction of rMoAChE with other OP compounds that afford neutral or monoanionic phosphoserine groups thereby indicating a high specificity for a precise OP conjugate. Antisera recognition correlated well with the rates of enzyme inhibition, aging, and oxime-induced reactivation indicating these antisera can both quantify the extent and type of inhibition and also differentiate between select mechanisms of inhibition. The ability to discern mechanistic differences between native AChE and OP-AChE conjugates suggests that these antisera can be used to identify biomarkers of OP exposure in a mechanism-based approach.