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)

1. Cell cultures propagated from foetal bovine ligamentum nuchae synthesized and secreted two glycoproteins, designated MFP I and MFP II, that are closely related to elastic-fibre microfibrils. Glycoproteins MFP I (apparent mol.wt. 150 000) and MFP II (apparent mol.wt. 300 000) were metabolically labelled, separated from other culture-medium components by immunoprecipitation with a specific anti-(microfibrillar protein) serum, and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and sodium dodecyl sulphate/gel-filtration chromatography. 2. Ligament cells also synthesized and secreted fibronectin, but salt-fractionation and immunoprecipitation studies with a specific anti-(cold-insoluble globulin) serum established that neither glycoprotein MFP I nor glycoprotein MFP II was related to fibronectin. 3. The secretion of glycoprotein MFP I, but not that of glycoprotein MFP II, was enhanced by the addition of ascorbate to the culture medium. 4. Ascorbate-supplemented ligament cells incorporated [3H]proline into glycoprotein MFP I, and 36% of the nondiffusible proline residues were hydroxylated, exclusively as 4-hydroxy[3H]proline. Less than 1% of the total proline residues in [3H]proline-labelled glycoprotein MFP II were hydroxylated. 5. Ascorbate-supplemented cells incorporated [14C]lysine into glycoprotein MFP I and 30% of the non-diffusible lysine residues were hydroxylated. 6. Newly secreted glycoprotein MFP I was digested by highly purified bacterial collagenase to yield polypeptide fragments of apparent mol.wts. 50 000 and 30 000. Glycoprotein MFP II was not digested by bacterial collagenase. 7. The results suggest that elastic-fibre microfibrils are composed of a novel collagenous glycoprotein MFP I in association, as yet undefined, with a non-collagenous glycoprotein MFP II.
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PMID:The nature of the microfibrillar glycoproteins of elastic fibres. A biosynthetic study. 627 35

During the growth of Empedobacter collagenolyticum on a medium with gelatin, only one proteinase, a collagenase, was excreted in the culture medium. No other proteolytic activity was detected in the extracellular medium or in acellular extracts. The other proteases of this bacteria are principally intracellular peptidases. By electrophoresis of an acellular extract five peptidases were separated; they were aminopeptidases and dipeptidases. Some of them exhibited a specificity towards peptides with aminoacid frequently found in collagen; Gly-Leu, Gly-Pro, Pro-Gly-Gly. Two other peptidases seem to have special specificity, one of them hydrolyses peptides with lysine residues at the NH2 terminal end, the other one hydrolyses dipeptides of the structure Lys-X. These enzymes were also found in the culture medium; they certainly play an important role in bacterial nutrition.
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PMID:[Inventory of proteolytic activity of a new collagenolytic bacteria Empedobacter collagenolyticum]. 627 74

Collagenolytic activity of rat kidneys with streptozotocin diabetes was estimated by means of a biological collagenase assay and compared to healthy controls. Collagenolytic activity was found significantly decreased in rat kidneys with diabetes correlating with blood glucose levels (r = -0.82, p less than 0.001). Elevated blood glucose levels seem to be responsible for the inhibition. This is supported by our experiment of incubating bacterial collagenase with several carbohydrates as glucose, galactose and saccharose: glucose and galactose significantly inhibited the collagenolytic activity, while saccharose failed to inhibit the enzymatic reaction. The interpretation of the results is that glucose is able to bind to the enzyme as Schiff base, which could be shown by tritiated sodium borohydride reduction of the Schiff base formed between collagenase and glucose. Another support of the hypothesis is that blocking of the amino group of lysine at the active site either by glucose or trifluoroacetylation of collagenase is reducing the collagenolytic activity. The biological significance could be the decreased catabolism of collageneous material of the extracellular matrix, as, e.g., the glomerular basement membrane, which was reported in a previous publication.
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PMID:Reduced collagenolytic activity of rat kidneys with steptozotocin diabetes. 628 20

Medium conditioned by the culture of porcine gingival explants was shown to contain, in addition to collagenase, proteolytic activity capable of releasing small fragments, devoid of hydroxyproline but containing hydroxynorleucine, from reduced (tritiated) type I collagen in solution at neutral pH. Quantitative comparison of this effect with that of cathepsin D, at pH 4, revealed that the fragments were derived at least in part from the carboxy-terminal, extra-helical portion of the collagen alpha 1-chains. Incubation of concentrated conditioned medium with fibrillar acetic acid-insoluble collagen resulted in the solubilization of the TC 3/4 and TC 1/4 fragments characteristic of the action of collagenase. However, alpha 1-chain fragments isolated from the latter were found to lack the antigenic determinant normally present on the amino-terminal side of the (hydroxy-)lysine residue which is known to be involved in intermolecular cross-linking. It is therefore suggested that the proteolytic activity described above was involved in the solubilization process. Both the release of low molecular fragments from soluble collagen and the solubilization effect were abolished by ethylenediaminetetra-acetic acid.
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PMID:Cleavage of the carboxy-terminal cross-linking region of type I collagen by proteolytic activity from cultured porcine gingival explants. 631 80

