Neurochemistry International Vol.2, pp.135-147. Pergamon Press Ltd. 1980. Printed in Great Britain. THE COLLAGEN-LIKE SUBUNITS OF ACETYLCHOLINESTERASE FROM THE EEL ELECTROPHORUS ELECTRICUS Terrone L. Rosenberry, Philip Barnett, and Carol Mays The Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106 and the Department of Neurology, Columbia University, New York, New York 10032 ABSTRACT Pepsin-resistant fragments of the tail subunits of 14S and 18S acetylcholinesterase from eel electric organ have been isolated and characterized. The native fragments are composed of three 24,000 molecular weight polypeptides linked by intersubunit disulfide bonds in a collagen-like triple helix. Intact tail subunits have also been isolated. These subunits appear to contain both the triple-helical domain and a noncollagenous domain that is linked to catalytic subunits by disulfide bonds. KEYWORDS Acetylcholinesterase; collagen-like subunits; pepsin-resistant fragments; basement membrane; neuromuscular junction INTROOUCTION In 1937, David Nachmansohn observed that acetylcholinesterase activity in muscle appeared at least three to six times higher in endplate regions than in extrajunctional regions (Marnay and Nachmansohn, 1937). This observation led Nachmansohn to investigate the enzyme activity in electric organs of electric fish, tissues known to be rich in synaptic innervation. Extraordinary concentrations were found, and the firstsoluble extract of acetylcholinesterase from the electric organ of the ray Torpedo marmorata was obtained in 1938 (Nachmansohn and Lederer, 1939). A 16S form of acetylcholinesterase was later shown to be specifically associated with rat diaphragm endplates (Hall, 1973) after Massoulie and Rieger (1969) had demonstrated the u t i l i t y of sucrose gradient sedimentation in characterizing the distribution of acetylcholinesterase forms in eel electric organ extracts. 14S and 18S Eel forms of the enzyme appear to be closely analogous to the 16S rat form, and the protein structure of these 14S and 18S forms has been studied in some detail (Rosenberry and Richardson, 1977). Collagen-like subunits have been identified that appear to anchor these enzyme forms in the extracellular basement membrane at peripheral cholinergic synapses (McMahan and colleagues, 1978). Because basement membrane components may serve important regulatory roles in developing synapses (Sanes and colleagues,1978), we have characterized in some detail the very unusual collagen-like tail subunits of 14S and 18S eel acetylcholinesterase. ISOLATION OF PEPSIN-RESISTANT FRAGMENTS OF ACETYLCHOLINESTERASE The digestion of tissue or tissue extracts with pepsin is frequently employed to solubilize collagen-like proteins or to selectively degrade noBcollagen-like regions in these proteins. The technique was introduced by Rubin and colleagues (1963), who showed that pepsin digestion of acid-soluble calf skin collagen at low pH released small peptides to the dialysate with amino acid compositions not typical of triple-helical collagen while retaining most of the protein in native triple-helical form. The collagen-like regions that resist degradation by pepsin can be fractionated into various collagen types by differential salt precipitation (Orkin and colleagues, 1977; Sage and Bornstein, 1979). In preliminary experiments we established that salt precipitation was not required for the isolation of pepsin-resistant fragments of 18S and 14S eel acetylcholinesterase and that gel exclusion chromatography in a nondenaturing solvent was more direct and provided a better yield. The isolation procedure is ]35