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)

Rat submandibular gland cells have been obtained through enzymatic dispersion using chromatographically purified collagenase (EC 3.4.24.3) and hyaluronidase (EC 3.2.1.35) and gentle mechanical force. The recovery of viable cells after the isolation procedure was 59% on the basis of total glandular DNA content. Approximately 60% of the total cell population consisted of acinar cells; less than 8% were immature granular duct cells; and the remainder were intercalated duct, striated duct, and myoepithelial cells. Most of the acinar cells were in acinar-intercalated duct complexes. The integrity of the isolated cells was substantiated by their exclusion of trypan blue, intracellular electrolyte composition, incorporation of [14C]glucosamine into trichloroacetic acid + phosphotungstic acid precipitable material at a linear rate for 1.5 hr, secretory responses to parasympathomimetic and sympathomimetic stimulation, and morphologic integrity as determined by light and electron microscopy. The cholinergic receptors were characterized through investigation of the net transmembrane flux of K+ in response to carbamoylcholine. The alpha-adrenergic receptors were characterized by investigating the net transmembrane flux of K+ in response to norepinephrine stimulation and the beta-adrenergic receptors were characterized by determining the rate of secretion of 14C-labeled mucin after isoproterenol stimulation. A high degree of sensitivity to both cholinergic and adrenergic secretagogues was observed.
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PMID:Functional characteristics of dispersed rat submandibular cells. 22 58

To test the hypothesis that idiopathic pulmonary fibrosis (IPF) is mediated through collagenase present in the lower respiratory tract, we used the fiberoptic bronchoscope to obtain fluid from the lower respiratory tract of 24 patients with IPF, 18 controls and nine patients with sarcoidosis. The fluid was analyzed for a variety of enzymes, including collagenase. Fifteen of 21 patients with IPF showed collagenase activity, whereas normal controls and patients with sarcoidosis showed none (P greater than 0.001, for all comparisons). In two patients with IPF who were re-evaluated after eight to 24 months, the collagenase activity was persistent. Fluid from patients with IPF also contained elevated levels of a non-specific neutral protease (P greater than 0.01 compared with controls), but there was no elastase activity in fluid from patients with IPF or from controls. The collagenase found in lavage fluid in IPF cleaved lung collagen into collagenase-specific TCA and TCB fragments. We conclude that in IPF the collagen of the lung is subjected to sustained lysis, followed by disordered resynthesis, and that the presence of active collagenase in the lower respiratory tract is a specific feature of the alveolitis associated with this disease.
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PMID:Collagenase in the lower respiratory tract of patients with idiopathic pulmonary fibrosis. 22 66

We have surveyed the amount and types of collagen synthesized in two regions of the chick heart during embryonic development. Cardiac tissues from successive periods of development were labeled with 3H-proline in short-term organ culture. The fraction of incorporated label present as collagen was estimated by comparison of TCA-soluble radioactivity before and after digestion with purified bacterial collagenase. This measure of collagen synthesis varied only slightly with the length of the labeling period and agreed with values obtained by labeling in ovo. In the developing outflow tract, the fraction of label present as collagen increased sharply during the period of truncal septation (5-9 days), rising from initial values of 6% at 3 days of incubation to a plateau of about 25% (10-19 days). In ventricular myocardium, this fraction rose gradually from 3 to 20% between 3 and 19 days of incubation. The types of collagen synthesized in developing heart were analyzed by limited pepsin digestion and acrylamide gel electrophoresis, using collagens synthesized in tendon, cartilage, skin, and lens capsule for comparison. The types of collagen synthesized in both cardiac regions changed in similar manner during development. During the first week of cardiac function, a substantial but progressively smaller fraction of total collagen synthesized was identified as Type IV. Synthesis of Type I collagen increased sharply during this period and predominated during the second half of development. Type III collagen was synthesized in trace amounts by the middle of development and constituted approximately one-sixth of total collagen synthesis just before hatching. Minor amounts of collagen identified as Type B collagen were synthesized throughout the latter two-thirds of development.
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PMID:Collagen synthesis in the developing chick heart. 23 22

Effects of the calcium antagonist verapamil on the synthesis of fetal rat bone collagen and noncollagen protein were investigated in tissue culture. Protein synthesis was quantitated by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP) using bacterial collagenase; [3H]proline was added for the last 2 h of culture. Verapamil (10(-5)--10(-4) M) decreased the incorporation of label into CDP and NCP after 24 h of culture; CDP formation was inhibited to a greater extent than NCP. The inhibitory response was observed in the presence and absence of unlabeled medium proline and was not associated with changes in trichloroacetic acid-extractable radioactivity. Increasing medium calcium from 1.0 to 5.0 mM did not affect the response to 10(-4) M verapamil, whereas 3.0 mM calcium abolished the response to 10(-5) M verapamil. The inhibitory effect was reversed by 48 h of control treatment subsequent to 24-h treatment with the antagonist. Verapamil did not decrease the incorporation of [3H]thymidine into DNA or [3H]uridine into RNA, nor was there any effect of the antagonist on the DNA content of cultured bones. The prostaglandin synthetase inhibitor indomethacin did not affect the response to verapamil. We conclude that a critical concentration of intracellular calcium is necessary for normal synthesis of skeletal protein in tissue culture, and that collagen may be more sensitive to changes in intracellular calcium than NCP. In addition, other ions (e.g. sodium and potassium) may also be involved in the control of skeletal protein synthesis.
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PMID:Effects of the calcium antagonist verapamil on in vitro synthesis of skeletal collagen and noncollagen protein. 48 4

