PubMed · 891
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T L Rosenberry. 1975. Acetylcholinesterase.. https://doi.org/10.1002/9780470122884.ch3
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Modification of Torpedo californica acetylcholinesterase (AChE) both by bis(1-oxy-2,2,5,5-tetramethyl-3-imidazolin-4-yl)disulfide (biradical) and by 4,4'-dithiopyridine, via a thiol-disulfide exchange reaction, was monitored by EPR and optical spectroscopy, respectively. Incubation with these reagents caused complete loss of enzymic activity. Treatment with glutathione of AChE modified by either of the two disulfides led to rapid release of the bound reagent with simultaneous regeneration of the single free thiol group of the enzyme. However, no concomitant recovery of catalytic activity was observed. SDS-PAGE showed that both the modified and demodified enzymes retained their structure as a disulfide-linked dimer. Circular dichroism revealed that modification of AChE by the disulfide agents with or without demodification by glutathione led to a complete disappearance of the ellipticity in the near-UV and to a much smaller decrease in ellipticity in the far-UV. The CD spectra observed are typical of the "molten globule" state of proteins. 1-Anilino-8-naphthalenesulfonate binding measurements and an enhanced susceptibility to trypsinolysis supported the supposition that chemical modification had transformed native AChE to a "molten globule".
The type of membrane association of acetylcholinesterase (AChE, EC 3.1.1.7) was studied in rabbit lymphocytes and erythrocytes. In both cases, the unique AChE molecular form was an amphiphilic dimer (referred to as G2a) anchored in the membrane by a glycosylphosphatidylinositol. In lymphocytes, G2a AChE was directly converted into its hydrophilic G2h counterpart by a treatment with Bacillus thuringiensis phosphatidylinositol-phospholipase C (PI-PLC, EC 3.1.4.10). In erythrocytes, AChE was resistant to PI-PLC but was rendered sensitive by a prior deacylation with alkaline hydroxylamine. This observation suggests that, as previously reported for human erythrocyte AChE, an acylation of the inositol ring in the glycolipid anchor of rabbit erythrocyte AChE (that does not occur in lymphocytes) prevents the cleavage.
To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.