Hydrolytic and Autolytic Behavior of Two Forms of Calcium-Activated Neutral Protease (CANP)

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Abstract
Some endogenous substrates were incubated with two forms of calcium-activated neutral protease (CANP) with high (μCANP) and low (mCANP) sensitivities to calcium ions. In addition to analyses of the processes of their degradation, changes in the molecular properties of these CANPs were also examined. Among the tested substrate proteins, the myosin heavy chain of rabbit skeletal muscle myofibrils and spectrin or band 3 protein of human erythrocyte membranes were degraded relatively rapidly. So far as these proteins were concerned, a higher degradation velocity was observed for μCANP than for mCANP. Vimentin from ascites tumor cells was degraded most rapidly and no difference was observed in degradation velocity between μCANP and mCANP. In all cases, μCANP and mCANP produced different proteolytic peptide fragments, suggesting the different substrate-specificities of these CANPs. The degradation of substrates always accompanied the autodigestion of CANPs, and the small subunits of both CANPs were degraded in the early stage of the auto-digestion. The large subunit of μCANP (79K) was converted to a 76K polypeptide via a 77K polypeptide as an intermediate. The autodigested μCANP with 76K polypeptide retained sufficient protease activity and, moreover, its calcium-sensitivity was higher than that of intact μCANP. The possibility is thus proposed that restricted autodigestion is a necessary activation step for the appearance of activity of μCANP. No such transition was observed for mCANP.

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