Structure of human erythrocyte acetylcholinesterase. Characterization of intersubunit disulfide bonding and detergent interaction.

A large scale purification procedure for human erythrocyte acetylcholinesterase that involved affinity chromatography on an acridinium resin permitted the routine isolation of about 5 mg of enzyme from 10 liters of outdated erythrocytes. The purified enzyme had a specific activity of 5000-5800 units/mg of protein and was free of polypeptide contaminants by gel electrophoresis criteria. In detergents, the isolated enzyme corresponded to a disulfide-linked dimer (G2) that was converted to 75-kDa subunit monomers (G1) by reduction with dithiothreitol. No free sulfhydryl groups were detected prior to reduction, but reduction under nondenaturing conditions generated active G1 and produced 1.7 mol of free sulfhydryl groups/mol of subunit. These data were interpreted as indicating a single intersubunit disulfide bond in the G2 enzyme. In the absence of nonionic detergents, both the G2 and the G1 enzymes formed aggregates with average Stokes radii of 10 nm. Introduction of Triton X-100 gave enzyme-detergent complexes according to hydrodynamic criteria. Quantitative determination of [3H]Triton X-100 binding to G2 and G1 by a novel affinity chromatography procedure revealed that each G2 molecule bound about 140 detergent molecules and each G1, about 80. These observations indicated that each subunit in both G2 and G1 interacted individually with a Triton X-100 micelle. Molecular weight estimates for the protein components of the G2- and G1-detergent complexes were obtained from the hydrodynamic properties and the detergent binding data and corresponded to 160,000 and 85,000, respectively. Data in this and the accompanying paper (Dutta-Choudhury, T.A., and Rosenberry, T. L. (1984) J. Biol. Chem. 259, 5653-5660) provide strong evidence that erythrocyte acetylcholinesterase is an amphipathic protein.