Limited proteolysis by chymotrypsin of midkine and inhibition by heparin binding

When digested with a low concentration of chymotrypsin, midkine (MK) underwent limited proteolysis and produced two fragments with Mr of 11,000 and 6,000 Da. The cleavage site was identified as on the carboxyl side of Phe55. This limited proteolysis was specifically inhibited by heparin, but not by other glycosaminoglycans. Using various heparin-derived oligosaccharides with different chain lengths or chemically desulfated heparin derivatives, it was shown that a minimum of 6 monosaccharide units was necessary for the inhibition, and that sulfonyl groups of the heparin disaccharide unit were required for inhibition. The present study showed that MK consists of two domains, N- and C-domains, and that Phe55 localized to the hinge region is exposed on the surface: of the molecule. It was also suggested that the N-domain may function as a stabilizing domain against proteolytic degradation of the C-domain in the intact molecule.