Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Feeder-cell-independent serially propagating keratinocytes from rat oral mucosa (tongue) dissolved reconstituted type I [3H]collagen fibrils, although rather slowly. Analysis of the conditioned medium from such cultures revealed secretion of a Mr = 65,000 collagenase which remained almost entirely latent in the absence of exogenous protease activity. Addition of trypsin (0.1-1.0 microgram/ml) or plasmin (1.0-4.0 micrograms/ml) resulted in substantial acceleration of the collagenolytic process in stimulated secretion of latent collagenase and, at higher concentrations, in conversion of the latent enzyme to the catalytic form. The keratinocyte collagenase was indistinguishable from interstitial, fibroblast-type collagenases by several criteria including: cleavage of native type I collagen in solution at the characteristic collagenase-sensitive locus at 22 degrees C and dissolution of reconstituted type I collagen fibrils at 35 degrees C; activation by trypsin and by organomercurials and inhibition by Zn2+ and Ca2+ chelators; and cross-reaction with antibody to fibroblast-type procollagenase. Expression of collagenolytic activity in keratinocyte cultures was effectively regulated by cell density. The activity (on a per cell basis) was maximal at 10-20% confluence and was more than 95% "contact-inhibited" at subconfluent and early confluent densities (2-4 X 10(5)/cm2). Our findings show that mucosal keratinocytes possess a potent enzymatic apparatus for degradation of interstitial collagen fibrils which includes a classical vertebrate collagenase.
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PMID:Degradation of type I collagen by rat mucosal keratinocytes. Evidence for secretion of a specific epithelial collagenase. 243 24

Connective tissue metabolism was studied in detail in three human skin fibroblast lines, demonstrating low, medium, or high levels of glucocorticoid receptor densities. In the cell lines with low and medium receptor density, dexamethasone, in the range of 10(-5)-10(-9) M, had no effect on collagen production, using short incubation time periods and high (20%) fetal calf serum concentration, while in the cells with highest receptor density, a slight stimulation of collagen synthesis was noted in the concentration range 10(-6)-10(-9) M. In the presence of low concentration (0.5%) of serum, dexamethasone markedly inhibited collagen production. The production of collagenase, assayed by degradation of 3H-labelled type I collagen substrate with a brief trypsin activation of the enzyme, was reduced in a dose dependent manner in all 3 cell lines, the inhibition with 10(-5) dexamethasone being up to 56% of the control. Similarly, the activity of an elastase-like neutral protease was decreased in the presence of dexamethasone. Thus the results indicate that glucocorticoids may have profound effects on the degradation of connective tissue components, while the effects on collagen synthesis may be more variable depending on the environmental milieu.
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PMID:Modulation of collagen metabolism in cultured human skin fibroblasts by dexamethasone: correlation with glucocorticoid receptor density. 243 73

A quantitative collagenase assay detecting soluble collagen fragments is described in this paper. Using the reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) type I collagen was conjugated with horseradish peroxidase (POD) which was employed as a reporter enzyme. POD was preferentially linked to the TC B fragment in a ratio of 1.4 mol POD/mol collagen. The conjugation product was immobilized on AH-Sepharose via carbodiimide coupling to form the final collagenase substrate used in the assay. POD activity in the supernatants caused by liberated TC B fragments exhibited a linear relationship for collagenase concentrations up to 100 micrograms/ml bacterial collagenase. Over an incubation period of 4 h the lowest detection limits found were 20 ng/100 microliters for bacterial collagenase and 60 ng/100 microliters for human leukocyte collagenase. Incubation of the assay mixture with 5 micrograms trypsin resulted in 3.8% of the activity released by the equivalent amount of leukocyte collagenase. The assay developed here has been shown to be sensitive and specific for collagenase, with the additional advantage that this method is suited for simple and economic handling.
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PMID:A spectroscopic collagenase assay using peroxidase-labeled collagen. 254 Jun 72

The specific role of proteolytic enzymes in the degradation by live cells of fibrillar model matrices (fibrin, collagen) was studied using monoclonal and polyclonal inhibitory (anti-catalytic) antibodies. Dissolution of fibrin by plasminogen-supplemented human HT-1080 cells was blocked by (1) omission of plasminogen, (2) inhibitory anti-plasmin antibody, and (3) inhibitory anti-u-PA antibody but not by non-inhibitory control antibodies. Using a similar approach, it was shown that the dissolution of reconstituted type I collagen fibrils by trypsin-supplemented live human skin fibroblasts was blocked by inhibitory antibodies to fibroblast-type procollagenase but not by noninhibitory control antibodies. These findings permit us to deduce that, at least in culture, the dissolution of fibrin by plasminogen-supplemented HT-1080 cells was mediated by plasminogen-assisted proteolysis which entailed the extracellular conversion of plasminogen to plasmin by cell-derived u-PA, and that the dissolution of collagen fibrils by trypsin-supplemented skin fibroblasts was mediated by a collagenase-dependent pathway.
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PMID:Use of inhibitory (anti-catalytic) antibodies to study extracellular proteolysis. 254 25

