Limited Autolysis of Calcium-Activated Neutral Protease (CANP): Reduction of the Ca2+-Requirement Is Due to the NK2-Tenninal Processing of the Large Subunit1

Abstract

Calcium-activated neutral protease (rabbit mCANP), composed of large and small subunits, was converted to a lower-Ca ²⁺ -requiring form (derived μCANP) by limited autolysis in the presence of Ca ²⁺ . The NH ₂ -terminal regions of the two subunits of mCANP were cleaved by autolysis, but the COOH-termini remained intact after autolysis. When native mCANP or derived μCANP was dissociated into subunits, the proteolytic activity of the large subunit was reduced to 2–5% of that of the native dimeric enzyme. The Ca ²⁺ -sensitivity of one hybrid CANP reconstituted from the large subunit of derived μCANP and the small subunit of native mCANP was similar to that of derived μCANP. However, the other hybrid molecule composed of the large subunit of native mCANP and the small subunit of derived μCANP required a high concentration of Ca ²⁺ for activity, like native mCANP. These results indicate that the Ca ²⁺ -sensitivity of derived μCANP is determined by the structural change of the large subunit resulting from loss of its NH ₂ -terminal region. The autolysis of the small subunit apparently has no effect on the reduction of the Ca ²⁺ -requirement.