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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)

Culture properties of bovine aortic endothelial cells and bovine aortic smooth muscle cells have been examined in relation to collagen gels. Endothelial cells grown on collagen maintain typical monolayer morphology not only in relationship to the overlying medium but also with respect to the collagen substrate. Endothelial cells placed within a collagen matrix assume a mycelial pattern resembling that of the microvasculature. Overlayment of confluent endothelial cells with collagen induces separation of the cells, changes in morphology, and reinitiation of growth. These shape changes do not require protein synthesis, appear to be independent of fibronectin, are not inhibited by cytochalasin D, but are inhibited by colchicine. Endothelial cell growth can be reinitiated by collagen. This phenomenon appears to be related to a change in cell shape and perhaps to separation of cells at the intercellular junction. In contrast, smooth muscle cells plated on collagen infiltrate the gels and assume a spindle-like, elongated morphologic appearance. Overlayment with collagen does not alter smooth muscle cell shape. Migration into the collagen gels is significantly enhanced when cells are cultured in medium containing platelet-released products, independent of growth stimulation itself. Migration is accompanied by collagen gel degradation. Release of labeled collagen into the medium by smooth muscle cells and appearance of TCA fragments in collagenase assay suggest secretion of collagenase. In summary, endothelial cells, but not smooth muscle cells, are restricted to the surface of collagen gels. The ability of the smooth muscle cells to invade is stimulated by platelet products and may be related to the synthesis of a smooth muscle collagenase.
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PMID:Interactions of vascular wall cells with collagen gels. 627 6

A new method for the assay of collagenase activity has been developed, whereby the collagen cleavage products, after initial collagenase digestion, are degraded further by a mixture of trypsin and alpha-chymotrypsin. The degradation products are soluble in TCA and can be conveniently separated from the remaining uncleaved collagen substrate by rapid filtration. The enzyme assay is shown to be reproducible and sensitive, and it lends itself to a convenient and rapid determination of collagenase activity in relatively large numbers of samples. The applicability of this method is demonstrated by the detection of increased collagenase activity in skin fibroblast cultures derived from a patient with recessive dystrophic epidermolysis bullosa.
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PMID:Assay of collagenase activity by a rapid, sensitive, and specific method. 628 40

The gelatinolytic activity of human skin fibroblast collagenase was examined on denatured collagen types I-V. All denatured substrates were cleaved, including types IV and V, which are resistant to collagenase in native form. Interestingly, the earliest major cleavage in denatured collagen types I-III occurred at a 3/4-1/4 locus, resulting in products electrophoretically identical with TCA and TCB fragments of mammalian collagenase action on these native collagens. However, in the denatured substrates, multiple additional proteolytic cleavages followed. The propensity for cleavage at a 3/4-1/4 site in denatured collagen, where sequence is the major specifier of enzymatic action, would seem to indicate that the most favorable amino acid sequence of gamma chains for catalysis is located in this region. The peptide bond specificity of human fibroblast collagenase on gelatin was examined by amino acid sequencing of extensively cleaved denatured type I collagen. Analysis of the NH2-terminal amino acid residues from the resultant gelatin peptides showed sequences of "-H2N-Ile-Y-Gly" and "H2N-Leu-Y-Gly" only (where Y indicates that any amino acid can be found in that position), indicating that Gly-Ile and Gly-Leu bonds are the only sites of collagenase cleavage in this substrate. Whereas the gamma1 chains of denatured collagen types I-III were cleaved at similar rates, fibroblast collagenase was a much better gamma2-gelatinase than gamm1-gelatinase on denatured type 1 collagen. This preference for the cleavage of gamma2(I) was the result of both a higher kcat (750 versus 230 h-1) and lower Km (3.7 versus 7.0 microM) than for a gamma1(1), resulting in an overall selectivity (kcat/Km) of greater than 6-fold. Compared to such kinetic parameters on native collagen, these values indicate that gelatinolysis is somewhat slower than collagenolysis.
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PMID:The gelatinolytic activity of human skin fibroblast collagenase. 628 90

