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

Abstract

The effects of Ca²⁺ ions on the structure and activity of Ca²⁺ 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. This 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.