Inhibitors of human neutrophil elastase in extracts of Streptococcus pneumoniae.
Alveolar architecture is spared during most pneumococcal pneumonias, despite the presence in pneumonic exudate of many neutrophils containing a potent elastase. We explored the possibility that pneumococci might contain an inhibitor of this enzyme. We found that pneumococcal extracts prepared by sonication or by lysis with sodium deoxycholate contained 2 different inhibitors of human neutrophil elastase. Both inhibitors were specific for neutrophil elastase and did not affect pancreatic elastase or trypsin. Inhibitor I was partly purified by affinity chromatography and preparative acrylamide gel electrophoresis and shown to be a negatively charged, low molecular weight substance that inhibited competitively (Lineweaver-Burk analysis). Inhibition depended on ionic interaction with the cationic enzyme and could be blocked by 0.15 M NaCl. For this reason, the first agent seemed unlikely to play an important role in modulating neutrophil elastase activity in inflammatory exudates and was not studied further. The second agent (Inhibitor II) eluted in the high molecular weight fraction during Sephacryl S-300 chromatography. Gradient SDS-polyacrylamide gel electrophoresis of partly purified Inhibitor II revealed an apparent molecular weight of 140,000 daltons. This agent inhibited noncompetitively and remained active in the presence of 0.15 M NaCl. Prolonged incubation with TPCK-trypsin resulted in cleavage of Inhibitor II into smaller fragments, which could be further dissociated by reduction with dithiothreitol. Inactivation of neutrophil elastase with N-acetyl-alanyl-alanyl-prolyl-valyl-chloromethyl ketone prevented complex formation between this enzyme and Inhibitor II, suggesting that an unblocked binding pocket in neutrophil elastase is required for its complexation to the noncompetitive pneumococcal inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)
D001696:Biomechanical Phenomena; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009504:Neutrophils / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D013296:Streptococcus pneumoniae / Q000032:analysis*; D014018:Tissue Distribution; D014020:Tissue Extracts / Q000494:pharmacology*
D014020:Tissue Extracts; D010196:Pancreatic Elastase