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)

Several small collagenous apatite binding (SCAB) proteins have been extracted from the mineralized matrix of fetal porcine calvarial bone. One protein (SCAB 3), released on demineralization of bone with 0.5 M EDTA, appears to represent the alpha 1 pN-propeptide that is normally released during proteolytic processing of type I procollagen. The 28 Kd protein, which stains blue with "Stains-all", is reduced to a 19 Kd fragment by bacterial collagenase digestion, but is not susceptible to cyanogen bromide. The amino acid composition, blocked amino-terminus and immunological properties are all consistent with properties of alpha 1 (I) pN-propeptide. Fractionation on hydroxylapatite in the presence of urea has revealed a nonbinding (SCAB 3a) and a binding (SCAB 3b) form. Extraction of the demineralized matrix of bone with 4 M GuHCl revealed a third form (G2-28) which was similar to SCAB 3a on hydroxylapatite chromatography but showed differences on FPLC "Mono Q" resin. The occurrence of these different forms of pN-propeptide in bone may be of significance in collagen fibril-associated hydroxylapatite formation and in the regulation of osteoblastic function during bone resorption.
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PMID:Identification of small collagenous proteins with properties of procollagen alpha 1 (I) pN-propeptide in fetal porcine calvarial bone. 339 4

Damage to interstitial connective tissue is associated with the rapid accumulation of monocytes and neutrophils at the site of injury. To study the role of collagen fragments in neutrophil migration, we analyzed the chemotactic properties of peptide fragments of bovine collagen digested with bacterial collagenase or cyanogen bromide and small molecular weight synthetic polypeptides containing proline (Pro), hydroxyproline (Hyp), and glycine (Gly), the major amino acids that comprise collagen. Using the Boyden chamber and under agarose techniques, we found that collagen fragments were as potent in inducing chemotaxis in neutrophils as the bacterial-derived peptide f-met-leu-phe. The synthetic polytripeptides (Pro-Pro-Gly)5 and (Pro-Hyp-Gly)5 were found to be equipotent in inducing chemotaxis, producing a maximal induction of chemotaxis at 5-10 nM. This suggests that Hyp, the unique imino acid found in collagen, is not required for chemotactic activity. Increasing the length of the synthetic tripeptide from 5 to 10 subunits decreased its chemotactic activity, while the single tripeptide subunit (Pro-Hyp-Gly)1 was the least active peptide, inducing a maximal response at 100 nM. To study the structural requirements for chemotaxis, Pro-Hyp-Gly tripeptides were synthesized with modifications at the N and C terminals ends. Addition of a methyl group to the carboxyl of Gly to form an ester enhanced the chemotactic activity of the peptide by 50%, while substitutions on the amino terminus with an acetyl group decreased the chemotactic activity by 50%. Substitution on the amino terminus with a Boc group decreased the chemotactic activity by 100%. These results indicate that there are specific structural requirements for chemotaxis induced by peptides having a collagen-like sequence of amino acids.
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PMID:Chemotactic activity of collagen-like polypeptides for human peripheral blood neutrophils. 345 7

A comparative study was undertaken to evaluate the effectiveness of various procedures (e.g., direct harvest vs. short-term culture) for the cytogenetic analysis of human solid tumors. A total of 51 specimens (38 primary tumors, 13 effusions) were examined from 45 patients with tumors of the ovary, lung, breast, colon, and testicles. Sufficient numbers (greater than or equal to 3) of metaphase cells to be useful for karyotypic analysis were obtained in 42 of 51 (82.3%) specimens. More than 10 analyzable metaphases were found in each of 32 specimens (62.7%), but in some cases it was necessary to examine many slide preparations to achieve this number. The rate of successful chromosome preparations was markedly better for short-term cultures than for specimens harvested directly. Short-term cultures generally showed a mitotic peak after about 3 days of growth in vitro. Generally, the quality of metaphase cells was better in specimens disaggregated enzymatically with collagenase than in those dissociated mechanically. Exposure to ethidium bromide (EB) for the final 2 hours of culture yielded more cells with elongated chromosomes than cultures harvested by conventional methods without EB. Overall, the findings indicate that successful karyotypic analysis can be performed in a high percentage of human solid tumors with the use of techniques that can be readily applied in most cytogenetics laboratories. However, further methodological advances in tissue culture are warranted to routinely provide large numbers of mitotic cells for karyotypic analysis.
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PMID:Assessment of methods for the cytogenetic analysis of human solid tumors. 348 Mar 80