The existing forms of neutral proteases present in inflamed human gingiva were examined. Neutral 2 M K Cl extracts of inflamed human gingival tissue were fractionated by gel filtration on Sephacryl S-200 and the fractions were assayed for collagenase, trypsin-, chymotrypsin-, and elastase-like proteases. Apparent molecular weights of 80-85 kDa were obtained for trypsin-, chymotrypsin-, and elastase-like proteases, and 70-75 kDa for latent collagenase. Further fractionation of high molecular weight proteases on Con A-Sepharose revealed that, unlike collagenase, chymotrypsin- and elastase-like proteases, the trypsin-like protease was bound by the affinity column. Native human placental type IV (basement membrane) collagen was degraded by chymotrypsin-like and elastase-like proteases but not by the trypsin-like protease. This degradation was inhibited by phenylmethyl sulfonyl fluoride and EDTA. The serine proteases also degraded efficiently denatured type I collagen. No correlation of the activities of trypsin-like protease and the other proteolytic enzymes was found in extracts of 18 individual gingival specimens. Significant correlation, however, was noted between collagenase and gelatinase. The gingival culture studies showed that, while the highest activity of the trypsin-, chymotrypsin-, and elastase-like enzymes were measured in medium during first days of the culture, collagenase and gelatinase activities increased up to the fourth day of culture and stayed high until the end of the culture. These results suggest that the neutral proteases that may participate in the periodontal tissue destruction are produced by different cell types of gingiva.
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PMID:Characteristics of neutral proteases present in inflamed human gingiva. 255 68

Mouse primordial germ cells (PGCs) isolated from the dorsal mesentery and gonadal ridges of 10.5-12.5 days post coitum (dpc) embryos showed a progressively increasing adhesiveness to laminin and fibronectin coated substrates, whereas type I collagen and various glycosaminoglycans (hyaluronic acid, heparin and chondroitin-sulphates) were poor adhesive substrates. At later stages germ cells appeared to lose their adhesiveness to fibronectin and laminin substrates; the ability to adhere to laminin decreased very rapidly in male and slowly in female germ cells. Oocytes and prospermatogonia from 15.5 dpc fetal gonads showed poor adhesiveness to all substrates tested. PGC adhesion to laminin and fibronectin substrates did not require calcium but was markedly trypsin sensitive. Antibodies against the fibronectin receptor of CHO fibroblasts and short peptides containing the Arg-Gly-Asp sequence greatly reduced PGC adhesion to fibronectin. Following adhesion to laminin or fibronectin, most PGCs did not exhibit a morphology typical of motile cells, but remained spherical. A significant proportion (about 30%) of oocytes from 13.5-14.5 dpc embryos appeared, however, able to spread and elongate following attachment to laminin. The results support the hypothesis that mouse PGCs may utilize laminin and/or fibronectin as adhesive substrates during migration and gonad colonization, but indicate that additional factors are probably required to promote PGC motility. In addition, our data provide indirect evidence that binding sites for specific components of extracellular matrix are present in PGCs, and that their expression may be developmentally regulated.
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PMID:In vitro adhesion of mouse fetal germ cells to extracellular matrix components. 270 69

Two cyanogen bromide fragments (alpha 1-CB7 and alpha 1-CB8) of bovine corneal stromal collagen have been isolated and characterized. These added to those characterized in our previous work account for 95% of the amino acid sequence of the alpha 1(1)-chain. The hydroxylysine glycoside content of each fragment was determined and in this way the general distribution of glycoside over the entire molecule was deduced accounting for all the galactosylhydroxylysine and most of the glucosylgalactosylhydroxylysine of this heavily glycosylated type I collagen. The characterization of fragments alpha 1-CB7 and alpha 1-CB8 has enabled us to resolve the controversy over the relative mobilities of these fragments on SDS gels. Fragment alpha 1-CB7 of bovine corneal collagen was digested by trypsin and by staphylococcal proteinase V8. The resultant peptides were isolated by gel and ion-exchange chromatography and identified in relation to the known amino acid sequence of type I collagen. The hydroxylysine glycosides were determined in the relevant peptides providing a complete account of their distribution along this part of the collagen molecule. Most of the glycoside was found in the gap region of collagen especially near the edges of the axial holes where it could act as a peg to facilitate fibre formation. In addition, some glycoside was found in the overlap region where, being unable to fit into axial holes, it might impede the growth of the fibre and, with other glycoside of the overlap region, might be responsible for the narrow fibres of corneal collagen that are essential for corneal transparency. This glycoside, with that previously found in the peptide alpha 1-CB3 is the only hydroxylysine glycoside identified in the overlap region of a type I collagen.
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PMID:Pinpointing the sites of hydroxylysine glycosides in peptide alpha 1-CB7 of bovine corneal collagen, and their possible role in determining fibril diameter and thus transparency. 275 43

