PROPROTEIN CONVERTASE PC1 3-RELATED PEPTIDES ARE POTENT SLOW TIGHT-BINDING INHIBITORS OF MURINE PC1/3 AND HFURIN/

The proprotein convertase PC1/3 belongs to the subtilisin/kexin-like e ndoprotease family and is synthesized as a preproenzyme. To investigat e the function of its propeptide, murine proPC1/3 and preproPC1/3 were isolated from the inclusion bodies of recombinant preproPC1/3 baculov irus-infected insect cells, rendered soluble with 6 M guanidine HCl an d 20 mM dithiothreitol, and purified by gel filtration and metal-bindi ng affinity chromatography. Two NH2-terminal fragments containing the complete propeptide 1-84 region were obtained after CNBr cleavage, pur ified, and chemically characterized, Progress curve kinetic analysis w ith enzymatically active murine 71-kDa PC1/3 or 50-kDa human furin dem onstrated that both fragments were potent slow tight-binding inhibitor s of either enzyme with K-i in the low nanomolar range. Additional cle avages at Trp residues yielded fragment(9-71), which no longer represe nts a potent inhibitor, Upon incubation at pH 5.5 in the presence of e xcess 71-kDa murine PC1/3, NH2-terminal fragment(1-98) is cleaved at t wo sites, as revealed through Western blotting using NH2-terminal-dire cted PC1/3 antibodies. Finally, murine PC2 is inhibited by the proPC1/ 3(1-98) peptide, albeit at a much lesser extent with a micromolar Ki a nd in a strictly competitive manner. These results suggest that the pr oregion of PC1/3 is an important feature in regulating its activity.