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Query: EC:3.4.21.37 (neutrophil elastase)
 4,078 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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)
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PMID:Inhibitors of human neutrophil elastase in extracts of Streptococcus pneumoniae. 656 7

The enzymatic degradation of insoluble elastin has been studied at several pH values using purified pepsin and cathepsin D, and neutrophil extracts. Pepsin degraded elastin throughout the pH range of 1.2-4.0 with the optimum pH below 2.0. Molecular sieve chromatography and gel electrophoresis indicated that a spectrum of molecular weight degradation products was produced. The degradation by pepsin was inhibited by sodium dodecyl sulfate (SDS), NaCl and pepstatin. Cathepsin D, which, like pepsin, degrades hemoglobin at acid pH and is inhibited by pepstatin, had no activity against insoluble elastin in the pH range of 3.2-7.2. Extracts of neutrophils degraded elastin above pH 4.0. The pH profile of elastin degradation by neutrophil extracts generally followed that of purified human leukocyte elastase. Our results suggest that during alimentation or pulmonary aspiration of gastric contents, extracellular elastin may be digested by gastric juice at acid pH. Inflammatory cells would not appear to be capable of contributing to such actions until local pH approaches neutrality. Cathepsin D, a major constituent of inflammatory cells, does not digest all types of connective tissue proteins.
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PMID:The enzymatic digestion of elastin at acidic pH. 678 96

As an approach to facilitating the understanding of proteinases associated with monocytes we have studied U-937 monocytelike cells. Elastase activity was identified in U-937 cell extracts and compared to monocyte elastase activity, neutrophil elastase, and the elastase activity from a continuous line of murine macrophagelike cells (P388D1). Serine proteinase activity which solubilized (14)C-labeled elastin accounted for >90% of the neutral proteinase activity of both U-937 cells and monocyte extracts. U-937 cell and monocyte elastase activities were similar catalytically, resembling neutrophil elastase. U-937 cells and monocytes showed other similarities: (a) both had activities reacting with [(3)H]diisopropylfluorophosphate that migrated in sodium dodecyl sulfate (SDS) polyacrylamide gels at approximately 30,000 and 60,000 daltons and (b) both contained material that cross-reacted with antiserum raised to neutrophil elastase. Preliminary characterization of U-937 cell elastase activity by affinity chromatography and ion-exchange chromatography suggested the presence of at least two distinct elastases. Minimal elastase activity was found in U-937 cell-conditioned medium, indicating that the activity is not spontaneously released by the cells. In contrast to the elastase activity associated with U-937 cells and monocytes, the elastase activity associated with P388D1 cells was a metalloproteinase and was found principally in the culture medium. These results indicate (a) U-937 cells will be useful for further investigation of proteinases associated with normal monocytes; (b) monocytes and U-937 cells contain material with catalytic and immunologic similarities to neutrophil elastase; (c) monocyte elastase activity differs from elastase activity secreted by murine macrophages and murine macrophagelike cells of the P388D1 line.
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PMID:Elastase of U-937 monocytelike cells. Comparisons with elastases derived from human monocytes and neutrophils and murine macrophagelike cells. 691 40

Complexes of alpha 1-antitrypsin with trypsin, alpha-chymotrypsin, and human leukocyte elastase were purified and examined for amino-terminal sequences. These complexes were shown to possess the expected N-terminal sequences for alpha 1-antitrypsin and the corresponding enzymes; no newly generated amino groups could be detected. Each of these three complexes was dissociated at pH 10, and the inhibitor component was isolated. When the latter was subjected to sodium dodecyl sulfate gel electrophoresis a single band was obtained in all cases, and its molecular weight was judged to be 45 000 compared to 52 000 for alpha 1-antitrypsin. Examination of the N-terminal sequence of these modified inhibitors, however, disclosed the presence of two molecular species with different N-termini. The predominant species had the N-terminal sequence previously reported for post-complex alpha 1-antitrypsin (Johnson, D. and Travis, J. (1978) J. Biol. Chem. 253, 7142-7144) and the same carboxyl sequence as alpha 1-antitrypsin. Present in lesser amounts was a species which had retained the same N-terminal sequence as alpha 1-antitrypsin, but of which the C-terminus was resistant to the action of carboxypeptidases A and B. From these results it is concluded that (1) alpha 1-antitrypsin is a double-headed inhibitor with identical but overlapping binding sites; (2) binding of the enzyme may occur at one of these two sites but not at both simultaneously, and (3) peptide cleavage does not occur as a consequence of the binding process but can be demonstrated only if the complex is dissociated.
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PMID:The interaction of alpha 1-antitrypsin with trypsin, chymotrypsin and human leukocyte elastase as revealed by end group analysis. 697 Nov 28

