Highly Efficient Inhibition of Human Chymase by α(2)-Macroglobulin

Abstract

The inhibition of human chymase by the protease inhibitor α(2)-macroglobulin (α2M) was investigated. Titration of chymase hydrolytic activity with purified α2M showed that approximately 1 mol of α2M tetramer inhibits 1 mol of chymase. Inhibition was associated with cleavage of the α2M bait region and formation of a 200-kDa covalent complex. NH2-terminal sequencing of chymase-treated α2M revealed cleavage at bonds Phe684–Tyr685 and Tyr685–Glu686 of the bait region. α2M pretreated with methylamine, an inactivator of α2M, did not inhibit chymase. The apparent second-order rate constant for inhibition (kass) was 5 × 106 M−1 s−1, making α2M the most efficient natural protein protease inhibitor of chymase so far described. The kass value for inhibition was decreased approximately 10-fold by addition of heparin, a glycosaminoglycan produced by mast cells that binds to chymase. Heparin did not change significantly the stoichiometry of inhibition or block covalent complex formation. These results indicate that α2M is an important inhibitor to consider in the regulation of human chymase.