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 high molecular weight extracellular protein has been purified from cell culture medium of Ewing's sarcoma cell lines, by high performance liquid chromatography and electroelution from SDS-PAGE electrophoresis. This protein has an apparent molecular mass of about 500,000 Da on SDS-PAGE. Immunoprecipitation studies with several extracellular matrix glycoproteins (laminin, fibronectin) specific antisera indicate it is a separate protein. Reduction of disulphide bonds with 2-ME or DTT fails to significantly alter its migration on SDS-PAGE gels, other than a slight apparent increase in molecular mass, indicating an apparent single polypeptide chain structure. The slightly greater mobility observed in unreduced gels suggests one or more regions of intrachain disulfide bonding. It is sensitive to pepsin and trypsin, but resistant to bacterial collagenase indicating that it does not contain collagenous domains. Metabolic labelling with 3H-proline, 3H-leucine, and 35S-methionine indicate that this protein is proline-poor, but leucine, and especially methionine, rich. Sodium 35S-sulfate incorporation is totally negative and treatment with glycosaminoglycan degrading enzymes has no effect on the mobility of the protein on gels, unlike typical proteoglycans. This protein appears by rotary shadowing electron microscopy as a long, thin, filamentous molecule at least 500 nm (0.5 um) in length. The tissue localization and function are unknown at this time, but are under active investigation.
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PMID:A novel 500,000 Da, linear, single chain extracellular protein synthesized by several childhood tumors. 263 60

A cDNA library constructed from chick aorta poly(A+) RNA in the expression vector pEX1 was screened with rabbit polyclonal antisera. Additional clones were obtained by DNA-DNA hybridization with subclones from the most 5'- and 3'-ends. The overlapping clones span 4.6 kilobases and code for the entire alpha 1 (VI) chain. The nucleotide sequence reveals a 3057-base pair open reading frame that codes for 1019 amino acids. Analysis of the deduced amino acid sequence predicts that alpha 1 (VI) has one collagenous domain (COL) of 336 residues flanked by three repeated domains of about 200 residues each, one at the amino (A'3) and two at the carboxyl ends (A'2 and A'1), respectively, that are similar to the type A repeats of von Willebrand Factor. The COL domain presents two short interruptions near the carboxyl end of the triple helix and three of the six potential N-asparaginyl-linked carbohydrate attachment sites (Asn-Xaa-Ser/Thr). Furthermore, it contains 1 cysteine at position 89 that could participate in the formation of dimers and 3 Arg-Gly-Asp sequences that might be potential sites for cell adhesion. The COL domain shows an extended region, starting from position 40, within the triple helix, made of 14 Gly-Xaa-Yaa triplets that lack proline in the Y position, suggesting that it might be more flexible than the rest of the domain. At the junction of the COL with the N- and C-terminal domains, there are several cysteines that could confer the well known resistance of type VI collagen to pepsin and collagenase digestion under nonreducing conditions. The present sequence data allow a structural model for type VI collagen assembly to be proposed that is consistent with the structure implied from previous electron microscopic observation by Furthmayr et al. (Furthmayr, H., Wiedemann, H., Timpl, R., Odermatt, R., and Engel, J. (1983) Biochem. J. 221, 303-311).
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PMID:Alpha 1 chain of chick type VI collagen. The complete cDNA sequence reveals a hybrid molecule made of one short collagen and three von Willebrand factor type A-like domains. 278 34

