Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Four new fluorogenic heptapeptide substrates have been synthesized with sequences that are optimized for five human matrix metalloproteinases (MMP). All four substrates are similar to one recently reported by Stack and Gray (1989, J. Biol. Chem. 264, 4277-4281) and have the fluorescent Trp residue in subsite P'2 and the dinitrophenol (DNP) quenching group on the N-terminus. The quenching of the Trp fluorescence in the intact substrate is relieved on hydrolysis of the P1-P'1 bond, giving rise to a continuously recording fluorescence assay. The residues placed in subsites P3-P'1 and P'3 have been optimized for each MMP, while Arg has been placed in P'4 to enhance solubility. Thus, DNP-Pro-Leu-Ala-Leu-Trp-Ala-Arg has been prepared as a substrate for fibroblast collagenase, DNP-Pro-Leu-Ala-Tyr-Trp-Ala-Arg for neutrophil collagenase, DNP-Pro-Tyr-Ala-Tyr-Trp-Met-Arg for neutrophil collagenase, DNP-Pro-Tyr-Ala-Tyr-Trp-Met-Arg for stromelysin, and DNP-Pro-Leu-Gly-Met-Trp-Ser-Arg for both 72-kDa fibroblast gelatinase and 92-kDa neutrophil gelatinase. These substrates have been characterized with respect to their composition, solubility, optical and fluorescence spectra, and hydrolysis by their target MMP. The hydrolysis rates rival or exceed those of either their natural protein substrates or other synthetic peptides. The solubility of each substrate in assay buffer exceeds the KM value for each reaction, allowing accurate determination of the kinetic parameters. These new substrates should greatly facilitate kinetic studies of the MMP.
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PMID:Continuously recording fluorescent assays optimized for five human matrix metalloproteinases. 188 20

Collagenase (matrix metalloproteinase 1) cleaves type I, II, and III collagen helices at a specific site between Gly-Ile or Gly-Leu bonds (residues 775 and 776, P1-P1'). To understand the mechanism of collagen processing, mutations around the cleavage site have been introduced into the cloned murine pro alpha 1(I) collagen (Col1a1) gene. These mutant constructs have been transfected into homozygous Mov13 fibroblasts that do not express the endogenous Col1a1 gene due to a retroviral insertion. Secreted triple-helical type I collagens containing substitutions of Pro for Ile (position 776) (P1') were not cleaved by human rheumatoid synovial collagenase, whereas those containing substitutions of Met for Ile (position 776) were cleaved. Type I collagens containing double substitutions of Pro for Gln-774 (P2) and Ala-777 (P2') were not cleaved regardless of whether they contained the wild-type residue Ile at position 776 or the substitution of Met for Ile at position 776. The wild-type alpha 2(I) chains derived from the endogenous Col1a2 gene were also resistant to enzyme digestion when they were complexed with the mutant alpha 1(I) chains, indicating that the presence of normal alpha 1(I) sequences is critical for cleavage of the alpha 2(I) chains in the type I heterotrimer.
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PMID:Generation of collagenase-resistant collagen by site-directed mutagenesis of murine pro alpha 1(I) collagen gene. 216 7

Granulomas are chronic, usually focal, tissue-destructive inflammatory reactions that usually form around slowly degradable, poorly soluble substances. They are dynamic lesions, regulated by complex immune mechanisms. Tachykinins are a family of neuropeptides characterized by the common C-terminal amino acid sequence -Phe-X-Gly-Leu-Met-NH2. One such tachykinin, substance P, has been reported to modulate immunologic responses. In this investigation, granulomas were examined for substance P. Granulomas were isolated from the livers of mice infected with murine schistosomiasis, and substance P was extracted. Immunoreactive substance P was detected by RIA. The authenticity of the molecule was confirmed by elution profile on HPLC. Immunoreactive substance P, identified by immunostaining, localized to eosinophils derived from collagenase-dispersed granulomas. Granulomas were then probed for expression of the gene for substance P (preprotachykinin). Preprotachykinin mRNA was localized to granuloma eosinophils by in situ oligonucleotide hybridization. It is concluded that substance P is present within the granuloma as a result of preprotachykinin production by eosinophils.
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PMID:Eosinophils from granulomas in murine schistosomiasis mansoni produce substance P. 245 38

Polymorphonuclear leukocytes (PMNs) recovered from 4-h lapine peritoneal exudates contained factors which provoked the synthesis of collagenase, gelatinase, caseinase, and prostaglandin E2 by lapine articular chondrocytes. Rapid secretion of these factors occurred after exposing the polymorphs to phorbol myristate acetate or formyl-Met-Leu-Phe. Fractionation of polymorph lysates by HPLC size exclusion chromatography provided a molecular weight of approximately 14,000 for the active principle. Examination of the most active fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining, confirmed the presence of a single band with this apparent molecular weight. Isoelectric focusing of this fraction revealed the presence of four distinct bands with the pI values 6.90, 7.05, 7.45, and 7.55. This fraction tested positive in a bioassay for interleukin-1. We were unable to activate chondrocytes by exposure to extracts of human PMNs from either peripheral blood or inflammatory synovial fluid.
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PMID:Chondrocyte activation by a putative interleukin-1 derived from lapine polymorphonuclear leukocytes. 253 51

