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)

Antihemorrhagic proteins from Crotalus atrox serum were tested for their ability to inhibit the proteolytic activity of the hemorrhagic toxin-e from Crotalus atrox venom and of several other proteolytic enzymes: trypsin, collagenase and thermolysin. The antihemorrhagic proteins inhibited the proteolytic activity of hemorrhagin-e when tested on gelatin type I and collagen type IV, the proteolytic activity of trypsin on photofilm gelatin and the proteolytic activity of whole venom when tested on azocollagen and photofilm gelatin. The antihemorrhagins failed to inhibit the proteolytic activity of trypsin when tested on the specific synthetic substrate N-acetyl-DL-phenylalanine-beta-naphthyl ester (APNE), the activity of microbial collagenase on N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) or on azocollagen and the activity of thermolysin on N-(3-[2-furyl]acryloyl)-Gly-Leu amide (FAGLA). It is tentatively suggested that the antihemorrhagins from snake blood serum are proteinase inhibitors that underwent specialization towards the neutralization of the proteolytic activity of hemorrhagic toxins.
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PMID:Inhibition of the proteolytic activity of hemorrhagin-e from Crotalus atrox venom by antihemorrhagins from homologous serum. 151 50

Several N-carboxyalkyl peptides were synthesized and tested as inhibitors of pig synovial collagenase, 72-kDa gelatinase and stromelysin (matrix metalloproteinases MMP-1, MMP-2, and MMP-3). The most potent of the series, CH3CH2CH2(R,S)CH(COOH)-NH-Leu-Phe-Ala-NH2, competitively inhibited cleavage of dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by MMP-1 and MMP-2 (KI = 30 and 40 microM, respectively). A similar inhibitory potency was found for MMP-1 with soluble Type I collagen and MMP-3 with substance P as substrate. The inhibitor was coupled to EAH-Sepharose 4B through a C-terminal amide. In the presence of 2 M NaCl at pH 7.2, this matrix bound MMP-1, MMP-2, and MMP-3 from concentrated culture medium of pig synovial membranes. The enzymes coeluted at pH 4.1 and subsequently were resolved by chromatography on DEAE-Sephacel and heparin-Sepharose. Purified MMP-1 catalyzed the o-phenanthroline-sensitive cleavage of collagen into TCA and TCB fragments as well as slower hydrolysis of the alpha 2 chain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of MMP-1 indicated a predominant polypeptide of approximately 44 kDa and minor species of approximately 24 and 21 kDa. The 44-kDa species and one of the smaller polypeptides reacted with an antiserum to residues 195-207 of human fibroblast MMP-1, indicating that porcine MMP-1 contains a similar sequence and that the smaller components were probably derived from MMP-1. Neither MMP-2 nor MMP-3 reacted with this antiserum. Purified porcine MMP-2 degraded gelatin but not collagen and exhibited an apparent Mr of approximately 71 kDa. Additional smaller polypeptides were present, one of which may correspond to tissue inhibitor of metalloproteinases. MMP-3 showed doublets of approximately 47/46 and 26/25 kDa and cleaved substance P at the Gly6-Phe7 bond. This procedure provides a rapid means of obtaining all three MMPs from one source in approximately 15% yield each.
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PMID:Application of N-carboxyalkyl peptides to the inhibition and affinity purification of the porcine matrix metalloproteinases collagenase, gelatinase, and stromelysin. 165 8

The midgut chymotrypsins (EC 3.4.4.5) of three species of shrimps, Penaeus monodon, Penaeus japonicus and Penaeus penicillatus were purified and studied in detail to clarify previous ambiguity in their identification. In each of the species there are two major forms of chymotrypsin, both single-chained with three disulfide bonds. One has a pI of 3.2 and Mr 27,000 or 28,000, while the other has a pI of 3.0 and Mr 25,000 or 26,000. The N-terminal amino acid sequences of the P. monodon enzymes are homologous to those of the crab (Uca pugilator) collagenase and to the other chymotrypsins. However, the active sites of the shrimp chymotrypsins are different from that of the well studied bovine alpha-chymotrypsin in some respects: (1) in spite of showing the typical specificity of chymotrypsin, the shrimp enzymes are more stringently selective for substrates with extended polypeptide chain; (2) some titration agents of alpha-chymotrypsin, including t-cinnamoylimidazole, 4-nitrophenyl guanidinobenzoate and its fluorescent derivative, do not react with the shrimp enzymes, neither do some of the alpha-chymotrypsin inhibitors: Tosyl-PheCH2Cl, methyl-4-nitrobenzenesulfonate and benzeneboronic acid; (3) the shrimp chymotrypsins are more reactive than the bovine enzyme toward native protein substrates including collagen; (4) the kinetic-salt-effects of the shrimp enzyme toward N-succinyl- and acetyl-Ala-Ala-Pro-Phe-4-nitroanilide mainly reflect electrostatic rather than hydrophobic interactions between the substrates and the enzyme. The shrimp enzymes are acid-labile but resistent to autolysis. Our results suggest that most Crustacea decapods contain chymotrypsins as one of the major digestive endopeptidases.
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PMID:The midgut chymotrypsins of shrimps (Penaeus monodon, Penaeus japonicus and Penaeus penicillatus). 165 78