Using nondegradative isolation procedures, we have purified and characterized the Mr 24,000 phosphoprotein from developing bovine and human bone where it constitutes 5% of the noncollagenous protein in the mineral compartment. This hydroxyproline-containing protein could not be cleaved by cyanogen bromide. The purified, intact product spontaneously formed a complex consistent with a collagen-like trimer that remained a trimer even in sodium dodecyl sulfate-polyacrylamide gels. The ability to form the complex was lost upon treatment with bacterial collagenase, a treatment that resulted in an NH2-terminally blocked fragment of Mr 17,000. After deblocking, the NH2-terminus of the intact, Mr 24,000 bovine product was shown to have virtually the same amino acid sequence (residues 1-24 with asparagine rather than aspartic acid at position 20 as reported earlier by Horlein et al. (Horlein, D., Fietzek, P. P., Wachter, E., Lapiere, C. M., and Kuhn, K. (1979) Eur. J. Biochem. 90, 31-38) as the amino-terminal segment of dermatosparatic calf skin alpha 1 type I procollagen. Furthermore, pulse-chase studies showed a precursor-product relationship between procollagen and the Mr 24,000 protein. Anti-serum made against the bovine bone protein bound to bands on electrotransfers that were consistent with the positions of both alpha 1(I) procollagen and the procollagen chain missing its COOH-terminal extension peptide (pN-alpha 1(I), as well as the original Mr 24,000 product in extracts of bone, skin, tendon, cornea, and other type I collagen-containing tissues. Fetal calf serum contained an average of 106 micrograms/ml of the Mr 24,000 protein as determined by quantitative enzyme-linked immunosorbent assay. The only serine residue in the bovine bone protein was phosphorylated. It is unknown whether the corresponding collagen NH2-terminal pro-peptides in other tissues and serum are similarly phosphorylated.
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PMID:The Mr 24,000 phosphoprotein from developing bone is the NH2-terminal propeptide of the alpha 1 chain of type I collagen. 365 22

Fibrosis, characterized by accumulation of collagen, is a delayed result of radiation injury in many tissues, including lung. To investigate its development, synthesis and degradation of collagen were measured in lungs of mice after X irradiation of the whole thorax. The ratio of type I (coarse fibered) to type III (meshwork) collagen was also determined. Synthesis of procollagen, measured as the activities of prolyl-4-hydroxylase and protein disulfide isomerase in lung tissue, was increased at 2 months after X-ray doses of 5, 7.5, and 9 Gy. Maximal increases were observed 6 to 7 months after doses of 9 Gy and persisted up to 15 months after exposure. Increases after 5 and 7.5 Gy were more gradual, but by 1 year after irradiation they had reached levels similar to those after 9 Gy. X irradiation had no effect on the degradation of collagen as assessed by collagenase activity in lung. The ratio of type I to type III collagen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of collagen-derived cyanogen bromide peptides, was the same in irradiated lungs as in age-matched controls. Therefore, increased synthesis of procollagen, rather than decreased degradation of collagen or changes in collagen type, is an important factor in the accumulation of collagen in irradiated lung.
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PMID:Biosynthesis and degradation of collagen in X-irradiated mouse lung. 368 61

