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 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 mineral and non-collagenous organic components of normal human femoral cortex were examined following powdering, demineralization with EDTA and digestion with bacterial collagenase. The protein, hexose, sialic acid and uronic acid contents of the matrix were determined. Neonatal bone had lower levels of mineral and calcium and higher levels of organic material and sialic acid than adult bone, suggesting increased glycoprotein content in neonatal bone. The soluble non-collagenous matrix of human femoral cortex was examined by gel filtration on Sephadex G100 and by ion-exchange chromatography on DEAE-cellulose. Four fractions were eluted off Sephadex-G100: a large molecular weight fraction, a shoulder on the descending portion of this, both of which contained sialic acid and two smaller molecular weight fractions. The material eluted off DEAE-cellulose was separated into 6 fractions which were similar to those found for beef and rabbit bone matrix. Human bone matrix appeared more resistant to collagenase digestion than beef bone, soluble collagen eluted later off DEAE-cellulose than beef bone; sialic acid gave 3 peaks: a major and two lesser ones. The sialic acid-containing material in the fifth fraction was probably bound to proteoglycan. Rabbit bone has 2 to 3 sialic acid peaks whereas beef bone has one, indicating species differences in cortical bone matrix.
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PMID:Chemical composition of human bone. 631 48

A highly unusual collagen was secreted by fibroblasts cultured from 150- and 270-d-old fetal calf nuchal ligaments. Purification revealed that this protein (which may be synthesized in a higher molecular weight form) was precipitated at unusually high concentrations of ammonium sulfate and was also eluted from DEAE-cellulose at greater salt concentrations than were types I and III procollagens. On SDS PAGE, the collagenous protein exhibited an Mr of approximately 12,750 that was not altered in the presence of reducing agent. The low molecular weight collagen (FCL-1) was sensitive to bacterial collagenase and had a [3H]glycine content comparable to that found in type I procollagen, although the [3H]Hyp to [3H]Pro ratio was 0.43. FCL-1 was not cleaved by human skin collagenase, mast cell protease, trypsin, Staphylococcal V8 protease, or proteinase K at 37 degrees C. The collagen was susceptible to trypsin, but not to V8 protease, only after heating at 80 degrees C for 30 min. Preliminary structural studies indicate that FCL-1 was resistant to cleavage by CNBr but exhibited limited proteolysis with pepsin. Both 150- and 270-d-old fibroblasts produced comparable levels of interstitial (types I and III) procollagens, which comprised approximately 70% of the total protein secreted into the culture medium. However, 270-d-old (term) fibroblasts secreted approximately 50% more FCL-1, as percent of total culture medium protein, in comparison to the cells from the earlier gestational stage. This collagen may therefore play a role in the development of the nuchal ligament.
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PMID:Fetal calf ligament fibroblasts in culture secrete a low molecular weight collagen with a unique resistance to proteolytic degradation. 631 46

Crude bacterial collagenase was chromatographed on DEAE-cellulose to yield three peaks with proteolytic activity: an arginine esterase (DEAE-1), gelatinase (DEAE-2) and a caseinolytic activity (DEAE-3). The arginine esterase and gelatinase activity fractions were slightly contaminated with each other but neither possessed caseinolytic activity; the caseinolytic fraction was devoid of arginine esterase and gelatinase activities. In addition, crude collagenase was fractionated by ZnII-affinity chromatography to produce a gelatinase peak (ZnII peak 1), which was free from arginine esterase and caseinolytic activities. The four fractions were compared to crude collagenase in their ability to liberate rat hepatocytes by using either liver slices or a standard perfusion technique. Compared to crude collagenase (0.05-0.1% w/v), which produced 70-80% liver digestion with approximately 80% cell viability, digestion with equivalent quantities of the isolated enzymic activities was relatively poor. Gelatinase activity (ZnII peak 1) was wholly ineffective and DEAE-1 and DEAE-2 each possessed only slight digestive properties. Hepatocyte liberation by the caseinolytic activity, DEAE-3, was partially successful (30-40% digestion, 25-30% viability) but only a portion of liver tissue was digested regardless of the quantity of DEAE-3 used. However, by mixing certain fractions before perfusion two gelatinase-dependent, cell-releasing mechanisms were identified: (a) DEAE-3 with ZnII peak 1 and (b) DEAE-1 mixed with either DEAE-2 or ZnII peak 1. Each system compared creditably with the digestive properties of an equivalent activity of crude collagenase. At present we are attempting to determine any differences between hepatocytes produced by the two enzymic mechanisms.
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PMID:The role of proteolytic enzymes derived from crude bacterial collagenase in the liberation of hepatocytes from rat liver. Identification of two cell-liberating mechanisms. 631 90

