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)

Bone proteins in alveolar bone of mandibles from young adult rabbits (3-month-old) were extracted with 4.0 M guanidine hydrochloride (GuHCl), followed by 0.5 M ethylenediaminetetraacetate, and again with 4.0 M GuHCl (G2-ext). The proteins in the G2-ext were fractionated on a gel-filtration column, followed by an anion-exchange column in the presence of 7.0 M urea. A 28-kDa protein was isolated from the G2-ext. The purified 28-kDa protein showed intense staining with silver on SDS-PAGE slab-gel under reducing conditions. This protein was digested with bacterial collagenase, and a 19-kDa fragment appeared on the gel. However, the protein was not susceptible to reduction with cyanogen bromide. The protein did not bind to hydroxyapatite crystals in the presence of 7.0 M urea, and also did not bind to some lectins. On SDS-PAGE under non-reducing conditions, the protein migrated as two bands; a new band appeared at approximately the 85-kDa region in addition to the original 28-kDa band. The amino acid compositions of the protein were similar to those of the alpha 1-pN-propeptide of type I procollagen obtained from other tissues.
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PMID:Characteristics of a 28-kDa collagenous protein extracted with guanidine from EDTA-demineralized rabbit alveolar bone. 815 87

The present study was undertaken to identify the cartilage matrix molecules that are bound with intermolecular disulfide bonds to IgG and serum albumin molecules recovered from the articular cartilage of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). The cartilage specimens were extracted sequentially with three changes of neutral buffer, three changes of 6 M guanidine hydrochloride and then partially degraded with bacterial collagenase. The extracted IgG and albumin, along with matrix molecules bound to these proteins, were isolated with affinity chromatography using antibodies to IgG or human serum albumin conjugated to agarose beads. The isolated materials were characterized with sodium dodecyl sulfate polyacrylamide gel electrophoresis and transfer blotting, using specific antibodies to IgG, albumin, and proteoglycans. In the isolated materials, heteropolymers with IgG or albumin were identified. These polymers contained keratan sulfate and less frequently chondroitin-4-sulfate and chondroitin-6-sulfate. These findings identified the keratan sulfate rich proteoglycans, prevalent at the surface of joint cartilage, as the most common cartilage matrix molecules that are covalently bound to IgG or to serum albumin by disulfide bonds in the articular cartilage of patients with RA or OA.
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PMID:Immunoglobulin G and serum albumin isolated from the articular cartilage of patients with rheumatoid arthritis or osteoarthritis contain covalent heteropolymers with proteoglycans. 823 91

The structure of the extension peptides retained on the tissue form of type V collagen molecules was determined. Type V collagen alpha chains containing extension peptides were extracted from fetal calf skin and bone by 4 M guanidine-HCl and 0.5 M acetic acid, respectively. Collagens present in both extracts were fractionated by sodium chloride precipitation. The collagen alpha(V) chains were then resolved by reverse-phase high performance liquid chromatography. The N-terminal extension peptides were characterized by direct sequence analysis after deblocking with pyroglutamate amino-peptidase and analysis of the products of digestion by bacterial collagenase, chymotrypsin, V8 protease and endoproteinase Lys-C. The results showed that the retained extension peptides on type V collagen molecules in the extracellular matrix of skin and bone were amino-propeptides and that the alpha 2(V) chain retains an intact amino-propeptide while the alpha 1(V) chain appears to be partially processed. The extended alpha 1(V) chain isolated from fetal calf bone gave an identical amino-terminal sequence to that of the alpha 1(V) chain isolated from fetal calf skin, suggesting that a specific enzyme may be involved in processing the alpha 1(V) amino-propeptide.
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PMID:Structural analysis of the extension peptides on matrix forms of type V collagen in fetal calf bone and skin. 826 15

The tectorial membrane is a gel-like, acellular connective tissue overlying the microscopic organ of Corti--the auditory sensory structure. It is instrumental in the sound-synchronous deflection of the stereocilia of the hair cells, a central event in auditory transduction. It is well established that collagen, primarily type II, constitutes the major protein of the tectorial membrane, with smaller amounts of types IX and XI also present. However, conclusive information on the proteoglycans in this structure is lacking. Tectorial membranes were extracted with a 4 M guanidine--HCl solvent, and proteoglycans isolated after ethanol precipitation and collagenase treatment. A colorimetric assay based on the binding of the cationic dye safranin O to glycosaminoglycans, in combination with enzymatic techniques, detected significant amounts of chondroitin sulfate and keratan sulfate (0.29 and 0.17% on a wet weight basis, respectively). Agarose-polyacrylamide electrophoresis of chondroitinase-digested samples revealed a core protein with a similar molecular mass to that of the large cartilage proteoglycan aggrecan. This proteoglycan reacted with the antibody 3-B-3 (recognizing modified chondroitin 6-sulfate linkage region oligosaccharides). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several low molecular mass proteins which reacted with 5-D-4, specific for keratan sulfate, one of which showed characteristics of fibromodulin. Comparison of the quantitative aspects of various connective tissue components of tectorial membrane with other type II collagen-containing structures revealed that this tissue resembles highly hydrated cartilage.
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PMID:Uronic acid-containing glycosaminoglycans and keratan sulfate are present in the tectorial membrane of the inner ear: functional implications. 827 27