Canine jejunal epithelial cells were isolated and maintained in short-term culture to study cholecystokinin (CCK) release. Sequential digestion of jejunal mucosa with collagenase and ethylenediaminetetraacetic acid was followed by counterflow elutriation to enrich CCK-containing cells. After 40 hours in culture on collagen-coated plates, 8.4% of the initially seeded cells were attached; 8.7% of them stained positive with a C-terminal CCK/gastrin antibody and 2.5% stained positive with a gastrin-specific antibody. Basal release of CCK into the culture medium amounted to 1.3% of total cell content over 105 minutes. Receptor-independent stimulation of protein kinase C by the phorbol ester beta-phorbol-12-myristate-13-acetate caused significant CCK release. The inactive form, 4 alpha-phorbol-12-myristate-13-acetate, had no effect. Activation of adenylate cyclase by 10(-5) mol/L forskolin evoked a 2.5-fold increase in CCK concentrations, which was completely abolished by 10(-8) mol/L somatostatin. L-phenylalanine stimulated CCK release at 20 and 50 mmol/L, whereas D-phenylalanine caused significant hormone output only at 50 mmol/L. L-tryptophan had no effect. Cholecystokinin release stimulated by L-phenylalanine was not influenced by the addition of either somatostatin or somatostatin antibody. In conclusion, a system of isolated canine jejunal epithelial cells was developed in short-term culture. This preparation proved suitable for the study of CCK release on a cellular basis.
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PMID:Cholecystokinin release from isolated canine epithelial cells in short-term culture. 172 60

The sequence specificities of human fibroblast and neutrophil collagenases have been investigated by measuring the rate of hydrolysis of 60 synthetic oligopeptides covering the P4 through P'5 subsites of the substrate. The choice of peptides was patterned after both known cleavage sites in noncollagenous proteins and potential cleavage sites (those containing Gly-Ile-Ala, Gly-Leu-Ala, or Gly-Ile-Leu sequences) found in types I, II, III, and IV collagens. The initial rate of hydrolysis of the P1-P'1 bond of each peptide has been measured under first-order conditions ([SO] much less than KM), and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P4 through P'4 all influence the hydrolysis rates for both collagenases. However, the effects of substitutions at each site are distinctive and are consistent with the view that human fibroblast and neutrophil collagenases are homologous but nonidentical enzymes. For peptides with unblocked NH2 and COOH termini, occupancy of subsites P3 through P'3 is necessary for rapid hydrolysis. Compared with the alpha 1(I) cleavage sequence, none of the substitutions investigated at subsites P3, P2, and P'4 produces markedly improved substrates. In contrast, many substitutions at subsites P1, P'1, and P'2 improve specificity. The preferences of both collagenases for alanine in subsite P1 and tryptophan or phenylalanine in subsite P'2, is noteworthy. Human neutrophil collagenase accommodates aromatic residues in subsite P'1 much better than human fibroblast collagenase. The subsite preferences observed for human fibroblast collagenase in these studies agree well with the residues found at cleavage sites in noncollagenous substrates. However, the sequence specificities of these collagenases cannot explain the failure of these enzymes to hydrolyze many potentially cleavable but apparently protected sites in intact collagens. This represents additional support for the notion that the local structure of collagen is important in determining the location of collagenase cleavage sites.
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PMID:Sequence specificities of human fibroblast and neutrophil collagenases. 184 91

