Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

1. An elastolytic enzyme has been isolated from dog granulocyte leukocytes. The purification procedure included preparation of the granula fraction, chromatography on Sephadex G-75 and ion-exchange chromatography on SP-Sephadex C-50 at pH 6.0. 2. The elastase isolated was homogeneous in analytical disc electrophoresis and showed in sodium dodecylsulfate electrophoresis a single protein component with the molecular weight of 24800. The enzyme lacked tyrosine and lysine and the N-terminal amino acid was phenylalanine. No carbohydrate or sialic acid were detected. 3. The dog granulocyte elastase showed similar activities as human granulocyte elastase on elastin and fibrin. The Km value for 3-carboxypropionyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide was 2.50 mM and the pH optimum 8.5. The elastase preparation obtained was 99.5% active as judged from active-site titration. 4. The enzyme is a cationic protein and shows pronounced trailing on agarose gel electrophoresis. 5. A monospecific antiserum against the purified enzyme was produced in rabbits.
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PMID:Isolation and partial characterization of elastase from dog granulocytes. 99 51

Poly-L-lysine with molecular masses of 3.3-290 kDa increased the amidolytic activities of leukocyte elastase and cathepsin G at low concentration, but had little effect on the activities of pancreatic elastase, alpha-chymotrypsin, plasmin and thrombin. Highly purified cathepsin G was obtained from column of EAH Sepharose 4B or Suc-L-Tyr-D-Leu-D-Val-pNA-Sepharose (affinity chromatography) by elution with poly-L-lysine solution (0.4 mg/ml, molecular weight (MW.) 290000 or 2.2 mg/ml, MW. 3300). Leukocyte elastase, adsorbed to Suc-L-Tyr-D-Leu-D-Val-pNA-Sepharose, was not eluted with poly-L-lysine solution. The amino acid composition of purified cathepsin G has been determined.
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PMID:Application of poly-L-lysine to purification of leukocyte cathepsin G by affinity chromatography. 139 85

A series of tripeptides which contain alpha,alpha-difluorostatone residues at P1-P1' and span the S3-S1' subsites have been shown to be potent inhibitors of human leukocyte elastase (HLE). The tripeptides described contain the nonproteinogenic achiral residue N-(2,3-dihydro-1H-inden-2-yl)glycine at the P2-position. This redidue has previously been shown in the case of HLE to be a good bioisosteric replacement for L-proline. Of the peptides prepared, those which contain the alpha,alpha-difluoromethylene keton derivative of L-valine (difluorostatone) are the preferred residue at the P1-primary specificity position. Substitution at P1 by the corresponding alpha,alpha-difluoromethylene ketones of L-leucine and L-phenylalanine gives inactive compounds. Of the tripeptides described the most potent in vitro compound is ethyl N-[N-CBZ-L-valyl-N-(2,3-dihydro-1H-inden-2-yl)glycyl]- 4(S)-amino-2,2-difluoro-3-oxo-5-methylhexanoate (17B) (IC50 = 0.635 microM). It is presumed that the inhibitor 17b interacts with the S3-S1' binding regions of HLE. Additionally extended binding inhibitors were prepared which interact with the S3-S3' binding subsites of HLE. In order to effect interaction with the S1'-S3' subsites of HLE, the leaving group side of cleaved peptides, spacers based upon Gly-Gly, and those linked via the N epsilon of L-lysine were utilized. One of the most potent extended compounds (P3-P3') in vitro is methyl N6-[4(S)-[[N-[N-CBZ-L-valyl-N- (2,3-dihydro-1H-inden-2-yl)glycyl]amino]-2,2-difluoro-3-oxo-5- methylhexanoyl]-2(S)-(acetylamino)-6-aminohexanoate (24b) (IC50 = 0.057 microM). The described in vitro active inhibitors were also evaluated in hamsters in an elastase-induced pulmonary hemorrhage (EPH) model. In this model, intratracheal (it.) administration of 22c, 5 min prior to HLE challenge (10 micrograms, it.) effectively inhibited hemorrhage (94.6%) in a dose-dependent manner. The described alpha,alpha-difluoromethylene ketone inhibitors are assumed to act as transition-state analogs. The inhibition process presumably acts via hemiketal formation with the active site Ser195 of HLE, and is facilitated by the strongly electron withdrawing effect of the alpha,alpha-difluoromethylene functionality.
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PMID:Inhibition of human leukocyte elastase by N-substituted peptides containing alpha,alpha-difluorostatone residues at P1. 147 81

