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)

Interlobular and intralobular ducts isolated from the pancreas of the rat by digestion with collagenase and chymotrypsin were cultured in an agarose matrix containing CMRL-1066 supplemented with insulin, dexamethasone, L-glutamine, soybean trypsin inhibitor, antibiotics, and fetal bovine serum. The cut ends of most interlobular ducts sealed to create enclosed lumina. Some ducts retained their original cylindrical organization; others enlarged to varying degrees, resulting in structures that ranged from cylindrical to spherical in shape. The duct walls consisted of viable epithelium and connective tissue, although the amount of connective tissue declined with age. Both epithelial and connective tissue cells became flattened in the enlarged ducts. Intralobular and small interlobular ducts often remained associated with the larger interlobular ducts. These duct fragments have been cultured for as long as 6 weeks.
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PMID:Ducts of the rat pancreas in a agarose matrix culture. 740 38

Primitive biliary cells are known to migrate from the ductal plate into the mesenchyme during human intrahepatic bile duct development, and this migration process is essential for normal development of intrahepatic bile ducts. However, its molecular mechanism is unknown. Matrix proteinases play an important role in cell migration during cancer invasion and organ development. In this study, we therefore investigated in situ expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMP) during human intrahepatic bile duct development, using 32 human fetal livers. We also examined in situ expression of trypsinogen/trypsin, chymotrypsinogen/chymotrypsin, and cathepsin B, which are matrix proteinases and activators of MMP. MMP-1 expression was noted in the ductal plate and migrating primitive biliary cells. MMP-2, MMP-3, and MMP-9 were expressed in the ductal plate. TIMP-1 and TIMP-2 were expressed in the ductal plate and migrating primitive biliary cells. Trypsinogen/trypsin, chymotrypsinogen/chymotrypsin, and cathepsin B were also expressed in primitive biliary cells. These data suggest that MMP, trypsinogen/trypsin, chymotrypsinogen/chymotrypsin, and cathepsin B play a critical role in biliary cell migration during human intrahepatic bile duct development by degrading extracellular matrix proteins. The data also suggest that MMP inhibitors (TIMP-1 and TIMP-2) and MMP activators (trypsin, chymotrypsin, and cathepsin B) play an important role in biliary cell migration. The coordinated expression of MMP, MMP inhibitors, and MMP activators may be necessary for the normal development of human intrahepatic bile ducts.
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PMID:Expression of matrix proteinases during human intrahepatic bile duct development. A possible role in biliary cell migration. 748 84

Most of the pancreatic exocrine epithelium consists of acinar and intralobular duct (ductular) cells, with the balance consisting of interlobular and main duct cells. Fragments of mouse acinar/ductular epithelium can be isolated by partial digestion with collagenase and purified by Ficoll density gradient centrifugation. We investigated whether previously developed culture conditions used for duct epithelium would result in the selective survival and proliferation of ductular cells from the acinar/ductular fragments. The fragments were cultured on nitrocellulose filters coated with extracellular matrix. After 2 to 4 wk the filters were covered with proliferating cells resembling parallel cultures of duct epithelium by the following criteria: protein/DNA ratio, light and electron microscopic appearance, the presence of duct markers (carbonic anhydrase [CA] activity, CA II mRNA, the cystic fibrosis transmembrane conductance regulator), the near absence of acinar cell markers (amylase and chymotrypsin), a similar polypeptide profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of spontaneous and secretin-stimulated electrogenic ion transport. Both duct and ductular epithelia formed fluid-filled cysts in collagen gels and both could be subcultured. We conclude that acinar/ductular tissue gives rise to ductular cells in culture by some combination of acinar cell death and/or transdifferentiation to a ductular phenotype, accompanied by proliferation of these cells and preexisting ductular cells. These cultures may be used to investigate the properties of this part of the pancreatic duct system, from which most of the pancreatic juice water and electrolytes probably originates.
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PMID:Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures. 752 26

We investigated the effect of gonadotropins on protease that were suggested to be implicated in the invasive activity of the trophoblast. hCG levels ranging from 10 x 10(3) to 333 x 10(3) IU/L produced a dose-dependent inhibition of the in vitro globinolytic activity of the purified proteases trypsin, chymotrypsin, and urokinase, but failed to inhibit plasmin, collagenase, elastase, and tissue-type plasminogen activator. Likewise, FSH inhibited purified trypsin and urokinase, but not plasmin or tissue-type plasminogen activator. Culture medium conditioned with human trophoblast displayed serine protease and urokinase-like activities; exposure of the cultured trophoblast to exogenous hCG markedly suppressed serine protease and urokinase activities in the conditioned medium. A short treatment of the conditioned medium with trypsin abolished the hCG-mediated inhibition of urokinase activity. The present findings offer an explanation for earlier observations that hCG reduced collagenase activity in trophoblasts without affecting the level of collagenase-specific mRNA. The present results are also consistent with the concept that hCG, by its direct ability to inhibit certain serine proteases and urokinase in trophoblast, suppresses a protease-mediated conversion of procollagenase to active collagenase. The ability of hCG to prevent initiation of the collagenolytic cascade suggests that gonadotropins may regulate the transient invasive activity of the trophoblast.
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PMID:Gonadotropin-mediated inhibition of proteolytic enzymes produced by human trophoblast in culture. 768 89

A collagenolytic proteinase was purified from the intestines of Atlantic cod by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The proteinase has an estimated molecular weight of 24.1 (+/- 0.5) kDa as determined by SDS-PAGE and belongs to the chymotrypsin family of serine proteinases. The enzyme cleaves native collagen types I, III, IV and V, and also readily hydrolyzes succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin, as well as succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, a reported elastase substrate, but had no detectable activity towards several other substrates of these proteinases or of trypsin. The pH optimum of the enzyme was between pH 8.0 and 9.5 and it was unstable at pH values below 7. Maximal activity of the enzyme when assayed against sAAPFpna was centered between 45 and 50 degrees C. Calcium binding stabilized the cod collagenase against thermal inactivation, but even in the presence of calcium, the enzyme was unstable at temperatures above 30 degrees C.
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PMID:Characterization of a collagenolytic serine proteinase from the Atlantic cod (Gadus morhua). 774 22

Heparin was extracted and purified from normal human plasma, and full characterization of its structure and physico-chemical properties was achieved for the first time. Plasma was submitted to exhaustive proteolytic treatment with papain, trypsin, chymotrypsin, collagenase and pepsin, anion-exchange chromatography and precipitation with organic solvents. By this procedure, we recovered heparin (about 0.7 mg/100 ml of plasma) and chondroitin sulfate (about 0.1 mg/100 ml of plasma). Chondroitin sulfate has a peak molecular mass of about 15,630, and it is composed of about 60% nonsulfated disaccharide, 3.5% disaccharide 6-monosulfate and about 40% disaccharide 4-monosulfate, with a sulfate-to-carboxyl ratio of 0.41. Heparin, identified by agarose-gel electrophoresis, is constituted by about 40% slow-moving component and about 60% fast-moving species. This glycosaminoglycan had a peak molecular mass of about 7000, and was identified as 'typical' heparin by its constituent disaccharide composition. About 70% of disaccharides were identified as trisulfated disaccharide, and about 18% as disulfated disaccharides, 3% as monosulfated disaccharides and 10% as nonsulfated disaccharide. Heparin extracted from normal human plasma has a high sulfate-to-carboxyl ratio (2.47) and in vitro anticoagulant activity of about 70 I.U. A more quantitative and statistical analysis performed on 10 ml of plasma obtained from 10 human healthy volunteers revealed a heparin level of 0.54 +/- 0.17 mg/100 ml plasma (mean +/- standard deviation) with a coefficient of variation of about +/- 32%. These findings demonstrate for the first time the presence of heparin molecules in normal human plasma and confirm the importance of adequate extraction processes to purify a molecule that strongly interacts with plasma protein components. This is discussed in light of other authors that described a polysaccharide molecule named heparan sulfate in human plasma.
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PMID:Qualitative and quantitative studies of heparin and chondroitin sulfates in normal human plasma. 782 7

Active digestive enzymes are involved in the pathophysiology of acute pancreatitis. Previous studies have mainly focused on the role of trypsin in the autodigestive process. The present study compares the noxious potential of different pancreatic enzymes to damage acinar cells. Acinar cells were isolated from rat pancreas by collagenase digestion. Cell viability was studied by (1) exclusion of trypan blue, (2) release of lactate dehydrogenase, and (3) release of newly synthesized proteins identified with methionine labeled with sulfur 35. Cells were then incubated in oxygenated N-2-hydroxyethylpiperazine-N-'-2-ethanesulfonic acid-Ringer solution containing different concentrations of various active digestive enzymes. Uptake of trypan blue was the most sensitive and reliable test of cell damage when compared with release of lactate dehydrogenase or radiolabeled newly synthesized proteins. All active digestive enzymes studied caused dose-dependent cell damage. The noxious potential, however, was strikingly different for the various enzymes. Pancreatic elastase in nanomolar concentrations caused marked cell damage after 45 to 90 minutes of incubation. Lipase and chymotrypsin caused a similar damage only at micromolar concentrations, whereas even millimolar concentrations of trypsin failed to cause significant damage. The present results confirmed recent work showing that lipase and phospholipase A2 probably cause cell damage through release of free fatty acids and lysolecithin. Although activation of trypsin might be the trigger to start the activation cascade in acute pancreatitis, trypsin itself is markedly less noxious to acinar cells when compared with other digestive enzymes. Elastase by far had the greatest noxious potential of all enzymes evaluated. Studies analyzing therapeutic effects of protease inhibitors should evaluate not only the inhibitory potential against trypsin but also that against other digestive enzymes, particularly elastase.
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PMID:Active pancreatic digestive enzymes show striking differences in their potential to damage isolated rat pancreatic acinar cells. 784 75

Affinity-based purification and characterization of the collagenolytic serine protease 1 from Uca pugilator (fiddler crab) hepatopancreas shows that the enzyme cleaves the native bovine alpha 1(I) collagen chain carboxyl-terminal to Gln and Arg residues adjacent to the metallocollagenase site. Cleavage carboxyl-terminal to Leu residues is observed in the alpha 2(I) chain and at a secondary site in alpha 1(I). These sites correlate with the preferences observed toward p-nitroanilide substrates varying at the P1 position, for which the specificity (kcat/Km) is Arg > Leu, Phe, Lys > Gln > Ala. Furthermore, collagen cleavage after Gln was found exclusively between two Gln-Arg bonds. The P'1-P'3 specificity of collagenase, as determined by nucleophile acyl transfer, indicated a strong preference for Arg in the P'1 position. Crab collagenase cleaves peptide bonds adjacent to Leu and Gln at the P1 position more efficiently than trypsin, chymotrypsin, or elastase. Moreover, the efficiency of collagenase toward P1-Arg substrates is equivalent to that of trypsin. Crystals of crab collagenase have been grown complexed with the protein inhibitor ecotin. These crystals diffract to better than 2.8 A resolution and belong to the space group P3(2)21 with unit cell dimensions of a = b = 89.0 A, c = 291.7 A.
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PMID:The substrate specificity of Uca pugilator collagenolytic serine protease 1 correlates with the bovine type I collagen cleavage sites. 803 25

Matrix vesicles (MV), microstructures which rapidly accumulate Ca2+ and induce mineral formation in vitro, are linked to type II and X collagens and proteoglycans in the hypertrophic cartilage. However, the roles of these matrix proteins on MV function are not known. This led us to investigate the influence of type II and X collagen binding on Ca2+ uptake by MV. MV isolated from chicken growth plate cartilage were treated with pure bacterial collagenase and 1 M NaCl in synthetic cartilage lymph to selectively and completely remove associated type II and X collagens. Uptake of 45Ca2+ by these collagen-depleted vesicles was markedly reduced. Further treatment with detergent, which disrupted the membrane, restored Ca2+ uptake, indicating that the vesicle membrane structure and the nucleational core inside the vesicle lumen were still intact after the collagenase and 1 M NaCl treatments. Readdition of either native type II or X collagen to the collagenase, 1 M NaCl-treated MV stimulated their Ca2+ uptake to levels similar to those of untreated vesicles. Pepsin-treated type II and X collagens were less effective in stimulating Ca2+ uptake, indicating that non-triple helical domains of these collagens were involved. The pepsin treatment of these collagens also decreased their binding to annexin V (anchorin CII), one of three annexins found in MV, suggesting that annexin V is involved in mediating the binding of type II and X collagens to the MV surface. Furthermore, treatment of collagenase, 1 M NaCl-treated MV with chymotrypsin, which damaged annexin V as well as many other MV proteins, prevented the stimulation of Ca2+ uptake by these collagens. Thus, the interaction between type II and X collagens with MV activates the influx of Ca2+ into MV and may play an important role in calcification of the vesicles.
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PMID:Stimulation of calcification of growth plate cartilage matrix vesicles by binding to type II and X collagens. 815 77

We describe here a cell-free system which will carry out the initial stages in the synthesis, post-translational modification and assembly of type-X collagen. The mRNA coding for bovine type-X collagen was synthesized in vitro and translated in a rabbit reticulocyte lysate to yield a protein that was collagenase sensitive and could be immunoprecipitated with antibodies raised to purified avian type-X collagen. When type-X collagen was synthesized in the absence of added microsomes or in the presence of canine pancreas microsomes, the translation products showed partial resistance to digestion with pepsin but were completely degraded with a mixture of chymotrypsin and trypsin, suggesting that only incorrectly aligned non-native collagen molecules were synthesized under these conditions. When the protein was synthesized in the presence of microsomes derived from avian fibroblasts or a human fibrosarcoma cell line, the translocated product migrated as a diffuse band characteristic of hydroxylated collagen. The synthesized polypeptides were also resistant to both pepsin and trypsin/chymotrypsin digestion, demonstrating the formation of correctly aligned native collagen. Furthermore, the collagen polypeptides assembled into higher-order structures, possibly trimers, which were stabilized by interchain disulphide bonds. The collagen helix synthesized in vitro had a melting temperature of 41 degrees C which is comparable with the protein synthesized in vivo, further demonstrating that the polypeptides were hydroxylated and that the triple helix formed was correctly aligned.
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PMID:Reconstitution of the folding pathway of collagen in a cell-free system: formation of correctly aligned and hydroxylated triple helices. 825 44


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