The secretion of matrix-degrading proteinases and protein components involved in the production of cytotoxic metabolites is an important step in the sequence of defense reactions executed by polymorphonuclear leukocytes (PMNL) in response to stimulation. In the present report, we have analyzed degranulation of PMNL stimulated either with soluble synthetic peptides fLeu-Phe (fMet, formylmethionyl), or fAhx-Leu-Phe-Ahx-Tyr-Phe (Ahx, aminohexyl) which trigger chemotaxis and degranulation, or with opsonized zymosan which induces phagocytosis. The release of elastase, myeloperoxidase and lactoferrin-containing granules was not at all or only slightly (less than 6%) induced either by fAhx-Leu-Phe-Ahx-Tyr-Leu or by zymosan particles. In contrast, type-I collagenase and gelatinase were secreted in significant amounts after treatment with these agents. The disruption of microfilaments by cytochalasin B and subsequent stimulation of PMNL with the formyl-peptide led to the secretion of elastase, myeloperoxidase and lactoferrin, and enhanced the release of gelatinase. Disruption of microtubules by incubation with colcemid resulted in inhibition of fAhx-Leu-Phe-Ahx-Thyr-Leu and fAhx-Leu-Phe-Ahx-Tyr-Leu/cytochalasin-B-induced granule release. Furthermore, we found different patterns of enzyme distribution after fractionation by centrifugation: most (greater than 90%) type-I collagenase and gelatinase was measured in the supernatant whereas 60-90% of elastase, myeloperoxidase and lactoferrin had partitioned into the cytoskeleton-containing pellet. Our results suggest that the two main types of secretory vesicles identified in PMNL (specific and azurophilic granules) consist of subpopulations. The differential association of the various types of granules with cytoskeletal elements may serve to control their sequential discharge.
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PMID:Release of proteinases from stimulated polymorphonuclear leukocytes. Evidence for subclasses of the main granule types and their association with cytoskeletal components. 201 20

Pseudomonas aeruginosa elastase is a zinc metalloproteinase which is released during P. aeruginosa infections. Pseudomonas keratitis, which occurs following contact lens-induced corneal trauma, can lead to rapid, liquefactive necrosis of the cornea. This destruction has been attributed to the release of both host-derived enzymes and the bacterial products P. aeruginosa elastase, alkaline protease, exotoxin A, and lipopolysaccharide endotoxin. A synthetic metalloproteinase inhibitor, HSCH2 (DL)CH[CH2CH(CH3)2]CO-Phe-Ala-NH2, which we previously showed to be a potent inhibitor of corneal collagenase and alkali-induced corneal ulceration, was tested as a potential inhibitor of P. aeruginosa elastase. Inhibition constants (Kis) for the resolved diastereomers were determined with the chromogenic substrate furylacryloyl-glycyl-L-leucyl-L-alanine. One isomer had a Ki of 0.3 microM, while the other had a Ki of 0.4 microM. The more potent diastereomer was evaluated in vivo in experimentally induced Pseudomonas keratitis in rabbits. Following inoculation of one cornea of each rabbit, topical treatment with a 1 mM solution of the inhibitor significantly delayed the onset of corneal melting and perforation, as compared with the results for the control and gentamicin-treated groups. This protective effect suggests that the inhibitor may have a therapeutic application by delaying the progression of corneal destruction in Pseudomonas keratitis.
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PMID:Inhibition of Pseudomonas aeruginosa elastase and Pseudomonas keratitis using a thiol-based peptide. 212 41

Effects of transforming growth factor (TGF)-beta, epidermal growth factor (EGF), insulin, 1, 25-dihydroxyvitamin D3 (1, 25 (OH)2D3), and parathyroid hormone (PTH) on the proliferation and differentiation of clonal dental pulp cells of rats were investigated. Interaction between growth factors (TGF-beta and EGF) and two hormones insulin and 1, 25 (OH)2D3, which have been noticed to accelerate the differentiation of the cells, were also studied, and the following results were obtained: 1) TGF-beta decreased alkaline phosphatase (ALPase) activity in a dose-dependent manner, and the inhibitory effect was not blocked by indomethacin, suggesting that the effect of TGF-beta on the cells may not be mediated by prostaglandins. Inhibitory effects of ALPase antagonists (L-phenylalanine, L-homoarginine, levamisole) on the activity were not affected by TGF-beta. TGF-beta showed no evident effect on the DNA synthesis (incorporation of [3H] thymidine) and collagen synthesis (incorporation of [2, 3-3H] proline into the collagenase-digestible protein) of the cells. 2) EGF stimulated the incorporation of [3H] thymidine and inhibited the ALPase activity. The inhibitory effect was not blocked by indomethacin, indicating that the EGF effect is not mediated by prostaglandins. Collagen synthesis was significantly inhibited by EGF. 3) Insulin showed a weak but significant inhibition of the DNA synthesis. Insulin increased the ALPase activity evidently, and accelerated the collagen synthesis significantly. 4) The vitamin 1, 25 (OH)2D3 significantly increased the ALPase activity though no significant changes were observed in the DNA synthesis and collagen synthesis. 5) PTH had no evident effect on the DNA synthesis and ALPase activity, but did tend to accelerate the collagen synthesis. 6) A study on the interaction between insulin and EGF or TGF-beta revealed that the acceleration of DNA synthesis induced by EGF was inhibited when the factor was combined with insulin, and the increase in ALPase activity elicited by insulin was inhibited by EGF and weakened by TGF-beta significantly when these factors were added simultaneously with the insulin. Or viewed another way, the inhibitory effect of EGF or TGF-beta on the ALPase activity was antagonized by insulin. The accelerative action of insulin on collagen synthesis was antagonized by EGF and potentiated by TGF-beta. 7) A study on the interaction between 1, 25 (OH)2D3 and EGF or TGF-beta revealed that 1, 25 (OH)2D3 inhibited the accelerating effect of EGF on the DNA synthesis and that the increasing effect of 1, 25 (OH)2D3 on ALPase activity was strongly inhibited by EGF.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[Effects of various growth factors and hormones on clonal rat pulp cells]. 213 79