The biosynthesis of type V collagen and its regulation were studied using diploid human gingival fibroblasts. Cells were metabolically labeled with radioactive amino acids and labeled proteins were subjected to limited pepsin digestion, DEAE-cellulose chromatography at 15 degrees C, and polyacrylamide gel electrophoresis. Proteins eluted from DEAE-cellulose columns by 0.25 M NaCl contained a collagen species which was resistant to mammalian collagenase and had alpha chains with hydroxylysine/lysine ratios and CNBr peptide patterns similar to alpha 1(V) and alpha 2(V). Procollagen(V) fractions obtained by DEAE-cellulose chromatography and immunoprecipitates of type V collagen antibody contained polypeptides with Mr = 239,000, 219,000, 198,000, 174,000, 157,000, and 132,000. By comparing the CNBr peptide maps of these proteins with those of standard alpha 1(V) and alpha 2(V) chains, the first three polypeptides were shown to be related to alpha 1(V) and the others to alpha 2(V). It was concluded that the gingival fibroblasts synthesize type V collagen, that the pro alpha 1(V) and the pro alpha 2(V) chains have Mr = 239,000 and 174,000, respectively, and that the alpha 1(V) and alpha 2(V) chains laid in the form of fibrils have Mr = 198,000 and 132,000, respectively. A detectable amount of type V collagen was synthesized only at high cell density, and it was associated with the cell layer. The amount and proportion of type V synthesized were increased when the cells were labeled in the presence of serum, and the increase was accompanied by a decrease in type III. This effect was dependent on serum concentration. Serum obtained from platelet-poor plasma failed to elicit this effect, and it was restored by the addition of platelet-derived growth factor. Platelet-derived growth factor was effective in medium with and without platelet-poor serum. Thus, it appears that platelet-derived growth factor may be an important regulatory factor in the synthesis of types V and III collagens.
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PMID:Biosynthesis and regulation of type V collagen in diploid human fibroblasts. 631 21

The incorporation of methionine, lysine, and leucine into protein was studied in Ca2+-depleted and Ca2+-restored preparations of C-6 glial tumor cells in minimal medium. Although incorporation proceeded at linear rates in both preparations for more than 1 h and into the same spectrum of proteins, Ca2+-restored cells incorporated amino acid 5- to 10-fold more rapidly than Ca2+-depleted cells. Addition of approximately 200 microM Ca2+ in excess of chelator was required to achieve maximal rates of incorporation in Ca2+-depleted preparations. Stimulation by Ca2+ was rapid in onset (several minutes) and slowly reversible by chelator. Ca2+ was uniquely potent and specific among physiologically occurring cations in conferring such stimulation. Stimulation of amino acid incorporation by Ca2+ occurred over a broad range of pH and osmolarities and was facilitated by Mg2+. The effects of Ca2+ in stimulating amino acid incorporation were not traceable to changes in cAMP metabolism, amino acid uptake, protein catabolism, cell ATP or GTP content, or aminoacylation of transfer RNA. Actinomycin D (1 microgram/ml) did not block the stimulatory effects of Ca2+ although puromycin and cycloheximide did. The stimulatory effects of Ca2+ on protein synthesis were not restricted to C-6 in minimal medium. Protein synthesis was reduced by ethylene glycol bis(B-aminoethyl ether)-N,N,N',N'-tetraacetic acid 40 to 75% in C-6 glioma, GH3 pituitary tumor, PC-12 adrenal tumor, N2A neuroblastoma, and HeLa cells incubated under simulated growth conditions with various enriched media and sera. Ca2+-depleted S49 lymphoma, CHO ovarian tumor, and normal, dispersed chicken embryo cells in enriched medium responded to Ca2+ restoration with increased rates of protein synthesis as did collagenase-dispersed normal rat liver cells in minimal medium. Protein synthesis in rabbit reticulocyte lysates was also inhibited by Ca2+-selective chelators or by Ca2+ removal by parvalbumin affinity chromatography and the inhibition was reversed by Ca2+. These findings are consistent with the existence of a Ca2+ requirement in the translational phase of protein synthesis in eukaryotic cells.
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PMID:Identification of a Ca2+ requirement for protein synthesis in eukaryotic cells. 631 27

