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)

We have earlier demonstrated that human growth hormone stimulates DNA synthesis and proteoglycan production in cultured chondrocytes. The present study is concerned with the effects of somatostatin and other neuropeptides on cell proliferation by cultured rat rib growth plate chondrocytes. Chondrocytes were isolated from the growth plates by collagenase digestion and cultured as monolayers in multiwell plates. The cells were allowed to attach overnight and subsequently incubated for 24 h under serum-free conditions to establish growth arrest. Somatostatin and other peptides were then added and the cultures were incubated for 18 h. Finally, the cultures were labelled for 6 h with tritiated thymidine in the presence of peptide. For screening purposes, the effect on DNA-synthesis was assayed as incorporation of [3H]-thymidine into acid-insoluble material. For a more exact estimate, parallel cultures were prepared for autoradiography and the fraction of labelled nuclei was determined by counting. Among the peptides we tested (somatostatin, GRF, TRH, SP, mENK, PHI, VIP, hCT) only somatostatin had any discernible effect on DNA synthesis, with an apparently optimal effect at 10 fM. This concentration is well within the range found in various tissues in vivo and suggests a physiological role for somatostatin in chondrocyte growth regulation. Further experiments are required, however, to clarify by which mechanism somatostatin influences the cells and whether the peptide interacts with other growth factors such as the IGFs.
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PMID:Stimulative effect of somatostatin on cell proliferation in cultured chondrocytes. 288 5

When calf aortic tissue, preincubated under organ culture conditions in the presence of [35S]sulfate, was submitted to a sequential collagenase and elastase digestion and guanidinium chloride extraction, the bulk of proteoheparan sulfate was obtained in the elastase fraction. Ion-exchange chromatography on DEAE-cellulose of the elastase digest under dissociative conditions yielded a proteoglycan fraction that contained heparan sulfate as the sole glycosaminoglycan. The proteoheparan sulfate fraction was resolved into a high-molecular-mass (P-HS 1) and a low-molecular-mass (P-HS 2) fraction by gel filtration on Sephacryl S-400. P-HS 1 has a Mr of 175,000 and possesses four heparan sulfate side-chains (Mr 32,000) covalently bound to the protein core via a galactose- and xylose-containing polysaccharide-protein binding region. The protein core (Mr 38,000), which was obtained after deglycosylation of PG-HS 1 with trifluormethane sulfonic acid, contained in addition a few N-glycosidically linked oligosaccharide units representing a complex type with terminal neuraminic acid residues. P-HS 2 is a single-chain peptidoheparan sulfate of Mr of 38,000 containing one heparan sulfate chain (Mr 32,000) linked to a polypeptide (Mr 6000). The ratio of specific radioactivities of P-HS 1 and P-HS 2 was 1:0.66.
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PMID:Isolation and characterization of two proteoheparan sulfate species of calf arterial tissue. 296 81

In osteoarthritis, despite increased matrix synthesis, there is a reduction of both major matrix components, proteoglycan and collagen. This study suggests that this is the result of enhanced degradative activity intrinsic to the cartilage. Because osteoarthritis is a focal disease, histologic controls were used to measure the severity in different areas of the cartilage and in different specimens. Neutral proteoglycanase and collagenase were both described in human cartilage, and their levels matched the severity of the disease as did acid phosphatase, a marker of lysosomal enzymes. Articular cartilage collagenase has an inhibitor in the cartilage and has negligible activity in normal cartilage. This was found not to be a lysosomal enzyme. A model of osteoarthritis was studied and found to have the same biochemical pattern as human disease. Using this method, inhibitors of degradative enzymes were used as a treatment. The chelator of metallic cations EDTA was found to have a significant effect on reduction of degradative enzyme activity and altered the arthritic process.
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PMID:Degradative enzyme systems in osteoarthritic cartilage. 298 65