The uptake of formaldehyde-treated 125I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20-30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.
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PMID:Uptake and degradation of formaldehyde-treated 125I-labelled human serum albumin in rat liver cells in vivo and in vitro. 55 48

Suspensions of oviduct cells were prepared by subjecting oviduct tissue to sequential incubations with EDTA, alpha-chymotrypsin, and crude collagenase, followed by a final incubation with EDTA. Cells isolated in this way incorporate mannose from exogenous GDP-mannose into mannosyl-lipid, oligosaccharide-lipid, and glycoprotein(s). Based on several criteria, the mannosyl-lipid is identical with mannosyl-phosphoryldolichol. Similarly, the oligosaccharide-lipid appears to be identical with the oligosaccharide-lipid synthesized in vitro (Lucas, J. J., Waechter, C. J., and Lennarz, W. J. (1975) J. Biol. Chem. 250, 1992-2002). In contrast, the glycoproteins are much lower in molecular weight than those labeled in cell-free preparations. Using intact oviduct cell suspensions it was found that: (a) exogenous GDP-mannose, not its breakdown products, serves as the direct mannosyl donor; (b) experiments using mixtures of known proportions of broken and intact cells, as well as studies with metabolic inhibitors, indicate that greater than 50% of the observed incorporation of mannose from GDP-mannose was catalyzed by enzymes associated with intact cells, rather than broken cells or membrane fragments; (c) incorporation of mannose from GDP-mannose into the mannosyl acceptors does not require energy and proceeds without significant uptake of GDP-mannose into trichloroacetic acid-soluble components of the cells; (d) under conditions where labeled guanosine incorporation into nucleic acids is readily detected, no incorporation of the guanosine moiety of [3H]GDP-mannose is observed. These results indicate that the enzymes catalyzing synthesis of lipid-linked intermediates involved in glycoprotein synthesis are not only associated with intracellular membranes, but with the plasma membrane as well.
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PMID:Utilization of exogenous GDP-mannose for the synthesis of mannose-containing lipids and glycoproteins by oviduct cells. 77 Apr 69

The biosynthetic activity of the B-cells of obese hyperglycemic mice (obob) was measured by the incorporation of [3H]leucine into proteins in collagenase-isolated pancreatic islets. To quantitate the incorporation into proinsulin and insulin, an immune binding method was used. For this purpose, anti-insulin serum was coupled to cyanogen bromide-activated SepharoseR 4B. This turned out to be a specific and versatile technique for the measurement of newly synthesized proinsulin and insulin in the B-cells. The B-cells of obob mice appear to be well adapted to a high rate of hormone biosynthesis, since at 16.7 mM glucose 44% of [3H]leucine incorporated into TCA-precipitable proteins was bound to the insulin antibodies coupled to Sepharose 4B. The insulin biosynthetic rate was stimulated 9 times at 16.7 mM glucose, during a 3-h incubation, compared with the basal insulin biosynthesis rate.
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PMID:Anti-insulin serum coupled to Sepharose 4B as a tool for the investigation of insulin biosynthesis in the B-cells of obese hyperglycemic mice. 110 96