Pregnancy-associated plasma protein-A (PAPP-A), like alpha 2-macroglobulin (alpha 2M), is a large protein with homotetrameric molecular conformation. Each monomer has Mr 205 kDa. Total carbohydrate content of PAPP-A (19.4%) exceeded that of alpha 2M (8.6%). In addition to glucose (9.4%), fucose (3.1%), mannose (2.3%) and galactose (0.8%), PAPP-A contained glucuronic acid (3.8%). The amino acid composition of PAPP-A differed most significantly from alpha 2M, in the content of glutamate, glycine and lysine. Although the peptide core of both proteins were of similar size, the difference in size, of native molecules was due to the carbohydrate moiety. Whereas alpha 2M monomer was autolytically cleaved into two smaller non-identical subunits (Mr 128 and 65 kDa), no such breakdown products were observed with PAPP-A. Unlike alpha 2M, PAPP-A is not a broad spectrum protease inhibitor. Both proteins inhibited human granulocyte elastase (HGE) in a dose dependent relationship, with PAPP-A (Ki 0.2 x 10(-6) M) being a more potent inhibitor than alpha 2M (Ki 1.02 x 10(-6) M). Since PAPP-A lacked internal thiolester groups, the mechanism of HGE inhibition was unlikely to be entrapment, as defined for alpha 2M. Inhibition kinetics of HGE for PAPP-A (noncompetitive inhibitor) and alpha 2M (uncompetitive inhibitor) were distinct. Thus, these findings do not support the tenet of a common ancestral protein for PAPP-A and alpha 2M.
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PMID:Characterization of pregnancy-associated plasma protein-A: comparison with alpha 2-macroglobulin. 169 71

In recent years, many studies have suggested a direct role for alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor, in growth factor regulation. When coincubated in the presence of either trypsin, pancreatic elastase, human neutrophil elastase, or plasmin, 125I-insulin rapidly formed a complex with alpha 2M which was greater than 80% covalent. The covalent binding was stable to reduction but abolished by competition with beta-aminopropionitrile. Neither native alpha 2M nor alpha 2M pretreated with proteinase or methylamine incorporated 125I-insulin. Experiments utilizing alpha 2M cross-linked with cis-dichlorodiammineplatinum(II) indicated that 125I-insulin must be present during alpha 2M conformational change to covalently bind. A maximum stoichiometry of 4 mol of insulin bound per mole of alpha 2M and the short half-life of the alpha 2M intermediate capable of covalent incorporation were consistent with thiol ester involvement. Protein sequence analysis of unlabeled insulin-alpha 2M complexes, together with results of beta-aminopropionitrile competition, confirmed that insulin incorporation occurs via the same gamma-glutamyl amide linkage responsible for covalent proteinase and methylamine binding to alpha 2M. Although intact insulin apparently incorporated through its sole lysine residue on the B chain, we found that isolated A chain also bound covalently to alpha 2M. Phenyl isothiocyanate derivatization of the N-terminus had no effect on A-chain binding, supporting the possibility of heretofore unreported gamma-glutamyl ester linkages to alpha 2M.
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PMID:Mechanism of insulin incorporation into alpha 2-macroglobulin: implications for the study of peptide and growth factor binding. 170 57

Fragmentation of lung matrix fibronectin by proteases released from activated phagocytic cells has been implicated in lung vascular injury. We examined whether denatured collagen (gelatin)-bound fibronectin can be degraded by peritoneal exudate mononuclear phagocytes harvested from rats 96 h after intraperitoneal casein injection. Microtiter plates were pretreated with gelatin and then supplemented with purified 125I rat plasma fibronectin, which readily bound to the gelatin. Stimulated inflammatory exudate cells were added and proteolysis of the bound fibronectin was studied by the release of [125I]fibronectin fragments into the media. Following 2 h of incubation, peritoneal exudate mononuclear macrophages stimulated with opsonized zymosan released three times more radiolabeled fibronectin into the medium as compared to background controls, and 1.5 times more radiolabeled fibronectin as compared to cells not stimulated with zymosan. Western blot analysis and autoradiography confirmed the presence of fragments of fibronectin in the culture medium. Some of these fragments were clearly derived from the radiolabeled matrix, but others that were not labeled were potentially released directly from the added stimulated macrophages. The release of radiolabeled fibronectin was inhibited by N-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin specific inhibitor, but not by methoxysuccinyl-alanine-alanine-proline-valine-chloromethyl-ketone (AAPVCK), a leukocyte elastase-specific inhibitor. These results suggest that fibronectin bound to denatured collagen is susceptible to leukocyte elastase-independent enzymatic degradation by stimulated inflammatory exudate mononuclear phagocytic cells. Such proteolysis may mimic a pathological process associated with lung vascular injury during the sequestration of activated macrophages in the lung microcirculation and interstitium.
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PMID:Leukocyte elastase-independent proteolysis of gelatin-bound fibronectin by inflammatory macrophages. 175 31