Rabbit synovial fibroblasts induced to undergo a specific switch in gene expression by agents that alter cell morphology secreted the neutral proteinase precursor procollagenase (apparent Mr of 53,000 and 57,000). A major Mr = 51,000 polypeptide that was always induced coordinately with procollagenase has now been identified as the proenzyme form of a metal-dependent proteinase active at neutral pH. We have named this proteinase stromelysin. Prostromelysin and procollagenase were the most prominent [35S]methionine-labeled secreted proteins of the induced fibroblasts. By the use of casein degradation as an assay for enzyme activity, stromelysin was isolated with high yield from the conditioned culture medium of 12-O-tetradecanoylphorbol 13-acetate-treated fibroblasts and migrated as an active form of Mr = 21,000 that was immunologically identical to the proteoglycan-degrading proteinase purified from rabbit bone. Immunoglobulin G from antiserum raised to purified rabbit bone proteoglycanase immunoprecipitated the Mr = 51,000 proenzyme form from conditioned medium of induced rabbit cells and also immunoprecipitated an Mr = 55,000 polypeptide from induced human fibroblasts. When rabbit prostromelysin was activated by trypsin or 4-aminophenylmercuric acetate, the proenzyme was converted to an active form of Mr = 41,000. During the course of the purification, prostromelysin was converted to an additional activatable form of Mr = 35,000 and additional active forms of Mr = 21,000-25,000, which had related peptide maps distinct from collagenase. All of these forms were immunologically cross-reactive. Purified stromelysin degraded casein, cartilage proteoglycans, fibronectin, alpha 1-proteinase inhibitor, and immunoglobulin G2a and had limited activity on laminin, elastin, type IV collagen, and gelatin, but did not degrade type I collagen. Stromelysin was inhibited by EDTA, 1,10-phenanthroline, and the specific glycoprotein tissue inhibitor of metalloproteinases isolated from human amniotic fluid and was therefore classified as a metalloproteinase.
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PMID:Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. Biosynthesis, isolation, characterization, and substrates. 299 74

Two forms of interleukin 1 (IL-1) were purified to homogeneity from the culture supernatants of pig buffy coat leukocytes stimulated with concanavalin A. The two proteins had identical Mr of 21,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but one, which had previously been purified as a cartilage-resorbing protein, had pI 5 (IL-1/5) and the other, pI 8.3 (IL-1/8). After initial gel filtration and separation by chromatofocusing IL-1/5 was purified by chromatography on hydroxyapatite and the ion exchangers, Mono S and Mono Q; IL-1/8 was purified by chromatography at pH 4.0 and pH 6.4 on Mono S. Purification was monitored by a cartilage proteoglycan release assay and both proteins had a final specific activity approximately 10(5) times that of the leukocyte culture medium. Medium conditioned by cells from 200 L of blood yielded approximately 15 micrograms of IL-1/5 and 50 micrograms IL-1/8. IL-1/8 augmented mouse thymocyte proliferation, stimulated synovial fibroblasts to produce prostaglandin E and latent collagenase, and was pyrogenic upon intracerebroventricular injection into rabbits. IL-1/5 has previously been shown to possess all these activities. An antiserum to each IL-1 was raised in rabbits. Each antiserum inhibited its respective IL-1 in a cartilage bioassay and stained it upon Western blotting. Neither antiserum inhibited or stained the other IL-1. We conclude that pig leukocytes make two immunologically distinct forms of IL-1 that have identical Mr, demonstrate the same range of biological activity, but differ in isoelectric point.
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PMID:Pig interleukin 1. Purification of two immunologically different leukocyte proteins that cause cartilage resorption, lymphocyte activation, and fever. 299 35

Pig synovial and human skin fibroblast procollagenases were treated with highly purified tryptase, the major proteinase of human mast cells, to determine whether this trypsin-like proteinase could activate the latent form of collagenase and so be involved in connective tissue breakdown. No significant activation of either human or pig procollagenase was found, but the highest concentration of tryptase partially destroyed procollagenase. Tryptase did not degrade type I collagen or proteoglycan. These data indicate that human mast cell tryptase does not contribute to connective tissue breakdown via procollagenase activation or via proteoglycan degradation.
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PMID:Human lung mast cell tryptase fails to activate procollagenase or degrade proteoglycan. 299 69

Human bone cell cultures were established by maintaining collagenase-treated bone fragments in low Ca++ medium. The resulting cell cultures exhibited a high level of alkaline phosphatase activity and produced a significant increase in intracellular cAMP when exposed to the 1-34 fragment of human parathyroid hormone. With continued culture, the cells formed a thick, extracellular matrix that mineralized when cultures were provided daily with normal levels of calcium, fresh ascorbic acid (50 micrograms/ml) and 10 mM beta-glycerol phosphate. Biosynthetically, these cells produced type I collagen (without any type III collagen), and the bone-specific protein, osteonectin. In addition, the cells produced sulfated macromolecules electrophoretically identical to those positively identified as the bone proteoglycan in parallel cultures of fetal bovine bone cells. This technique provides a useful system for the study of osteoblast metabolism in vitro.
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PMID:Human bone cells in vitro. 299 72