A newly synthesized compound, the non-steroidal antiinflammatory agent N-methoxy-3-(3,5-di-tert-butyl-4-hydroxybenzylidene)pyrrolidin-2-o ne (E-5110) was investigated. E-5110 inhibited prostaglandin E2 (PGE2) generation by cultured rat synovial cells upon stimulation with interleukin-1-like factor of rat polymorphonuclear leucocytes (PMN). The IC50 values (mumol/l) for PGE2 generation were 0.026 for E-5110, 0.008 for indometacin, 0.112 for piroxicam and 0.667 for the compound B (3-amino-1-(M-trifluoromethyl-phenyl-2-pyrazoline). Calcium ionophore A23187-stimulated leukotriene B4 generation by human PMN was inhibited by E-5110 with an IC50 value of 0.20 mumol/l; E-5110 was as inhibitory as nordihydroguaiaretic acid and was more potent than the compound B (IC50 of 2.58 mumol/l). E-5110 suppressed superoxide generation by human PMN stimulated with opsonized zymosan, f-Met-Leu-Phe and phorbol myristate acetate. E-5110 also inhibited the generation of leucocytic pyrogen and leucocyte factor(s) which stimulated collagenase production by cultured synovial cells.
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PMID:Effect of the novel non-steroidal antiinflammatory agent N-methoxy-3-(3,5-di-tert-butyl-4-hydroxybenzylidene)pyrrolidin-2-one on in vitro generation of some inflammatory mediators. 282 44

Leukocyte chemoattractants markedly alter the morphology and membrane functions of leukocytes. Bacterial collagenase causes a change in cell shape similar to that seen with the leukocyte chemoattractant, f-Met-Leu-Phe, and also promotes capping of concanavalin A. Human neutrophils in suspension or adherent to cover glasses were exposed to clostridial collagenase (10-250 units/ml) for up to 30 min at 37 degrees C and then fixed. Collagenase (125 units/ml) caused more than 85% of PMNs to assume an asymmetric or motile morphology even in the presence of 1% gelatin or 10 mg/ml bovine serum albumin. Trypsin alone (0.01-1%) did not induce a shape change. A similar morphology was seen in some untreated PMNs (less than 5% of all cells) and is characteristic of f-Met-Leu-Phe-treated cells (more than 90%). Collagenase inhibitors (i.e., reduced glutathione, cysteine, and acid-soluble collagen), however, prevented the shape change induced by collagenase but not by f-Met-Leu-Phe. At 4 degrees C, fluorescein-Con A (20 micrograms/ml) bound uniformly to both untreated and collagenase-treated cells. Upon further incubation at 37 degrees C, Con A was internalized over the entire cell periphery of the rounded, untreated cells but on collagenase-treated PMNs was rapidly gathered into a cap overlying the uropod or protuberant region of cytoplasm where it was subsequently internalized. Checkerboard Boyden chamber assays showed clostridial collagenase to be chemokinetic and chemotactic for human PMNs. In receptor binding experiments, the clostridial collagenase preparation competed poorly with [125I]formylhexapeptide for binding to PMN formylpeptide receptors (less than 15% reduction in binding at 200 units/ml collagenase). Thus, collagenase does not seem to interact strongly with the neutrophil formylpeptide receptor and may stimulate PMN motility by interacting at an altogether different site.
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PMID:Clostridial collagenase. A chemoattractant for human neutrophils. 302 90

