11. Component Peptides Obtained by Selective Proteolysis and Disulfide Bond Reduction*

The asymmetric forms (17 S and 13 S ) of acetylcholinesterase isolated from Torpedo californica electric organs have been subjected to collagenolytic and tryptic digestions and the hydrodynamic properties and component peptides of the products analyzed. The asymmetric forms contain the catalytic subunits and a trypsin-sensitive structural subunit attached to a filamentous collagen-like tail unit. Digestion with a purified Clostridial collagenase at 25 "C removes a segment of the tail unit leading to formation of stable acetylcholinesterase species with larger sedimentation coefficients (20 S and 16 S). Exposure to 37 "C results in a partial unfolding of the structure such that more extensive proteolysis by collagenase or trypsin occurs. At 37 "C, collagenase digestion of the 17 S species leads to formation of 20 S, 16 S, and 11 S species in a 1:2:1 ratio. Subsequent disulfide bond reduction then leads to formation of an 11 S species. Analysis of the peptide composition of acetylcholinesterase following collagenase digestion shows that the multiplicity of high molecular weight disulfide-linked polypeptides seen with the native enzyme is retained, but upon the removal of the tail unit their molecular weights are reduced. Following exposure to dithiothreitol, the high molecular weight disulfide-linked polypeptides are found to be composed of varying ratios of the catalytic subunit of 68,000 daltons and a 100,000-dalton structural subunit. The latter subunit is unusually sensitive to tryptic digestion and concomitant with the formation of an 85,000-dalton fragment from the 100,000-dalton subunit is the conversion of the larger polypeptides to monomers and oligomers of the catalytic subunit. Thus, the 100,000-dalton peptide appears to disulfide-link terminal portions of the individual chains in the tail unit and is in close apposition to the catalytic subunits. This peptide may be analogous to a procollagen or noncollagenous basement membrane peptide and during assembly of the asymmetric forms of acetylcholinesterase it is probably removed by proteolysis enabling replacement by the catalytic subunits.