Studies on a Ca2+-activated neutral proteinase of rabbit skeletal muscle. II. Characterization of sulfhydryl groups and a role of Ca2+ ions in this enzyme.

The effects of Ca2+ ions on the structure and activity of Ca2+-activated neutral proteinase (CANP) were examined by means of carboxymethylation. CANP was inactivated by carboxymethylation of 1 mol of sulfhydryl group in the 80 K subunit. The sulfhydryl group was exposed to the solvent, and the buried single sulfhydryl group, which was exposed by addition of Ca2+ ions, was not essential for activity. The carboxymethylated CANP (Cm-CANP), though inactive, was indistinguishable from native CANP with respect to conformation. The structural change of Cm-CANP induced by Ca2+ at various Ca2+ concentrations and pH values corresponded well to the activity-Ca2+ and activity-pH relationships of native CANP. It is suggested that the observed conformational changes were essential for the appearance of enzyme activity. The induced structural changes occurred only around a cysteine residue and Trp and/or Tyr residues, and no significant changes in the secondary structures were observed.