This study has examined the interaction between human leukocyte elastase and alpha 2-plasmin inhibitor, or C1 inactivator, inhibitors of proteases of the complement, kinin, coagulation, and fibrinolytic enzyme systems. Leukocyte elastase, in catalytic concentrations, progressively inactivates the plasmin inhibitory activity of both inhibitors. The C1s binding function of C1 inactivator is also destroyed by leukocyte elastase. The nature of the molecular events underlying the inactivation of these protease inhibitors was examined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Loss of functional activity was accompanied by limited proteolytic cleavage of both inhibitors with the production of several characteristic derivative peptide chains. Leukocyte elastase cleaved alpha 2-plasmin inhibitor at two separate sites and generated lower molecular weight fragments similar to those produced by bovine beta-trypsin. C1 inactivator was hydrolyzed at three different regions on the molecule whereas beta-trypsin cleaved two regions in common with leukocyte elastase. These findings suggest that inactivation of alpha 2-plasmin inhibitor and C1 inactivator by leukocyte elastase released in the inflammatory reaction may potentiate pathological proteolysis. The limited digestion of these inhibitory proteins by leukocyte elastase may prove useful in studies of their primary structure.
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PMID:Proteolytic cleavage and inactivation of alpha 2-plasmin inhibitor and C1 inactivator by human polymorphonuclear leukocyte elastase. 698 Aug 81

The human neutrophil peptide-generating protease, which generates a low molecular weight vasoactive peptide from a plasma protein substrate, is directly fibrinolytic and cleaves human fibrinogen in a manner distinct from plasmin. Fibrinogen was reduced from 340,000 Mr to derivatives of 270,000-325,000 Mr during interaction with the protease at enzyme-to-substrate ratios of 0.3 or 1.0 microgram/1.0 mg. The 310,000-325,000 Mr cleavage fragments exhibited prolonged thrombin-induced clotting activity but were able to be coagulated, whereas the 270,000-290,000 Mr fragments were not able to be coagulated. Anticoagulants were not generated at either enzyme dose. As analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis in 4-30% gradient gels and 10% gels stained for protein and carbohydrate, the diminution to 310,000-325,000 Mr and the prolongation of thrombin-induced clotting time resulted from cleavage of the fibrinogen A alpha chain. The further decrease in size to 270,000-290,000 Mr was associated with B beta-chain and gamma-chain cleavage and an inability to form gamma-gamma dimers. The neutral peptide-generating protease, a distinct human neutrophil neutral protease with fibrinolytic and fibrinogenolytic activities comparable to those of plasmin on a weight basis, cleaves fibrinogen in a manner that is distinct from the action of plasmin, leukocyte elastase, and leukocyte granule extracts. It may be that the concerted action of this neutrophil protease to generate a vasoactive peptide and to digest fibrinogen and fibrin facilitates neutrophil movement through vascular and extravascular sites.
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PMID:Cleavage of fibrinogen by the human neutrophil neutral peptide-generating protease. 700 79

In this article, the authors describe a new method using phosphate-buffered saline for the initial extraction of extracellular matrix (ECM) and soluble proteins from hematologic tissues. Direct comparisons between this method and previously reported methods demonstrate superior total protein yields with the novel technique in a fraction of the time for these ovine hematologic tissues: bone marrow, marrow aspirate, spleen, liver, and blood. Analysis by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining demonstrate comparable protein patterns with this method and a previously reported method. This method also successfully extracts the ECM glycoproteins fibronectin and laminin as well as the proteoglycan, chondroitin sulfate from marrow. These findings are demonstrated by Western and dot blotting. Bone marrow ECM and soluble proteins extracted by the novel method stimulate myeloid progenitor growth in a methylcellulose assay. Using an assay for elastase inhibitory capacity, the authors demonstrate that alpha 1-proteinase inhibitor, the principal inhibitor of neutrophil elastase, is present and that its activity is preserved in bone marrow samples extracted with this team's method. In contrast, very low total protein yields are obtained with the method used previously to recover hemonectin, a rabbit bone marrow ECM granulocytic cytoadhesion molecule. This team's novel procedure, which extracts ECM and soluble proteins from small samples of tissue in a rapid, efficient, and reproducible manner, greatly enhances the analysis of these proteins from tissue culture, animal, and human clinical samples. In addition, with purification by chromatofocusing chromatography and molecular sieving gel electrophoresis, N-terminal amino acid sequencing could be performed on a developmentally regulated marrow protein with biochemical properties similar to those of hemonectin and the plasma protein fetuin. The authors propose that this novel technique be used for the initial extraction of ECM and soluble proteins from hematologic tissues and that subsequent, definitive recovery of insoluble proteins be accomplished using previously reported, though less efficient, methods.
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PMID:Ovine bone marrow extracellular matrix and soluble protein extraction: fetuin amino terminus microheterogeneity. 753 48