Pretreatment of articular cartilage with a highly purified collagenase in the presence of selected protease inhibitors allowed the extraction under nondissociative conditions of 65% of the tissue hexuronate. Extracted proteoglycans were purified by two successive equilibrium centrifugations in Cs2SO4 and CsCl, respectively, and then characterized by their sedimentation properties. The use of labeled proteoglycan preparations demonstrated that no detectable degradation was introduced by the new extraction procedure. When applied to growth cartilage of rachitic rats the sedimentation profile of the purified proteoglycans was practically identical to that of the proteoglycan molecules recovered by micropuncture-aspiration. Proteoglycans were extracted from normal articular cartilage of rabbits and dogs with either the new procedure or 4.0 M guanidine HCl. The purified aA1 and A1 preparations were characterized by their sedimentation properties. The aA1 contained a higher proportion of aggregates which sedimented as two distinctive populations of molecules. This bimodal distribution of the aggregates was never observed in the A1 preparations even when the dissociative extraction was performed after collagenase pretreatment of cartilages. The two extraction procedures, however, extracted the same proteoglycan monomers since the aA1-D1 and A1-D1 preparations had similar biochemical composition and g(s) distribution functions. These observations and additional in vitro aggregation studies suggested that the differences in the size and proportion of aggregates between the aA1 and A1 preparations result from a more efficient recovery of link glycoproteins in nondissociative extractions that could have determined two structurally different hyaluronate molecules.
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PMID:Characterization of the proteoglycans recovered under nondissociative conditions from normal articular cartilage of rabbits and dogs. 300 3

Proteoglycans were isolated from young and mature human articular cartilage 4 different ways: by direct extraction with 4M guanidine hydrochloride (GuHCl); after digestion of the residue from this first extraction with collagenase, by extraction with 4M GuHCl; associatively with 0.5M GuHCl after digestion of the cartilage with collagenase; and dissociatively with 4M GuHCl after digestion of the cartilage with collagenase. The structural properties of these proteoglycans were compared. Proteoglycan aggregates and monomers isolated from second extractions and from young cartilage were of larger hydrodynamic size than proteoglycans isolated from first extractions and mature cartilage, respectively. The same applied to the chondroitin sulfate chain lengths of these proteoglycans. The proteoglycan fraction from second extractions of cartilage contained a larger proportion of monomers than the fraction from first extractions. Associative extraction of mature collagenase-digested cartilage yielded mainly proteoglycan monomers, whereas an appreciable amount of proteoglycan aggregate was also liberated from young collagenase-digested cartilage. Our results indicate that, because of their larger size, proteoglycans from second extractions of cartilage are more entrapped in the collagen network. These large proteoglycans can only be liberated from the matrix after extraction of the smaller proteoglycans, followed by digestion of the residue with collagenase. This indicates that proteoglycans overlap and entangle with the collagen and protect it from degradation by collagenase.
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PMID:Heterogeneity of proteoglycans extracted before and after collagenase treatment of human articular cartilage. I. Physical properties related to age. 302 Nov 76

Proteoglycans (A1 fractions) were extracted with 4M guanidine hydrochloride (GuHCl) from human articular cartilage samples of a wide age range. Distinctions were made between hip and knee, and upper and lower layers. The residues of these extractions were digested with purified collagenase, and a second extraction with 4M GuHCl was performed, which yielded appreciable amounts of proteoglycans. When proteoglycans from second extractions were compared with those from first extractions, the following changes were observed: an increase in chondroitin sulfate; a relative decrease in keratan sulfate; a decrease in protein content; and a decrease in the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate. The same changes were found when nonaggregating proteoglycans were compared with proteoglycan aggregates, when proteoglycans from young cartilage were compared with those from mature cartilage, when proteoglycans from knee cartilage were compared with those from hip cartilage, and when proteoglycans from upper layers of cartilage were compared with those from deeper layers. It is suggested that the differences found between first and second extractions of cartilage, between upper and lower layers of cartilage, and between knee and hip cartilage are caused by variations in the relative amount of nonaggregating proteoglycans and/or variations in proteoglycan size.
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PMID:Heterogeneity of proteoglycans extracted before and after collagenase treatment of human articular cartilage. II. Variations in composition with age and tissue source. 302 Nov 77


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