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 proteins of the cuticle of adult Ascaris lumbricoides suum were characterized with respect to heterogeneity, glycosylation, and susceptibility to collagenase. Pepsin digestion of intact cuticles was used to determine the extent of stable triple-helical structures of the cuticular components. With sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, it was shown that treatment of purified cuticles with beta-mercaptoethanol released three components (99, 90, and 68 kDa) which comprise 95% of the total solubilized material. The remaining fraction consists of at least four components (16, 28, 154, and 173 kDa). Periodic acid-Schiff staining showed that the only glycoprotein was the 173-kDa component. All cuticular components, except the 173-kDa protein, were degraded by bacterial collagenase. Pepsin digestion of intact cuticles for 24 hr at 4 C produced, after reduction, a 95-kDa fragment; by 96 hr, four fragments (95, 90, 83, and 77 kDa) were evident. When the 96-hr pepsin digest was treated with fresh pepsin, the 77-kDa fragment became the major constituent. With agarose gel electrophoresis, analysis of non-reduced, pepsin-released material revealed intact aggregates that were greater than 2 X 10(3) kDa. The enzyme digestion studies indicate that, with the exception of the 173-kDa component, each cuticular protein contains collagenous domains and that, within the cuticle, the longest contiguous collagen chain in a triple-helical conformation has a uniform molecular size of 77 kDa.
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PMID:Ascaris lumbricoides: characterization of the collagenous components of the adult cuticle. 298 39

We have previously shown that G8-1, a murine clonal skeletal-muscle cell line, produces a substrate-attached extracellular matrix [Beach, Burton, Hendricks & Festoff (1982) J. Biol. Chem. 257, 11437-11442]. To examine further the expression of extracellular-matrix proteins by muscle cells, we have analysed the collagenous proteins secreted by G8-1 myoblasts. We have found that collagens and/or procollagens, corresponding to genetic types I, III and IV (and possibly V), are produced and secreted by G8-1 myoblasts. The major secreted collagenous polypeptides were identified as alpha 1 type I and its precursors by using pulse-chase studies, pepsin and collagenase digestions and CNBr fragmentation. The presence of lesser amounts of the other collagens was determined by immunoprecipitation. These results demonstrate that clonal skeletal-muscle cells, in the absence of fibroblasts and an exogenous collagen substrate, are able to synthesize and secrete several extracellular-matrix collagenous proteins in proportions similar to those which are commonly found in muscle tissue and mixed cultures of muscle cells and fibroblasts.
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PMID:Extracellular-matrix synthesis by skeletal muscle in culture. Major secreted collagenous proteins of clonal myoblasts. 298 79

CL glycoprotein (CLGP), the 140,000-dalton collagenous glycoprotein, has been isolated from fetal bovine aorta and nuchal ligament, in milligram amounts in its reduced and alkylated form, using a multistage procedure. This material exhibited a characteristic amino acid composition with a consistent ratio of hydroxylysine to hydroxyproline (approximately 1:1). Digestion of CLGP with bacterial collagenase yielded three discrete noncollagenous fragments. Monospecific anti-CLGP antiserum exhibited strong cross-reactivity with the pepsin-resistant polypeptides of type VI collagen. CLGP was also prepared in the unreduced disulfide-bonded form and in a partially reduced form, using brief treatment with cysteine. On treatment with pepsin these preparations yielded resistant peptides corresponding in size to the longer and shorter forms, respectively, of type VI collagen. A slightly larger, soluble form of CLGP (Mr = 150,000) was detected in the media from cultures of aortic smooth muscle cells and nuchal ligament fibroblasts. The evidence indicates that CLGP is the native form in which type VI collagen is present in the tissues and that it consists of three structurally distinct polypeptide chains, each about 140 kDa in mass, which are disulfide bonded into a triple-helical molecule. The CLGP molecules appear to be present in the tissues as dimers and larger aggregates, stabilized by intermolecular disulfide bonding. The distribution of type VI collagen will thus be as described in our earlier immunofluorescence studies with anti-CLGP antiserum (Gibson, M.A., and Cleary, E.G. (1983) Collagen Relat. Res. 3, 469-488).
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PMID:CL glycoprotein is the tissue form of type VI collagen. 299 3