Recent studies of murine tumor models and certain human tumor cell lines have provided evidence for intratumor heterogeneity in expression of extracellular matrix receptors and in the elaboration of matrix-degrading enzymes. However, little is known about possible intratumoral heterogeneity in the production of matrix macromolecules. We have, therefore, examined the biosynthesis and secretion of matrix proteins by cells derived from a polyclonal human cell line (JH-17) established from a large cell undifferentiated carcinoma of the lung. For the present studies, we focused on the production of collagens and structural glycoproteins by two phenotypically different aneuploid clones, designated C13 and C22. These clones were distinctive in their inability to grow in soft agar or to form tumors in nude mice and had identical DNA contents. Tumor cells were labeled with [3H]proline and the newly synthesized proteins accumulating in the culture medium were identified using biochemical and immunologic techniques. Clone C13 secreted at least three genetically distinct collagens, including type V procollagen (PC), type IV procollagen, and a type VIII-like collagen. By contrast, the clone C22 synthesized fibronectin, and a single bacterial collagenase-sensitive and pepsin-resistant component consistent with type I trimer. These studies emphasize the potential diversity of matrix proteins synthesized by neoplastic cells and suggest that there is intratumoral heterogeneity in matrix protein biosynthesis in vivo. These studies further suggest that tumor-derived matrix may be altered during tumor progression or cell selection in vivo.
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PMID:Heterogeneity in the production of collagens and fibronectin by morphologically distinct clones of a human tumor cell line: evidence for intratumoral diversity in matrix protein biosynthesis. 282 40

A new method was developed for the measurement of collagenase activity using the enzyme-linked immunosorbent assay (ELISA). Rabbit colon wall collagenase, pepsin-soluble rat skin type I collagen, and its antisera were used in the present experiment. After the collagenase-degraded portion of the collagen coated on the microwell was released, the immunoreaction of the residual collagen on the microwell to anticollagen sera was determined by ELISA. This method was approximately 10 times more sensitive than the conventional assay procedure using [14C]-glycine-labeled reconstituted collagen fibrils as substrate. It was suitable for screening a large number of samples without radioisotopes.
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PMID:An assay of collagenase activity using enzyme-linked immunosorbent assay for mammalian collagenase. 282 39

H-ras-transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form of 72 kDa, which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on its ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors, which has the same molecular mass and has been linked to their metastatic potential. Type IV collagenase consists of three domains. Two of them, the amino-terminal domain and the carboxyl-terminal domain, are homologous to interstitial collagenase and human and rat stromelysin. The middle domain, of 175 residues, is organized into three 58-residue head-to-tail repeats which are homologous to the type II motif of the collagen-binding domain of fibronectin. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin.
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PMID:H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen. 283 83

Arterial microfibrils contain a 128 Kd collagenase and pepsin resistant glycoprotein (GP 128) essential for their ability to induce platelet aggregation. A previous report (Fauvel F. et al, (1984) Biochem. Biophys. Res. Comm., 123, 114-120) showed that GP 128 and thrombospondin (TSP) synthetized by endothelial cells each inhibited the aggregation of platelets by microfibrils and not by collagen. We used a monospecific antiplatelet TSP IgG in an immunoblotting assay for the identification of a TSP-like structure in untreated, collagenase-treated and pepsin-treated arterial microfibrils. The only constituent recognized in the three samples of microfibrils was GP 128. Fab fragments of this IgG provoked a dose dependent inhibition of the microfibril induced platelet aggregation (50% inhibition with 0.25 mg, 100% inhibition with 1 mg); in contrast, they did not affect collagen induced aggregation. The results indicate that a glycoprotein constituent with a thrombospondin-like antigenicity is involved in the thrombogenic properties of arterial microfibrils.
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PMID:Immunochemical identification of a thrombospondin-like structure in an arterial microfibrillar extract. 283 11

The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25 degrees C, collagenase attacked type II and type X (45-kDa pepsin-solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35 degrees C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin-solubilized type M) and collagen type XI were resistant to collagenase attack at both 25 degrees C and 35 degrees C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25 degrees C. Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25 degrees C or 35 degrees C. At 25 degrees C collagen types IX (pepsin-solubilized type M) and XI were also resistant to elastase, but at 35 degrees C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45-kDa pepsin-solubilized) was susceptible to elastase attack at 25 degrees C and 35 degrees C as judged by the production of specific products that corresponded closely with those produced by collagenase. Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35 degrees C by conditioned culture medium from IL1-activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte-derived collagenase(s) has yet to be resolved.
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PMID:Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase. 284 Nov 21

