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)

A disulfide-cross-linked collagen has been extracted with neutral salt solutions from organ cultures of embryonic chick sternal cartilage. This collagen, which we term pM collagen, is presumed to be the native extracellular precursor molecule to disulfide-cross-linked collagen fragments recently described. Cleavage of pM collagen under native conditions with pepsin gives rise to the collagen fragments M1 and M2, which had also been isolated from pepsin extracts of chick hyaline cartilage [K. von der Mark, M. van Menxel & H. Wiedemann (1982) Eur. J. Biochem. 124, 57-62]. Native pM collagen was purified by DEAE-cellulose chromatography and agarose gel filtration. On agarose and following polyacrylamide gel electrophoresis, the unreduced molecule migrates with an apparent Mr of 300 000. Reduction of disulfide bridges produces two subunits with Mr 80 000 (pMa) and 60 000 (pMb) when compared with collagen standards. Cyanogen bromide cleavage of pMa and pMb, excised from dodecyl sulfate gels, resulted in different peptide maps, indicating that both components are genetically distinct polypeptide chains. The occasional appearance of the unreduced pM collagen as a doublet band on dodecyl sulfate gels and the observation that pMa and pMb occur in non-stoichiometric ratios suggests that pMa and pMb form separate native molecules, although their incorporation into a single pM molecule cannot be excluded. Native pM collagen was completely digested with bacterial collagenase, and contained hydroxyproline and proline in a ratio of 1.15:1, indicating the absence of significant non-collagenous domains. Thus it represents, despite several pepsinlabile sites, more likely a largely triplehelical, processed form of collagen rather than a procollagen-like molecule containing globular domains. Processing of pM collagen to M1 and M2 fragments or other intermediate forms was not observed in cartilage organ culture or in chondrocyte cell cultures within 18 h.
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PMID:Isolation and characterization of a precursor form of M collagen from embryonic chicken cartilage. 669 38

Various dermal fibrotic conditions, such as progressive systemic sclerosis, localized morphea and familial cutaneous collagenoma, are characterized by excessive deposition of collagen in the skin. In the present study, we examined the possibility that a circulating serum factor(s) is responsible for increased collagen production in these diseases. The effects of human serum on the synthesis of procollagen were examined by incubating normal human dermal fibroblasts with [3H]proline and varying concentrations of dialyzed heat-inactivated serum. The synthesis of procollagen was measured as formation of nondialyzable [3H]hydroxyproline and collagenase-digestible 3H]polypeptides. In the absence of serum little procollagen was formed but the synthesis was markedly stimulated by the addition of normal serum in a concentration-dependent manner. THe ratio of genetically distinct 3H-procollagens of type I and type III, assayed by DEAE-cellulose chromatography and SDS-polyacrylamide gel electrophoresis after limited pepsin proteolysis, was unaffected by the addition of serum. Thus, normal human serum contains a nondialyzable factor(s) which stimulates the synthesis of procollagens type I and type III equally. Sera from 5 patients with progressive systemic sclerosis, 3 with localized scleroderma, and 2 with familial cutaneous collagenoma were also tested. Sera from these patients failed to stimulate 3H-procollagen production more than sera from healthy age-matched controls. Therefore, no increased quantities or qualitatively aberrant factors were shown to be present in the sera of these patients.
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PMID:Human skin fibroblasts in culture: procollagen synthesis in the presence of sera from normal human subjects and from patients with dermal fibroses. 724 Jul 93