In addition to releasing collagenase and proteoglycanase activity, rabbit articular chondrocytes in monolayer culture released into the culture medium, latent, neutral enzyme activity which when activated by p-aminophenylmercuric acetate degraded fluorescein-labeled polymeric rat tail tendon Type I collagen and the tropocollagen TCA and TCB fragments of human Type II collagen into smaller peptides at 37 degrees C. Enzyme activity was abolished if p-aminophenylmercuric acetate-activated culture medium was preincubated with 1.10-phenanthroline, a metal chelator. Thus, articular chondrocytes in monolayer culture are capable of producing neutral proteinases which acting together can result in complete degradation of tendon and cartilage collagen to small peptides.
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PMID:'Gelatinase-like' activity from articular chondrocytes in monolayer culture. 629 87

Human gingival fibroblast procollagenase has been purified to apparent homogeneity from serum-free and serum-supplemented fibroblast culture medium by a combination of ammonium sulfate precipitation, CM-cellulose chromatography, and gel-filtration on Bio-Gel P-150. Sodium dodecylsulfate polyacrylamide gel electrophoretic studies suggests that the purified fibroblast proenzyme is comprised of two closely related zymogens with the estimated Mr of 57,000 and 52,000. Upon densitometric scanning of the gels, the ratio of the two proenzyme forms was about 1 : 4 (57 : 52 kdal). Limited proteolysis of the fibroblast procollagenase with trypsin resulted in the conversion of both proenzyme forms into active enzyme forms of Mr 48,000 and 44,000, respectively. Amino acid analysis of the active enzymes and proenzyme forms revealed that the active enzymes contained fewer basic amino acids than do the proenzyme forms. The purified trypsin-activated fibroblast collagenase hydrolyzed type I collagen fibrils, cleaved tropocollagen in solution at 24 degrees C into TCA and TCB fragments, and cleaved the synthetic peptide substrate, DNP-peptide III, at the Gly-Ile bond. The gingival fibroblast collagenase exhibited a pH optimum of 7.5, was completely inhibited by EDTA or dithioerythritol but was not inhibited by N-ethylmaleimide or phenylmethylsulfonyl fluoride, and appeared to cleave human type III collagen approximately 10-fold faster than homologous type I collagen. In addition, comparison of the biochemical properties of the precursor and active forms of human gingival fibroblast collagenase with the precursor and active forms of human skin fibroblast collagenase, previously characterized by Stricklin and co-workers (Biochemistry 17: 2331-2337, 1978), revealed that they were similar in Mr, amino acid composition, and substrate specificity. Furthermore, the human gingival and skin fibroblast procollagenases were immunologically identical.
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PMID:Human gingival fibroblast collagenase: purification and properties of precursor and active forms. 632 84

Nonenzymatic glucosylation of type I and type II collagens was examined by incubating collagen substrates with D-glucose in vitro. In one set of experiments, unlabeled collagen was incubated with [14C]-glucose and the incorporation of [14C]-radioactivity into protein was determined by TCA precipitation. The incorporation was dependent on the concentration of glucose and the time of incubation. The glucosylated product was also examined by SDS-polyacrylamide slab gel electrophoresis. The results indicated that both alpha 1(I)- and alpha 2(I)-chains of type I collagen were glucosylated and the glucosylation occurred both with native and denatured collagen as substrate. In further studies [3H]-lysine-labeled collagens were glucosylated, the products reduced by NaBH4, and the [3H]-lysine-derived residues were separated by amino acid analyzer. After a 144 h incubation in vitro, 18.9% of [3H]-lysyl residues and 36.5% of [3H]-hydroxylysyl residues in type I collagen were substituted with glucose. In contrast, 47.9% of [3H]-lysyl residues and 68.1% of [3H]-hydroxylysyl residues in type II collagen were glucosylated after 144 h incubation. Based on quantitative amino acid analyses of the substrates, these values represent 27.6 lysine plus hydroxylysine residues substituted per triple-helical type I collagen molecule and 65.3 residues per triple-helical type II collagen molecule. Thus, type I and type II collagens display differential susceptibilities to nonenzymatic glucosylation. Finally, [3H]-proline-labeled type I collagen was glucosylated to varying extents, and the glucosylated products were used as substrates for human polymorphonuclear leukocyte collagenase. No difference in susceptibility to this collagenase was noted, irrespective of the extent of glucosylation.
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PMID:Nonenzymatic glucosylation of lysyl and hydroxylysyl residues in type I and type II collagens. 644 73

