Acetylcholinesterase in Mouse Neuroblastoma NB₂A Cells: Analysis of Production, Secretion, and Molecular Forms

Abstract

The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.

Association of the HNK‐1 epitope with the detergent‐soluble G4 isoform of acetylcholinesterase from human neuroblastoma cells

“…Previous work on this cell line (Johnson and Moore, 2000) has shown that there are no high salt-soluble isoforms present. These results on the distribution of AChE isoforms confirm others on neuroblastoma cell lines (Kimhi et al, 1980;Lazar and Vigny, 1980;Melone et al, 1987;Stieger et al, 1989). The increase in AChE levels seen on differentiation is well documented, and is frequently used as a differentiation marker in many cell types.…”

Cholinesterases modulate cell adhesion in human neuroblastoma cells in vitro

“…There is no chemical or structural basis for a difference in substrate affinity: butyrylcholine is simply a larger molecule that cannot access the more constrained active site of AChE. These results on neuroblastoma AChE and BChE levels, both cell-associated and in the medium, confirm those of other workers [45][46][47].…”

Hierarchy of Post-translational Modifications Involved in the Circulatory Longevity of Glycoproteins

“…In Vitro Tetramerization of BoAChE and Its Effect on Circulatory Longevity-In contrast to the rBoAChE that was characterized by the predominant presence of dimeric and monomeric forms, naturally occurring forms of AChE, whether circulatory or membrane-bound, are arranged in multimeric complexes (16,39,(57)(58)(59)(60). Dimerization of catalytic subunits of AChE occurs invariably by covalent disulfide bridging involving cysteine residues situated in the C-terminal domains of the enzyme (14).…”