The amino acid sequences surrounding three major phosphorylation sites in rat and bovine synapsin I have been determined by employing automated gas-phase sequencing and manual Edman degradation of purified phosphopeptide fragments. Site 1 is a serine residue phosphorylated by cAMP-dependent protein kinase and by calcium/calmodulin-dependent protein kinase I. The sequence around site 1 was derived from tryptic/chymotryptic phosphopeptides and overlapping cyanogen bromide cleavage fragments. This sequence, identical in rat and bovine synapsin I, is Asn-Tyr-Leu-Arg-Arg-Arg-Leu-Ser(P)-Asp-Ser-Asn-Phe-Met. Site 1 is located at the NH2 terminus of the protein, within the collagenase-resistant head region. Sites 2 and 3 are serine residues phosphorylated by calcium/calmodulin-dependent protein kinase II. The sequences surrounding bovine site 2 and site 3 were derived from tryptic phosphopeptides and overlapping fragments generated by cleavage with chymotrypsin, collagenase, and endoproteinase Lys-C. The sequence around bovine site 2 is Thr-Arg-Gln-Thr-Ser(P)-Val-Ser-Gly-Gln-Ala-Pro-Pro-Lys, and the sequence around bovine site 3 is Thr-Arg-Gln-Ala-Ser(P)-Gln-Ala-Gly-Pro-Met-Pro-Arg. Sites 2 and 3 are located within the COOH-terminal, collagenase-sensitive tail region of the molecule, separated by 36 amino acids. The sequences surrounding rat site 2 and site 3 were derived from tryptic phosphopeptides. The sequence around rat site 2 is Gln-Ala-Ser(P)-Ile-Ser-Gly-Pro-Ala-Pro-Pro-Lys, and the sequence around rat site 3 is Gln-Ala-Ser(P)-Gln-Ala-Gly-Pro-Gly-Pro-Arg. Thus, the sequences surrounding the four sites that are phosphorylated by calcium/calmodulin-dependent protein kinase II, namely sites 2 and 3 in rat and bovine synapsin I, exhibit a high degree of homology.
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PMID:Amino acid sequences surrounding the cAMP-dependent and calcium/calmodulin-dependent phosphorylation sites in rat and bovine synapsin I. 311 71

Human lung specimens were minced and treated for 30 min with collagenase (1 mg ml-1) and DNase (0.1 mg ml-1) to obtain a suspension of viable (approximately 80%) and metabolically active lung cells (5 x 10(6) cells per gram of tissue). Treatment of these mixed lung cells with bradykinin (1.25 x 10(-6) to 1 x 10(-5) M) and f-Met-Leu-Phe (f-MLP; 1 x 10(-8) to 5 x 10(-6) M) did not stimulate to a substantial extent the release of prostaglandins and thromboxanes (measured with novel Enzyme Immunoassays). The only concentration of PAF that stimulated significantly the release of icosanoids from lung cells was 5 x 10(-7) M. Phorbol myristate (PMA; 5 x 10(-8) to 2 x 10(-6) M) and ionophore a-21387 (2.5 x 10(-6) to 2 x 10(-5) M) strongly stimulated the release of prostaglandins and thromboxanes by dispersed human lung cells. These findings support previous observations showing that human lungs have the enzymes necessary for the synthesis and release of prostaglandins and thromboxanes but stimulation of the release of these mediators is not obtained with the hormonal stimuli that are active in guinea pigs. Studies in progress will purify the cell populations and characterize the cells responsible for the release of these icosanoids.
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PMID:Release of prostaglandin E2 and thromboxane B2 by mixed isolated human lung cells. 313 66

A proteinase inhibitor which has strong anti-collagenase activity was found in chicken egg white. The inhibitor (pI = 4.9) was purified by poly(ethylene glycol) (5.5-10%) precipitation and chromatography on Ultrogel AcA 34, DEAE-cellulose, and Sephacryl S-300. The final product was homogeneous on 5% polyacrylamide gel electrophoresis. Stoichiometric inhibition was observed with the inhibitor and rabbit synovial collagenase and thermolysin (1:1 molar ratio with thermolysin). The inhibitor ran on sodium dodecyl sulfate-gel electrophoresis with reduction as a single protein band of Mr = 165,000. The molecular weight of the native inhibitor was estimated to be 780,000 by sedimentation equilibrium centrifugation. Centrifugation analysis in 6 M guanidine hydrochloride and of the reduced sample gave M omega = 380,000 and M omega = 195,000, respectively, where M omega is the weight-average molecular weight determined by equilibrium ultra-centrifugation. The results indicated that the inhibitor molecule is a tetramer of identical subunits linked in pairs by disulfide bonds. Since the molecular weight and the quaternary structure of the inhibitor were similar to those of alpha 2-macroglobulin (alpha 2M) in plasma, chicken alpha 2M was isolated and compared with the inhibitor. The inhibitor was not sensitive to methylamine, whereas chicken alpha 2M was. No immunocross-reactivity was observed between the inhibitor and chicken alpha 2M. The NH2-terminal sequence of the egg white inhibitor is Lys-Glu-Pro-Glu-Pro-Gln-Tyr-Val-Leu-Met-Val-Pro-Ala. The sequence of chicken alpha 2M is Ser-Thr-Val-Thr-Glu-Pro-Gln-Tyr-Met-Val-Leu-Leu-Pro-Phe. Considerable homology was found between the two sequences and to the NH2-terminal sequence of human alpha 2M. Monospecific antibody raised against the egg white inhibitor was employed to examine the tissue distribution of the inhibitor. The inhibitor was found only in oviduct and egg white, but not in other tissues or serum of chickens.
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PMID:Ovostatin: a novel proteinase inhibitor from chicken egg white. I. Purification, physicochemical properties, and tissue distribution of ovostatin. 640 74


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