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)

The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15,000 units/mg (1 unit = 1 microgram of gelatin degraded/min at 37 degrees C) by titration with alpha 2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.
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PMID:Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells. 137 48

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).
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PMID:Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties. 140 Apr 81

alpha 1-Antitrypsin (AAT) is a potent fluid-phase inhibitor of serine proteases. It forms a tightly bound, stoichiometric complex with these enzymes and is inactivated by cleavage within its reactive center. Evidence is here presented, that the 44-residue C-terminal fragment of AAT, termed SPAAT (short peptide from AAT), is found in human tissue, where it is apparently bound to the extracellular matrix (ECM). The identity of SPAAT was established by amino acid sequence analysis through its 40 N-terminal residues. Placental SPAAT inhibits chymotrypsin, human neutrophil elastase (HNE) and pancreatic elastase, but has no effect on trypsin. Unlike AAT, both placental and chemically-synthesized SPAAT are reversible, competitive inhibitors of chymotrypsin with Kl's of 0.92 and 7.5 microM, respectively. Both AAT and placental SPAAT also bind to diisopropyl fluorophosphate (DFP)-treated HNE as well as cathepsin G. SPAAT may therefore play an important role in the protection of ECM proteins, such as elastin, proteoglycans (PG) and/or collagen, from inappropriate attack by serine proteases.
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PMID:Isolation and serine protease inhibitory activity of the 44-residue, C-terminal fragment of alpha 1-antitrypsin from human placenta. 140 56

In this report, the susceptibility of type VIII collagen to human neutrophil elastase is compared to other extracellular matrix components. Type X collagen is degraded to specific fragments at a substrate to enzyme ratio of 5:1 after 20 h at room temperature, but type VIII collagen is almost completely degraded after only 4 h incubation at a substrate to enzyme ratio of 50:1 and partly degraded after only 15 min. Laminin, merosin and types I, III, IV and V collagen exhibit no susceptibility to neutrophil elastase under the latter conditions, while fibronectin is degraded.
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PMID:Cleavage of type VIII collagen by human neutrophil elastase. 151 54

Reversible binding of calcium ions to a single high-affinity binding site in the 40-kDa basement membrane protein (BM-40) caused a 33% increase of alpha-helicity, an about 60% change in intrinsic fluorescence and a dramatic increase of the rate of cleavage by alpha-chymotrypsin. All these effects exhibited identical dependencies on calcium concentration from which a dissociation constant Kd = 0.6 microM was determined. Calcium release was accompanied by an increase of the frictional ratio in solution but not by denaturation which occurred at about equal guanidine.HCl concentration for both calcium-saturated and calcium-depleted protein (midpoint 1.5 M). The cleavage sites for alpha-chymotrypsin are located in or near to the EF-hand domain IV of calcium-depleted BM-40 (also known as SPARC, i.e. secreted protein acidic and rich in cysteine, and osteonectin). These and other data indicate that binding occurs in the EF-hand domain from which a large conformational change is transmitted. Low-affinity calcium-binding sites in the N-terminal glutamic-acid-rich domain I of BM-40 were identified by human leukocyte elastase which was found to cleave very specifically in the middle of this domain. From the increase of cleavage rate with increasing calcium concentration a Kd greater than or equal to 10 mM was estimated. It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM-40 such as collagen binding and that high-affinity binding is important for stabilization, folding and secretion during biosynthesis.
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PMID:High-affinity and low-affinity calcium binding and stability of the multidomain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/osteonectin). 155 84

The effects of proteoglycan and collagen digestion on the transient response of human articular cartilage when tested in unconfined compression were determined. Small cylindrical specimens of cartilage, isolated from the femoral head of the hip joint and from the femoral condyles of the knee joint, were subjected to a suddenly applied compressive load using a test apparatus designed to yield a transient oscillatory response. From this response values of the elastic stiffness and the viscous damping coefficient were determined. Cathepsin D and cathepsin B1 were used to digest the proteoglycan in some specimens, while in other specimens leukocyte elastase was used to attack the non-helical terminal regions of the Type II tropocollagen molecules and possibly the Type IX collagen molecule and thereby disturb the integrity of the collagen mesh. The results showed that proteoglycan digestion alone reduced the viscous damping coefficient but it did not significantly alter the elastic stiffness as determined from the oscillatory response. In contrast, the action of elastase reduced both the damping coefficient and the elastic stiffness of the cartilage. The results demonstrated the role of proteoglycans in regulating fluid transport in cartilage and hence controlling the time-dependent viscous properties. The elastic stiffness was shown to be dependent on the integrity of the collagen fibre network and not on the proteoglycans.
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PMID:The effects of selective matrix degradation on the short-term compressive properties of adult human articular cartilage. 158 43

