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)

Fluorescent probes have been used to obtain dissociation constants for the fluid-phase interaction of human plasma fibronectin and several of its gelatin-binding fragments with purified alpha chains of type I rat tail collagen, as well as with a cyanogen bromide fragment (CB7) of the alpha 1 chain in 0.02 M Tris buffer containing 0.15 M NaCl at pH 7.4. Addition of fibronectin to fluorescein-labeled collagen chains caused a dose-dependent increase in the fluorescence anisotropy which continued over several logs of titrant concentration. Scatchard-type plots of the anisotropy response were biphasic indicating the presence of one or more weak sites (Kd greater than microM) along the collagen chain in addition to a strong site characterized by Kd = 1.3 X 10(-8) M at 25 degrees C. Gelatin-binding fragments with Mr = 42,000, 60,000, and 72,000 also produced a biphasic response with Kd values for the high affinity site being 10- to 20-fold greater than for intact fibronectin. Binding of fibronectin and its fragments to fluorescent-labeled CB7 was essentially the same as to the whole alpha 1 chain. In all cases, the anisotropy response could be reversed or prevented by addition of excess unlabeled gelatin or CB7, but not by synthetic peptides spanning the collagenase cleavage site of alpha 1 (I). Studies of the temperature dependence of Kd for binding of fibronectin to the high affinity site on alpha 1 produced a value of +16 kcal/mol for the enthalpy of dissociation below 30 degrees C. Above this temperature, fibronectin appeared to undergo a subtle conformational transition characterization by a reduced affinity for collagen. This transition occurred in whole fibronectin but not in the gelatin-binding fragments and may involve disruption of intramolecular interactions between different domains.
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PMID:Interaction of fibronectin and its gelatin-binding domains with fluorescent-labeled chains of type I collagen. 312 86

Hot phenol-water-extracted lipopolysaccharide (LPS) from Bacteroides gingivalis 381 was purified by Sephadex G-100 chromatography with Tris buffer supplemented with sodium deoxycholate and EDTA (B-LPS). In the present study, B-LPS was examined for its ability to induce interleukin 1 (IL-1) production, a mitogenic response, and macrophage activation in LPS high-responder C3H/HeN and low-responder C3H/HeJ mice. A significant increase in IL-1 production was observed in C3H/HeN and C3H/HeJ peritoneal macrophages treated with various doses (1.0 to 50 micrograms/ml) of B-LPS. IL-1 production by C3H/HeN macrophages treated with B-LPS (10 micrograms/ml) was about seven times greater than that by C3H/HeJ macrophages. However, the IL-1 production induced by B-LPS (10 micrograms/ml) in C3H/HeN macrophages was four times lower compared with that induced by Escherichia coli O111 B4 LPS. Also, a significant increase in IL-1 production was found in human monocytes stimulated with B-LPS. That B-LPS-induced IL-1 exhibits some molecular weight heterogeneity was indicated from Sephadex G-75 gel filtration profiles. A significant, high mitogenic response by whole spleen cells with 1 X 10(5) to 5 X 10(4) cells of either mouse strain per well treated with B-LPS (10 to 50 micrograms/ml) was observed. However, the response of C3H/HeJ mice was less than that of the C3H/HeN strain. Also, glucose consumption assays indicated that enhanced macrophage activation occurred in C3H/HeN but not in C3H/HeJ mice treated with B-LPS. In light of recent studies showing that IL-1 stimulates bone resorption in a mouse calvaria system and collagenase production in fibroblasts, we suggest that B-LPS-induced IL-1 may play a significant role in the pathogenesis of adult periodontal disease.
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PMID:Functional role of interleukin 1 in periodontal disease: induction of interleukin 1 production by Bacteroides gingivalis lipopolysaccharide in peritoneal macrophages from C3H/HeN and C3H/HeJ mice. 387 85

Insoluble bone gelatin with inclusions of insoluble noncollagenous protein produces new bone when implanted in muscle in allogeneic rats. The implanted residue provides the milieu for expression of bone morphogenetic potential of migratory mesenchymal cells. Neutral buffer solutions activate endogenous enzymes that degrade components essential for cell interactions and differentiation of bone. Chloroform-methanol either denatures or extracts constituents responsible for degradation. Insoluble bone gelatin produces new bone after extraction at 2 degrees with neutral salts, 0.5 M EDTA, 0.1 M Tris.HCl, 4 M urea, 0.5 M hydroxylamine, and 10 M KCNS, as well as after limited digestion with pepsin or collagenase, but not after extraction with 5 M guanidine, 7 M urea, water saturated with phenol, or after alkali hydrolysis with 0.1 N NaOH. The specific activity of cell populations interacting with insoluble bone gelatin suggests that a chemical bond between collagen and a noncollagenous protein or part of a protein, cleaved by a neutral proteinase, controls the bone morphogenetic reaction.
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PMID:Bone morphogenesis in implants of insoluble bone gelatin. 435 76

