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Inhibition of Proteinase 3 by ₁-Antitrypsin In Vitro Predicts Very Fast Inhibition In Vivo

Laboratoire d'Enzymologie, INSERM Unité 392, Université Louis Pasteur de Strasbourg, Illkirch, France

Address correspondence to: Joseph G. Bieth, INSERM U 392, Faculté de Pharmacie, 74 route du Rhin, F-67400 Illkirch, France. E-mail: jgbieth{at}aspirine.u-strasbg.fr

Neutrophil proteinase 3 (Pr3) cleaves elastin and other matrix proteins, and is thought to cause lung tissue destruction in emphysema and cystic fibrosis. Its deleterious action is theoretically prevented by ₁-antitrypsin, a serpin present in lung secretions. We have evaluated the anti-Pr3 activity of this inhibitor to decide whether it may play a physiologic proteolysis-preventing function in vivo. We show that (i) the oxidized inhibitor does not inhibit Pr3; (ii) the inhibitor competes favorably with elastin for the binding of Pr3, but is less efficient for inhibiting elastin-bound proteinase than for complexing free enzyme; and (iii) the inhibition takes place in at least two steps: the enzyme and the inhibitor first form a high-affinity reversible inhibitory complex EI* with an equilibrium dissociation constant K^*_i of 38 nM; EI* subsequently transforms into an irreversible complex EI with a first-order rate constant k₂ of 0.04 s^-1. Because the ₁-antitrypsin concentration in the epithelial lining fluid is much higher than K^*_i, any Pr3 molecule released from neutrophils will be taken up as an EI* complex within much less than 1 s, indicating very efficient inhibition in vivo.

Abbreviations: ₁-antitrypsin, 1-AT • ₁-proteinase inhibitor, ₁PI • human neutrophil proteinase 3, Pr3 • remazol-brilliant-blue-labeled bovine neck ligamentum elastin, RBB-elastin • methoxysuccinyl-lysine-(2-picolinoyl)-alanine-proline-valine-thiobenzyl ester, TBE

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