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)

1. Catecholamines, adenosine, gonadotrophins, vasoactive intestinal peptide (VIP) and E-series prostaglandins all elicit K+ currents in follicle-enclosed Xenopus oocytes. Evidence suggests that cyclic nucleotides act as intracellular messengers in the activation of this K+ conductance. Muscarinic agonists and some divalent cations (e.g. Co2+, Mn2+, Ni2+ and Cd2+) elicit slow oscillatory Cl- currents, which are activated through hydrolysis of inositol phospholipids and mobilization of intracellular calcium by inositol phosphates. 2. We investigated whether these membrane current responses were generated in the oocyte itself or in enveloping follicular cells which are coupled to the oocyte by gap junctions. Oocytes were defolliculated, either enzymatically using collagenase, or by manual dissection combined with rolling over poly-L-lysine-coated slides. Removal of follicular cells was checked using scanning electron microscopy. Membrane current responses of defolliculated oocytes were compared with responses seen in follicle-enclosed oocytes taken from the same ovary. 3. The K+ responses evoked by all the various hormones/neurotransmitters were either drastically reduced (greater than 90%) or abolished by defolliculation. K+ currents generated by the adenylate cyclase activator forskolin and by intraoocyte injection of adenosine 3',5'-cyclic monophosphate (cyclic AMP), or guanosine 3',5'-cyclic monophosphate were similarly reduced in defolliculated oocytes. In contrast, oscillatory Cl- currents to acetylcholine and divalent cations were selectively preserved through defolliculation. 4. Injection of cyclic AMP (1-20 pmol) into defolliculated oocytes had little or no effect on oscillatory Cl- currents elicited by ACh. However, the calcium-dependent transient Cl- current, activated by depolarization of the oocyte membrane, was consistently potentiated (100-900%) by injections of cyclic AMP (1-10 pmol). 5. These experiments suggest that cyclic nucleotide-activated K+ currents arise essentially in follicular cells and are monitored within the oocyte through electrical coupling by gap junctions. Oscillatory Cl- responses evoked by ACh and divalent cations are produced largely or wholly in the oocyte itself.
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PMID:Effects of defolliculation on membrane current responses of Xenopus oocytes. 255 77

The major collagenolytic proteinase present in the culture filtrate of Bacillus cereus (strain Soc 67, isolated from the human oral cavity) has been purified to homogeneity by a procedure that comprised concentration of ultrafiltered growth medium on a Millipore PTTK00005 membrane, precipitation with ammonium sulfate, gel permeation chromatography, chromatofocusing, fast protein liquid chromatography on an anion-exchange column, and finally fast protein liquid chromatography on a gel column. The enzyme hydrolyzed, with decreasing rates, phenylazobenzyloxy-carbonyl-L-Pro-L-Leu Gly-L-Pro-D-Arg (PZ-PLGPA), furylacrylolyl-L-Leu-Gly-L-Pro-L-Ala, and furylacryloyl-L-Phe-Gly-Gly, while furylacryloyl-Gly-L-Leu-NH2 was not hydrolyzed. The enzyme degraded soluble and insoluble collagens, Azocoll and gelatin. Bradykinin was hydrolyzed at a high rate at the Phe-Ser bond. The enzyme was sensitive to pyrophosphate, L-cysteine, and L-histidine and could be totally inactivated in the presence of metal chelators. The enzyme contains 1 mol of Zn/mol and the hydrolysis of PZ-PLGPA is slightly increased by Ca2+. The enzyme is readily inhibited by heavy metal cations, but Cu2+ and Ni2+ affected the catalysis in opposite ways: increasing levels of Cu2+ decreased the affinity of the enzyme for PZ-PLGPA, whereas Ni2+ had no effect. The effect of Cu2+ also depended on the pH and type of buffer used. Detailed chemical modification experiments suggested that the active site of the enzyme contains at least 1 tyrosyl and 1 lysyl residue, and 1 carboxyl group. The enzyme was not sensitive to sulfhydryl reagents and thiols did not activate the enzyme. The modification studies were unable to reveal active histidyl residues. The ability of the enzyme to hydrolyze PZ-PLGPA, furylacryloyl-L-Leu-Gly-L-Pro-L-Ala, furylacryloyl-L-Phe-Gly-Gly, and various collagenous materials, its inactivity toward furylacryloyl-Gly-L-Leu-NH2, and the results from the chemical modification studies suggest that the B. cereus (Soc 67) collagenolytic enzyme can be regarded as a true collagenase which resembles the Clostridium histolyticum collagenase(s).
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PMID:Purification and properties of an extracellular collagenolytic protease produced by the human oral bacterium Bacillus cereus (strain Soc 67). 304 Jul 51