The collagenolytic protease from Uca pugilator was studied with respect to its catalytic properties on collagen types I-V. The crab protease degraded all five collagen types, producing multiple cleavages in the triple helix of each native collagen at 25 degrees C. The major early cleavage in the alpha 1 polypeptide chain of collagen types I-III occurred at a 3/4:1/4 locus, resulting in fragments electrophoretically similar to the TCA and TCB products of mammalian collagenase action. Interestingly, a propensity toward this same cleavage was observed even following thermal denaturation of the substrates. The ability of the crab protease to degrade all native collagen types and to catalyze cleavages at multiple loci in the triple helix distinguishes its action from that of mammalian collagenases. The collagenolytic activity of the crab protease was also examined on fibrillar collagen and compared to that of human skin fibroblast collagenase. Enzyme concentrations of fibroblast collagenase which resulted in the saturation of available substrate sites failed to show such an effect in the case of the crab protease. Binding studies of the crab protease to fibrillar collagen likewise indicated substantially reduced levels of enzyme binding in comparison to fibroblast collagenase. These data suggest that the affinity of the crab protease for native collagen is considerably less than the affinity of mammalian collagenase for this substrate.
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PMID:Substrate specificity of the collagenolytic serine protease from Uca pugilator: studies with collagenous substrates. 675 69

A peptide mapping technique is described which uses a two dimensional format to display CNBr peptides of collagen chains. Biosynthetically-labeled products from 250,000 cells are analyzed in each map by a rapid procedure which does not require preliminary purification steps. Proteins trapped within polyacrylamide gels are digested with CNBr under conditions where diffusion of radiolabeled peptides from gels is negligible, and the reaction products are recovered quantitatively by electroelution. Peptide maps of pro alpha chains, alpha chains, and TCA chains cleaved with mammalian collagenase are presented with the identification of specific fragments. This method is useful for the analysis of structural abnormalities in collagen proteins from patients with certain genetic disorders, examination of collagenous proteins produced by primary cultures which exhibit phenotypic switching, and identification of new collagen types.
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PMID:Peptide mapping of collagen chains using CNBr cleavage of proteins within polyacrylamide gels. 734 34

We investigated the alterations of major fatty acid components in epidermis by natural aging and photoaging processes, and by acute ultraviolet (UV) irradiation in human skin. Interestingly, we found that 11,14,17-eicosatrienoic acid (ETA), which is one of the omega-3 polyunsaturated acids, was significantly increased in photoaged human epidermis in vivo and also in the acutely UV-irradiated human skin in vivo, while it was significantly decreased in intrinsically aged human epidermis. The increased ETA content in the epidermis of photoaged human skin and acute UV-irradiated human skin is associated with enhanced expression of human elongase 1 and calcium-independent phosphodiesterase A(2). We demonstrated that ETA inhibited matrix metalloproteinase (MMP)-1 expression after UV-irradiation, and that inhibition of ETA synthesis using EPTC and NA-TCA, which are elongase inhibitors, increased MMP-1 expression. Therefore, our results suggest that the UV increases the ETA levels, which may have a photoprotective effect in the human skin.
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PMID:Skin aging and photoaging alter fatty acids composition, including 11,14,17-eicosatrienoic acid, in the epidermis of human skin. 2051 27


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