An extensive series of N-(monoethylphosphoryl)peptides was synthesized and their inhibition of purified human skin fibroblast collagenase examined. At the cleavage site S1 all reported compounds have the (EtO)(OK)P(O) group and the peptide side chain extended toward the C-terminal end (up to P5') of the substrate sequence. These phosphoramidates with a tetrahedrally hybridized phosphorus atom are thought to be transition state analogue inhibitors. They exhibited fair inhibitory potency against this vertebrate collagenase having Ki values in the micromolar range. The most potent of these, (EtO)(OK)P(O)-Ile-TrpNHCH3 (68), inhibits with a Ki value of 1.5 microM and is nearly 100 times stronger than (EtO)(OK)P(O)-Ile-Ala-GlyOK (51) (Ki of 140 microM), which has the sequence matching that of the alpha 1 (I) chain of collagen in P1', P2', P3' after the cleavage site. Several compounds were prepared in an attempt to identify the nature of the S2', S3', and S4' binding sites. Alanine at the P2' position was replaced by leucine, phenylalanine, tryptophan, or tyrosine derivatives, resulting in Ki values in a significantly lower range, 1.0-40 microM, compared to 51. No upper size limitation or specificity has been found at this position, yet similar replacements at the P3' position, which is occupied naturally by a glycine residue, gave weaker inhibitors: (EtO)(OK)P(O)-Ile-Tyr(OBzl)-PheOK (57) had a Ki of 120 microM. Hexapeptide derivatives had weaker activities in the 270 microM-2 mM range. All inhibitors were evaluated by using the synthetic thio peptolide spectrophotometric assay.
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PMID:Phosphoramidate peptide inhibitors of human skin fibroblast collagenase. 215 7

To define the inhibitory requirements of mammalian collagenase, several N-substituted amide and peptide derivatives of the mercaptomethyl analogue of leucine, 2-[(R,S)mercaptomethyl]-4-methylpentanoic acid (H psi[SCH2]-DL-leucine), were synthesized and tested as inhibitors of pig synovial collagenase with soluble type I collagen as substrate. H psi[SCH2]-DL-leucine (IC50 = 320 microM) was about 10 times more potent than the beta-mercaptomethyl compound, N-acetylcysteine. The amide of H psi[SCH2]-DL-leucine was six times more potent than the parent thiol acid. Aliphatic N-substituted amides were less potent than the unsubstituted amide, whereas the N-benzyl amide was slightly more potent. Dipeptides, particularly those with an aromatic group at P2', were up to 20-fold more potent, while tripeptides with an aromatic L-amino acid at P2' and Ala-NH2 at P3' were up to 2200 times more potent than H psi[SCH2]-DL-leucine. The resolved diastereomers of H psi[SCH2]-DL-Leu-Phe-Ala-NH2 inhibited by 50% at 0.3 and 0.04 microM, respectively. The most potent inhibitor synthesized, an isomer of H psi[SCH2]-DL-Leu-L-3-(2'-naphthyl)alanyl-Ala-NH2, exhibited an IC50 of 0.014 microM, a value about 300 times less than similar thiol-based analogues of the P'-cleavage sequence of type I collagen, H psi[SCH2]-DL-Leu-Ala-Gly-Gln-. These structure-function studies establish within the present series of compounds that the most effective inhibitors of mammalian collagenase are not closely related to the P2'-P3' elements of the cleavage site of the natural substrate but rather have an aromatic group at the P2' position and Ala-NH2 at the P3' position.
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PMID:Thiol-based inhibitors of mammalian collagenase. Substituted amide and peptide derivatives of the leucine analogue, 2-[(R,S)-mercaptomethyl]-4-methylpentanoic acid. 215 64


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