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)

The biosynthetic activity of the B-cells of obese hyperglycemic mice (obob) was measured by the incorporation of [3H]leucine into proteins in collagenase-isolated pancreatic islets. To quantitate the incorporation into proinsulin and insulin, an immune binding method was used. For this purpose, anti-insulin serum was coupled to cyanogen bromide-activated SepharoseR 4B. This turned out to be a specific and versatile technique for the measurement of newly synthesized proinsulin and insulin in the B-cells. The B-cells of obob mice appear to be well adapted to a high rate of hormone biosynthesis, since at 16.7 mM glucose 44% of [3H]leucine incorporated into TCA-precipitable proteins was bound to the insulin antibodies coupled to Sepharose 4B. The insulin biosynthetic rate was stimulated 9 times at 16.7 mM glucose, during a 3-h incubation, compared with the basal insulin biosynthesis rate.
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PMID:Anti-insulin serum coupled to Sepharose 4B as a tool for the investigation of insulin biosynthesis in the B-cells of obese hyperglycemic mice. 110 96

Monoclonal antibodies were prepared that were specific for chicken type I and type III collagens. The specificity of these antibodies was determined by ELISA, inhibition ELISA, and immunoblot assays. The results showed that the monoclonal antibodies were specific for their respective antigens without significant cross reactivity to other types of collagen. An analysis of the location of the epitopes by rotary shadowing that a monoclonal antibody for type I collagen (called DD4) recognized type I procollagen close to the large globular domain at the carboxyl terminus of the molecule. A monoclonal antibody for type III collagen (called 3B2) recognized both the intact type III molecule and also the TCA fragment of type III collagen after mammalian collagenase digestion. The epitope was located approximately one-fifth of the distance from the amino-terminus of the intact molecule. The monoclonal antibodies were used for immunolocalization of type I and type III collagens in cryosections of heart, aorta, kidney, liver, thymus, skin, gizzard and myotendinous junction. In heart, aorta, kidney, liver, thymus and skin, type I and III collagens were colocalized in the connective tissue of each organ. In contrast, gizzard and myotendinous junction showed distinctly different staining patterns for the distribution of type I and type III collagen. The two monoclonal antibodies reported here are potentially useful reagents to study fibril formation involving type I and type III collagens.
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PMID:Monoclonal antibodies that distinguish avian type I and type III collagens: isolation, characterization and immunolocalization in various tissues. 156 Jul 90

Several N-carboxyalkyl peptides were synthesized and tested as inhibitors of pig synovial collagenase, 72-kDa gelatinase and stromelysin (matrix metalloproteinases MMP-1, MMP-2, and MMP-3). The most potent of the series, CH3CH2CH2(R,S)CH(COOH)-NH-Leu-Phe-Ala-NH2, competitively inhibited cleavage of dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by MMP-1 and MMP-2 (KI = 30 and 40 microM, respectively). A similar inhibitory potency was found for MMP-1 with soluble Type I collagen and MMP-3 with substance P as substrate. The inhibitor was coupled to EAH-Sepharose 4B through a C-terminal amide. In the presence of 2 M NaCl at pH 7.2, this matrix bound MMP-1, MMP-2, and MMP-3 from concentrated culture medium of pig synovial membranes. The enzymes coeluted at pH 4.1 and subsequently were resolved by chromatography on DEAE-Sephacel and heparin-Sepharose. Purified MMP-1 catalyzed the o-phenanthroline-sensitive cleavage of collagen into TCA and TCB fragments as well as slower hydrolysis of the alpha 2 chain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of MMP-1 indicated a predominant polypeptide of approximately 44 kDa and minor species of approximately 24 and 21 kDa. The 44-kDa species and one of the smaller polypeptides reacted with an antiserum to residues 195-207 of human fibroblast MMP-1, indicating that porcine MMP-1 contains a similar sequence and that the smaller components were probably derived from MMP-1. Neither MMP-2 nor MMP-3 reacted with this antiserum. Purified porcine MMP-2 degraded gelatin but not collagen and exhibited an apparent Mr of approximately 71 kDa. Additional smaller polypeptides were present, one of which may correspond to tissue inhibitor of metalloproteinases. MMP-3 showed doublets of approximately 47/46 and 26/25 kDa and cleaved substance P at the Gly6-Phe7 bond. This procedure provides a rapid means of obtaining all three MMPs from one source in approximately 15% yield each.
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PMID:Application of N-carboxyalkyl peptides to the inhibition and affinity purification of the porcine matrix metalloproteinases collagenase, gelatinase, and stromelysin. 165 8