Skin fibroblasts in culture can provide a convenient means to study aberrations of collagen metabolism in a variety of clinical conditions. In the present study, the culture conditions for the synthesis of procollagen by cultured human skin fibroblasts were optimized by independently varying parameters in the cell culture environment. To study the synthesis of procollagen the cell cultures were labeled with [3H]proline and the collagenous polypeptides were determined either by measuring the synthesis of hydroxy[3H]proline or by assaying the 3H-labeled proteins digested into dialyzable 3H-labeled peptides by bacterial collagenase. On the basis of the experimental results, the following culture conditions are suggested for optimal synthesis of procollagen: (a) cell culture medium should be supplemented with ascorbic acid (25--50 micrograms/ml) and fetal calf serum (20%); (b) the pH of the culture medium should be kept above 7.2 and preferably in the pH range 7.5--7.8; (c) the cell cultures should be used one to two days after reaching visual confluency. Under these conditions the synthesis and secretion of [3H]procollagen was found to be linear through a 24 h incubation period, and procollagen was demonstrated to be a major gene product of the fibroblasts. The relative synthesis of type I and type III procollagens was also monitored by isolating these genetically distinct procollagens by DEAE-cellulose chromatography or by measuring type I and III collagens by sodium dodecyl sulfate polyacrylamide gel electrophoresis after limited pepsin proteolysis. No marked changes were observed in type I/III procollagen ratios in situations where the total formation of hydroxy[3H]proline was significantly affected. The average coefficient of variance for procollagen synthesis between replicate cultures was found to be relatively small (14%), and the optimization of the culture conditions for the control cells has, therefore, created a reliable and reproducible basis for employing human skin fibroblasts to study collagen metabolism in acquired and inherited diseases.
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PMID:Collagen biosynthesis by human skin fibroblasts. I. Optimization of the culture conditions for synthesis of type I and type III procollagens. 737 Feb 60

Human skin fibroblasts in culture have previously been shown to synthesize genetically distinct procollagens type I and type III. In the present study, cultured human skin fibroblasts were incubated under conditions optimized for synthesis of these procollagens in medium containing [3H]proline. The newly synthesized type I and type III 3H-labeled procollagens in the culture medium were then isolated as native proteins by DEAE-cellulose chromatography, or by gel filtration and SDS-polyacrylamide slab gel electrophoresis under denaturing conditons after limited pepsin proteolysis. The chromatographic procedures were optimized to yield reliable and reporducible results with good recoveries. The isolated procollagens were identified by cyanogen bromide peptide mapping and characterized by cleavage with highly purified collagenase synthesized by human skin fibroblasts. Assay of the relative synthesis of type I/III procollagens by normal human skin fibroblasts using DEAE-cellulose chromatography indicated that 80% of the procollagen in the medium was type I while the remaining 20% consisted of type III. When the ratio of newly-synthesized type I/III collagens was estimated by gel filtration or using SDS-polyacrylamide slab gel electrophoresis after limited pepsin proteolysis, relatively fewer type III collagen alpha-chains were recovered. This observation suggests that some of the type of the type III collagen molecules are in a conformation which is less resistant to digestion by pepsin than the triple-helix of type I procollagen. The coefficient of variation for the relative synthesis of type I and type III procollagens by control cultures was relatively small (16%), indicating that the phenotypic expression of type I and type III procollagen genes, under optimized culture conditions, is under a relatively tight control. The results further suggest that the optimized methodology developed for assay of the relative synthesis of type I and type III procollagens and collagens by cultured human skin fibroblasts can be utilized in studies on collagen aberrations in acquired and inherited diseases of connective tissue.
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PMID:Collagen biosynthesis by human skin fibroblasts. II. Isolation and further characterization of type I and type III procollagens synthesized in culture. 741 91

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.
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PMID:Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels. 747 51

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

The snake venom of Echis coloratus was found to abolish the hemagglutinating activity, hemolytic activity and in vivo infectivity of Sendai virus. The active factor (Echinhibin-1) was purified by gel filtration on Sephadex G-50, followed by chromatography on DEAE-Sepharose and CM-Sepharose. Echinhibin-1 is a protease with a molecular weight of about 25 kDa, an isoelectric point of 7 and is stained by PAS, indicating that it is a glycoprotein. It showed a strong azocollase activity that was stable up to 68 degrees C and at pH values of 4.5-10.5. Ten micrograms/ml were sufficient to abolish the hemolytic effect of the virus on human erythrocytes when incubation was at 37 degrees C for 2 h, while 20 micrograms/ml abolished the hemagglutinating activity. Addition of Echinhibin-1 after the adsorption of Sendai virions onto washed erythrocytes at 4 degrees C did not inhibit the subsequently hemolytic activity at 37 degrees C, indicating that Echinhibin-1 interferes with virus adsorption to the cells. Of various protease inhibitors, only Na2 EDTA and o-phenanthroline inhibited the antiviral activity of the purified factor, indicating that it is a metalloproteinase. In vivo, mice inoculated intranasally with the virus pretreated with Echinhibin-1 developed well and gained weight, whereas untreated virus-infected mice lost weight and died within 1 week. Intravenous administrations of the purified factor up to 80 micrograms/mouse produced no signs of toxicity and subcutaneous injections caused no hemorrhagic activity, while the whole venom is very hemorrhagic with an LD50 of 250 micrograms/kg for mice.
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PMID:Echinhibin-1--an inhibitor of Sendai virus isolated from the venom of the snake Echis coloratus. 814 82

