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Inhibition of neutrophil elastase by recombinant human proteinase inhibitor 9
J R Dahlen1, D C Foster, W Kisiel
1Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.
Abstract:
Proteinase inhibitor PI9 (PI9) is an intracellular 42-kDa member of the ovalbumin family of serpins that is found primarily in placenta, lung and lymphocytes. PI9 has been shown to be a fast-acting inhibitor of granzyme B in vitro, presumably through the utilization of Glu(340) as the P(1) inhibitory residue in its reactive site loop. In this report, we describe the inhibition of human neutrophil elastase by recombinant human PI9. Inhibition occurred with an overall K(i)' of 221 pM and a second-order association rate constant of 1.5 x 10(5) M(-1) s(-1), indicating that PI9 is a potent inhibitor of this serine proteinase in vitro. In addition, incubation of recombinant PI9 with native neutrophil elastase resulted in the formation of an SDS-resistant 62-kDa complex. Amino-terminal sequence analyses provided evidence that inhibition of elastase occurred through the use of Cys(342) as the reactive P(1) amino acid residue in the PI9 reactive site loop. Thus, PI9 joins its close relatives PI6 and PI8 as having the ability to utilize multiple reactive site loop residues as the inhibitory P(1) residue to expand its inhibitory spectrum.
Insights
Proteinase inhibitor PI9 (PI9) potently inhibits human neutrophil elastase in vitro. This serpin utilizes a novel reactive site residue, expanding its inhibitory capabilities against serine proteinases.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Proteinase inhibitor PI9 (PI9) is an intracellular serpin.
- PI9 is known to inhibit granzyme B.
- Its inhibitory mechanism involves specific reactive site residues.
Purpose of the Study:
- To investigate the inhibition of human neutrophil elastase by recombinant human PI9.
- To characterize the inhibitory kinetics and mechanism.
Main Methods:
- Recombinant human PI9 production.
- In vitro inhibition assays with human neutrophil elastase.
- Determination of inhibition constants (Ki') and association rate constants.
- SDS-PAGE and amino-terminal sequencing to identify the reactive site.
Main Results:
- PI9 is a potent inhibitor of human neutrophil elastase with a Ki' of 221 pM.
- The second-order association rate constant was 1.5 x 10(5) M(-1) s(-1).
- SDS-resistant complex formation and sequencing confirmed Cys(342) as the P(1) inhibitory residue.
Conclusions:
- Human PI9 effectively inhibits human neutrophil elastase.
- PI9 demonstrates flexibility in utilizing different reactive site residues (e.g., Glu(340) for granzyme B, Cys(342) for elastase).
- This expands the inhibitory spectrum of PI9 and related serpins.