The serum protein alpha 1-antitrypsin (alpha 1-AT) serves as the major inhibitor of neutrophil elastase. The most common allele of the alpha 1-AT gene is designated as PiM. The Z mutation is a single-base substitution of the normal M allele, causing a Glu----Lys change at position 342 in the molecule. The ZZ phenotype is associated with a severe deficiency of alpha 1-AT, serum concentrations of the protein being 10% of normal. Individuals with an alpha 1-AT deficiency are at an increased risk of developing emphysema. To generate antibodies that specifically detect the 342 position in the context of the flanking sequences, we synthesized several peptides that included the 342 position for both the M and the Z variant. Immunization with variant-specific peptide-carrier conjugates elicited alpha 1-AT variant-specific responses, as determined in a direct enzyme-linked immunoassay. Monoclonal antibodies (MAbs) were selected with different specificity for the 342 region: MAbs F43 recognize only the alpha 1-AT sequence with 342Glu, i.e., all variant proteins that are non-Z, either from hetero- or homozygous individuals; MAbs F50 recognize only the sequence with 342Lys, i.e., all Z-variant proteins in ZZ or heterozygous individuals; MAbs F46 recognize alpha 1-AT with either 342Lys or 342Glu, all variant proteins with sequences as in the peptides used. Z homo- and heterozygotes were detected with our MAbs in a rapid and simple immunoblot assay. Other variants (M, S, and F) can also be assigned on the basis of the electrophoretic pattern. This sensitive detection method is very easy, rapid, and straightforward and provides a powerful tool for diagnosis of the alpha 1-AT deficiencies, allowing early treatment (augmentation of alpha 1-AT) and proper advice on lifestyle practices.
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PMID:Detection of genetic variants of alpha 1-antitrypsin with site-specific monoclonal antibodies. 189 97

The microvascular response to two polycationic proteins, poly-L-lysine (mol wt 104,000) and leukocyte elastase, was studied in the hamster cheek pouch microcirculation model. A 2-min topical application of polylysine (100 micrograms/ml) induced vigorous macromolecular leakage from venules only that declined within 30 min. A second application induced significantly less leakage. The leakage was inhibited by admixing polylysine with dextran sulfate prior to application or by giving hamsters an intravenous injection of dextran sulfate. The histamine antagonist pyrilamine did not interfere with the leakage, and only a few degranulated mast cells were found after polylysine application. No intravascular adhesion of leukocytes could be detected. Elastase (100 micrograms/ml) was deposited adjacent to venules with micropipets. The resulting leakage response was not inhibited by L658,758, an inhibitor of elastase enzymatic activity, but by dextran sulfate. These results may prove significant in light of the numerous polycationic proteins present within neutrophil granules.
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PMID:Polycations induce microvascular leakage of macromolecules in hamster cheek pouch. 193 92

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits the coagulant proteinase alpha-thrombin. We have recently demonstrated that proteolysis of HC by catalytic amounts of polymorphonuclear leukocyte proteinases (elastase or cathepsin G) generates leukocyte chemotaxins (Hoffman, M., Pratt, C. W., Brown, R. L., and Church, F. C. (1989) Blood 73, 1682-1685). One of four peptides produced when HC is degraded by neutrophil elastase has chemotactic activity for both monocytes and neutrophils with maximal migration comparable to formyl-Met-Leu-Phe, the "gold standard" bacterially derived chemotaxin. The amino-terminal sequence of this HC peptide is Asp-Phe-His-Lys-Glu-Asn-Thr-Val-... and the peptide corresponds to Asp-39 to Ile-66 of HC. A variety of synthetic peptides derived from this sequence were evaluated for leukocyte migration activity, and a dodecapeptide from Asp-49 to Tyr-60 (Asp-Trp-Ile-Pro-Glu-Gly-Glu-Glu-Asp-Asp-Asp-Tyr) was identified as the active site for leukocyte chemotactic action. The 12-mer synthetic peptide possesses significant neutrophil chemotactic action at 1 nM (60% of the maximal activity of formyl-Met-Leu-Phe), while a peptide with the reverse sequence has essentially no chemotactic activity. Cross-desensitization experiments also show that pretreatment of neutrophils with a 19-mer peptide (Asn-48 to Ile-66) greatly reduces subsequent chemotaxis to HC-neutrophil elastase proteolysis reaction products. When injected intraperitoneally in mice, the HC-neutrophil elastase digest elicits neutrophil migration. Our results demonstrate that not only does HC function as a thrombin inhibitor, but that limited proteolysis of HC near the amino terminus yields biologically active peptide(s) which might participate in inflammation and in wound healing and tissue repair processes.
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PMID:Leukocyte chemoattractant peptides from the serpin heparin cofactor II. 198 58

Secretory leukocyte protease inhibitor (SLPI) is a two-domain protein that inhibits a wide range of proteases including chymotrypsin, leukocyte elastase, and trypsin. Based on its homology to other protease inhibitors and on x-ray crystallography of an SLPI-chymotrypsin complex it has been proposed that the elastase and chymotrypsin-inhibitory site is in the COOH-terminal domain and that the trypsin-inhibitory site is in the NH2-terminal domain. We have prepared muteins of SLPI by site-directed mutagenesis of a synthetic gene for the protein, followed by expression in Escherichia coli. The protease-inhibitory activities of these muteins indicate that leucine 72 in the COOH-terminal domain is at the inhibitory site for elastase and chymotrypsin. Unexpectedly, our measurements indicate that the trypsin-inhibitory site is not in the NH2-terminal domain. Instead they suggest that leucine 72 is also the inhibitory site for trypsin, even though the amino acid residues at the inhibitory sites of other trypsin inhibitors are almost always either lysine or arginine.
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PMID:Location of the protease-inhibitory region of secretory leukocyte protease inhibitor. 211 May 63


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