title: A cytosolic inhibitor of human neutrophil elastase and cathepsin G.
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 alpha 1-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 or 125I-cathepsin G with neutrophil cytosol or alpha 1-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 alpha 1-proteinase inhibitor the relative size was 85 kDa. Enzyme-inhibitor complexes were stable in sodium dodecyl sulfate at 100 degrees C but were dissociated by hydrolysis in ammonium hydroxide (1.5 mol/L) at 37 degrees 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 alpha 1-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 serpinlike protein that inhibits elastase and cathepsin G. The inhibitor may be important in protecting the intracellular environment from proteolytic injury during degranulation.
authors.affiliation: Department of Medicine, VA Medical Center, Iowa City.
authors.forename: R M
authors.initials: RM
authors.lastname: Thomas
authors.forename: W M
authors.initials: WM
authors.lastname: Nauseef
authors.forename: S S
authors.initials: SS
authors.lastname: Iyer
authors.forename: M W
authors.initials: MW
authors.lastname: Peterson
authors.forename: P J
authors.initials: PJ
authors.lastname: Stone
authors.forename: R A
authors.initials: RA
authors.lastname: Clark
mesh_terms: D064753:Ammonium Hydroxide; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006878:Hydroxides / Q000737:chemistry; D066298:In Vitro Techniques; D007526:Isoelectric Point; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D009195:Peroxidase / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases; D014407:Tumor Cells, Cultured
chemical_list: D006878:Hydroxides; D011480:Protease Inhibitors; D064753:Ammonium Hydroxide; D006861:Hydrogen Peroxide; D009195:Peroxidase; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase