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)

The collagenase from the larvae Hypoderma lineatum, with a molecular weight of 24 000 and isoelectric point of 4.1, was obtained in homogeneous form by ion-exchange chromatography. It is stoichiometrically inhibited by diisopropylfluorophosphate. On the other hand it is unaffected by ethylenediaminetetraacetate, p-chloromercuribenzoate, dithiothreitol, N-tosyllysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone and ovomucoid trypsin inhibitor. The enzyme which degrades native collagen in its helical parts, has a specific activity on thermally reconstituted collagen fibrils of 150 micrograms collagen degraded x min-1 x (mg enzyme)-1 at 37 degrees C. It hydrolyses casein but has no esterolytic activity characteristic of trypsin, chymotrypsin nor elastase. It has no action on the synthetic peptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-L-glycyl-L-prolyl-D-arginine. The amino acid composition of Hypoderma collagenase indicates a distinct similarity with the serine proteinases of the trypsin family and with another athropode serine collagenase, that of the fiddler crab Uca pugilator. This suggests that eucaryotic collagenases with digestive rather than morphogenic function represent a new category of members of the trypsin family.
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PMID:Chemical and enzymatic characterization of the collagenase from the insect Hypoderma lineatum. 23 30

1. Rat livers were dissociated into their constituent cells by perfusion through the portal vein with a medium containing collagenase, and hepatocytes separated from non-parenchymal cells. 2. It is shown that the procedure described by Wisher & Evans [(1975) Biochem. J. 146, 375-388] for preparation of plasma membranes from liver tissue when applied to isolated hepatocytes also yielded subfractions of similar morphology and marker-enzyme distribution. 3. Thus the distribution of alkaline phosphodiesterase, 5'-nucleotidase and the basal and glucagon-stimulated adenylate cyclase among two 'light' vesicular and one 'heavy' junction-containing plasma-membrane subfractions paralleled that reported for tissue-derived plasma-membrane subfractions. 4. Increased recoveries and specific activities of plasma-membrane marker enzymes were obtained when soya-bean trypsin inhibitor was included in the collagenase-containing perfusion media used to dissociate the liver. 5. Polyacrylamide-gel-electrophoretic analysis of the corresponding plasma-membrane subfractions prepared from liver tissue and isolated hepatocytes were generally similar. 6. The results indicate that the functional polarity of the hepatocyte's plasma membrane is retained after tissue dissociation. The damage occurring to plasma-membrane ectoenzymes by the collagenase-perfusion procedure is discussed.
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PMID:Preparation of plasma-membrane subfractions from isolated rat hepatocytes. 88 Feb 46

A novel model of arterial thrombosis was developed. A mechanical endothelium-denuding injury was created (using a scalpel blade) on harvested, freezer-stored rat carotid arteries. Vessel length of 5 mm. were grafted into the femoral arteries of recipient Sprague-Dawley rats using microvascular anastomotic technique. Patency rates in untreated animals were compared with those in animals receiving systemic aspirin or heparin. The control group patency after 2 hours of flow was 15%, while grafts in aspirin- and heparin-treated animals achieved 35% and 95% patency rates, respectively. Uninjured non-frozen carotid grafts in untreated animals yielded a 95% patency rate, while frozen grafts achieved an 80% patency. Therapeutic levels of aspirin, heparin, and urokinase were confirmed through tail bleeding and whole blood clotting tests, as well as platelet aggregation studies and scanning electron microscopy of the graft lumenal surfaces. A long-term series using syngeneic grafts placed in recipients (Lewis-to-Lewis) and employing systemic heparinization demonstrated maintenance of patency for 4 weeks. Scanning electron microscopy revealed good re-endothelialization, well advanced by one week. Histology confirmed the regrowth of endothelial cells, but showed sparse cellular repopulation of medial and adventitial layers. The mechanical injury model was compared to enzymatic de-endothelialization (using trypsin or collagenase), for which patency rates were similar (10% and 0%, respectively). Trypsin de-endothelialized vessels were tested in vitro for the amount of active trypsin remaining bound to the lumenal surface; no detectable activity was found when trypsin inhibitor was applied following trypsin treatment. The versatility of allowing both in vitro evaluation and in vivo patency assessment demonstrates the uniqueness and value of this new model, offering an avenue toward more direct investigations of surface-mediated thrombotic processes.
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PMID:The de-endothelialized rat carotid arterial graft: a versatile experimental model for the investigation of arterial thrombosis. 144 May 9

Stimulation of polymorphonuclear leukocytes with kallikrein demonstrated that enzyme acts selectively on the release of lysosomal enzymes of these cells. The release of collagenase, similarly to the release of lysozyme into the incubation medium increased proportionally to kallikrein concentration and the duration of incubation. Kallikrein had a small effect on beta-glucuronidase secretion. No effect on cytoplasm lactate dehydrogenase release was detected. These results suggest that kallikrein, as a soluble stimulus, predominantly induces degranulation of specific granules containing collagenase capable of degrading the connective tissue. Secretion of lysozyme and collagenase requires the presence of active kallikrein. Soybean trypsin inhibitor diminished the enzyme release.
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PMID:Influence of human plasma kallikrein on lysosomal enzyme release from polymorphonuclear leukocytes. 165 May 20

Interstitial collagenase either obtained from human neutrophils by phorbol myristate acetate (PMA) induced degranulation or isolated from human gingival crevicular fluid was found to be activated by addition of an oxidative agent, hypochlorous acid (HOCl). Collagenase released by PMA stimulated neutrophils was completely in latent form but underwent partial autoactivation during 16 h incubation at 22 degrees C in the presence of soy bean trypsin inhibitor. The partial autoactivation was potentiated to complete activation of released collagenase after addition of exogenous HOCl. Ascorbate prevented this activation of neutrophil collagenase. Isolated human gingival crevicular fluid collagenase represented an apparent Mr of 70 kD in completely latent form, whereas 70/54 kD enzyme species were detected for partially autoactive form of the enzyme. Western blot analysis of gingival crevicular fluid using a polyclonal antibody raised against purified human neutrophil collagenase revealed the same 70/54 kD molecular forms of the enzyme. The latent gingival crevicular fluid collagenase was also activated by HOCl and this activation could be prevented by ascorbate. Activation of the 70 kD latent collagenase by HOCl as well as by other non-proteolytic activators such as an organomercurial compound (phenylmercuric chloride) and a gold(I) compound (gold thioglucose) was not associated with detectable changes in apparent Mr, whereas trypsin activation resulted in fragmentation of 70 kD enzyme to 54 kD species. Our results provide further evidence for the neutrophil origin of gingival crevicular fluid collagenase and suggest that, in addition to proteolytic activation, oxidative and antioxidative agents seem to be able to regulate neutrophil collagenase activity.
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PMID:Hypochlorous acid induced activation of human neutrophil and gingival crevicular fluid collagenase can be inhibited by ascorbate. 166 35

The application of flow cytometry to enrich airway epithelial cell subpopulations is described. A complementary epithelial cell preparative technique is also outlined. The ability of the airway epithelium to protect the lung from environmental insults results from a complex interaction among the different cells that form its matrix. The separation of the different epithelial cell types is an essential step in the studies of mechanisms of the controlling factors of cell repair, cell differentiation, and neoplastic transformation. Epithelial cells of the New Zealand white rabbit trachea are prepared using enzymatic digestion and microdissection. Small sections of tracheal wall are dissected into pieces approximately 10 mm2. The mucosa is dissected and placed in 0.15% hyaluronidase for 40 min at 22 degrees C. Mucus is removed, and the mucosa is then placed in 0.1% pronase at 37 degrees C for 30 min. With careful dissection, the epithelium can be dissected from the mucosa in 10-mm2 sheets. Sheets of epithelial cells are placed in 6 ml of an enzymatic solution containing collagenase, 0.2% bovine serum albumin, 0.04% soya bean trypsin inhibitor, 0.06 ml of 1 M Hepes buffer for 3 h at 37 degrees C. The cells are gently pipetted during the 3-h period, yielding a suspension of viable cells. Subpopulations of these different cell types are enriched using an Orthocytofluorograph 50111. A krypton ion laser was used for excitation of cells at 488 nm. Forward-angle and 90 degrees scatter were gated on the histogram. The purification of the ciliated, basal, and secretory cells was 90%, 97%, and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enrichment of subpopulations of respiratory epithelial cells using flow cytometry. 184 47

The effects of oxidative damage were assessed in rat proximal tubule fragments (isolated by collagenase perfusion) by monitoring lactate dehydrogenase release (LDH-R) to measure cell viability and thiobarbituric acid (TBA) reactive material to follow oxidative damage. Increasing the oxygen content in the incubation atmosphere from 10 to 95% significantly increased LDH-R and TBA reactants. Addition of butylated hydroxytoluene or deferoxamine (DF) to the medium prevented these changes, but ascorbic acid or mannitol had no positive effect. Lima bean trypsin inhibitor also reduced LDH leakage significantly when added to the medium, but not when added to the perfusion buffers. In contrast, adding DF to the perfusate during tubule isolation produced the most pronounced benefit; net LDH-R after 4 hr was about 10% in tubules prepared this way compared to 20% when DF was omitted. Basal oxygen consumption declined to approximately the same extent as LDH-R increased. Maintenance of nystatin-stimulated respiration, ATP/ADP, GSH content and total adenine nucleotides indicated good cell function. These results suggest that oxidative damage initiated during the tubule isolation procedure limits cell survival but this effect can be counteracted substantially by the addition of DF to the perfusion buffer.
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PMID:Stress initiated during isolation of rat renal proximal tubules limits in vitro survival. 196 72

Isolation and culture techniques for hepatocytes from whole livers of the cynomolgus monkey, Macaca fascicularis, are described. Hepatocytes were isolated by two-step perfusion of livers, using collagenase with hyaluronidase; fructose and trypsin inhibitor were included to reduce cell loss. Yields from a single liver average 4 X 10(9) cells with viabilities of 90.8 +/- 5.7%. Cells, plated on collagen substrates, were assessed for changes in morphology and various marker enzyme activities over a period of 7 d in culture. Cells exhibited a morphology similar to that observed for this species in vivo; little change in attached and spread cells was observed over the length of time monitored. Enzyme activities for catalase, succinate dehydrogenase, and tyrosine aminotransferase were observed to decrease significantly (though considerable activity remained), whereas acid phosphatase and 5'-nucleotide phosphodiesterase remained unchanged. Activity of cytochrome P-450 reductase was observed to increase slightly for the first 2 d, then decrease to about 60% of initial levels. Activity of alpha-mannosidase was stable for 4 d but was observed to be increased at Day 7. Cells were observed to retain metabolic responsiveness, demonstrated by glucose production by both gluconeogenesis and glycogenolysis in response to glucagon stimulation. The monkey hepatocytes obtained by methods described here thus retain hepatocellular morphology and activity through at least 1 wk in culture without medium or culture modification.
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PMID:Isolation and culture of hepatocytes from the cynomolgus monkey (Macaca fascicularis). 197 77

We have previously reported a bioassay for human plasma ACTH based upon trypsin dispersed guinea-pig adrenal cells which was sensitive to 100 ng/L ACTH in unextracted human plasma when measured against human pituitary ACTH (1-39) standard 74/555. We now present a bioassay of increased sensitivity (12 ng/L) which incorporates three major changes. The trypsin/trypsin inhibitor step in the cell dispersion protocol has been replaced with collagenase, donor calf serum (3%) has been incorporated into the standard curve and ACTH has been extracted from human plasma and dilutions of standard hormone by a sephacryl bound monoclonal antibody (2A3) directed towards the 25-39 sequence. The extracted standard curve has a detection limit of 6 ng/L and the cells can tolerate up to 50% plasma equivalent concentration. Thus, the improved assay has a detection limit of 12 ng/L ACTH in plasma. The assay can now measure bioactive plasma ACTH levels reliably in the normal range.
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PMID:ACTH adrenal cell bioassay: improved sensitivity (12 ng/L) achieved by immunoextraction of ACTH from human plasma by a monoclonal antibody. 215 72

The relationship between the sensitivity (the pD2 value) of carbachol and the density (the total concentration of receptors) of muscarinic receptors using single cells from the guinea pig taenia caecum prepared with a mixture of crude collagenase and trypsin inhibitor, purified collagenase alone, and a mixture of purified collagenase and papain was examined. The sensitivity of the single cells prepared with a mixture of purified collagenase and papain was about 10 times more effective than that of the single cells prepared under other conditions. The dissociation constant of [3H]quinuclidinyl benzilate (QNB) and Hill's coefficient did not change in the single cells prepared under the three conditions, though the maximum binding sites were significantly greater in the cells prepared with the mixture of purified collagenase and papain than in those prepared by other means. These results suggest that the increase in the sensitivity of carbachol obtained in the single cells prepared with this mixture is due to the increase in the density of muscarinic receptors and also suggest that the effects of this enzyme mixture may be due to an increase in the incorporation of newly synthesized receptors and (or) changes in receptor turnover.
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PMID:Relationship between sensitivity and density of muscarinic receptors in single smooth muscle cells of guinea pig taenia caecum prepared under three conditions. 216 66


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