The structural heterogeneity of rabbit lung collagen was examined by extracting labeled collagen from short-term cultures of lung minces with 1 M NaCl-50 mM Tris.HCl (pH 7.4), 0.5 M acetic acid, or 0.4 ionic strength phosphate buffer. The extracted collagens were purified by carboxymethyl-cellulose chromatography, and their cyanogen bromide peptides were mapped by ion exchange chromatography and acrylamide gels. Rabbit skin alpha1(I) and alpha2 chains and rabbit sternal cartilage alpha1(II) chains were used as markers. The peripheral lung, containing alveoli, small blood vessels, and small airways, synthesized alpha1(I) and alpha2 chains. The trachea and the bronchial tree (first through seventh order branches) both synthesized alpha1(II) chains. Lung alpha1(I), alpha2, and alpha1(II) chains all have a molecular weight of about 100,000 and are all sensitive to Clostridial collagenase. The extraction and purification methods used isolate only 50% of the collagen synthesized by these structures in vitro. Once all collagen types in lung can be described and quantitated, it should be possible to utilize lung collagen types as biochemical markers to study normal lung development and to define the lung fibrotic diseases.
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PMID:Lung collagen heterogeneity. 452 88

The existing forms of collagenase [EC 3.4.24.7] in the human uterine cervix were examined. The latent collagenase extracted by homogenization in 0.25% Triton X-100 containing 0.01 M CaCl2 was indicated to be a complex of collagenase with alpha 2-macroglobulin by the behavior of the fraction of this enzyme before and after treatment with NaSCN on Sephadex G-150 column chromatography and an immunodiffusion method. The active collagenase was extracted by rehomogenization in 50 mM Tris-HCl buffer, pH 7.4, containing 0.1 M M CaCl2 from the insoluble residue at 0 degrees C. Another latent collagenase was extracted from the insoluble fraction in the same buffer by heating at 60 degrees C for 4 min and this enzyme was activated by 4-aminophenylmercuric acetate or trypsin. The molecular weights of the active and the latent forms were approximately 7.3 x 10(4) and 9.4 x 10(4), respectively. This indicates that the latency is due to the formation of a low molecular weight inhibitor enzyme complex. These results clarified that the human uterine cervix contains three existing forms (alpha 2-macroglobulin complex, active form and low molecular weight inhibitor complex) of collagenase under these experimental conditions.
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PMID:The existing forms of collagenase in the human uterine cervix. 624 99

Neutral collagenase (EC 3.4.24.7) has been detected in human liver biopsies, baboon liver for the first time. The reaction mixtures were composed of collagen in solution and total liver homogenate in 0.5 M Tris-HCl, pH 7.5 at 25 degree C/0.2 M NaCl/10 mM CaCl2/3 mM p-chloromercuribenzoic acid. Collagenase activity was found by directly subjecting the reaction mixtures to viscometric assay and the activity was confirmed to be due to neutral collagenase by identifying the products using disc gel electrophoresis. It proved necessary to use p-chloromercuribenzoic acid in order to reveal collagenase activity in total liver homogenates from these species. The p-chloromercuribenzoic acid served to inhibit thiol proteinases and all other signs of nonspecific collagenolysis on disc gel electrophoresis, and it was able to activate latent collagenase which trypsin could not.
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PMID:Direct measurement of neutral collagenase activity in homogenates from baboon and human liver. 626 Feb 7

Most effects of thyroid hormones appear to be mediated by the binding of triiodothyronine (T3) to nuclear triiodothyronine binding sites (NT3BS). Although thyroid hormones influence adipocyte metabolism, NT3BS have not been described in mature adipocytes yet. This report describes T3 nuclear binding in isolated nuclei from rat epididymal fat pad adipocytes. Nuclei were isolated by exposing collagenase-dispersed adipocytes to STM (sucrose, 0.25 mol/L; TRIS, 20 mmol/L; MgCl2, 1.1 mmol/L, pH 7.85) containing 0.5% (vol/vol) Triton X-100. Incubation of nuclei suspended in STM/EDTA (2 mmol/L)/DTT (5 mmol/L) with 125I-T3 and varying concentrations of unlabeled T3 at 37 degrees C for one hour revealed the presence of high-affinity, low-capacity NT3BS. Their MBC was 0.39 +/- 0.04 (SD) ng of T3 per milligram of DNA and their Kd was 1.4 +/- 0.5 (SD) X 10(-10) mol/L T3. Specific binding reached a plateau between 30 minutes and two hours of incubation. The addition of 5 X 10(-7) mol/L T3 to nuclei incubated for one hour with 2 X 10(-11) mol/L T3 completely displaced the specifically bound 125I-T3 within 30 minutes. Thyroxine (T4) and 3, 3', 5'-triiodothyronine (rT3) could displace 125I-T3 from the NT3BS but were less than 10% and 1% as effective, respectively, as T3. Rat epididymal fat pad adipocytes contain NT3BS, the binding characteristics of which are similar to those of rat hepatic NT3BS.
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PMID:Nuclear triiodothyronine binding sites in rat adipocytes. 632 15