Reduction of a commercially available, pepsin-solubilized, bovine dermal collagen (Vitrogen 100) with sodium [3H]borohydride provided radiolabeled collagen preparations with specific activities ranging from 7.1-12.0 muCi/mg collagen. These specific activities were 2-3 times greater than those obtained by reduction of intact rat tail tendon collagen under similar conditions. The alpha, beta, and higher aggregate components of type I collagen were radiolabeled as well as the alpha component of a small amount of type III collagen present in the samples. Fractionation of cyanogen bromide peptides showed that alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB3,5 were the predominant peptides labeled by this procedure. Amino acid analysis indicated that the majority of the radioactivity was in reducible cross-links, precursors of these cross-links, and in hexosyllysine residues. Reconstitution experiments comparing this radiolabeled collagen with nonlabeled collagen showed them to be indistinguishable. Bacterial collagenase digestion of this reconstituted fibrillar collagen in both a lightly cross-linked (glutaraldehyde 0.0075%) and noncross-linked form provided evidence that digestion of labeled and nonlabeled collagens proceeded at similar rates. Thus, labeling did not change the properties of the collagen. Cross-linking made the preparation refractory to proteolytic degradation. Injection of fibrillar collagen preparations, spiked with radiolabeled collagen, into the guinea pig dermis followed by quantitation of the amount of radioactivity recovered from implant sites as a function of time, indicated that the lightly cross-linked samples also were more resistant to degradation in vivo than the noncross-linked preparation. The half-life of noncross-linked collagen was about 4 days while that of the cross-linked collagen was about 25 days. These degradation rates were much faster than observed for similar, nonlabeled samples injected into the dermis of humans, presumably due to a higher metabolic activity in the guinea pig dermis.
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PMID:Preparation of [3H]collagen for studies of the biologic fate of xenogenic collagen implants in vivo. 371 80

In an effort to elucidate the nature of the collagen-platelet interaction, the effects of collagen modification on platelet aggregation have been studied. We have shown that purified rat skin (salt) soluble collagen is effective at about 20 nM in mediating platelet aggregation in human platelet-rich plasma. This concentration is somewhat greater than that required of several skin insoluble collagens (ca. 10 nM). Both the alpha1(I) and alpha2 chains from rat skin soluble collagen produced platelet aggregation, but only at concentrations of about 13 muM and 55 muM, respectively. In contrast, heat-denatured collagen and chains (e.g., 65 muM alpha1(I) and 160 muM alpha2) failed to induce platelet aggregation and to inhibit platelet aggregation by native collagen. Glycopeptides were prepared from human skin insoluble collagen by extended digestion with bacterial collagenase and trypsin, and were purified by gel filtration into two classes. One class of higher molecular weight contained sialic acid, glucosamine, galactosamine, fucose, mannose, galactose, and glucose, and the other of lower molecular weight consisted primarily of a mixture of galactose and galactosyl-glucose units O-glycosidically linked to hydroxylysine-containing peptides. We found that, after the residual tryptic activity contaminating the higher molecular weight fraction was inhibited, neither of the glycopeptide classes produced nor inhibited native human skin insoluble collagen-mediated platelet aggregation at the highest concentration examined (ca. 1-2 mg glycopeptide per ml of platelet-rich plasma). Highly purified samples of the hydroxylysyl glycosides, hydroxylysylgalactose and hydroxylysylgalactosylglucose (Hyl-Gal and Hyl-Gal-Glc, respectively), were prepared from human urine and labeled at galactose using galactose oxidase followed by reduction with tritiated borohydride. Binding studies with platelet-rich plasma showed that, at concentrations greater than 50 nM, Hyl-Gal gives apparent binding to platelets, but there was no evidence of Hyl-Gal-Glc binding to platelets at concentrations up to 250 nM. At concentrations several hundredfold higher than the equivalents present in the minimum concentration of rat skin soluble collagen required for platelet aggregation, neither Hyl-Gal (at 29 muM) nor Hyl-Gal-Glc (at 18 muM) caused platelet aggregation or inhibited platelet aggregation by native collagen. Also, at a concentration of 85 muM (which represents a concentration about two thousandfold higher than the equivalents in the minimum concentration of soluble collagen required for platelet aggregation) the Gal-Glc-containing 36 residue rat skin soluble collagen alpha1(I)cyanogen bromide #5 peptide had no platelet aggregating or inhibiting activity. Modification of at least 90% of the rat skin soluble collagen carbohydrate by mild periodate oxidation had no effect on the platelet aggregating activity. Human skin insoluble collagen was reacted with periodate under the same conditions, and this had no demonstrable effect on its ability to induce platelet aggregation. This indicates that the normal carbohydrate side chains of these collagens are not required for the platelet interaction that produces the release of ADP and other metabolic constituents and leads to aggregation.Thus, collagen-platelet interactions appear to involve at least two distinct binding sites on the platelet plasma membrane. One is a protein binding site that activates platelet aggregation and has high specificity and affinity for the collagen triple-helical fold or perhaps even for a particular amino acid sequence in the triple helix.
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PMID:Collagen-mediated platelet aggregation. Effects of collagen modification involving the protein and carbohydrate moieties. 435