Polymorphonuclear leukocytes have been shown to contain proteolytic enzymes which are capable of degrading connective tissue proteins such as native collagen. In this study, proteolytic enzymes were extracted from human polymorphonuclear leukocytes and a neutral proteinase was extensively purified and characterized. The activity of this enzyme was monitored by degradation of denatured [ 3H ]proline-labeled type I collagen or by cleavage of a synthetic dinitrophenylated peptide with a Gly-Ile sequence. The enzyme was readily separated from leukocyte collagenase by concanavalin-A--Sepharose affinity chromatography and further purified by QAE-Sephadex ion-exchange chromatography and gel filtration on Sephacryl S-200. The purified enzyme had a molecular weight of approximately 105000, its pH optimum was about 7.8, and it was inhibited by Na2EDTA and dithiothreitol, but not by fetal calf serum. The enzyme degraded genetically distinct type I, II, III, IV and V collagens, when in a non-helical form, but not when in native triple-helical conformation. Dansyl-monitored end-group analyses, combined with digestion by carboxypeptidase A, indicated that the enzyme cleaved denaturated type I collagen at Gly-Xaa sequences, in which Xaa can be leucine, isoleucine, valine, phenylalanine, lysine, or methionine. Thus, the purified enzyme referred to here as Gly-Xaa proteinase, is a neutral proteinase, which may be of importance in inflammatory disease processes by degrading further collagen peptides which have been rendered non-helical as a result of collagenase cleavage.
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PMID:Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence. 634 59

The extent of the odontoblast cell process has been the subject of controversy for many years. Using SEM we have examined the extent and morphology of the process on dentine surfaces of human teeth which were partially demineralized and collagenase digested. Third molars were extracted and split; the dentine surface was demineralized, digested by bacterial collagenase, fixed with glutaraldehyde, postfixed in osmium tetroxide, and prepared for SEM investigation. The SEM study revealed the presence of many processlike structures which extended from the odontoblast cell bodies up to the dentinoenamel junction (DEJ). These processes demonstrated lateral and terminal branching and some of them terminated in distended spheres. We have also applied an immunofluorescence technique at the light microscope level to these exposed dentinal surfaces to localize the intracellular microtubules. For this, a second series of third molars was processed in the same manner as for the SEM up to the fixation stage. Teeth were then fixed in periodate-lysine-paraformaldehyde, postfixed in -20 degrees C acetone, and then incubated with affinity-purified rabbit antitubulin antibodies, followed by fluorescein-conjugated goat antirabbit IgGs. Intratubular immunofluorescence labelling for tubulin was evident from the odontoblast cell bodies up to the DEJ. The presence of the tubulin-containing structures extending to the DEJ supports the hypothesis that the structures observed with the SEM are odontoblast processes and that the odontoblast processes do extend to the DEJ.
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PMID:A combined scanning electron microscopy and immunofluorescence study demonstrating that the odontoblast process extends to the dentinoenamel junction in human teeth. 639 20

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 production of [14C]proline-labeled collagen by embryonic chick tendon cells in suspension culture is reduced when the cells are incubated in the presence of lysosomotropic agents NH4Cl or chloroquine. Since these agents have multiple effects on fibroblasts, including inhibition of collagen secretion, specific proteinase inhibitors were tested for their effect on collagen production. Here the proteinase inhibitors N-p-tosyl-L-lysine chloromethylketone (TLCK) and leupeptin, specific for certain cysteine and serine proteinases, and pepstatin A, specific for aspartic proteinases, were tested for their effects on both the production and secretion of collagen. When treated with the proteinase inhibitor TLCK, the percentage of protein synthesis devoted to collagen decreased from control levels of 19.0 +/- 1.4% to 10.5 +/- 2.4% with 10 microM TLCK. Collagen synthesis was further reduced to only 1.2% of total protein synthesis with 100 microM TLCK. The incorporation of [14C]proline into collagenase-digestible peptides was only slightly decreased in the samples treated separately with 50 micrograms/ml leupeptin or 60 micrograms/ml pepstatin A. However, the production of collagen was reduced to 10.9 +/- 1.4% of total protein synthesis in samples treated with leupeptin and pepstatin A together. The basal intracellular degradation of newly synthesized, [14C]proline-labeled collagen was not significantly altered by any of the reagents tested, and secretion of the collagen which was produced was not impaired except in samples treated with 100 microM TLCK. The data presented are consistent with the hypothesis that a proteolytic mechanism utilizing some combination of cysteine, serine, and aspartic proteinases is necessary for continued collagen synthesis in freshly isolated embryonic chick tendon fibroblasts, and suggests that a heretofore unknown regulatory system may be operative in controlling the synthesis of collagen in fibroblasts.
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PMID:The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture. 648 92


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