The effects of various calcium-channel blockers on androgen production by collagenase-dispersed mouse testicular interstitial cells were investigated. Cobalt caused a dose-dependent inhibition of the maximum rate of luteinizing hormone (LH)-stimulated androgen production without altering the concentration of LH required for half maximum stimulation (EC50). Nickel and manganese also inhibited LH-stimulated steroidogenesis but were less potent than cobalt. The major site at which cobalt treatment inhibited steroidogenesis was beyond cAMP formation and before 3 beta-hydroxysteroid dehydrogenase. This conclusion was based on the observation that cobalt inhibited dibutyryl cAMP-stimulated androgen production but did not affect protein synthesis and pregnenolone-supported androgen production. Androgen production was unaffected by the organic calcium-channel blockers verapamil and the (+) and (-) enantiomers of D600 at concentrations less than 0.1 mM. At a concentration of 0.1 mM the organic calcium-channel blockers inhibited LH- and dibutyryl cAMP-stimulated androgen production. Unlike cobalt, the organic calcium-channel blockers also inhibited pregnenolone-supported androgen production and reduced the rate of protein synthesis. Similarities between the effects of cobalt in the present study and previous reports of the effects of reduced extracellular calcium concentrations on androgen production suggest that cobalt inhibits androgen production as a result of its ability to block calcium influx. The calcium channels involved in the steroidogenic process appear, however, to be relatively insensitive to the organic calcium-channel blockers.
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PMID:Effects of the calcium-channel blockers cobalt, verapamil, and D600 on Leydig cell steroidogenesis. 630 61

Isolated chick embryo tendon cells were used in [14C]proline and [14C]lysine labelling experiments to investigate the effect of divalent cations on collagen biosynthesis with a special reference to prolyl hydroxylation and lysyl modifications. The following metals were studied by adding them to the incubation medium of the cells: Ca2+, Cd2+, Co2+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+ and Zn2+. Zn2+ caused a potent reductin in collagen prolyl hydroxylation with a concomitant increased cellular retention of collagenase-digestible material. These effects were detectable even at physiological concentrations. At the same concentrations of Zn2+, lysyl hydroxylation was considerably less inhibited than prolyl hydroxylation, and the extent of hydroxylysyl glycosylation was even increased. Co2+ was also an efficient inhibitor of collagen prolyl hydroxylation, but at concentrations ten times higher than those of Zn2+. In the presence of other metal ions, no or only up to 10% inhibition of prolyl hydroxylation was noted even at those concentrations at which [14C]proline incorporation into the protein was decreased. However, an increased cellular retention of collagen was detected in the presence of some metal ions. No reduction in lysyl hydroxylation was found in the presence of Ca2+ or Mg2+.
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PMID:Effects of divalent cations on collagen biosynthesis in isolated chick embryo tendon cells. 677 78

We examined intracellular Ca2+ responses of nasal gland acinar cells in order to clarify cellular responses and molecular events with regard to the regulatory mechanism of nasal secretion. The acinar cells of the serous gland, in the guinea-pig nasal septum, were obtained by meticulous and selective dissection with minimal contamination of epithelial lining cells followed by collagenase treatment. The dispersed acini were incubated in an oxygenated solution supplemented with fura -2 acetoxymethyl ester and the intracellular Ca2+ concentration ([Ca2+]i) was measured using fluorescence ratio imaging microscopy. The application of acetylcholine (ACh) to the nasal gland acinar cells induced an initially rapid increase in [Ca2+]i followed by a sustained plateau. The increase in [Ca2+]i induced by ACh was concentration-dependent and ranged from 10(-8) to 10(-5) M. The intracellular Ca2+ response was completely inhibited by atropine, indicating the presence of muscarinic cholinergic receptors. Removal of external Ca2+ with addition of EGTA resulted in a transient increase without a sustained phase. The sustained phase of the [Ca2+]i increase induced by ACh was inhibited by Ni2+, but not by nifedipine. The initial phase seems to be due to mobilization from cytosolic Ca2+ stores while the subsequent sustained phase is dependent on the influx of external Ca2+ ions sensitive to Ni2+. We have demonstrated that increasing the Ca2+ gradient by elevating external Ca2+ accelerates Ca2+ entry, and that depolarization of cells due to elevated external K+ attenuates Ca2+ entry. These findings suggest that the Ca2+ entry process in nasal gland acinar cells is dependent on the electrochemical gradient across the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Intracellular Ca2+ response induced by acetylcholine in the submucosal nasal gland acinar cells of guinea pigs]. 760 11