Type I collagen is highly susceptible to proteolytic cleavage by neutral mammalian collagenase. Following an initial site specific cleavage of the substrate, two characteristic products are generated, TCA and TCB. These two products then spontaneously denature and are degraded into multiple smaller molecular weight peptides. We prepared TCA and TCB from native type I collagen by the action of rat uterine fibroblast neutral collagenase. In addition we prepared denatured type I alpha chains and exposed them to the action of collagenase under controlled conditions in order to generate small molecular weight peptides. We then examined intact type I collagen, TCA and TCB and type I gelatin peptides for chemotactic activity in a Boyden chamber assay using both human peripheral monocytes and polymorphonuclear leucocytes as target cells. Intact type I collagen, while chemotactic for neutrophils, failed to elicit any chemotactic response in mononuclear cells. In addition, the results demonstrate an absence of any detectable chemotactic activity for either TCA or TCB when human peripheral monocytes were used as the target cells. However, type I collagen peptides demonstrated chemotactic activity for peripheral monocytes. Maximum cell migration was found with digests which had been exposed to neutral mammalian collagenase for three to four hours. No chemotactic activity was found using the same peptides, when neutrophils were used as the target cells. The data strongly suggest that chemotactic activity for mononuclear cells, normally suppressed in intact type I collagen, is revealed and/or activated by neutral collagenase digestion. Conversely, chemotactic activity for neutrophils is lost when intact type I collagen is digested into smaller molecular weight fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Recruitment of peripheral mononuclear cells by mammalian collagenase digests of type I collagen. 165 75

Collagenolytic enzymes were quantitated by a method based on spectrophotometry of suspended reconstituted collagen fibrils. To obtain optically stable suspensions it was necessary to perform a short sonication of the aggregated fibrils at 10 degrees C. When fibrils were cleaved with mammalian fibroblast collagenase at 35 degrees C the triple helical collagen fragments (TCA and TCB) would uncoil spontaneously and the decreasing turbidity was used as an estimate of enzyme activity. The method is a specific collagenase assay since a possible cleavage in the non-helical parts of the collagen molecule with contaminating proteinases is without effect on the turbidity of the suspension and the collagen substrate is not converted to gelatin at 35 degrees C. After 1 h of incubation 0.2 U (equivalent to 0.2 micrograms) of fibroblast collagenase could be detected. In purification procedures with microbial collagenases many fractions were tested by overnight incubations in disposable cuvettes. Sealing of cuvettes with square silicone stoppers allowed rotation of enzyme-substrate mixtures directly in the cuvettes. Only standard laboratory equipment is required for this assay, which is not dependent on radiolabeling or preparation of specific immunologic reagents.
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PMID:Collagenolytic enzymes assayed by spectrophotometry with suspensions of reconstituted collagen fibrils. 166 Aug

In view of the possible link between collagenase and the formation of aortic aneurysms we have determined whether cells within the aorta are able to synthesize this enzyme. Explanted cells obtained from fragments of lapine abdominal aorta secreted little or no collagenase. Two related metalloproteinases, gelatinase and stromelysin, were also produced at very low levels. Treatment with purified human monocyte interleukin-1 beta, partially purified lapine, synovial IL-1 or phorbol myristate acetate strongly induced the synthesis of all these enzymes. These activators also increased synthesis of prostaglandin E2. The identity of collagenase was confirmed by detection of the characteristic TCA and TCB breakdown fragments of collagen and by demonstration of collagenase mRNA within activated aortic cells. Unactivated aortic cells contained no detectable collagenase mRNA, suggesting a pretranslational level of regulation. Aortic cells thus possess the ability to express several neutral metalloproteinases and, if a sufficient inflammatory stimulus was present, they might do so in arteries undergoing aneurysmal degeneration.
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PMID:Inducible synthesis of collagenase and other neutral metalloproteinases by cells of aortic origin. 166 97

We have recently presented biochemical evidence for collagen and gelatin degrading activities associated with plasma membranes of various human cancer cell lines. In this report we describe the localization of interstitial collagenase at the basal plasma membrane of the human pancreatic cancer cell line RWP-I, using immunofluorescence and ultrastructural immunogold labeling techniques. Collagenase was expressed on the extracellular face of the plasma membrane. Furthermore, the immunogold labeling was concentrated on the long, finger-like microvillous projections typically seen on the basal cell surface, while the short, brush-like projections characteristic of the apical cell surface were unlabeled. When the cytoplasmic face of the membrane was made accessible, the number of reactive sites increased markedly, indicating a high concentration of enzyme at the inner surface of the plasma membrane. When plasma membrane fractions of RWP-I cells were prepared by differential centrifugation, high salt washes virtually failed to extract collagenase activity from the membrane, while detergent extraction with n-octyl glucoside, a detergent used in the purification of integral membrane proteins, yielded soluble collagenase activity. When detergent extracted membrane fractions were passed over an anticollagenase immunoaffinity column, collagenase was specifically bound, as demonstrated by the TCA and TCB degradation of type I collagen by the bound material. Gelatinolytic activity did not bind to the column. Furthermore, immunoprecipitation of 125I-labeled detergent extracts of tumor membranes yielded a single Mr 55,000 band consistent with the zymogen form of the connective tissue collagenase. These morphological and biochemical findings suggest that collagenase is a tightly associated component of the basal plasma membrane, where it occupies a strategic location for directional proteolysis during cell migration and invasion.
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PMID:Localization of collagenase at the basal plasma membrane of a human pancreatic carcinoma cell line. 217 14

