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)

Osteogenesis in the embryonic long bone rudiment occurs initially within an outer periosteal membrane and subsequently inside the cartilaginous core as a consequence of the endochondral ossification process. In order to investigate the development of these two different mechanisms of bone formation, embryonic chick tibial cell isolates were prepared from sites of first periosteal bone formation and from the immediately underlying hypertrophic cartilaginous core region. Mid-diaphyseal periosteal collars and the corresponding cartilage core were microdissected free from Hamburger-Hamilton stage 35 (Day 9) chick tibias and separately digested with a trypsin-collagenase enzyme mixture. The released cell populations were cultivated in vitro and characterized by morphological analysis, histochemical localization of alkaline phosphatase, alizarin red S staining for mineral deposition, growth rate [( 3H]thymidine uptake), and proteoglycan content. Results of these studies showed that periosteal collar cell cultures form nodule-like structures that stain positive with alkaline phosphatase and alizarin red S. Light and electron microscopic observation revealed cell and matrix morphologies similar to that of intact periosteum. The nodules were composed of plump cell types embedded within a mineralized matrix surrounded by a fibroblastic cell layer. Core cartilage cell cultures displayed typical characteristics of the hypertrophic state in their visual appearance and proteoglycan composition. The formation of osseous-like structures in periosteal collar cell cultures but not in core chondrocyte cell cultures demonstrates the relatively autonomous nature of intramembranous ossification while emphasizing the dependence of the endochondral ossification process upon an intact vascularized environment present in the developing tibia.
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PMID:Isolation and characterization of osteogenic cells derived from first bone of the embryonic tibia. 401 99

The effects of bone morphogenetic protein (BMP), a molecule extracted from demineralized bone, were observed in organ cultures of 21-day fetal rat calvariae. The effects of BMP on cell replication in cultures of normal rat kidney (NRK) fibroblasts were studied for comparison. At concentrations of 0.1-10 micrograms/ml for periods of 24-96 hours, BMP stimulated the incorporation of 3H-thymidine into acid-insoluble residues (DNA) in calvariae by 25%-159%, and at 1-10 micrograms/ml it increased bone DNA content by 20%-23%. BMP at 1 micrograms/ml also increased the number of calvarial mitoses after colcemid arrest by 1.5-1.8-fold. The effect of BMP on calvarial DNA synthesis was observed in the periosteal bone. In contrast to its effects on DNA synthesis, BMP did not stimulate the incorporation of 3H-proline into collagenase-digestible and noncollagen protein and did not alter calvarial alkaline phosphatase activity. BMP at 1-10 micrograms/ml caused a marked increase in 3H-thymidine incorporation into DNA in cultured NRK fibroblasts and increased DNA content and cell number by 1.5-2-fold. These studies indicate that BMP stimulates DNA synthesis and cell replication in calvarial and fibroblast cultures but does not stimulate postdifferentiated bone cells in incubated calvariae.
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PMID:Effect of partially purified bone morphogenetic protein on DNA synthesis and cell replication in calvarial and fibroblast cultures. 402 62

In situ studies with the mouse macrophage (M phi)-specific antibody, F4/80, have shown that resident M phi in femoral bone marrow (RBMM) form hematopoietic islands with immature myelomonocytic and erythroid cells (Hume, D. A., et al. 1983. J. Exp. Med. 158: 1522). We have isolated these islands (clusters) by collagenase digestion, purified them from single cells by velocity sedimentation, and analyzed their cellular content. The clusters, ranging from 5- to 100 cells, constituted approximately 7% of the total nucleated cells, and greater than 70% contained at least one strongly staining, F4/80+ central M phi. In comparison, less than 26% showed reactivity for alkaline phosphatase, a marker of fibroblastoid reticulum cells. Compared with the nonclustering population, clusters were enriched with RBMM, fibroblastoid cells, and immature hematopoietic cells, but depleted of mature granulocytes and erythrocytes. The RBMM population was purified from other cells in clusters by selective adherence to glass and was compared with resident peritoneal M phi (RPM) for morphology and the presence of antigens, receptors, and enzymes. RBMM spread more extensively than RPM and frequently extended delicate plasma membrane processes. These and subsequent differences were not attributable to the collagenase treatment. Both M phi populations stained positively with antibodies F4/80 and 2.4G2 (Fc receptor IgG1/2b), bore mannosyl/fucosyl receptors, and showed reactivity for acid phosphatase and nonspecific esterase I. In contrast to RPM, RBMM had no detectable Mac-1 antigen (CR3) or complement receptors, but bore higher levels of Fc receptors (IgG2a and IgG2b) and Ia antigens. In addition, RBMM possessed a novel hemagglutinin activity for unopsonized sheep erythrocytes, which was not present on RPM. RBMM showed no respiratory burst activity in response to zymosan particles, but ingested them avidly. The growth properties of clustering and nonclustered populations were compared by measurement of [3H]thymidine incorporation and progenitor assays. Cells in clusters incorporated three- to fourfold more thymidine than nonclustered cells even in the absence of exogenous growth factors, and autoradiography demonstrated that RBMM made contact with proliferating cells. In contrast, the clusters contained over threefold fewer granulocyte/M phi progenitors compared with nonclustering cells. When clusters were cultivated for up to 3 d, there was rapid outgrowth of monocytes and fibroblastoid cells. These studies demonstrate that RBMM bear a distinct morphology and phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Isolation and characterization of resident stromal macrophages and hematopoietic cell clusters from mouse bone marrow. 403 89