The organ culture of neonatal mouse calvaria produced both collagenase and collagenase inhibitor. The inhibitor was purified by a series of column chromatographies: DEAE-cellulose and CM-cellulose ion-exchange chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, and finally by Sephacryl S-200 gel filtration. The purified inhibitor migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular mass of 28,000. The inhibitor was purified 140-fold to a specific activity of 163 units/mg with a yield of 18% over the first step of the purification by DEAE-cellulose chromatography. The inhibitor stained positively for carbohydrate with periodic acid-Schiff's reagent indicating, in conjunction with its affinity to concanavalin A, that the inhibitor is a glycoprotein. In addition to mouse bone collagenase, this inhibitor also inhibited chick bone, rat bone, rabbit corneal, and human gingival collagenase, but did not inhibit bacterial collagenase.
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PMID:Mouse bone collagenase inhibitor: purification and partial characterization of the inhibitor from mouse calvaria cultures. 632 Jul 46

Digestion of the cuticle collagen from the annelid Nereis virens with Clostridium histolyticum collagenase yields a native, collagenase-resistant fragment (CCRF) of the molecule with an Mr of about 900,000 (Kimura and Tanzer, J. Biol. Chem. 252: 8018, 1977). We have produced 940 nm long, SLS-crystallites from the collagenase-resistant fragment; the SLS pattern matches a region at one end of the 2,400 nm SLS obtained from intact cuticle collagen. Upon denaturation, the fragment yields two subunits, CCRFA and CCRFB, which can be separated by SDS polyacrylamide gel electrophoresis or by chromatography on DEAE-cellulose; the subunits are in about a 2:1 ratio. The subunits have amino acid compositions which are similar to those of the original A and B chains, although the fragments have a higher content of acidic residues and a lower content of hydroxyl residues. Previous studies of the intact B chain have shown that there is about one methionine in the chain, and that it is located near the COOH terminus. The CCRFB subunit also contains about one methionine, indicating that CCRFB is probably derived from the COOH end of the intact molecule. Based on these composite data, we have provisionally defined the amino and carboxyl ends of the cuticle collagen SLS and provide additional evidence that the molecule is a heteropolymer with the formula A(B)2.
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PMID:Characterization of a large fragment from annelid cuticle collagen and its relationship to the intact molecule. 632 Oct 95

The purification and properties of an estradiol-sensitive hydrolytic activity from mouse uterus which fits several criteria for being an induced protein are described. The activity in the uteri of immature animals can be stimulated 2--4-fold by estradiol to that approaching the adult level. Stimulation is blocked by puromycin. The enzyme which we have designated hydrolase II, was purified approx. 400-fold to apparent homogeneity by chromatography on Affigel Blue, DEAE-cellulose and octyl-Sepharose. Hydrolase II is a single chain polypeptide with an estimated mol. wt = 65,000 daltons and has an N-terminal serine residue. A variety of N-blocked L-amino acid nitrophenyl esters are cleaved by the enzyme. Km's at pH 7.2 were all approx. 40 microns. Of substrates tested, phenylalanine nitrophenyl ester had the highest Vmax. Cbz-beta-alanine nitrophenyl ester, which is not a normal protease substrate was cleaved with a Km of 145 microM. The enzyme had no detectable activity against peptide nitroanilide substrates for trypsin-, chymotrypsin- or elastase-like enzymes. It is inhibited by ZPCK and DIFP but not by TLCK and Ala-Ala-Pro-Ala chloromethyl ketone, a potent inhibitor of elastase-like enzymes. Mouse plasma protein protease inhibitors were without effect as was SBTI. Our results rule out hydrolase II being a carnosinase, non-serine esterase, plasminogen activator, collagenase or collagenase activator and suggest that it is a chymotrypsin-like protease.
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PMID:Properties of an estrogen-induced hydrolytic enzyme from mouse uterus. 635 Jul 23

