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)

Chondrocytes were isolated from bovine growth-plate cartilage and cultured within type I collagen gels. A major collagen with chains of Mr 59,000, decreasing to 47,000 on pepsinization, was synthesized and identified as type X collagen. This collagen was cleaved at two sites by mammalian collagenase, resulting in a major triple-helical fragment with chains of Mr 32,000. The species of Mr 59,000, 47,000 and 32,000 were not detected by SDS-polyacrylamide gel electrophoresis before reduction, indicating the presence of disulphide bonds within the triple helix. In contrast, similar biosynthetic studies with human growth-plate cartilage in organ culture, indicated that human type X collagen does not contain disulphide bonds. A polyclonal antiserum was raised to bovine type X collagen and used in immunolocalization studies to provide direct evidence for the association of type X collagen with the hypertrophic chondrocytes in both bovine and human growth plates during development.
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PMID:The synthesis of type X collagen by bovine and human growth-plate chondrocytes. 193 74

Previous studies demonstrated that tetracyclines (TCs) inhibited Type I (interstitial) and Type IV collagenases from different mammalian sources, but there are no studies of TCs effect on osteoblast collagenase (C'ase). The present study assessed the effect of TCs on C'ase activity from osteosarcoma cells. Semiconfluent UMR 106-01 cells were treated with minocycline or chemically modified tetracycline (CMT) at 10 micrograms/ml in the presence or absence of bovine parathyroid hormone, b-PTH-(1-34), at 10(-7)M for 24, 48, 72 and 96 hours. Media were collected at each time point and assayed following concentration, destruction of TIMP by reduction/alkylation, activation with p-aminophenylmercuric acetate (APMA), and incubation with 3H-methylated collagen substrate (approximately 100,000 dpm) at 27 degrees C for 18 hours. Collagenase activity from media was also analyzed by SDS-PAGE and fluorography. b-PTH appeared to stimulate C'ase 60-fold compared to controls; minocycline and CMT reduced PTH stimulation approximately 65% and 90%, respectively. Moreover, TCs incubated with partially purified osteoblastic collagenase directly, inhibited its activity in vitro as indicated by a lack of degradation to collagen alpha A chains. Therefore, TCs ability to inhibit bone resorption in organ culture, reported previously, may be due, in part, to reduced osteoblast collagenase activity.
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PMID:The effect of tetracyclines on collagenase activity in UMR 106-01 rat osteoblastic osteosarcoma cells. 196 17

The cuticles from distinct developmental stages of Ascaris suum were isolated by a combination of mechanical disruption and detergent treatment of larvae or by surgical removal of cuticle from adults. Proteins from the isolated cuticles were solubilized with SDS and 2-mercaptoethanol (2ME) and analyzed by PAGE. Cuticular proteins from the third and fourth larval stages (L3 and L4) were comparable to adult, but differences in the number of bands were observed. The soluble proteins from the adult, L3 and L4 were readily degraded by bacterial collagenase, suggesting that these proteins are collagen-like structural elements of the cuticle. The soluble proteins from the L2 differed from the adult and other larval stages in both the number and molecular weight of protein bands and their lack of collagenase sensitivity. Antibodies made against the soluble cuticular proteins reacted with the medial and basal layers of the cuticle but not the external cortical or epicuticular regions. A significant amount of the cuticle was not solubilized by 2ME and was not digested by bacterial collagenase. These insoluble cuticular proteins were probably derived from the epicuticular and external cortical regions of the cuticle. Different developmental stages of A. suum were biotinylated and examined by electron microscopy. An organic soluble biotin reagent labeled all stages in a transcuticular pattern, while an aqueous soluble biotin labeled only the external cortical and epicuticular regions of the L4 and adult cuticle. These data indicate the presence of a hydrophobic barrier in the cuticle of later stages of the parasite.
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PMID:The cuticular biology in developmental stages of Ascaris suum. 197 29

