Characterization of Pepsin-Resistant Collagen-like Tail Subunit Fragments of 18S and 14S Acetylcholinesterase from Electrophorus electricus

Digestion of 18S and 14S acetylcholinesterase from eel electric organ with pepsin at 15 °C for 6 h results in extensive degradation of the catalytic subunits, but a major portion of the collagen-like tail structure associated with these enzyme forms resists degradation. The pepsin-resistant structures partially aggregate and can be isolated by gel exclusion chromatography on Sepharose CL-6B in buffered 1 M sodium chloride. The largest structure, denoted F₃, has a molecular weight of 72000 according to gel electrophoresis in sodium dodecyl sulfate and is composed of three 24 000 molecular weight polypeptides linked by intersubunit disulfide bonds. This structure is largely, but not completely, a collagen-like triple helix as indicated by a circular dichroism spectrum typical of triple-helical collagen and an amino acid composition characterized by 27% glycine, 5% hydroxyproline, and 5% hydroxylysine. Continued pepsin action results in degradation of the disulfide linkage region such that disulfide-linked dimers F₂ and finally F₁ monomers become the predominant forms in sodium dodecyl sulfate. Digested samples in which either F₃ or F₂ predominate have virtually identical circular dichroic spectra and amino acid compositions and generate similar diffuse 24 000 molecular weight polypeptides following disulfide reduction. Thus the intersubunit disulfide linkages in F₃ must occur close to the end(s) of the fragment polypeptide chains. Pepsin conversion of F₃ to F₂ is particularly accelerated between 25 and 30 °C, suggesting that the triple-helical structure in the disulfide linkage region undergoes thermal destabilization in this temperature range. Digestion at 40 °C yields presumably triple-helical F₁ structures devoid of disulfide linkages, although their degradation to small fragments can be detected at this temperature. The question of whether the three tail subunits that give rise to F, polypeptides are identical remains open.