1. Collagenase from bovine nasal hyaline cartilage was extracted with 1 and 3 M NaCl in Tris-CaCl2 buffer. 2. Two peaks of collagenase activity were revealed on DE52 ion exchange column, collagenase 1 and collagenase 2. 3. The apparent mol. wt of collagenase 1 and 2 as determined by SDS-PAGE were 68 and 43 kDa, respectively. 4. Both enzymes degrade native collagen type II into two characteristic products, TCA(3/4) and TCB(1/4), at 25 degrees C and pH 7.6. 5. Trypsin and aminophenylmercuric acetate were capable of increasing the collagenase 1 activity. 6. The two enzymes can be characterized as metalloproteinases since they were inhibited by EGTA and 1,10-phenanthroline. The use of proteinase inhibitors (N-ethylmaleimide, iodoacetic acid, phenylmethylsulphonyl fluoride, soybean trypsin inhibitor, pepstatin, dithiothreitol) showed that the enzymes do not contain serine, cysteine or aspartic acid in their active sites.
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PMID:Purification and properties of bovine nasal hyaline cartilage collagenase. 217 74

The binding of fn to collagen (type I) fibres has been found to resemble that of vWf in the following respects: Binding is rapid, specific, saturable, similar at 4 and 37 degrees C, and reduced by increasing ionic strength. Binding is not inhibited by native, monomeric collagen, suggesting a multivalent mechanism of interaction. Binding of fn occurs to a variety of collagen fragments (after their renaturation and polymerization), including, for example, the collagenase-derived TCA and TCB 3/4 and 1/4 molecular fragments and the peptides alpha 1(I)CB3, 6b, 7 and 8 obtained by cleavage with cyanogen bromide (CB), suggesting a wide distribution of binding sites on the native collagen molecule. As judged by the effect of heat-treatment, the native conformation of fn is required. Chemical modification indicates the involvement of arginyl residues in collagen and carboxyl groups in fn. However, fn and vWf did not compete with one another in binding to collagen, suggesting the participation of different collagen arginyl residues in the two interactions. Fn-binding differed from that of vWf in that the former was inhibited by denatured monomeric collagen (gelatin). Fn-binding was also inhibited by the fragment TCA in denatured form. The inhibitory activity was lost after chemical modification of arginyl residues in gelatin. Our results suggest that fn binding to collagen fibres and gelatin involves the same widely-distributed spectrum of binding sites.
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PMID:The interaction of fibronectin (fn) with native, polymeric collagen (collagen fibres): comparison with von Willebrand factor (vWf)-binding by collagen. 279 52

Rat skin type-I and type-III collagens were degraded by human fibroblast collagenase at a temperature below the 'melting' temperature for the two resulting fragments, namely the N-terminal three-fourths, TCA, and the C-terminal one-fourth, TCB. The specific cleavage of the collagen was confirmed by electrophoresis and determination of molecular length by electron microscopy. The two fragments were separated by gel filtration and the thermal stabilities of the isolated fragments were determined. For type-I collagen, the 'melting' temperatures of the two fragments were found to differ by only 0.5 degrees C and were 4.5-5.0 degrees C below that of the uncleaved molecule. The 'melting' temperatures of the uncleaved molecule and the N-terminal fragment were independent of the extent of N-terminal intramolecular cross-linking. For type-III collagen, the 'melting' temperatures of the fragments were found to differ by 1.3 degrees C. The small fragments of the two types of collagen 'melted' at the same temperature, whereas the large type-III fragment 'melted' at a slightly higher temperature than did the large type-I fragment. Reduction of the disulphide bonds located in the C-terminal type-III fragment did not affect the thermal stability of this fragment. The thermal stability of uncleaved type-III collagen was found to be variable, but the reason for this is not known at present.
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PMID:Thermal stability of human-fibroblast-collagenase-cleavage products of type-I and type-III collagens. 282 36


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