To examine structure-activity relationships of human IL-6, we have determined the effects of specific mutations on the biologic activity of a human rIL-6 expressed in bacteria. Three types of mutants were examined: 1) a variant that contains serines in place of the four naturally occurring cysteines; 2) a series of cysteine-containing deletion mutants, each having a single internal 20 amino acid deletion; and 3) a cysteine-free variant containing a single 20 amino acid deletion. The mutants of the second type constitute a set of nonoverlapping, adjacent deletions spanning amino acids 4 through 183 of the 184 amino acids in natural human IL-6. All of the mutants were expressed, along with the full length, cysteine-containing analogue, in Escherichia coli as fusion proteins, joined to beta-galactosidase through a collagen linker. This system allows microgram quantities of the rIL-6 variants to be partially purified from small bacterial cultures without chromatographic or refolding steps. Each of the rIL-6 variants was released from the beta-galactosidase fusion protein with collagenase, and the recovered rIL-6 was quantitated by laser densitometry of Coomassie-stained, SDS polyacrylamide gels. The sp. ac. of each of the rIL-6 variants was determined using four assays: induction of IgM secretion from an EBV transformed human B cell line, induction of fibrinogen secretion from a human hepatoma cell line, induction of fibrinogen secretion from a rat hepatoma cell line, and induction of proliferation of a murine hybridoma cell line. Replacement of cysteines with serines reduced activity relative to cysteine-containing rIL-6 to about 20% in the rat hepatoma assay and about 3% in the mouse hybridoma assay, whereas activity in both of the human cell lines was reduced to less than 0.1%. These data suggest that the murine and rat cell lines are less selective than the human cell lines in their requirements for recognition of biologically active IL-6. Each of the deletions, except that of amino acids 4 through 23, resulted in loss of activity in all four assays. These results suggest that the information necessary for activity is not contained within any one portion of the IL-6 molecule, but rather that multiple segments of the protein are required for each of the biologic activities that we tested.
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PMID:Effects of site-specific mutations on biologic activities of recombinant human IL-6. 198 78

Type X collagen was extracted with 1 M NaCl and 10 mM dithiothreitol at neutral pH from fetal human growth plate cartilage and purified to homogeneity by gel filtration and anion-exchange chromatography. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 66,000 under reducing conditions, and as a high-Mr oligomer under non-reducing conditions. Purified collagenase digests most of the molecule; pepsin digestion at 4 degrees C decreases the Mr of the monomer to 53,000. A rabbit antiserum was raised against purified human type X collagen; the IgG fraction was specific for this collagen by criteria of ELISA and immunoblotting after absorption with collagen types I, II, VI, IX and XI. Immunohistological studies localized type X collagen exclusively in the zone of hypertrophic and calcifying cartilage.
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PMID:Isolation of human type X collagen and immunolocalization in fetal human cartilage. 201 80

The v-sis oncogene of simian sarcoma virus encodes a protein which is homologous to the human platelet-derived growth factor B-chain. The v-sis protein undergoes a series of processing steps including dimer formation and proteolytic digestion to generate several molecular sizes of the protein. Two of these v-sis proteins were expressed alone or as polyhedrin-sis fusion proteins using the Bombyx mori nuclear polyhedrosis virus vector. The polyhedrin-sis fusion proteins contained a collagenase-sensitive site at the junction. The expression levels of the fusion proteins whose polyhedrin portions consisted of only 8 amino-terminal amino acids were 3-4 times higher than those of non-fusion proteins. One of these fusion proteins was expressed in silkworm larvae and the v-sis protein was isolated from the fusion protein by collagenolysis followed by chromatography. Because the purified v-sis protein exhibited the same molecular size on SDS-polyacrylamide gels under reducing and non-reducing conditions, it was concluded to be monomeric in structure. It possessed chemotactic activity but lacked mitogenic activity. In addition, a small amount (approximately 1%) of monomeric v-sis protein was converted in vitro to the mitogenically active v-sis protein, which could be a homo-dimer.
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PMID:Characterization of v-sis protein expressed in silkworm larvae using the Bombyx mori nuclear polyhedrosis virus vector. 201 72

Cementum forms the interface through which soft connective tissue of the periodontium is attached to the root surface. The interactions between cementum and connective tissue are not completely understood and whether cementum influences periodontal connective tissue formation and regeneration is not clear. We have examined the effect of cementum components on the attachment of gingival fibroblasts. Cementum was harvested from healthy human and bovine teeth and extracted sequentially in 0.5 M CH3COOH, 4 M guanidine and bacterial collagenase. Fibroblast attachment was measured using 51Cr-labelled human gingival fibroblasts on tissue culture plates previously incubated with cementum components. Results showed that all three extracts mediated fibroblast attachment and attachment was dependent on concentration and incubation time. The attachment activity was not destroyed by digestion with bacterial collagenase or by antibodies to fibronectin and laminin. However, it was inhibited by a peptide containing the amino acid sequence RGD. By gel filtration or HPLC using a DEAE-cellulose column several proteins with attachment activity were fractionated. SDS-polyacrylamide gel electrophoresis revealed that HPLC fraction eluted by 0.2-0.3 M NaCl contained a protein with molecular weight 55 kDa as a major component. This protein was isolated and shown to promote fibroblast attachment, and optimal attachment occurred at a concentration of 2 micrograms/ml. We conclude that cementum contains substances capable of mediating fibroblast attachment and that these substances play an important role in periodontal connective tissue formation and regeneration by facilitating fibroblast attachment to root surfaces.
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PMID:Isolation of a fibroblast attachment protein from cementum. 213 24