A structural defect in the alpha 2(I) chain of type I collagen was characterized in a new case of the Ehlers-Danlos syndrome type VII. The patient's skin, fascia, and bone collagens all showed an abnormal additional chain, pN-alpha 2(I)s, running slower than the alpha 2(I) chain on electrophoresis. The extension was shown to be on the amino-terminal fragment of pN-alpha (I)s by cleavage with human collagenase, but pepsin was unable to convert pN-alpha 2(I)s to alpha 2(I). Skin collagen was 4-fold more extractable and contained fewer beta-dimers and a lower concentration of cross-linking amino acids than control skin collagen. Electron micrographs of both dermis and bone showed markedly irregular ragged outlines of the collagen fibrils in cross-section, although the patient had no clinical signs of bone disease. Procollagen secreted by her skin fibroblasts in culture showed equal amounts of the normal and abnormal alpha 2(I) chains on pepsin digestion. Before pepsin, the pN-alpha 2(I) component ran as a doublet on electrophoresis; pepsin removed only the normal slower chain. The suspected deletion in pN-alpha 2(I)s was traced by CNBr peptide analysis to the N-propeptide fragment, which behaved on electrophoresis about 15-20 residues smaller than that from the normal pN-alpha 2(I) chain. The simplest genetic explanation is a spontaneous heterozygote in which one normal and one abnormal allele for the pro-alpha 2(I) gene are expressed, the protein defect being a deletion of the junction domain that spans the N-propeptidase cleavage site and the N-telopeptide cross-linking sequence.
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PMID:A heterozygous collagen defect in a variant of the Ehlers-Danlos syndrome type VII. Evidence for a deleted amino-telopeptide domain in the pro-alpha 2(I) chain. 299 7

Confluent cultures of normal human skin fibroblasts were labelled overnight with [35S]sulphate, and the incorporation of the isotope into type III procollagen, secreted into the medium, was verified by radioimmunoassay and immunoprecipitation after removing the heavily sulphated proteoglycans by anion-exchange chromatography. Type III procollagen and its pro and pN alpha chains were visualized in fluorographs of the immunoprecipitates. The labelled procollagen could be isolated by a combination of ion-exchange chromatography and gel filtration and was found to contain tyrosine O-sulphate, which was identified by thin-layer electrophoresis after Ba(OH)2 hydrolysis. The regions sulphated in the type III procollagen molecule were susceptible to pepsin digestion. Digestion with purified bacterial collagenase at +37 degrees C produced a labelled fragment that was recognized by antibodies against the aminoterminal propeptide of type III procollagen, indicating that the sulphated tyrosine residues are located either in this propeptide or in the non-helical telopeptide region of the type III collagen molecule proper. Sulphation of tyrosine residues is a new post-translational modification in procollagen, which could be involved in the regulation of the processing of type III procollagen into collagen and thus affect the formation of collagen fibres.
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PMID:Incorporation of sulphate into type III procollagen by cultured human fibroblasts. Identification of tyrosine O-sulphate. 300 90

Tubular basement membrane (TBM) was prepared from normal human kidneys and solubilized with various enzymes. Collagenase digestion released antigenic moieties from the TBM. All four anti-TBM antibodies we studied, three from patients with idiopathic tubulo-interstitial nephritis (TIN) and one from a renal allograft recipient, distinctively reacted with collagenase-digested (CD) TBM during enzyme-linked immunoassay and could discriminate among sera of normal controls or of other nephritis patients, including anti-glomerular basement membrane (anti-GBM) nephritis. When digested with pronase, trypsin, or pepsin, antigenicity of the TBM decreased. We studied the TBM antigens with immunoprecipitation and immunoblotting. After incubation of radio-iodinated CDTBM with anti-TBM sera, immunoprecipitates were identified by single-dimension SDS polyacrylamide gel electrophoresis or two-dimension gel electrophoresis, followed by autoradiography. All four antibodies had identical results on immunoprecipitation; under nonreducing conditions, they gave two protein bands with m.w. of 54,000 and 48,000 and with pI 7.0 to 8.0 and 6.5 to 7.0. Electrophoresis performed under reducing conditions disclosed only one band at the m.w. of 48,000 and pI of 6.5, suggesting that the 54-kDa component is composed of peptides linked by interchain disulfide bonds. Immunoblot analysis showed that the anti-TBM antibodies were heterogeneous; three antibodies from the idiopathic TIN patients reacted with the 54-kDa band, but the one from the renal allograft recipient reacted with neither band. This finding suggests that there are two antigenic determinants on the 54-kDa component. One such determinant that was resistant to denaturation with SDS was detected by the first three antibodies, and the other that was sensitive to such denaturation bound to the last antibody. The 48-kDa component seemed not to be immunoreactive after incubation with SDS. We studied TBM antigens reactive with anti-GBM antibodies. By immunoblotting, all four sera from patients with anti-GBM nephritis stained TBM proteins of 45 to 50 kDa and 25 to 27 kDa at pH 8.0 to 9.0; this was similar to the staining pattern of CDGBM with the same sera, but the highly cationic (pH greater than 9.0) components were specifically detected in the CDGBM. By inhibition ELISA, the binding of the anti-GBM sera to denatured CDTBM decreased with preincubation of the sera with CDGBM, suggesting that the anti-GBM antibodies recognize the same epitope(s) on the GBM and the TBM.
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PMID:Characterization of tubular basement membrane antigens in human kidney. 300 98

