Human erythrocyte acetylcholinesterase is an amphipathic protein whose short membrane-binding domain is removed by papain digestion.
Human erythrocyte acetylcholinesterase was shown to be an amphipathic protein in which proteases could cleave the hydrophobic domain from the enzymatically active hydrophilic domain. Papain and Pronase cleaved these domains with greatest efficiency, as measured by the disaggregation of purified acetylcholinesterase to disulfide-linked dimers (G2) on sucrose density gradients in the absence of detergent. Nonspecific proteolytic degradation was reduced both by the inclusion of edrophonium chloride, which protected acetylcholinesterase from inactivation, and by covalent attachment of papain to Sepharose CL-4B. In contrast to nondigested control acetylcholinesterase, the papain-disaggregated enzyme did not bind detergent according to hydrodynamic criteria and could not be reconstituted into liposomes. Thus, we conclude that the hydrophobic domain removed by papain digestion is in fact the membrane-binding domain in situ. This domain appeared largely inaccessible to proteases in intact erythrocytes, however, as less than 10% of the enzyme activity was solubilized by protease digestion. The hydrophobic domain removed by papain appeared very small, as nondigested control and disaggregated enzyme were identical in molecular weight and amino acid composition within experimental error. The fully reduced 75-kDa catalytic subunits of nondigested control enzyme appeared about 2 kDa larger than the corresponding subunits of disaggregated enzyme on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, an indication that the hydrophobic domain was cleaved from the COOH or NH2 terminus of the catalytic subunit primary structure. Studies in which the NH-terminal amino acid was labeled by reductive methylation suggested that the hydrophobic domain is at the COOH terminus.
D000110:Acetylcholinesterase / Q000097:blood*; D001665:Binding Sites; D010450:Endopeptidases / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D010206:Papain / Q000494:pharmacology; D011485:Protein Binding
D046911:Macromolecular Substances; D000110:Acetylcholinesterase; D010450:Endopeptidases; D010206:Papain