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 metalloproteinase similar or identical to stromelysin was shown to co-purify with interstitial collagenase from the rat mammary carcinoma cell line, BC1. The mixture of BC1 metalloproteinase and collagenase degraded casein, gelatin, fibronectin, fibrinogen, laminin, proteoglycan and type IV collagen, in addition to types I and II collagen. Using SDS-PAGE and zymography, the Mr of both enzymes was 51.10(3). During storage, the 51.10(3) protein converted to fragments of Mr 34.10(3) and 24.10(3), and isoelectric points of 4.6-5.3 and 5.7-6.0, respectively. The fragments were separated from the intact (Mr 51.10(3) enzymes by DEAE-Sepharose chromatography, but intact metalloproteinase and collagenase activities resisted separation by a range of chromatographic methods. The Mr 34.10(3) fragment retained the proteinolytic activities of the intact enzymes, excepting collagenase cleavage of collagen types I and II. The Mr 24.10(3) fragment had no proteinolytic activity, showed an increase in Mr of 6.10(3) upon reduction, in common with the intact enzymes, and also had similar chromatographic properties to the intact enzymes. The data presented are consistent with a pattern of breakdown which is common to both collagenase and the metalloproteinase, and suggest that both enzymes are comprised of two protein domains.
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PMID:Identification of a metalloproteinase co-purifying with rat tumour collagenase and the characteristics of fragments of both enzymes. 253 40

Pump-1 cDNA has recently been isolated by screening a human tumor cDNA library with a transin (rat stromelysin) probe under low-stringency hybridization conditions. The cDNA codes for a potential protein with significant sequence similarity to the metalloproteinases collagenase and stromelysin, but which lacks the hemopexin-like domain characteristic of these enzymes. Expression of pump-1 cDNA in cos cells using an expression vector leads to secretion of a protein of Mr 28,000 with latent, organomercurial-activatable proteinase activity. Cos cells transfected with a partial pump-1 cDNA in the vector pPROTA secrete a fusion protein between the IgG-binding domains of staphylococcal protein A and pump-1. The fusion protein binds to IgG-Sepharose, and the bound fusion protein undergoes apparent autocleavage in the presence of 4-aminophenylmercuric acetate with elution of active pump-1 species of Mr 21,000 and 19,000. Active pump-1 degrades casein, gelatins of types I, III, IV, and V, and fibronectin and can activate collagenase. Active pump-1 is inhibited by EDTA, 1,10-phenanthroline, and the tissue inhibitor of metalloproteinases. These results show that, despite the absence of a hemopexin-like domain, pump-1 is a latent secreted metalloproteinase. Postpartum rat uteri contain elevated levels of rat pump-1 mRNA. On the basis of this observation, its size, and its substrate specificity, we suggest that pump-1 might correspond to a previously described uterine metalloproteinase, matrix metalloproteinase 7.
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PMID:Pump-1 cDNA codes for a protein with characteristics similar to those of classical collagenase family members. 255 50

On purification, human fibroblast collagenase breaks down into two major forms (Mr22,000 and Mr 27,000) and one minor form (Mr 25,000). The most likely mechanism is autolysis, although the presence of contaminating enzymes cannot be excluded. From N-terminal sequencing studies, the 22,000-Mr fragment contains the active site; differential binding to concanavalin A shows the 25,000-Mr fragment is a glycosylated form of the 22,000-Mr fragment. These low-Mr forms can be separated by Zn2+-chelate chromatography. An activity profile of this column, combined with data from substrate gels, indicates no activity against collagen in the 22,000-Mr and 25,000-Mr forms, but rather, activity casein and gelatin. The 27,000-Mr form has no activity. The 22,000/25,000-Mr form can act as an activator for collagenase in a similar way to that reported for stromelysin. The activity of the 22,000/25,000-Mr form is not inhibited by the tissue inhibitor of metalloproteinases (TIMP). The 27,000-Mr C-terminal part of the collagenase molecule therefore appears to be important in maintaining the substrate-specificity of the enzyme, and also plays a role in the binding of TIMP.
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PMID:Fragments of human fibroblast collagenase. Purification and characterization. 255 22

