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

The inhibition of human chymase by the protease inhibitor alpha(2)-macroglo bulin (alpha 2M) was investigated. Titration of chymase hydrolytic activity with purified alpha 2M showed that approximately 1 mol of alpha 2M tetrame r 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-term inal sequencing of chymase-treated a2M revealed cleavage at bonds Phe684-Ty r685 and Tyr685-Glu686 of the bait region. alpha 2M pretreated with methyla mine, an inactivator of alpha 2M, did not inhibit chymase. The apparent sec ond-order rate constant for inhibition (K-ass) was 5 x 10(6) M-1 s(-1), mak ing alpha 2M the most efficient natural protein protease inhibitor of chyma se so far described. The k(ass) value for inhibition was decreased approxim ately 10-fold by addition of heparin, a glycosaminoglycan produced by mast cells that binds to chymase. Heparin did not change significantly the stoic hiometry of inhibition or block covalent complex formation. These results i ndicate that alpha 2M is an important inhibitor to consider in the regulati on of human chymase. (C) 1999 Academic Press.