The mammalian pulmonary blood-gas barrier is well known to be extremely thin. For example, in the human lung, half of the area of the barrier (the 'bulging' part) has a thickness of only 0.2-0.4 micron. We show here that the barrier is also immensely strong. This is an essential requirement because the capillary wall stresses during heavy exercise become very large (about 7 x 10(4) N/m2 = 70 kPa) when capillary pressure increases to 30 mmHg. Stress failure of the pulmonary capillary wall consistently occurs in experimental rabbit preparations at abnormally high pressures exceeding 40 mmHg and may be the cause of bleeding into the lung in galloping racehorses. The great strength of the thin side of the blood-gas barrier can be attributed to the extracellular matrix, especially the type IV collagen which is predominantly located in the very thin lamina densa. The alveolar wall is therefore particularly vulnerable to injurious agents which attack type IV collagen such as autoantibodies in Goodpasture's Syndrome and perhaps neutrophil elastase in emphysema. The combination of extreme thinness and great strength of the blood-gas barrier poses a unique design requirement.
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PMID:Strength of the pulmonary blood-gas barrier. 162 33

Fragmentation of subendothelial matrix-bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125I-labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I-fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose-dependent manner by the addition of both methoxysuccinyl-alanine-alanine-valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non-specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60-200 kD) were not labelled, but the smaller molecular weight fragments (less than 45 kD), derived from the artificial matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.
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PMID:Proteolysis of gelatin-bound fibronectin by activated leukocytes: a role for leukocyte elastase. 165 37

Arthritis was induced by injecting cationic amidated bovine serum albumin (aBSA) (pI approximately 9.2) into the knee joint of immunized guinea pigs and the mechanisms of articular cartilage destruction were studied morphologically and biochemically. Marked synovitis associated with polymorphonuclear leukocyte (PML) infiltration occurred within 1 day of the challenge. Articular cartilage infiltrated by PMLs was almost completely destroyed after 2 weeks. During the initial destructive process, proteoglycans were depleted from the cartilage and later collagen fibers disappeared. Granulation tissue growing in the inflamed synovium and bone marrow replaced the destroyed cartilage and joint cavity and formed fibrous scar tissue (fibrous ankylosis) by 8 weeks. Subsequently, the knee joints developed cartilagenous ankylosis by 12 weeks and finally bony ankylosis at 28 weeks. Autoradiography using 125I-aBSA and immunofluorescence studies for immunoglobulin (IgG) and complement (C3) demonstrated that the antigen is trapped in all zones of the articular cartilage and serves as a trigger for immune complex formation. Significantly increased neutral proteinase activities against substrates of proteoglycan subunits, [3H]carboxymethylated transferrin and L-pyroglutamyl-L-prolyl-L-valine-paranitroanilide were detected in homogenates of the synovium and cartilage from arthritic knee joints 1 and 2 weeks after induction. Inhibitor studies and pH curves suggested that the proteinase is leukocyte elastase. Measurable amounts of gelatinolytic activity, detected by activation with 4-aminophenylmercuric acetate and inhibited with EDTA, were also present in the same samples, but there was no detectable collagenase activity. The data on SDS-gelatin substrate gel showed that the proteinase is gelatinase derived from PMLs. These results suggest that in aBSA-induced arthritis, elastase and gelatinase from PMLs invading articular cartilage may play important roles in cartilage destruction.
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PMID:Arthritis induced immunologically with cationic amidated bovine serum albumin in the guinea pig. A morphological and biochemical study on the destruction of articular cartilage. 167 78

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


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