The structural heterogeneity of rabbit lung collagen was examined by extracting labeled collagen from short-term cultures of lung minces with 1 M NaCl-50 mM Tris.HCl (pH 7.4), 0.5 M acetic acid, or 0.4 ionic strength phosphate buffer. The extracted collagens were purified by carboxymethyl-cellulose chromatography, and their cyanogen bromide peptides were mapped by ion exchange chromatography and acrylamide gels. Rabbit skin alpha1(I) and alpha2 chains and rabbit sternal cartilage alpha1(II) chains were used as markers. The peripheral lung, containing alveoli, small blood vessels, and small airways, synthesized alpha1(I) and alpha2 chains. The trachea and the bronchial tree (first through seventh order branches) both synthesized alpha1(II) chains. Lung alpha1(I), alpha2, and alpha1(II) chains all have a molecular weight of about 100,000 and are all sensitive to Clostridial collagenase. The extraction and purification methods used isolate only 50% of the collagen synthesized by these structures in vitro. Once all collagen types in lung can be described and quantitated, it should be possible to utilize lung collagen types as biochemical markers to study normal lung development and to define the lung fibrotic diseases.
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PMID:Lung collagen heterogeneity. 452 88

The relationship between the six collagenases (alpha, beta, gamma, delta, epsilon, and zeta) isolated and characterized in the preceding papers [Bond, M.D., & Van Wart, H.E. (1984) Biochemistry (preceding two papers in this issue)] has been investigated. Chemical modification reactions establish that all six enzymes contain essential carboxyl, tyrosine, and lysine residues. Circular dichroism spectra of the peptide bond region show that the secondary structures of the collagenases are very similar. Ouchterlony double-immunodiffusion experiments carried out with antiserum prepared against beta-collagenase indicate that all six collagenases are cross-reactive. Reverse-phase high-pressure liquid chromatography elution profiles of tryptic digests of these collagenases and sodium dodecyl sulfate electrophoresis gels of the peptides formed on reaction with cyanogen bromide have been obtained. The results indicate that the class I collagenases have extensive sequence homology with each other and that the class II collagenases have extensive sequence homology with each other but that the enzymes in the two classes have substantially different sequences. In addition, the data show that beta-collagenase probably consists of domains that have homologous amino acid sequences, which may have arisen by full or partial intragenic gene duplication. This may account for the unusually high molecular weight of this and the other collagenases. Finally, on the basis of the similarities between the collagenases in the two classes, it is suggested that one class evolved from the other by gene duplication followed by independent evolution by point mutations to yield enzymes with different substrate specificities.
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PMID:Relationship between the individual collagenases of Clostridium histolyticum: evidence for evolution by gene duplication. 608 89

Synthesis of collagen types I, II, III, and IV in cells from the embryonic chick cornea was studied using specific antibodies and immunofluorescence. Synthesis of radioactively labeled collagen types I and III was followed by fluorographic detection of cyanogen bromide peptides on polyacrylamide slab gels and by carboxymethylcellulose chromatography followed by disc gel electrophoresis. Type III collagen had been detected previously by indirect immunofluorescence in the corneal epithelial cells at Hamburger-Hamilton stages 20--30 but not in the stroma at any age. Intact corneas from embryos older than stage 30 contain and synthesize type I collagen but no detectable type III collagen. However, whole stromata subjected to collagenase treatment and scraping (to remove epithelium and endothelium) and stromal fibroblasts from such corneas inoculated in vitro begin synthesis of type III collagen within a few hours while continuing to synthesize type I collagen. As demonstrated by double-antibody staining, most corneal fibroblasts contain collagen types I and III simultaneously. Collagen type III was identified biochemically in cell layers and media after chromatography on carboxymethylcellulose be detection of disulfide-linked alpha l (III)3 by SDS gel electrophoresis. The conditions under which the corneal fibroblasts gain the ability to synthesize type III collagen are the same as those under which they lose the ability to synthesize the specific proteoglycan of the cornea: the presence of corneal-type keratan sulfate.
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PMID:Synthesis of type III collagen by fibroblasts from the embryonic chick cornea. 624 16


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