1. A collagenase digest of rat kidney cortex was separated into four bands by zonal centrifugation. 2. Two of these bands were shown by light-microscopy to contain glomeruli and tubular fragments, which were free from each other and well separated from other renal material. 3. Protein, N-acetyl-beta-glucosaminidase, 5'-nucleotidase, l-leucine beta-naphthylamidase, leucine aminopeptidase, acid phosphatase and alkaline phosphatase were assayed across the gradient. 4. The greater proportion of these enzyme activities was recovered in the tubular fragments and acid phosphatase was the only enzyme detected in significant amounts in the glomeruli. 5. Tubular fragments and glomeruli were sedimented and multiple forms of beta-naphthylamidase, N-acetyl-beta-glucosaminidase, acid phosphatase and alkaline phosphatase were investigated by starch-gel electrophoresis.
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PMID:The distribution of some hydrolases in glomeruli and tubular fragments prepared from rat kidney by zonal centrifugation. 433 86

Matrix vesicles, associated with initial calcification in cartilage, have been isolated from bovine fetal epiphyseal cartilage. Cartilage was digested with collagenase, then partitioned into seven fractions by differential centrifugation. The cellular fractions contained over 80% of the DNA in the digest. The extracellular fraction that contained matrix vesicles, in which apatite crystals were often seen on electron microscopy, also displayed the highest specific activity for alkaline phosphatase, pyrophosphatase, ATPase, and 5'-AMPase (EC 3.1.3.1., 3.6.1.1, 3.6.1.3, and 3.1.3.5, respectively). Most of the acid phosphatase (EC 3.1.3.2) activity, on the other hand, was found in the cellular fractions, indicating that matrix vesicles are quite distinct from lysosomes. This appears to be the first instance of isolation of membrane-bounded extracellular particles from any normal tissue. The matrix vesicles possess enzymes that can increase the local concentration of orthophosphate and thus could lead to the formation of hydroxyapatite. The membrane-bounded matrix vesicles may also provide a mechanism for ATP-dependent transport of calcium or phosphate into the lumen of the vesicles with resultant mineralization.
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PMID:Isolation and characterization of calcifying matrix vesicles from epiphyseal cartilage. 527 75

An attempt was made to concentrate plasma membranes of homogenized chondrocytes isolated by collagenase digestion of rachitic rat epiphyseal growth plate cartilage. This study reports the characterization of enzymes in the plasma membrane of isolated chondrocytes and their comparison with extracellular matrix vesicle components. The plasma membrane-enriched fractions that were obtained showed a sevenfold increase in 5'-nucleotidase and a 15-fold increase in alkaline phosphatase, both of which are regarded as plasma membrane markers. SDS-polyacrylamide gel electrophoretic profiles of proteins extracted from membrane fractions contained several major protein bands also seen in isolated matrix vesicles. These studies indicate the usefulness of concentrating plasma membrane components from isolated chondrocytes, after the chondrocytes have been enzymatically freed from investing matrix and other stromal components by collagenase.
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PMID:Isolation of a plasma membrane-enriched fraction from collagenase-suspended rachitic rat growth plate chondrocytes. 609 Jun 23