The secretion of elevated levels of proteinases is considered to be a distinct property of most transformed cells. The cellular and secreted levels of plasminogen activators and collagenases have been examined in the nonmalignant human osteosarcoma (HOS), the malignant Kirsten murine sarcoma virus transformed (KHOS/NP), the temperature sensitive revertant of virus transformed HOS (KHOS-240S) and N-methyl-N'-nitro-N-nitrosoguanidine transformed HOS (MNNG/HOS) clones. Virus and MNNG transformed clones exhibit 100- and 7-fold higher cellular and and 270- and 30-fold higher extracellular plasminogen activator (PA) activity as compared with untransformed HOS controls. The cellular PA activity of the revertant clone is similar to but the secreted level is slightly higher than the HOS controls. SDS-PAGE in the presence of casein and plasminogen is consistent with the major PA species of urinary type (u-PA) and with the absence of PA inhibitor in the parent and revertant clones. The cellular levels of active collagenase are low in all the clones. However, on activation by trypsin, the two active collagenase bands of similar intensity are observed for all the lines in SDS-PAGE in the presence of gelatin. While there appears to be some elevation of secreted collagenase prior to trypsin activation, the activated collagenases appear to have the same size and activity in all of the clones.
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PMID:Synthesis and secretion of plasminogen activators and collagenases in human cells transformed by Kirsten murine sarcoma virus and N-methyl-N'-nitro-N-nitrosoguanidine. 256 62

Commercial Collagenase* prepared from Clostridium histolyticum is widely used in isolation of pancreatic islets. It is known that the enzyme is very impure and that there are substantial variations in effectiveness between batches. Our studies suggest that one of the impurities of importance in islet isolation is a protease that has not been very well characterized. Comparison of two batches of enzyme, one of which was known to give good yields of islets and the other poor yields, showed that they had very similar activity against collagen (measured by digestion of insoluble collagen followed by assay of soluble products with ninhydrin) but substantially different activities against azocasein as measured by optical density increase (measured by release of dye). Eighteen batches of Collagenase were examined for efficiency in islet isolation, and the yields obtained correlated with manufacturer's data of activity against casein. The data show that low caseinase activity is associated with performance in islet isolation (r = .5 after adjusting for collagenase activity). The effect of supplementing a batch of collagenase, known to be poor in isolating islets, with proteolytic enzymes was investigated. Trypsin and papain had apparently no effect, but dispase significantly increased yield. Dispase alone failed to digest pancreas. Size-exclusion high-performance liquid chromatography identified a peak associated with high protease activity and efficiency in islet isolation, having an Mr of approximately 30,000, compared to 78,000 for collagenase. The protease, like collagenase, is inhibited by EDTA. Increased Ca2+ and Mg2+ (up to 10 mM) did not affect activity. Both the protease and collagenase are stable under normal use but are inactivated by heating at 56 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protease activity in pancreatic islet isolation by enzymatic digestion. 264 34

A. niger LCF 9 synthesizes a new aspergillopeptidase of potential interest in therapeutics. The properties and operating range of the enzyme were determined. It is a semi-alkaline aspergillopeptidase (EC 3.4.23.4) with one endopeptidase activity. Its pI is 4.10, its molecular weight is 21000 Da and its A1%(1 cm) at 280 nm is 9.75. It rapidly hydrolyzes casein and hemoglobin. Its optimal pH is 7.8 and optimal temperature is 45 degrees C. It is thermally labile above 40 degrees C but can be stabilized by adding calcium ions. It is inhibited by phenylmethylsulfonyl fluoride (PMSF), by ethylenediaminetetraacetic acid (EDTA) and by certain metals ions, e.g. copper, manganese and cobalt ions. It has no dipeptidase or tripeptidase activity and its esterase activity is weak. It has a high collagenase activity and is to our knowledge the only aspergillopeptidase that is active towards benzoyl-arginine p-nitroanilide (BAPNA).
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PMID:Properties of a new alkaline proteinase from Aspergillus niger. 269 84

Many of the common substrate digestion assays have the disadvantage of being tedious and expensive, thus aggravating serial determinations of collagenase activity. In the assay presented here, the proteolytic reaction proceeds on a sheet of chromatographic paper bearing spots of dried gelatin or collagen solutions. After application of the enzyme sample, the sheets are incubated in a water-saturated atmosphere for 45 min and the reaction products are visualized by spraying with chromatographic, ninhydrin-containing peptide reagents. By its specific yellow staining, the activity of bacterial collagenase can be discriminated from the gray, brown, red, or violet spots which are produced by eight tested noncollagenolytic proteases. The detection limit for collagenase activity reaches down to 0.002 U in a sample volume of 1-2 microliters dropped onto a spot of 25 microliters gelatin solution (3%), thus corresponding with the sensitivity of a conventional radioassay. The sensitivity for noncollagenolytic proteases is better than 2 ng per sample, herewith exceeding a common casein-agar diffusion test. The method is also evaluated for native collagen. Within 1.5 h, 100 samples can be assayed. An example for the application in a collagenase purification step (ion exchange chromatography) is also presented.
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PMID:A rapid and sensitive microassay for bacterial collagenase and other proteolytic activities on collagenous substrates. 284 9