The present study was undertaken to provide a highly sensitive detection system for the identification and characterisation of serine proteinases separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Biotinylated aprotinin of high specific activity (88-92% active) was prepared (i) by reaction of aprotinin directly with N-hydroxy sulfosuccinimidyl-6-(biotinamido) hexanoate, and (ii) by reaction of aprotinin-trypsin complex with N-hydroxy succinimidobiotin. Both biotinylated aprotinin samples were suitable as probes for the detection of the serine proteinases, neutrophil elastase and cathepsin G, pancreatic trypsin and chymotrypsin and plasmin on nitrocellulose blots. Specific irreversible chloromethyl ketone proteinase inhibitors used in combination with this detection system enabled respective proteinases to be selectively inactivated and thus positively identified. The biotinylated aprotinin detection system was highly sensitive and could detect as little as 0.2 ng (8.5 fmol) of active proteinase (trypsin). In summary, a method has been developed for the sensitive detection of serine proteinases separated by SDS-PAGE. The method is more sensitive and convenient to perform than conventional zymography and significantly, when used in conjunction with specific serine proteinase inhibitors or specific antibodies can yield appreciable information on the identity of the respective serine proteinases being examined. Furthermore the molecular mass of the serine proteinase may be reliably obtained by this method. This method should find application in identifying the role that serine proteinases play in the etiopathogenesis of connective tissue disorders.
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PMID:Biotinylated aprotinin: a versatile probe for the detection of serine proteinases on western blots. 758 24

The interactions of bovine pancreatic chymotrypsin (Chtr) and recombinant alpha 1-antichymotrypsin (rACT) and rACT variants were studied by kinetic and gel electrophoretic analyses, leading to the formulation of a general kinetic scheme that accounts for all known results concerning this serpin-protease pair, as well as for results obtained with other such pairs. Incubation of rACT and Chtr leads rapidly to the formation of an inhibited complex, Chtr.rACT*, that is stable toward sodium dodecyl sulfate denaturation and boiling. The extent of release of active Chtr from this complex increases markedly as ionic strength, mu, is raised. The kinetic scheme quantitatively accounts for this effect on the basis of a partitioning of Chtr.rACT* between dissociation of the complex to yield active enzyme and cleaved rACT, and Chtr-catalyzed conversion of the complex to a form that is much more resistant to release of active enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of reaction mixtures of rACT and Chtr are consistent with the scheme. Also consistent are the results of experiments measuring the effects of 1) Chtr.rACT* concentration, 2) uncomplexed Chtr, and 3) added alpha 2-macroglobulin on active Chtr release from Chtr.rACT*. Proteolysis of Chtr.rACT* to give a resistant complex is also catalyzed by human neutrophil elastase, a process with potential physiological relevance. Comparison of the rates of Chtr dissociation from the complexes formed with rACT and with rACT variants mutated at the P1 site suggests that such rates are more sensitive to P1 substitution at low mu than at high mu. Several equivalents of the L358R-rACT variant are required for full inhibition of Chtr. This observation is also quantitatively accounted for by the proposed kinetic scheme, on the basis of another partitioning step between L358R-rACT acting as a substrate or as an inhibitor toward Chtr.
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PMID:Antichymotrypsin interaction with chymotrypsin. Partitioning of the complex. 769 93

Monolayer cultures of human epithelial and endothelial cells were used to study the association of latent transforming growth factor-beta 1 (TGF-beta 1) to extracellular matrices and its release and activation during matrix degradation. Human umbilical vein endothelial cells and embryonic lung fibroblasts produced relatively high levels of TGF-beta 1, its propeptide (beta 1-latency-associated protein), and latent TGF-beta-binding protein and incorporated latent TGF-beta 1 into their matrices as shown by immunoblotting. Amnion epithelial cells produced lower levels of these proteins. Confluent cultures of epithelial cells were exposed to matrix-degrading proteases and glycosidases. Mast cell chymase, leukocyte elastase, and plasmin efficiently released matrix-bound latent TGF-beta 1 complexes, while chondroitinase ABC and heparitinases were ineffective. The ability of the proteases to activate recombinant latent TGF-beta 1 was tested using growth inhibition assays and a novel sodium deoxycholate-polyacrylamide gel electrophoresis followed by immunoblotting. Sodium deoxycholate solubilized M(r) 25,000 TGF-beta 1 but did not dissociate high M(r) latent TGF-beta 1 complexes, allowing separation of these forms by polyacrylamide gel electrophoresis. Mast cell chymase and leukocyte elastase did not activate latent TGF-beta 1, suggesting that its release from matrix and activation are controlled by different mechanisms. The release of TGF-beta from the matrix by leukocyte and mast cell enzymes may contribute to the accumulation of connective tissue in inflammation.
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PMID:Human mast cell chymase and leukocyte elastase release latent transforming growth factor-beta 1 from the extracellular matrix of cultured human epithelial and endothelial cells. 787 40


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