Adult bovine aortic tissue was homogenized in a neutral phosphate buffer containing proteinase inhibitors. The insoluble residue was rehomogenized in Tris-buffered 6 mol/L guanidinium chloride (pH 7.4). An insoluble fibrillar protein, floating above the main pellet after recentrifugation, was harvested. This material agglutinated washed fixed human platelets in the presence of either normal human plasma or purified von Willebrand factor (vWF). No such reaction was seen when either buffer or plasma from patients with severe von Willebrand's disease was added instead. The extent of platelet agglutination was measured photometrically, similarly to the ristocetin cofactor assay. The agglutination reaction was strongest at neutral pH and was impaired after either addition of EDTA or previous digestion of the fibrillar material by collagenase or pepsin. By light microscopy platelets were seen to adhere onto isolated fibers. Amino acid composition, subunit polypeptides, substrate properties, and interaction with fibronectin of this fibrillar protein were comparable to those of collagen. Therefore, we tentatively denote the induction of platelet agglutination by vWF protein in the described test system as "vWF-collagen cofactor" activity. Comparison of this activity in 65 plasma samples, containing various concentrations of vWF, with ristocetin cofactor activity showed good correlation between results obtained in both tests (r = 0.91).
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PMID:Von Willebrand factor-dependent agglutination of washed fixed human platelets by insoluble collagen isolated from bovine aorta. 300 53

It has recently been shown that patients with IgA nephropathy have circulating IgA antibodies against extracts of human glomerular basement membrane. The present study extends those observations and demonstrates by using ELISA and immunoblotting techniques that patients with IgA nephropathy have circulating IgA antibodies against collagen IV alpha chains. The antigenicity of the alpha chains could be destroyed by digestion with collagenase, which indicates that the antigenic site(s) is located on the triple helical part of collagen IV. Furthermore, it was shown by inhibition tests that the IgA antibodies are directed against epitopes also present in collagen I and II isolated after pepsin digestion.
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PMID:Patients with IgA nephropathy have circulating anti-basement membrane antibodies reacting with structures common to collagen I, II, and IV. 301 44

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of pepsin-solubilized collagens from post-burn granulation tissues revealed that type V collagen consisted of 3 alpha chains: alpha 1(V), alpha 2(V), and alpha 3(V). The mean value (0.12 +/- 0.01 SD) of the type V/type I ratio in the granulation tissues was significantly higher (p less than 0.001) than that (0.03 +/- 0.01 SD) of the ratio in normal skin. The average ratio of alpha 1(V):alpha 2(V):alpha 3(V) of type V collagen purified from the granulation tissues was determined to be about 5:3:1. SDS-polyacrylamide gel electrophoresis patterns of 3 alpha chains were not affected in the presence or absence of 2-mercaptoethanol. Purified type V collagen was degraded by bacterial collagenase, but remained intact after tadpole collagenase digestion, in contrast to type I and type III collagens. Amino acid analyses of each alpha chain separated on SDS-gel electrophoresis of type V collagen revealed that all 3 alpha chains of type V collagen were poor in alanine, rich in hydroxylysine, and had high ratios of hydroxylysine/lysine, which are typical features of type V collagen. The purified type V collagen was further fractionated by ammonium sulfate into 2 molecular species, [alpha 1(V)]2 alpha 2(V) and alpha 1(V)alpha 2(V)alpha 3(V). Our data demonstrate that type V collagen in preparations from human post-burn granulation tissues consists of 3 alpha chains and can be resolved into 2 distinct heterotrimers.
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PMID:Isolation and characterization of type V collagen from human post-burn granulation tissues. 302 Jan 32

Normal adult rabbit corneas were digested with 5% pepsin and their collagens extracted with acetic acid. Collagen extracts were fractionated by differential salt precipitation. The 2.5 M NaCl fraction was then redissolved with tris buffer and precipitated with sodium acetate. The precipitate contained a high-molecular-weight disulfide-bonded aggregate which, upon reduction with mercaptoethanol, was converted into three distinct polypeptides having molecular weights between 45 and 66 Kd. These physical characteristics, together with the susceptibility of these polypeptides to collagenase and their amino acid composition, identified the high molecular weight aggregate as type VI collagen. Corneas from neonate rabbits and adult corneas containing 2-week-old scars were organ cultured in the presence of [14C] glycine to incorporate radiolabel into collagen. Tissues were digested with 0.02% pepsin and their collagens extracted with formic acid. The total radioactivity of the extracts and tissue residues was determined before the collagens were separated by SDS-polyacrylamide slab gel electrophoresis. Radioactive collagen polypeptides bands were then stained with Coomassie blue, processed for fluorography, and analyzed by densitometry. The results show that: (1) type VI collagen is synthesized by neonate corneas and healing adult corneas; (2) it is not readily solubilized from either corneal tissue by 0.02% pepsin digestion and formic acid extraction; and (3) the proportion of type VI collagen deposited in scar tissue is markedly lower than that found in neonate corneas.
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PMID:Heterogeneity of collagens in rabbit cornea: type VI collagen. 313 Mar 20


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