Abstract
To determine the substrate recognition mechanism in calcium-activated neutral protease (CANP), the hydrolytic velocities for some possible substrates were compared. In general, succinylated polypeptides were poorer substrates than unmodified ones, suggesting that CANP interacts with positively charged amino groups and/or repels negatively charged succinyl groups in substrates. Among the substrates examined, protamine was degraded quite rapidly in a restricted manner. This degradation of protamine was remarkably accelerated by the addition of salt, and, in the absence of salt, protamine was inhibitory as to the degradation of vimentin by CANP. Protamine was separated into components and the sites cleaved by CANP were determined. CANP cleaved the clupeine YII and Z components at two sites, both being arginyl-arginine bonds, and the amino acid sequences around these sites were almost identical between YII and Z. No other arginyl-arginine bond was cleaved at all. These results showed that CANP prefers basic amino acid side chains but its specificity is very restricted.
