Journal of Leukocyte Biology 50:568-579 (1991) © 1991 Wiley-Liss, Inc.

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 a1-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 1251-elastase or 1251-cathepsin G with neutrophil cytosol or a1-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 a1-proteinase inhibitor the relative size was 85 kDa. Enzyme-inhibitor complexes were stable in sodium dodecyl sulfate at 100#{176}Cbut were dissociated by hydrolysis in ammonium hydroxide (1.5 mol/L) at 37#{176}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 eithe