Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Digestion of human foreskin with collagenase and hyaluronidase disperses approximately 3.4 X 10(7) nucleated cells per gram of tissue, of which mast cells constitute 4.7%. These may be purified to 80% by use of density gradient centrifugation. The majority of mast cells (79%) measured between 9 and 13 micron in diameter, and the mean histamine content was 4.6 pg/cell. Viability was demonstrated by trypan blue exclusion by 93% of the cells and the low spontaneous histamine secretion of less than 7% in functional studies. Anti-IgE released up to 17.5% of cell-associated histamine within 5 to 7 min. Calcium ionophore-induced release was optimal with 0.3 microM A23187 when 28.6% histamine was released. Unlike human lung mast cells, skin mast cells released histamine in response to compound 48/80 and poly-L-lysine. This release, which was complete within 20 sec, was totally dependent on intact glycolysis and oxidative phosphorylation and partially dependent on extracellular calcium. The same characteristics were observed with secretion induced by substance P and morphine. The weak activity of eledoisin and physalaemin suggests that the substance P receptor, like that of the rat mast cell, is not of the classical types described for smooth muscle. Morphine-induced secretion was partially blocked by naloxone in a manner not compatible with competitive antagonism at a classical opioid receptor. The sensitivity of skin mast cells to nonimmunologic stimulation clearly distinguishes them from mast cells of the lung and lymphoid tissues and provides evidence of functional heterogeneity within human mast cells.
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PMID:Human skin mast cells: their dispersion, purification, and secretory characterization. 243 32

A new type of collageneous structure, tentatively named 7-S collagen, was isolated from a mouse tumor basement membrane, mouse and human placenta, bovine lens capsule and human kidney. The protein was solubilized from the tissues by limited digestion with pepsin or trypsin and could easily be separated from other collageneous protein because of its resistance towards further degradation by bacterial collagenase at 20 degrees C. 7-S collagen showed an amino acid composition typical of basement membrane collagen and contained 22% carbohydrate mainly as glucosyl-galactosyl bound to hydroxylysine but also some mannose and glucosamine. Ultracentrifugal analysis demonstrated that the proteins were homogeneous with a sedimentation coefficient of about 7.2 S and with a molecular weight of about 360,000 both in phosphate buffer pH 7 and 6 M guanidine. The peptide was triple helical as shown by circular dichroism and exhibited a biphasic melting profile indicating two conformationally distinct domains with tm = 48 degrees C and 70 degrees C. The more stable domain could be isolated as an homogeneous fragment (Mr = 225,000) after a second digestion with collagenase at 37 degrees C. This fragment contained all the disulfide bonds (42 Cys/1,000 residues) of the original molecule. Electron microscopy showed a rod-like structure in agreement with the hydrodynamic properties of 7-S collagen. The dimensions of these peptides were 3 X 95 nm (long form) and 2.4 X 40-50 nm (short form). Complete reduction of 7-S collagen under denaturing conditions produced several polypeptide chains in the molecular weight range of 27,000-153,000 which differ from each other by Mr increments 25,000-27,000. Separation of the chains on agarose did not reveal any simple stoichiometric relationship indicating that some chains are either cross-linked or represent fragments produced during proteolytic treatments. Complete reduction of 7-S collagen under non-denaturing conditions lowered the thermal transiton of the triple helix to 48 degrees C but did not change its molecular weight except when exposed to dissociating solvents. 7-S collagens were potent immunogens and could be characterized by radioimmunoassays. Antigenicity was slightly reduced by reduction and denaturation while collagenase at 37 degrees C produced a larger decrease. Proteins obtained from various sources showed distinct immunological relationships although interspecies differences in affinity exist. No or only little cross-reaction was observed with type IV and V collagens and some further fragments of basement membrane collagen. The data indicate that 7-S collagen is a unique component of basement membranes which shows a more compact and stable structure than other collageneous proteins.
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PMID:7-S collagen: characterization of an unusual basement membrane structure. 625 Aug 29

The coding region for human neutrophil short form procollagenase lacking the hemopexin like domain coding region was amplified by polymerase chain reaction. Recombinant short form procollagenase was expressed in E. coli and purified in a three step procedure. Renaturation of this proenzyme was carried out by an effective new method using Q-Sepharose chromatography. Treatment of short form procollagenase with mercurials resulted in active short form collagenase M(r) 21,000 and an intermediate product of M(r) 23,000. These two products were separated by hydroxamate affinity chromatography. The active, short form collagenase M(r) 21,000 is stable. Despite full proteolytic activity, it lacks type I collagen substrate specificity and forms the basis for crystallisation experiments.
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PMID:The recombinant catalytic domain of human neutrophil collagenase lacks type I collagen substrate specificity. 846 Sep 92