For the purpose of studying pepsinogen secretion from gastric chief cells, we established a monolayer culture system of guinea pig chief cells and an enzyme immunoassay (EIA) system specific for guinea pig pepsinogen. Dispersed chief cells were obtained from gastric mucosa of a guinea pig using collagenase, GEDTA, and Percoll solution, suspended in DMEM/F-12 (1/1 containing 10% FCS) media, and cultured for 70hr. Then the monolayer culture system was established. Pepsinogen was purified from gastric mucosa of a guinea pig using DEAE-Sephacel and Sephacryl S-200 columns. Antibody to pepsinogen was raised by immunizing rabbit with the purified pepsinogen. A two-site EIA system was then established using beta-galactosidase-labeled Fab' antibody. The EIA system showed sensitivity to measure above 1.5ng of guinea pig pepsinogen, and the monolayer culture system responded well to secretagogues. These systems are useful for studying pepsinogen secretion.
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PMID:[The establishment of a monolayer culture system of guinea pig chief cells and an enzyme immunoassay system for guinea pig pepsinogen]. 846 66

A serine collagenolytic protease was purified from a water soluble fraction of greenshore crab digestive gland by acidic precipitation, gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a Fractogel TSK DEAE column, immobilized metal ion affinity chromatography (IMAC) on IDA (Cu2+) Sepharose 6B and ion-exchange chromatography on Hyper D column. The molecular mass of the monomeric Carcinus serine collagenase (CSC) was estimated to be 23,000 by SDS PAGE and its isoelectric point was found to be 4.0. The CSC is optimally active at pH 7 and 30 degrees C and is stable over a month at room temperature. The CSC activity is strongly inhibited by PMSF, 3,4-DCI, soybean trypsin inhibitor, alpha 1 proteinase inhibitor and elastatinal. The CSC hydrolyzes native collagen (Type I and III). CSC N-terminal sequence is similar to shrimp chymotrypsin-like protease and crab collagenolytic protease sequences. Kinetic parameters of the CSC were determined using some peptidyl-p-nitroanilides. The catalytic efficiency (kcat/Km) is Leu > Phe > Ala.
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PMID:Purification, kinetical and molecular characterizations of a serine collagenolytic protease from greenshore crag (Carcinus maenas) digestive gland. 889 34

Using the monoclonal antibody GDA-J/F3, a 50-kDa noncollagenous component of human skin basement membrane zone was identified. Immunofluorescence stainings of normal human skin with the GDA-J/F3 antibody showed a linear fluorescence decorating the basement membrane zone. With immunoelectron microscopy, the epitope was localized to the insertion points of the anchoring fibrils into the lamina densa. The antigen is distinct from collagen VII, from the main structural protein of the anchoring fibrils, and from several other structural molecules of the basement membrane zone, because the GDA-J/F3 antibody did not react with purified basement membrane components in vitro. In serum-free cultures, the antigen was synthesized and secreted by normal and transformed human keratinocytes and to a lesser extent by normal human skin fibroblasts. Immunoprecipitation of radiolabeled epithelial cell-conditioned medium with the GDA-J/F3 antibody yielded two polypeptides that migrated on SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 46 and 50 kDa under nonreducing conditions. Using reducing gels, only the 50-kDa polypeptide was observed. The antigen was resistant to digestion with bacterial collagenase but sensitive to trypsin and pepsin. It also bound to heparin and DEAE cellulose at low ionic strength and alkaline pH. These findings indicate that the GDA-J/F3 antigen is a small globular disulphide-bonded protein with a potential to interact with basement membrane proteoglycans. Integration of the GDA-J/F3 antigen into the histoarchitecture of the dermo-epidermal junction is dependent on the presence of collagen VII, because the GDA-J/F3 epitope was missing in several patients with a genetic blistering disorder of the skin, epidermolysis bullosa dystrophica, who lacked collagen VII and anchoring fibrils.
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PMID:Characterization of a 50-kDa component of epithelial basement membranes using GDA-J/F3 monoclonal antibody. 908 95


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