A low molecular weight fraction (Mr = 500-1500) was isolated by membrane and gel filtration from rat embryonic brain extract. This fraction was shown to stimulate collagen production in muscle cultures. Procollagen, intermediate collagenous proteins, and collagens of types I, III, and V were identified by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE- and Cm-cellulose chromatography and by selective degradation by pepsin, bacterial collagenase, and cyanogen bromide. Immunofluorescent staining with antisera to collagen types I, III, IV, and V of muscle cultures treated with the collagen-inducing factor revealed a large increase in staining compared to control cultures and a distinct pattern of staining on the surface of the treated myotubes.
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PMID:Characterization and localization of collagens synthesized by cultured muscle cells stimulated with collagen-inducing factor from embryonic brain extracts. 635 23

A proteinase inhibitor which has strong anti-collagenase activity was found in chicken egg white. The inhibitor (pI = 4.9) was purified by poly(ethylene glycol) (5.5-10%) precipitation and chromatography on Ultrogel AcA 34, DEAE-cellulose, and Sephacryl S-300. The final product was homogeneous on 5% polyacrylamide gel electrophoresis. Stoichiometric inhibition was observed with the inhibitor and rabbit synovial collagenase and thermolysin (1:1 molar ratio with thermolysin). The inhibitor ran on sodium dodecyl sulfate-gel electrophoresis with reduction as a single protein band of Mr = 165,000. The molecular weight of the native inhibitor was estimated to be 780,000 by sedimentation equilibrium centrifugation. Centrifugation analysis in 6 M guanidine hydrochloride and of the reduced sample gave M omega = 380,000 and M omega = 195,000, respectively, where M omega is the weight-average molecular weight determined by equilibrium ultra-centrifugation. The results indicated that the inhibitor molecule is a tetramer of identical subunits linked in pairs by disulfide bonds. Since the molecular weight and the quaternary structure of the inhibitor were similar to those of alpha 2-macroglobulin (alpha 2M) in plasma, chicken alpha 2M was isolated and compared with the inhibitor. The inhibitor was not sensitive to methylamine, whereas chicken alpha 2M was. No immunocross-reactivity was observed between the inhibitor and chicken alpha 2M. The NH2-terminal sequence of the egg white inhibitor is Lys-Glu-Pro-Glu-Pro-Gln-Tyr-Val-Leu-Met-Val-Pro-Ala. The sequence of chicken alpha 2M is Ser-Thr-Val-Thr-Glu-Pro-Gln-Tyr-Met-Val-Leu-Leu-Pro-Phe. Considerable homology was found between the two sequences and to the NH2-terminal sequence of human alpha 2M. Monospecific antibody raised against the egg white inhibitor was employed to examine the tissue distribution of the inhibitor. The inhibitor was found only in oviduct and egg white, but not in other tissues or serum of chickens.
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PMID:Ovostatin: a novel proteinase inhibitor from chicken egg white. I. Purification, physicochemical properties, and tissue distribution of ovostatin. 640 74

Employing various radioactive amino acids, protein biosynthesis by guinea pig lung fibroblasts has been studied in monolayer culture. The cells were shown to synthesize and secrete several collagenous and noncollagenous proteins. The biosynthesized macromolecules have been characterized employing molecular sieve and ion exchange chromatography (DEAE-cellulose column), SDS-polyacrylamide gel electrophoresis, and enzymatic digestion. It was found that the guinea pig lung fibroblasts synthesized mainly type I procollagen molecules which appeared in the media in various stages of cleavage. The intact procollagen molecules and the various processed products were identified by their electrophoretic migration in SDS-polyacrylamide slab gels and further characterized by 1) elution position on SDS-agarose columns under reducing conditions; 2) hydroxyproline content; 3) change in elution position on SDS-agarose after pepsin digestion; 4) chromatographic separation on DEAE-cellulose columns and electrophoretic mobility of the various peaks; and 5) susceptibility to collagenase digestion. Slab gel electrophoresis under non-reducing conditions of aliquots of culture media after limited pepsin digestion indicated the presence of disulfide-bonded type III collagen alpha-chains. In addition, the lung fibroblasts synthesized a non-collagenous protein composed of two disulfide-bonded chains. The individual chains appeared to have a molecular weight of approximately 220,000 when examined under reducing conditions. This protein has been identified as fibronectin based on its molecular weight, its resistance to collagenase attack, its susceptibility to protease digestion and its precipitation with specific antisera to fibronectin.
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PMID:Biochemical characterization of collagens and of a non-collagenous protein synthesized by guinea pig lung fibroblasts in culture. 667 79


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