The synthesis of a series of N-phosphonalkyl dipeptides 6 is described. Syntheses were devised that allowed the preparation of single diastereoisomers and the assignment of stereochemistry. The compounds were evaluated in vitro for their ability to inhibit the degradation of radiolabeled collagen by purified human lung fibroblast collagenase. Several of the compounds were potent collagenase inhibitors and were at least 10-fold more potent than their corresponding N-carboxyalkyl analogues. Activity was lost when the phosphonic acid group P(O)(OH)2 was replaced by the phosphinic acid groups P(O)(H)(OH) and P(O)(Me)(OH). At the P1 position, (R)- or (S)-alkyl groups, especially ethyl and methyl (e.g., 12a,b, 52a,b, and 53a,b), or an (R)-phenethyl moiety (55a) conferred high potency (IC50 values in the range 0.23-0.47 microM). (S)-Stereochemistry was preferred for the P1' isobutyl side chain. Structure-activity relationships were also investigated at the P2' site, and interestingly, compounds with basic side chains, such as the guanidine 57a, were equipotent with more lipophilic compounds, such as 52a. As with other series of collagenase inhibitors, potency was enhanced by introducing bicyclic aromatic P2' substituents. The most potent phosphonic acid of the series was the bicyclic aromatic P2' tryptophan analogue 59a (IC50 0.05 microM).
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PMID:Synthesis of novel N-phosphonoalkyl dipeptide inhibitors of human collagenase. 828 90

1. In rat whole portal veins, guanabenz (100 nM to 10 microM) and antazoline (100 nM to 100 microM) each increased the amplitude, frequency and duration of spontaneous contractions. In addition, guanabenz (30 microM) and antazoline (30 microM) each antagonized the ability of levcromakalim (3 nM to 10 microM) to inhibit the spontaneous contractions of this tissue. 2. Whole-cell voltage-clamp recordings were made from freshly-isolated rat portal vein cells dispersed by a collagenase/pronase enzyme treatment. The ability of several agents (antazoline, cirazoline, clonidine, guanabenz and phentolamine, each containing an imidazoline or guanidine moiety), to modulate potassium (K) currents and to inhibit the actions of levcromakalim was investigated. 3. Antazoline, cirazoline, clonidine, guanabenz and phentolamine (each at a concentration of 30 microM) had little effect on control non-inactivating currents but inhibited the delayed-rectifier current, IK(V). 4. Levcromakalim (1 microM) induced a non-inactivating current, IK(ATP), and also inhibited the delayed rectifier current, IK(V). 5. Glibenclamide (1 microM) had no effect on control delayed rectifier or non-inactivating currents, but it inhibited the simultaneous induction of IK(ATP) and reduction of IK(V) produced by levcromakalim (1 microM). 6. Antazoline, cirazoline, clonidine and guanabenz (each at a concentration of 30 microM) prevented the induction of IK(ATP) by levcromakalim (1 microM). Phentolamine (30 microM) and clonidine (30 microM) each inhibited the IK(ATP) generated by levcromakalim (1 microM). 7. It is concluded that a variety of agents which possess either an imidazoline (antazoline, cirazoline, clonidine and phentolamine) or a guanidine (guanabenz) moiety within their structure inhibit the delayed rectifier current, IK(V). This action may thus be mediated via a so-called non-adrenoceptor imidazoline binding site. Furthermore, the ability of these ligands to inhibit IK(V) and to antagonize both the induction of IK(ATP) and the vasorelaxation produced by levcromakalim is consistent with the view that the channel (KATP) which underlies IK(ATP) is a voltage-insensitive state of the delayed rectifier K-channel (Kv).
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PMID:Antagonism of levcromakalim by imidazoline- and guanidine-derivatives in rat portal vein: involvement of the delayed rectifier. 830 1

Asymmetric forms of acetylcholinesterase (AChE) are thought to be the predominant forms of this enzyme at vertebrate neuromuscular junctions where they attach to the synaptic basal lamina via a collagen-like tail. High salt and heparin-containing buffers are capable of solubilizing asymmetric AChE molecules from skeletal muscle; however, detachment of AChE specifically from synaptic basal lamina using these procedures has not been demonstrated. To determine whether AChE can be solubilized from mature neuromuscular junctions, adult quail muscle fibers were extracted with buffered detergent solutions containing either 0.05 M NaCl, 1 m NaCl, 0.5-2 mg/ml heparin, 8 M urea, or 4 m guanidine HCl, and the remaining AChE molecules were localized by indirect immunofluorescence. Analysis of extracted AChE oligomeric forms showed that low salt buffers containing heparin and high salt buffers were capable of solubilizing substantial amounts of catalytically active collagen-tailed AChE, whereas none of these buffers were capable of detaching AChE from synaptic basal lamina. In contrast, digestion with purified collagenase detached asymmetric forms from the non-extractable fraction and removed the AChE from the neuromuscular junctions. Parallel experiments using rat gastrocnemius muscle and enzyme histochemistry to detect AChE gave similar results. These studies indicate that the junctional AChE molecules are firmly attached to the extracellular matrix and that all the conventional extraction buffers used to solubilize the asymmetric collagen-tailed forms of AChE are incapable of detaching this enzyme from the synaptic basal lamina.
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PMID:Localization of "non-extractable" acetylcholinesterase to the vertebrate neuromuscular junction. 836 Jan 97