The effects of alterations in extracellular calcium concentration on prostaglandin (PGE) and thromboxane (TXB2) syntheses were studied in isolated epithelial cells from the urinary bladder of the toad, Bufo marinus. In epithelial cells prepared using collagenase, basal iPGE synthesis was greater than iTXB2 synthesis. Increasing extracellular calcium from zero to 1 mM increased iPGE synthesis and decreased iTXB2 synthesis equivalently such that total conversion of endogenous arachidonate to these two metabolites was unaltered. Vasopressin stimulated iPGE and iTXB2 syntheses when the incubation buffer contained 1 mM calcium but had no effect in the presence of 0.4 microM calcium. In contrast, using an EDTA isolation method, basal iPGE and iTXB2 syntheses were equal in the presence of zero calcium. Increasing extracellular calcium concentration to 1 mM caused a greater enhancement in iTXB2 synthesis compared to iPGE. Increasing extracellular calcium to 2 mM was associated with a decline in iPGE and iTXB2 syntheses back to the levels observed with no calcium added to the medium. The effect of increasing the calcium concentration was greater in phosphate than in bicarbonate buffer. In a Tris buffer the effect of altered calcium was almost completely abrogated. These studies demonstrate that the choice of buffer and alterations in extracellular calcium concentration differentially alter basal arachidonic acid metabolism to prostaglandins and thromboxane in isolated toad urinary bladder cells. The results suggest that there may exist several endogenous pools of arachidonic acid which are differentially influenced by calcium. Furthermore, the pool sensitive to vasopressin has an absolute requirement for calcium.
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PMID:Alterations in extracellular calcium concentration differentially influence prostaglandin and thromboxane synthesis in epithelial cells from the toad urinary bladder. 642 55

A cylindrical segment, free of complex atherosclerotic lesions, was resected at autopsy from each of 59 descending human thoracic aortas by cutting just below the level of the first pair of intercostal arteries and 35 mm distal to this incision. Each isolated tunica media was defatted and subjected to successive treatment with EDTA-Tris, 5 M guanidine hydrochloride-Tris, 5 M guanidine hydrochloride-Tris-DTE, collagenase and either trypsin or hot alkali. After each extraction or digestion, the dimensions and weight of the segments were measured and the extracted materials were analyzed and quantitated. This allowed the total content of the various components of the tunica media to be assessed by both gravimetric and analytical means. An age-related rise was observed in the total content of the following components: proteins and glycoproteins soluble in chaotropic solvents (ranging from 24 mg/cm in the youngest samples to 46 mg/cm in the oldest) and collagen (38 mg/cm to 69 mg/cm). In contrast, the total content of elastin remained constant at 70 mg/cm at all ages, but its concentration decreased due to the rise in the concentration of the other tissue components as the tunica media thickened with age. It was also noted that with increasing age there was an accumulation of protein(s) which could not be solubilized by extraction with chaotropic agents or with collagenase, but which could be removed by treatment with either trypsin or hot alkali. Mechanical measurements conducted before and after trypsin digestion on samples previously subjected to purification with the first four agents used suggest that this accumulated protein(s) influenced the elastic response of the tissue to the applied stress by increasing the incremental modulus, the breaking stress, and the hysteresis. After the removal of this additional protein(s), the mechanical behavior of the elastin component was found to be identical in all samples, irrespective of age. It is therefore proposed that the morphological changes and the stiffening observed in the aging aortic wall are not due to degradation of its elastin network but to variations in the supramolecular organization of connective tissue components.
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PMID:Age-related changes in composition and mechanical properties of the tunica media of the upper thoracic human aorta. 682 97

Rabbit aortic intima-media fragments were incubated with [14C]mannose and [3H]fucose for 6 h to detect glycoproteins synthesized in situ. The radioactively labelled and the non-labelled samples were extracted with 0.2 mM-CaCl2/0.5 mM-dithiothreitol/0.5 mM-ATP and chloroform/methanol/water (4:4:1, by vol.). The delipidated residue was extracted with 5 M-guanidinium chloride/0.05 M-dithiothreitol/0.1 M-Tris/0.4% Na2EDTA, pH 7.5, before (extract 1) and after hydrolysis with collagenase (extract 2). The proteins in extracts 1 and 2 were S-carboxamidomethylated and separated by molecular-sieve chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing in sucrose gradients in urea. The apparent molecular weights of glycoproteins were 36 000 (glycoprotein I) from extract 1, 50 000 (glycoprotein II) and 130 000 (glycoprotein III) from extract 2. The molecular weights of the non-labelled and radioactively labelled glycoproteins were identical. Glycoproteins I, II and III contain large amounts of polar amino acids and methionine. They contain neither hydroxyproline nor 3-methylhistidine. A hydroxyproline-containing component of 160 000-apparent-mol.wt. relatively rich in polar amino acids and labelled with incorporated sugars was isolated from extract 1. The incorporation in vitro of radioactive sugars into glycoproteins I, II, III and collagenous glycoproteins indicates that they are synthesized in the surviving aorta by the smooth-muscle cells.
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PMID:Structural glycoproteins from rabbit aortic media. 687 Aug 24


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