Highly efficient inhibition of human chymase by α(2)-macroglobulin

The inhibition of human chymase by the protease inhibitor alpha(2)-macroglobulin (alpha 2M) was investigated. Titration of chymase hydrolytic activity with purified alpha 2M showed that approximately 1 mol of alpha 2M tetramer inhibits 1 mol of chymase. Inhibition was associated with cleavage of the alpha 2M bait region and formation of a 200-kDa covalent complex. NH2-terminal sequencing of chymase-treated a2M revealed cleavage at bonds Phe684-Tyr685 and Tyr685-Glu686 of the bait region. alpha 2M pretreated with methylamine, an inactivator of alpha 2M, did not inhibit chymase. The apparent second-order rate constant for inhibition (K-ass) was 5 x 10(6) M-1 s(-1), making alpha 2M the most efficient natural protein protease inhibitor of chymase so far described. The k(ass) 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 alpha 2M is an important inhibitor to consider in the regulation of human chymase. (C) 1999 Academic Press.