Procollagenase of human polymorphonuclear leucocytes was purified to homogeneity using a rapid and reproducible method. The purification procedure included affinity chromatography on zinc chelate Sepharose, ion exchange chromatography on Q-Sepharose fast flow, followed by affinity chromatography on orange Sepharose and finally a gel-permeation step on Sephacryl S-300. It was shown by SDS/PAGE, under reducing conditions, that the latent collagenase of human polymorphonuclear leucocytes consists of a single polypeptide chain with an apparent relative molecular mass of 85,000. Upon deglycosylation by endoglycosidase F digestion, the apparent relative molecular mass of the procollagenase was reduced to 53,000 which is similar to that of the fibroblast enzyme, and indicates a close relationship between both enzymes. Sequence data were determined by direct automated Edman degradation of the purified polymorphonuclear leucocyte procollagenase. The complete sequence of the propeptide region (residue 1-120) was thereby established. The proteolytic activation of the polymorphonuclear leucocyte procollagenase by various enzymes was investigated by determining the N-terminal sequences of the intermediate and final activated forms. Activation by chymotrypsin and cathepsin G led to the active form (Mr 64,000) by cleaving 79 N-terminal residues from the proenzyme. Trypsin activates in a two-step process. Cleavage of 48 N-terminal residues led to a still latent Mr 70,000 species. The final active form (Mr 65,000) was obtained by splitting off 20 additional N-terminal residues.
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PMID:Characterization and activation of procollagenase from human polymorphonuclear leucocytes. N-terminal sequence determination of the proenzyme and various proteolytically activated forms. 215 79

Phenytoin (PHT), a widely used anticonvulsant, has been shown to inhibit bone resorption in rodent organ cultures. The drug also has complex effects on bone metabolism including chronic clinical symptoms of osteomalacia. However, the precise mechanism of PHT action in bone is still unclear. Neutral collagenases that specifically cleave native collagen have been implicated in the turnover of connective tissue. The effect of PHT was assessed on collagenase and gelatinase activities from UMR 106-01 rat osteoblastic osteosarcoma cells. Semiconfluent cells were treated with PHT (50 and 10 micrograms/ml) in the presence of bovine parathyroid hormone, b-PTH-(1-34), at 10(-7) M for 24, 48, 72 and 96 h. The media were assayed following concentration, APMA activation, and incubation with native or denatured [3H]-methyl collagen substrate (approximately 100,000 dpm) at 27 degrees C for 18 h and 35 degrees C for 2 h, respectively. Enzyme activities were presented as primary counts per minute for each time point and calculated as % activity of PTH at 10(-7) M. Parathyroid hormone (10(-7) M) stimulated collagenase activity (approximately 65-fold) and gelatinase activity (approximately 400-fold). PHT (50 micrograms/ml) reduced the PTH-stimulated collagenase activity by 18-53% and the gelatinase activity by 58-72%. SDS PAGE and fluorography following PHT treatment indicated a PHT-induced partial inhibition of PTH-stimulated degradation to alpha A chains of Type I collagen. Phenytoin may inhibit bone resorption through its action on the transcription, synthesis, and/or secretion of the collagenolytic enzymes, collagenase and gelatinase.
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PMID:The effect of phenytoin on collagenase and gelatinase activities in UMR 106-01 rat osteoblastic osteosarcoma cells. 216 99

Saliva collected from subjects with healthy and with diseased periodontium was assayed for collagenase activity by incubation at 25 degrees C with soluble type I, II or III collagen. The degradation products were analyzed by separation in SDS-polyacrylamide gel electrophoresis followed either by protein staining or by exposure of the dried gel to X-ray film in the case of radioactively labeled type I collagen. Collagenase of vertebrate type was detected in the whole saliva of all subjects but not in parotid, sublingual or submandibular fluids. Most of the collagenase was in the soluble fraction of saliva that also contained factors which both activated and inhibited the enzyme. The salivary collagenase resembled the collagenase of human PMNs and gingival sulcular fluid in its molecular size of 70,000 daltons, in its activation by gold thioglucose and in its tendency to degrade types I and II collagens over type III collagen. Before periodontal treatment, the saliva of periodontitis patients had significantly higher collagenase than after treatment. In periodontitis, collagenase existed mainly in the active form, while in the healthy mouths most of the enzyme was latent but could be activated by sulfhydryl reagents or proteolytically with trypsin, and chymotrypsin but not by human plasma kallikrein or plasmin. In some of the samples from untreated periodontitis patients bacterial collagenase may have been present in small quantities. Most of the collagenase in the saliva from all subjects appeared to originate from PMNs entering the oral cavity through the gingival sulcus.
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PMID:Salivary collagenase. Origin, characteristics and relationship to periodontal health. 216 44


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