Bone cells isolated from mouse calvariae by a sequential digestion procedure have many osteoblast characteristics: they respond to PTH and prostaglandin E2 by activation of adenylate cyclase but not to calcitonin, they stain for alkaline phosphatase and they make only type I collagen. In confluent monolayer culture, they do not secrete collagenase in appreciable quantities, unless stimulated with resorptive substances such as PTH, prostaglandin E2, 1,25(OH)2 vitamin D-3 and monocyte-conditioned medium. This suggests they play a direct role in bone resorption.
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PMID:Mouse osteoblasts synthesize collagenase in response to bone resorbing agents. 609 72

Microvessels were isolated from rat brain using a double collagenase treatment which removed the endothelial basement membranes. The isolate was characterized by intact luminal and abluminal membranes and an absence of pericytes and astrocyte membranes. Minimal contamination by 5'-nucleotidase, an enzyme believed exclusively localized within the plasma membranes of neuroglia, established the purity of the isolated microvessels. Enrichment of alkaline phosphatase and gamma-glutamyl transpeptidase activity in microvessel preparations supports the endothelial localization of these enzymes.
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PMID:Isolation and characterization of brain endothelial cells: morphology and enzyme activity. 610 94

A method is described for the preparation of an enriched population of proximal tubules from the cortices of neonate (21-28 d old) rabbits. The method uses collagenase-hyaluronidase digestion, followed by gentle shear to yield a suspension of tubules and glomeruli. Tubular enrichment is achieved by discontinuous density gradient centrifugation in a Percoll gradient. Two fractions were obtained by this method. The denser fraction contained predominantly proximal tubules, was depleted of glomeruli and was enriched in the proximal tubule marker enzyme alkaline phosphatase. Qualitatively similar results to those obtained with neonate animals were obtained with adult tissue. Neonate tubules from the denser gradient fraction grew readily in tissue culture. When examined by electron microscopy the cells exhibited a marked polarity of ultrastructural organization and retained apical tight junctions. Despite this, there was an obvious loss of structure in comparison with in vivo proximal tubule cells. The use of primary culture techniques to study in vitro renal epithelial function is discussed.
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PMID:Isolation and culture of renal cortical tubules from neonate rabbit kidneys. 612 33

Nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.8) activity is associated with matrix vesicles purified from collagenase digests of fetal calf epiphyseal cartilage. This enzyme hydrolyzes nucleoside triphosphates to nucleotides and PPi, the latter inducing precipitation in the presence of Ca2+ and Pi. An assay for matrix vesicle nucleoside triphosphate pyrophosphohydrolase is developed using beta, gamma-methylene ATP as substrate. The assay is effective in the presence of matrix vesicle-associated ATPase, pyrophosphatase, and alkaline phosphatase activities. A soluble nucleoside triphosphate pyrophosphohydrolase is obtained from matrix vesicles by treatment with 5 mM sodium deoxycholate. The solubilized enzyme induced the precipitation of calcium phosphate in the presence of ATP, Ca2+, and Pi. Extraction of deoxycholate-solubilized enzymes from matrix vesicles with 1-butanol destroys nucleoside triphosphate pyrophosphohydrolase activity while enhancing the specific activities of ATPase, pyrophosphatase, and alkaline phosphatase. In solutions devoid of ATP and matrix vesicles, concentrations of PPi between 10 and 100 microM induce calcification in mixtures containing initial Ca2+ X P ion products of 3.5 to 7.9 mM2. This finding plus the discovery of nucleoside triphosphate pyrophosphohydrolase in matrix vesicles supports the view that these extracellular organelles induce calcium precipitation by the enzymatic production of PPi. Nucleoside triphosphate pyrophosphohydrolase is more active against pyrimidine nucleoside triphosphates than the corresponding purine derivatives. The pH optimum is 10.0 and the enzyme is neither activated nor inhibited by Mg2+ or Ca2+ ions or mixtures of the two. Vmax at pH 7.5 for beta, gamma-methylene ATP is 0.012 mumol of substrate hydrolyzed per min per mg of protein and Km is below 10 microM. The enzyme is irreversibly destroyed at pH 4 and is stable at pH 10.5.
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PMID:The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification. 613 31


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