The relationship of enzyme structure to substrate specificity for the matrix metalloproteinases interstitial collagenase and stromelysin-2 has been investigated by analysis of the cleavage specificity of recombinant human collagenase-stromelysin-2 hybrid proteins and C terminally truncated collagenase and stromelysin-2. Two series of chimeric proteins were devised by progressive substitution of exon-encoded domains. The recombinant proteins were expressed in COS-7 cells as protein A-fusion proteins and purified on an IgG affinity matrix. Treatment with 4-amino-phenylmercuric acetate released active metalloproteinase of the sizes predicted for the chimeric proteins. Active forms of both the chimeric protein series and the short form enzymes expressed both casein- and gelatin-degrading activities. Like stromelysin, the catalytic activity of stromelysin-2 was contained in the N-terminal domain (encoded by exons 1-5) and was apparently independent of the C-terminal domain (encoded by exons 6-10). Only full-length collagenase displayed a triple helicase (collagenolytic) activity; no combination of N- or C-terminal collagenase domains fused with stromelysin-2 domains had such activity. This suggests that the triple helicase activity is a composite of elements derived from both halves of the collagenase molecule. C terminally truncated collagenase (exons 1-5) and a hybrid of collagenase exons 1-5 and stromelysin-2 exons 6-10 cleaved denatured type I collagen (gelatin) to generate diagnostic peptides in gelatin fingerprint assays. When exon 5 (the exon encoding the zinc-binding domain) was derived from stromelysin-2, the enzyme specificity in the fingerprint assay changed to that of native stromelysin-2. In contrast, when exon 5 was derived from collagenase, the specificity reflected that of the parent enzyme. Our data also suggest that mismatching of exons 2 and 5 destabilizes the enzyme, presumably by altering the geometry of the propeptide-zinc-binding site interaction. We conclude that the loss of triple helicase collagenolytic activity is not accompanied by a shift to the broad specificity characteristic of stromelysin. Rather, the zinc-binding domain confers a distinct cleavage specificity on each metalloproteinase.
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PMID:Role of zinc-binding- and hemopexin domain-encoded sequences in the substrate specificity of collagenase and stromelysin-2 as revealed by chimeric proteins. 846 59

Collagen IX, a structural component of the extracellular matrix of connective tissues, is synthesized as long and short forms which contain or lack, respectively, a 27 kDa non-collagenous (NC) 4 domain at the N-terminus of the alpha 1(IX) chain of the molecule. The long form occurs in cartilage and developing cornea, but not in vitreous, suggesting a specialized function for the NC4 domain, perhaps by interacting with other macromolecules. To test this hypothesis, embryonic chick cartilage was treated with DTSSP, dissociated with bacterial collagenase, and the NC4-containing DTSSP-cross-linked protein complexes examined and purified. Analysis of cartilage extracts using an anti-NC4 antibody, and of purified NC4-containing complexes, identified a predominant NC4 dimer. A naturally-occurring N-terminal fragment of the alpha 1(IX) chain, whose size is equivalent to the NC4-COL3-NC3 domains of the chain, was identified. Association of collagen IX molecules via NC4 domains and the existence of a cleavage site close to the NC3 domain of the molecule are likely to be of primary importance in the growth and remodeling processes of cartilage, in health and disease.
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PMID:Collagen IX: evidence for a structural association between NC4 domains in cartilage and a novel cleavage site in the alpha 1(IX) chain. 955 Feb 66

The expression and distribution of the long form of Type XII collagen were investigated histochemically during chicken corneal development using a monoclonal antibody (P3D11) raised against the N-terminal domain of chicken Type XII collagen. Specificity of the antibody was confirmed by immunoprecipitation before and after bacterial collagenase digestion. Immunofluorescent microscopic studies showed that during chicken cornea formation, the long form of Type XII collagen is initially detected on Day 3 embryo (stage 19) in the sub-epithelial matrix of the corneal periphery and in the matrix around the optic cup. On Day 5 embryo (stage 27) the long form was expressed in the primary stroma. Thereafter, as the secondary stroma was formed, the long form localized in the sub-epithelial and sub-endothelial matrices and in the anterior region of the limbus (corneoscleral junction) before the formation of Descemet's and Bowman's membranes. After hatching, the immunoreactivity decreased predominantly in the sub-epithelial and sub-endothelial matrices but remained at the anterior region of the limbus. Immunoelectron microscopic examination demonstrated that the long form localizes in the Descemet's and Bowman's membranes and along the collagen fibrils in the stroma with a periodic repeat. Based on the distribution of the long form of Type XII collagen in the sub-epithelial and sub-endothelial matrices and limbus, it was suggested that the long form of Type XII collagen is involved in formation of the Descemet's and Bowman's membranes and in stabilization of the limbus.
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PMID:Changes in distribution of the long form of type XII collagen during chicken corneal development. 1201 1

Adiponectin is protective against hepatic fibrosis, whereas leptin promotes fibrosis. In HSCs (hepatic stellate cells), leptin signals via a JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) pathway, producing effects that enhance ECM (extracellular matrix) deposition. SOCS-3 (suppressor of cytokine signalling-3) and PTP1B (protein tyrosine phosphatase 1B) are both negative regulators of JAK/STAT signalling, and recent studies have demonstrated a role for adiponectin in regulating SOCS-3 expression. In the present study we investigate mechanisms whereby adiponectin dampens leptin signalling and prevents excess ECM production. We treated culture-activated rat HSCs with recombinant adiponectin, leptin, both or neither, and also treated adiponectin knockout (Ad-/-) and wild-type mice with leptin and/or carbon tetrachloride (CCl4) or saline. We analyse JAK2 and Ob-Rb (long form of the leptin receptor) phosphorylation, and PTP1B expression and activity. We also explore potential mechanisms through which adiponectin regulates SOCS-3-Ob-Rb association. Adiponectin inhibits leptin-stimulated JAK2 activation and Ob-Rb phosphorylation in HSCs, whereas both were increased in Ad-/- mice. Adiponectin stimulates PTP1B expression and activity in vitro, whereas PTP1B expression was lower in Ad-/-mice than in wild-type mice. Adiponectin also promotes SOCS-3-Ob-R association and blocks leptin-stimulated formation of extracellular TIMP-1 (tissue inhibitor of metalloproteinases-1)-MMP-1 (matrix metalloproteinase-1) complexes in vitro. These results suggest two novel mechanisms whereby adiponectin inhibits hepatic fibrosis: (i) by promoting binding of SOCS-3 to Ob-Rb, and (ii) by stimulating PTP1B expression and activity, thus inhibiting JAK2/STAT3 signalling at multiple points.
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PMID:Adiponectin inhibits leptin signalling via multiple mechanisms to exert protective effects against hepatic fibrosis. 2184 28