Hedgehog plasma was separated by gel filtration on Sephacryl S-200, the fractions resolved by electrophoresis and the electrophoretograms characterized for collagenase, papain and plasmin inhibiting activities with the high mol. wt substrate casein. The three inhibitors previously identified as alpha 2-, alpha 2-beta- and beta-macroglobulins were found to inhibit all three proteases. These were the only collagenase inhibitors found in plasma. Hedgehog alpha 2-chymotrypsin inhibitor and beta-protease inhibitor were both found to also inhibit papain. Three new inhibitors specific for papain (gamma-, alpha 2- and alpha 1-cysteine protease inhibitors) and one for plasmin (alpha 2-antiplasmin) were also found, bringing the number of protease inhibitors in hedgehog plasma to 14. Immunological cross-reactivity as studied by immunoelectrophoresis showed homology between hedgehog alpha 2-macroglobulin and rat murinoglobulin I and between hedgehog alpha 2-antithrombin and rat antithrombin III.
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PMID:Further studies of plasma protease inhibitors in the hedgehog, Erinaceus europaeus; collagenase, papain and plasmin inhibitors. 288 40

Rabbit synovial fibroblasts induced to undergo a specific switch in gene expression by agents that alter cell morphology secreted the neutral proteinase precursor procollagenase (apparent Mr of 53,000 and 57,000). A major Mr = 51,000 polypeptide that was always induced coordinately with procollagenase has now been identified as the proenzyme form of a metal-dependent proteinase active at neutral pH. We have named this proteinase stromelysin. Prostromelysin and procollagenase were the most prominent [35S]methionine-labeled secreted proteins of the induced fibroblasts. By the use of casein degradation as an assay for enzyme activity, stromelysin was isolated with high yield from the conditioned culture medium of 12-O-tetradecanoylphorbol 13-acetate-treated fibroblasts and migrated as an active form of Mr = 21,000 that was immunologically identical to the proteoglycan-degrading proteinase purified from rabbit bone. Immunoglobulin G from antiserum raised to purified rabbit bone proteoglycanase immunoprecipitated the Mr = 51,000 proenzyme form from conditioned medium of induced rabbit cells and also immunoprecipitated an Mr = 55,000 polypeptide from induced human fibroblasts. When rabbit prostromelysin was activated by trypsin or 4-aminophenylmercuric acetate, the proenzyme was converted to an active form of Mr = 41,000. During the course of the purification, prostromelysin was converted to an additional activatable form of Mr = 35,000 and additional active forms of Mr = 21,000-25,000, which had related peptide maps distinct from collagenase. All of these forms were immunologically cross-reactive. Purified stromelysin degraded casein, cartilage proteoglycans, fibronectin, alpha 1-proteinase inhibitor, and immunoglobulin G2a and had limited activity on laminin, elastin, type IV collagen, and gelatin, but did not degrade type I collagen. Stromelysin was inhibited by EDTA, 1,10-phenanthroline, and the specific glycoprotein tissue inhibitor of metalloproteinases isolated from human amniotic fluid and was therefore classified as a metalloproteinase.
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PMID:Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. Biosynthesis, isolation, characterization, and substrates. 299 74

We have isolated an activator of collagenase from medium conditioned with articular cartilage. The activity is contained in an acidic protein appearing as a doublet band of Mr 57,000 and 56,000 on sodium dodecyl sulfate polyacrylamide gels. Both components of the doublet have identical isoelectric points as demonstrated by gel electrophoresis. Purified synovial collagenase has a high dependence on the presence of this factor for activity. Other known activators of latent proteolytic enzymes such as trypsin and mercurials will stimulate collagenase but only if activator protein is present. The activator protein is itself a latent metalloprotease because in the presence of p-aminophenylmercuric acetate and calcium it will digest casein. The caseinase activity and collagenase activation activity have identical heat inactivation profiles, both being stable to a temperature of 60 degrees C and partially inactivated at 80 degrees C. The synthesis of the activator is localized in the superficial zone of articular cartilage.
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PMID:Purification and characterization of collagenase activator protein synthesized by articular cartilage. 302 50


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