Assembly of monomeric acetylcholinesterase into tetrameric and asymmetric forms.
A pulse-chase experiment was performed in embryonic rat myotube cultures to examine possible precursor-product relationships among the various molecular forms of acetylcholinesterase (AChE). AChE was labeled with paraoxon, a compound which diethylphosphorylates AChE at its active site. Diethylphosphorylated (labeled) AChE is inactive but can be reactivated by treatment with 1-methyl-2-hydroxyiminomethyl-pyridinium. Thus labeled enzyme could be followed as AChE that regained activity following treatment with 1-methyl-2-hydroxyiminomethylpyridium. To selectively label monomeric AChE (the hypothesized precursor form), cultures were treated with methanesulfonylfluoride which irreversibly inactivated more than 97% of total cellular AChE. Methylsulfonylfluoride was then washed from the cultures, and they were labeled with paraoxon during a 40-55-min recovery period. AChE appearing in the cultures during this recovery period is newly synthesized and consists almost entirely (92%) of the monomeric form. Immediately and 120-130 min after labeling, cultures were subjected to a sequential extraction procedure to separate globular from asymmetric forms. Individual forms were then separated by velocity sedimentation on sucrose gradients. In our first series of experiments, we observed a 55% decrease in labeled monomers during the chase, a 36% increase in labeled tetramers, and a 36% increase in labeled asymmetric forms. In a second series of experiments focused on individual asymmetric forms, we observed a 55% decrease in labeled monomers, a 58% increase in labeled tetramers, an overall increase of 81% in labeled asymmetric forms, and a 380% increase in labeled A12 AChE. These data provide the first uniequivocal proof that complex forms of AChE are assembled from active monomeric precursors.
D000110:Acetylcholinesterase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D005260:Female; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology; D010261:Paraoxon / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D046911:Macromolecular Substances; D011108:Polymers; D000110:Acetylcholinesterase; D010261:Paraoxon