The undifferentiated F9 embryonal carcinoma cells produce a unique collagen that decreases in amount during retinoic acid-induced differentiation of F9 cells into basement-membrane parietal endoderm. A bacterial-collagenase-sensitive protein of approx. 60,000 Da was resolved on polyacrylamide-gel electrophoresis. After pepsin digestion, two pepsin-resistant fragments containing hydroxyproline were demonstrated, suggesting that a portion of the molecule has a stable triple helix. The mRNA from the undifferentiated F9 cells translates a collagenase-sensitive protein with a molecular mass consistent with the 60,000 Da collagenous protein produced by undifferentiated F8 cells.
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PMID:Undifferentiated F9 embryonal carcinoma cells produce a short-chain collagen molecule. 284 13

Collagen IV dimers of two collagen IV molecules connected by their C-terminal globular NC1 domains were isolated by limited digestion with bacterial collagenase from mouse Engelbreth-Holm-Swarm (EHS) sarcoma tissue. The collagenous domains were only 300 nm long as compared to 400 nm of intact collagen IV but the disulfide bonds in the N-terminal region of the major triple helix were retained. Unfolding of the collagenous domains as monitored by circular dichroism occurred in a temperature range of 30 to 44 degrees C with a midpoint at 37 degrees C. The transition is significantly broader than that of the continuous triple helices in collagens I, II and III, a feature which can be explained by the frequent non-collagenous interruptions in the triple-helical domain of collagen IV. Refolding at 25 degrees C following complete unfolding at 50 degrees C was monitored by circular dichroism, selective proteolytic digestion of non-refolded segments and by a newly developed method in which the recovered triple-helical segments were visualized by electron microscopy. Triple-helix formation was found to proceed in a zipper-like fashion from the C-terminal NC1 domains towards the N-terminus, indicating that this domain is essential for nucleations. For collagen IV dimers with intact NC1 domains the rate of triple-helix growth was of comparable magnitude to that of collagen III, demonstrating that the non-collagenous interruptions do not slow down the refolding process where the rate-limiting step is the cis-trans isomerization of proline peptide bonds. Refolding was near to 100% and the refolding products were similar to the starting material as judged by thermal stability and electron microscopic appearance. Removal of the NC1 domains by pepsin or dissociation of their hexametric structures by acetic acid led to a loss of the refolding ability. Instead products with randomly dispersed short triple-helical segments were formed in a slow reaction. In no case, even when the disulfide bonds in the N-terminal region of the triple-helical domain were intact, was refolding from the N- towards the C-terminus observed. Taken together with results in other collagens, this suggests that C to N directionality might be an intrinsic property of triple-helix folding.
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PMID:Folding of collagen IV. 285 Jan 75

The results of a large number of studies indicate that pulmonary surfactant contains a unique protein whose principal isoform has a molecular weight of about 30,000, and whose presence in surfactant is associated with important metabolic and physicochemical properties. This protein, SP-A, as isolated from canine surfactant, contains a domain of 24 repeating triplets of Gly-X-Y, similar to that found in collagens. These studies were undertaken to determine whether SP-A forms a collagen-like triple helix when in solution, and to describe certain aspects of its size and shape. Our experiments were done on SP-A extracted by two different methods from canine surfactant, and on SP-A produced by molecular cloning. The results from all three preparations were similar. The circular dichroism of the complete protein was characterized by a relatively large negative ellipticity at 205 nm, with a negative shoulder ranging from 215 to 230 nm. There was no positive ellipticity, and the spectrum was not characteristic of collagen. Trypsin hydrolysis resulted in a fragment with peak negative ellipticity at about 200 nm, without the negative shoulder. Further hydrolysis of this fragment with pepsin resulted in a CD spectrum similar to that of collagen. The spectrum of the collagen-like fragment was reversibly sensitive to heating to 50 degrees C, and was irreversibly lost after treatment with bacterial collagenase. SP-A migrated on molecular sieving gels with an equivalent Stokes radius of 110 to 120 A, and had a sedimentation coefficient of 14 S. Using these data we calculate a molecular weight of about 700,000. The hydrodynamic characteristics can be approximated as a prolate ellipsoid of revolution having an axial ratio of about 20. We conclude that SP-A aggregates into a complex of 18 monomers, which may form six triple-helices. The shape of the complex is considerably more globular than collagen and is not consistent with end-to-end binding of the helices to form fibrous structures.
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PMID:Aspects of secondary and quaternary structure of surfactant protein A from canine lung. 291 54


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