Inhibition of neutrophil elastase by alpha-1-proteinase inhibitor oxidized by activated neutrophils.
The present study was aimed at testing whether neutrophil-oxidized alpha 1-proteinase inhibitor (alpha 1PI) is a slow-binding inhibitor of neutrophil elastase like N-chlorosuccinimide-oxidized alpha 1PI or whether it does not inhibit this enzyme at all as currently thought. alpha 1PI was reacted with phorbol-myristate-acetate-activated neutrophils and isolated from the oxidation medium by fast protein liquid chromatography on an anion exchange column. When sufficient time was allowed for oxidized alpha 1PI to react with neutrophil elastase (2 h), enzyme-inhibitor complex formation could be demonstrated by three means: (1) inhibition of elastase activity, (2) detection of the complex using an enzyme-linked immunosorbent assay specific for the native alpha 1PI-elastase complex, and (3) SDS-polyacrylamide gel electrophoresis, which evidenced a complex with a molecular weight of 80,000. Porcine pancreatic elastase was not inhibited. Neutrophil-oxidized alpha 1PI behaved as an irreversible inhibitor of neutrophil elastase, as revealed by the kinetic analysis of the inhibition reaction. The rate constant for the inhibition of neutrophil elastase by neutrophil-oxidized alpha 1PI (0.76 +/- 0.22 x 10(4) M-1 s-1) was close to that for the inhibition of the enzyme by N-chlorosuccinimide-oxidized alpha 1PI (0.96 +/- 0.24 x 10(4) M-1 s-1), but it was more than three orders of magnitude lower than that for the reaction of native alpha 1PI with neutrophil elastase. Both native and oxidized alpha 1PI were temporary elastase inhibitors: the enzyme slowly and spontaneously escaped the complex with formation of an inactive alpha 1PI derivative with a molecular weight of 49,000.(ABSTRACT TRUNCATED AT 250 WORDS)
Padrines
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Laboratoire d'Enzymologie, INSERM U 237, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, Illkirch, France.
Schneider-Pozzer
M
M
Bieth
J G
JG
D001798:Blood Proteins / Q000378:metabolism*; D003470:Culture Media; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D010084:Oxidation-Reduction; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D000515:alpha 1-Antitrypsin
D001798:Blood Proteins; D003470:Culture Media; D000515:alpha 1-Antitrypsin; D010196:Pancreatic Elastase