The mechanisms of organic matrix breakdown in the root caries process are not well understood. Therefore, the combined and separate effects of lactic acid and proteolytic enzymes on the degradation of human dentin collagen, glycoproteins, proteoglycans and phosphoproteins were investigated in the present study. Dentin powder was pretreated with lactic acid (pH 4.0), distilled and deionized (dd) water (pH 7.0) and EDTA/guanidine HCl (pH 7.4) for 24 h. Pellets of acid- or dd water-pretreated dentin powder were washed, dried, and then treated with trypsin, bacterial or mammalian tissue collagenase, or control buffer for 3 h. The released dentin proteins were analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify degraded type I collagen, proteoglycans, glycoproteins and phosphoproteins. All water and acid pretreatment and enzyme treatment groups demonstrated two collagen fragment bands with molecular weights at approximately 79 kD. Further studies showed that the 79 kD proteins from acid-pretreated dentin collagen were degraded by tissue collagenase, suggesting that endogenous collagenase may be involved in the degradation of root dentin collagen. Dentin proteoglycans were detectable in all the treatment groups by protein slot blotting. Relatively few distinct glycoproteins and proteoglycans, and no phosphoproteins were detected by immunoblotting. Results from this study suggest that both acids and proteolytic enzymes from either host or microbial origin are important in the degradation of human dentin matrix and the mechanisms involved in the release of various noncollagenous proteins may be different.
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PMID:Effect of lactic acid and proteolytic enzymes on the release of organic matrix components from human root dentin. 855 53

Recombinant interstitial collagenase (rMMP-1) forms insoluble inclusion bodies when over-expressed in Escherichia coli. We surveyed conditions for renaturation of purified rMMP-1 in 6 M guandine hydrochloride (GdnHCl) and found that optimal folding occurred when the denatured protein was diluted at 4 degrees C in approximately 2 M guanidine HCl, 20% glycerol, 2.5 mM reduced and oxidized glutathione, and 5 mM CaCl2, followed by buffer exchange to remove denaturant and thiols. The circular dichroism spectrum and catalytic constants of the refolded enzyme were similar to those of native MMP-1. The propeptide, which comprises approximately 20% of the mass of proMMP-1, was not required for folding to a functional enzyme. Size exclusion chromatography and spectroscopic measurements at intermediate [GdnHCl] revealed two intermediate folding states. The first, observed at 1 M GdnHCl, had a slightly larger Stokes' radius than the folded protein. CD and fluorescence analysis showed that it contained ordered tryptophan residues with a higher quantum yield than the fully folded state. The second intermediate, which appeared between 2 and 4 M GdnHCl, exhibited properties consistent with the molten globule, including secondary structure, lack of ordered tryptophan, exposed hydrophobic binding sites, and a Stokes' radius between that of the folded and unfolded states.
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PMID:Characterization of folded, intermediate, and unfolded states of recombinant human interstitial collagenase. 862 83

An enzyme-linked immunosorbent assay (ELISA) was developed that detects PrP(Sc) in crude extracts from brain and spleen tissue of scrapie-affected mice with high sensitivity and specificity. Brain tissue was homogenized in 8% Zwittergent 3-12 and 0.5% Sarkosyl. The homogenate was treated with collagenase and DNase I and then subjected to proteinase K digestion. Precipitates containing PrP(Sc) were obtained by ultracentrifugation. Spleen tissue was homogenized in 4% Triton X-100 and 0.5% Sarkosyl, and the homogenate was treated firstly with collagenase and DNase I, and secondly with proteinase K. PrP(Sc) was then extracted with 6.25% Sarkosyl and precipitated through salting-out with NaCl and by ultracentrifugation. When PrP(Sc) was dissolved in 3-4 M guanidine thiocyanate and adsorbed to microtiter plates, strong and specific reactions to the formation of antigen-antibody complexes could be detected by ELISA. The sensitivity of PrP(Sc)-detection for this ELISA, as measured by serial dilution of scrapie material in tissue homogenates from uninfected animals, was equal or higher than that attained by Western blot. This ELISA is more rapid than Western blot and seems to be more suitable for screening large numbers of animals. It also has potential application for the diagnosis of the transmissible spongiform encephalopathies.
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PMID:Sensitive enzyme-linked immunosorbent assay for detection of PrP(Sc) in crude tissue extracts from scrapie-affected mice. 907 66


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