The primary aim of this study was to develop a model system that uses epidermal cells (keratinocytes and accessory pigmented cells) cultured on a reconstituted basement membrane biomatrix for use in conjunction with type I collagen as a replacement dermis-epidermis in the treatment of recurrent ulcerative lesions. Type I collagen sponges (Collistat) have been shown to promote rapid healing of leg ulcers but with extensive scarring (Reindorf et al, 1989). Dermatome sections from patients undergoing elective plastic surgery were treated with dispase to dissociate epidermis from dermis, the inner epidermal cells were then dissociated with trypsin-EDTA and plated on the biomatrix. A 4 cm2 skin specimen yielded approximately 10 to 12 million inner epidermal cells. These cells were plated at a density of 500,000 cells/cm2, thereby covering an area of 20 to 24 cm2. At 24 hours attached cells were dispersed primarily in monolayers, and by day 3 most of the epidermal cells reassociated into bi- or multilayered aggregates. Cells containing melanin granules were distributed in the basal to middle layers of the aggregates and persisted throughout the culture period (up to 14 days).
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PMID:Model for phase III autografts of epidermal cells cultured on a collagen-proteoglycan biomatrix. 281 Mar 92

Evidence is presented that biopsy specimens from fibroadenomas, benign cystic lesions, and carcinomas of the human breast can produce in organ culture a neutral protease capable of digesting type I collagen. This enzyme activity, measured with the use of a radioactive release assay, was characterized as true vertebrate collagenase and occurred in both active and latent (requiring trypsin activation) forms. For the two types of benign breast lesion studied, collagenase secretion was significantly higher from fibroadenomas than from benign cystic tissue. Breast carcinomas, however, exhibited a wide quantitative spectrum of collagenase secretion, encompassing the extremes observed for the benign lesions and showing no correlation with histologic type. These results, while providing a plausible mechanism for the marked collagen degradation seen in disseminating neoplasms, demonstrate that high collagenase secretory activity is not pathognomonic of invasive behavior. The findings, however, indicate disordered regulation of collagenase activity in malignant tumors.
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PMID:Collagenase secretion by human breast neoplasms: a clinicopathologic investigation. 298 53

An assay for the degradation of type IV (basement membrane) collagen was developed as a biochemical marker for neoplastic cells from chemically transformed human skin fibroblasts. Type IV collagen was isolated from basement membrane of Syrian hamster lung and type I collagen was isolated from rat tails; the collagens were radioactively labelled by reductive alkylation. Both collagens were in the native form as shown by their low sensitivity to degradation by trypsin. The abilities of normal (KD) and chemically transformed (Hut-11A) human skin fibroblasts to degrade the collagens were studied. The normal cells released a collagenase into the medium which after activation by trypsin or oxidized glutathione degraded type I collagen. Hut-11A cells also produced a collagenolytic activity that degraded type I collagen; however, no activation of the medium was required for this activity. Type IV collagen was not degraded by medium conditioned with the normal (KD) cells with or without activation. In contrast, Hut-11A cells secreted an active collagenase into the medium that degraded type IV collagen extensively. Treatment of the medium from Hut-11A cells with trypsin resulted in only a loss of activity while treatment with oxidized glutathione was without effect. The degradation of type IV collagen by Hut-11A conditioned medium was linear for up to 1 h and the extent of degradation increased linearly with increasing amounts of conditioned medium. A cell-associated assay was performed by growing either normal or transformed cells in the presence of radioactively labelled type IV collagen and measuring the released soluble peptides in the medium. This assay also demonstrated that KD cells failed to synthesize an activity capable of degrading type IV collagen whereas Hut-11A cells degraded type IV collagen in a linear manner for up to 4 h. Human serum at very low concentrations, EDTA and L-cysteine inhibited the enzyme activity, whereas protease inhibitors like phenylmethyl sulfonyl fluoride, N-ethyl maleimide or soybean trypsin inhibitor did not inhibit the enzyme from Hut-11A cells. These results suggest that the ability to degrade specifically type IV collagen may be an important marker for neoplastic human fibroblasts and supports a role for this collagenase in tumor cell invasion.
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PMID:Degradation of type IV collagen by neoplastic human skin fibroblasts. 298 12


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