Acetylcholinesterase

This chapter presents procedures for isolation of 18 S and 14 S eel acetylcholinesterase, for the pepsin-resistant triple- helical tail subunit fragments, and for disulfide-reduced and denatured intact tail subunits. Isolation of 18 S and 14 S acetylcholinesterase from eel electric organ by affinity chromatography involves preparation of acridinium-linked resin. In isolation of pepsin-resistant fragments of 18 S and 14 S acetylcholinesterase, pepsin digestion of tissue or tissue extracts at low pH is frequently used to solubilize collagen-like proteins or selectively to degrade noncollagen-like protein domains. In isolation of intact tail subunits, dissociation of the intact tail subunits from catalytic subunits requires both reduction of the intersubunit disulfide bonds and exposure to a denaturing solvent. The isolation procedure involves: extraction of native enzyme; DEAE-cellulose column chromatography; and Sepharose CL 6B column chromatography. It is found that The 27% glycine content of the pepsin-resistant fragments is somewhat higher than that of the intact tail subunits, but this percentage is low enough to indicate that both preparations contain noncollagen-like domains. A major portion of the difference between the two preparations is contributed by the 8000-dalton residual tail subunit fragment generated by endogenous protease conversion of 18 S to 11 S acetylcholinesterase.