Basic fibroblast growth factor (basic FGF) is a potent mitogen for chondrocytes in vitro and is present in developing cartilage in vivo. Studies of intracellular basic FGF localization in other cell types have revealed a transient nuclear presence. We have examined ovine fetal growth plate chondrocytes for the presence of intracellular basic FGF by immunocytochemistry. Chondrocytes were isolated from the proximal tibial growth plate of lamb fetuses between 75 and 80 days' gestation using collagenase, and were cultured in monolayer before use between passages 3 and 6. In non-synchronized cell cultures 58 +/- 6% of cells (mean +/- s.d., n = 3) demonstrated cytoplasmic staining for immunoreactive basic FGF. Of these cells, 18 +/- 3% also exhibited strong nuclear staining. Chondrocytes were growth-restricted and restarted into the cell cycle with 2% (vol/vol) fetal calf serum. The timing of S phase was followed by nuclear labelling of nuclei with [3H] thymidine followed by autoradiography, or by the incorporation of [3H] thymidine into trichloroacetic acid-precipitable DNA in parallel cultures. A cytoplasmic presence of immunoreactive basic FGF did not appear, following immunocytochemistry, until the second half of G1 with 97% of cells immunopositive 2 hr prior to S phase. Nuclear staining for basic FGF appeared 2 hr before peak nuclear labelling index, and 56% of cell nuclei were immunopositive. Following entry into S phase cytoplasmic and nuclear basic FGF immunostaining rapidly disappeared. When these experiments were repeated with or without the presence of anti-basic FGF antibody or heparin, the presence of the antibody significantly reduced peak [3H] thymidine incorporation into DNA during S phase while exposure to heparin increased this. However, the proportion of cells demonstrating cytoplasmic or nuclear staining for immunoreactive basic FGF, and the time of onset of staining, were unaltered. Incubation of cells with suramin blocked subsequent DNA synthesis and no intracellular basic FGF was visualized. Cell-conditioned culture medium, extracellular matrix and cytoplasm from synchronized cultures of chondrocytes were taken at time points throughout the cell cycle and assessed for basic FGF content by radioimmunoassay. Basic FGF was detectable in each compartment and steadily rose throughout the second half of G1 to reach maximum values around the S phase. The accumulation of basic FGF in medium, matrix and cytoplasm was blocked by the presence of cycloheximide.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cell cycle-dependent localization of immunoreactive basic fibroblast growth factor to cytoplasm and nucleus of isolated ovine fetal growth plate chondrocytes. 145 27

Monoclonal antibodies were prepared that were specific for chicken type I and type III collagens. The specificity of these antibodies was determined by ELISA, inhibition ELISA, and immunoblot assays. The results showed that the monoclonal antibodies were specific for their respective antigens without significant cross reactivity to other types of collagen. An analysis of the location of the epitopes by rotary shadowing that a monoclonal antibody for type I collagen (called DD4) recognized type I procollagen close to the large globular domain at the carboxyl terminus of the molecule. A monoclonal antibody for type III collagen (called 3B2) recognized both the intact type III molecule and also the TCA fragment of type III collagen after mammalian collagenase digestion. The epitope was located approximately one-fifth of the distance from the amino-terminus of the intact molecule. The monoclonal antibodies were used for immunolocalization of type I and type III collagens in cryosections of heart, aorta, kidney, liver, thymus, skin, gizzard and myotendinous junction. In heart, aorta, kidney, liver, thymus and skin, type I and III collagens were colocalized in the connective tissue of each organ. In contrast, gizzard and myotendinous junction showed distinctly different staining patterns for the distribution of type I and type III collagen. The two monoclonal antibodies reported here are potentially useful reagents to study fibril formation involving type I and type III collagens.
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PMID:Monoclonal antibodies that distinguish avian type I and type III collagens: isolation, characterization and immunolocalization in various tissues. 156 Jul 90

Several N-carboxyalkyl peptides were synthesized and tested as inhibitors of pig synovial collagenase, 72-kDa gelatinase and stromelysin (matrix metalloproteinases MMP-1, MMP-2, and MMP-3). The most potent of the series, CH3CH2CH2(R,S)CH(COOH)-NH-Leu-Phe-Ala-NH2, competitively inhibited cleavage of dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by MMP-1 and MMP-2 (KI = 30 and 40 microM, respectively). A similar inhibitory potency was found for MMP-1 with soluble Type I collagen and MMP-3 with substance P as substrate. The inhibitor was coupled to EAH-Sepharose 4B through a C-terminal amide. In the presence of 2 M NaCl at pH 7.2, this matrix bound MMP-1, MMP-2, and MMP-3 from concentrated culture medium of pig synovial membranes. The enzymes coeluted at pH 4.1 and subsequently were resolved by chromatography on DEAE-Sephacel and heparin-Sepharose. Purified MMP-1 catalyzed the o-phenanthroline-sensitive cleavage of collagen into TCA and TCB fragments as well as slower hydrolysis of the alpha 2 chain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of MMP-1 indicated a predominant polypeptide of approximately 44 kDa and minor species of approximately 24 and 21 kDa. The 44-kDa species and one of the smaller polypeptides reacted with an antiserum to residues 195-207 of human fibroblast MMP-1, indicating that porcine MMP-1 contains a similar sequence and that the smaller components were probably derived from MMP-1. Neither MMP-2 nor MMP-3 reacted with this antiserum. Purified porcine MMP-2 degraded gelatin but not collagen and exhibited an apparent Mr of approximately 71 kDa. Additional smaller polypeptides were present, one of which may correspond to tissue inhibitor of metalloproteinases. MMP-3 showed doublets of approximately 47/46 and 26/25 kDa and cleaved substance P at the Gly6-Phe7 bond. This procedure provides a rapid means of obtaining all three MMPs from one source in approximately 15% yield each.
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PMID:Application of N-carboxyalkyl peptides to the inhibition and affinity purification of the porcine matrix metalloproteinases collagenase, gelatinase, and stromelysin. 165 8


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