A cytosolic inhibitor of human neutrophil elastase and cathepsin G

R. M. Thomas, W. M. Nauseef, S. S. Iyer, M. W. Peterson, P. J. Stone, Robert A Clark

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

The neutrophil serine proteinases elastase and cathepsin G produce connective tissue injury, the extent of which depends on the balance between these enzymes and their inhibitors. The most important of these inhibitors is α1-proteinase inhibitor, a member of a superfamily of homologous proteins known as serpins. Neutrophil cytosol inhibited the activities of human neutrophil elastase and cathepsin G in a dose-dependent fashion. To demonstrate formation of an enzyme-inhibitor complex, we combined 125I-elastase of 125I-cathepsin G with neutrophil cytosol or α1-proteinase inhibitor and analyzed the products by polyacrylamide gel electrophoresis. Unbound elastase and cathepsin G each migrated to an apparent molecular weight of 25 kDa. In the presence of cytosol from neutrophils both radiolabeled enzymes migrated with a relative size of 68 kDa, whereas in the presence of α1-proteinase inhibitor the relative size was 85 kDa. Enzyme-inhibitor complexes were stable in sodium dodecyl sulfate at 100°C but were dissociated by hydrolysis in ammonium hydroxide (1.5 mol/L) at 37°C. Formation of each complex was prevented by pretreatment of elastase or cathepsin G with diisopropylfluorophosphate, indicating that the inhibitor binds to the active site of the enzyme. Exposure of either α1-proteinase inhibitor or neutrophil cytosol to the myeloperoxidase-H2O2-halide system prevented complex formation, suggesting the presence of an oxidizable amino acid at the binding site of the inhibitor. By electrophoretic analysis, the molecular weight of the cytosolic inhibitor was 43 kDa and neutrophils contained approximately 1 attomol of inhibitor per cell. The isoelectric points of the elastase and cathepsin G inhibitor were 5.5-5.9 and inhibitors of the two proteinases coeluted using size exclusion chromatography. These data demonstrate that human neutrophil cytosol contains a single serpin-like protein that inhibits elastase and cathepsin G. The inhibitor may be important in protecting the intracellular environment from proteolytic injury during degranulation.

Original languageEnglish (US)
Pages (from-to)568-579
Number of pages12
JournalJournal of Leukocyte Biology
Volume50
Issue number6
StatePublished - 1991
Externally publishedYes

Fingerprint

Secretory Proteinase Inhibitory Proteins
Cathepsin G
Pancreatic Elastase
Neutrophils
Cytosol
Peptide Hydrolases
Enzyme Inhibitors
Serpins
Molecular Weight
Ammonium Hydroxide
Isoflurophate
Leukocyte Elastase
Wounds and Injuries
Isoelectric Point
Serine Proteases
Enzymes
Human Activities
Sodium Dodecyl Sulfate
Connective Tissue
Peroxidase

Keywords

  • Polymorphonuclear leukocytes
  • Serine proteinase
  • Serine proteinase inhibitor

ASJC Scopus subject areas

  • Cell Biology

Cite this

Thomas, R. M., Nauseef, W. M., Iyer, S. S., Peterson, M. W., Stone, P. J., & Clark, R. A. (1991). A cytosolic inhibitor of human neutrophil elastase and cathepsin G. Journal of Leukocyte Biology, 50(6), 568-579.

A cytosolic inhibitor of human neutrophil elastase and cathepsin G. / Thomas, R. M.; Nauseef, W. M.; Iyer, S. S.; Peterson, M. W.; Stone, P. J.; Clark, Robert A.

In: Journal of Leukocyte Biology, Vol. 50, No. 6, 1991, p. 568-579.

Research output: Contribution to journalArticle

Thomas, RM, Nauseef, WM, Iyer, SS, Peterson, MW, Stone, PJ & Clark, RA 1991, 'A cytosolic inhibitor of human neutrophil elastase and cathepsin G', Journal of Leukocyte Biology, vol. 50, no. 6, pp. 568-579.
Thomas RM, Nauseef WM, Iyer SS, Peterson MW, Stone PJ, Clark RA. A cytosolic inhibitor of human neutrophil elastase and cathepsin G. Journal of Leukocyte Biology. 1991;50(6):568-579.
Thomas, R. M. ; Nauseef, W. M. ; Iyer, S. S. ; Peterson, M. W. ; Stone, P. J. ; Clark, Robert A. / A cytosolic inhibitor of human neutrophil elastase and cathepsin G. In: Journal of Leukocyte Biology. 1991 ; Vol. 50, No. 6. pp. 568-579.
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AB - The neutrophil serine proteinases elastase and cathepsin G produce connective tissue injury, the extent of which depends on the balance between these enzymes and their inhibitors. The most important of these inhibitors is α1-proteinase inhibitor, a member of a superfamily of homologous proteins known as serpins. Neutrophil cytosol inhibited the activities of human neutrophil elastase and cathepsin G in a dose-dependent fashion. To demonstrate formation of an enzyme-inhibitor complex, we combined 125I-elastase of 125I-cathepsin G with neutrophil cytosol or α1-proteinase inhibitor and analyzed the products by polyacrylamide gel electrophoresis. Unbound elastase and cathepsin G each migrated to an apparent molecular weight of 25 kDa. In the presence of cytosol from neutrophils both radiolabeled enzymes migrated with a relative size of 68 kDa, whereas in the presence of α1-proteinase inhibitor the relative size was 85 kDa. Enzyme-inhibitor complexes were stable in sodium dodecyl sulfate at 100°C but were dissociated by hydrolysis in ammonium hydroxide (1.5 mol/L) at 37°C. Formation of each complex was prevented by pretreatment of elastase or cathepsin G with diisopropylfluorophosphate, indicating that the inhibitor binds to the active site of the enzyme. Exposure of either α1-proteinase inhibitor or neutrophil cytosol to the myeloperoxidase-H2O2-halide system prevented complex formation, suggesting the presence of an oxidizable amino acid at the binding site of the inhibitor. By electrophoretic analysis, the molecular weight of the cytosolic inhibitor was 43 kDa and neutrophils contained approximately 1 attomol of inhibitor per cell. The isoelectric points of the elastase and cathepsin G inhibitor were 5.5-5.9 and inhibitors of the two proteinases coeluted using size exclusion chromatography. These data demonstrate that human neutrophil cytosol contains a single serpin-like protein that inhibits elastase and cathepsin G. The inhibitor may be important in protecting the intracellular environment from proteolytic injury during degranulation.

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