We examined intracellular Ca2+ responses of the nasal gland acinar cells to clarify cellular responses and molecular events with regard to the regulatory mechanism of the nasal secretion. The acinar cells of the serous gland in the nasal septum of guinea pigs were obtained by meticulous and selective dissection with minimal contamination by epithelial lining cells after collagenase treatment. The dispersed acini were incubated in the oxygenated solution supplemented with fura 2 acetoxymethyl ester, and the intracellular Ca2+ concentration ([Ca2+]i) was measured by fluorescence ratio imaging microscopy. The application of acetylcholine (ACh) to the cells induced an initially rapid increased [Ca2+]i followed by a sustained plateau. The increase in [Ca2+]i induced by ACh was concentration dependent, ranging between 10(-8) and 10(-4) M. The [Ca2+]i response was completely inhibited by atropine, further indicating the involvement of muscarinic cholinergic receptors. Removal of external Ca2+ with addition of EGTA resulted in a transient increase without a sustained phase, and the transient increase was abolished by the intracellular Ca2+ antagonist 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, indicating that this increase in [Ca2+]i was due to release from internal stores. The initial peak was not altered by changes in external pH, addition of adenosine 3',5'-cyclic monophosphate (cAMP), nor addition of phorbol 12-myristate 13-acetate (PMA) but was augmented by external K(+)-induced depolarization, suggesting that the transient increase was due to a changing in the binding affinity to inositol 1,4,5-trisphosphate. The sustained Ca2+ entry induced by ACh was inhibited by Ni2+, but not by nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Intracellular Ca2+ responses induced by acetylcholine in the submucosal nasal gland acinar cells in guinea pigs. 790 Aug 16

The active N-terminal domain of the mouse tissue inhibitor of metalloproteinases-1 is a 14.1-kDa polypeptide with three disulfide bonds. When expressed using a T7 system in Escherichia coli, this truncated protein, in contrast to the WT protein, was found only in trace amounts in the cell. However, when the coding sequence was placed downstream of a 60-bp sequence that encoded an in-frame histidine-rich "tag," the fusion product (NF.TIMP*His) was expressed in considerably increased abundance. WT.TIMP-1 was expressed in abundance with or without the tag. The mRNAs encoding the various forms of TIMP were present in similar amounts in all four cases. NF.TIMP*His, renatured and purified on a nickel affinity column, was found to be about 10-fold less effective than native human TIMP-2 at inhibiting cleavage of collagen type I by human fibroblast collagenase. A thrombin cleavage site in the tag was susceptible to cleavage by low levels of a contaminating proteinase.
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PMID:Presence of an N-terminal polyhistidine tag facilitates stable expression of an otherwise unstable N-terminal domain of mouse tissue inhibitor of metalloproteinase-1 in Escherichia coli. 963 17

It has been shown that small proteoglycans containing leucine-rich repeats in their core proteins can form complexes with TGF-beta. Decorin, a ubiquitously found molecule of the extracellular matrix, is the best-studied example. Therefore, binding domains on its core protein were investigated using recombinant decorin fragments generated as fusion proteins in prokaryotes. The peptide Leu155-Val260 immobilized by the polyhistidine tag on a nickel chelate column bound TGF-beta1 and -beta2 almost as effectively as the largest fragment (Asp45-Lys359) studied. Other peptides were less effective. For the two peptides Asp45-Lys359 and Leu155-Val260 dissociation constants in the nanomolar range for high-affinity binding sites were calculated in a solid-phase assay with immobilized TGF-beta2. Peptide Asp45-Lys359 also contained a lower affinity binding site. Domains with lower affinity were also found in peptides Asp45-Leu155 and Arg63-Gly190. Peptide Leu155-Val260 also formed complexes with TGF-beta in the liquid phase as determined by equilibrium gel filtration. Furthermore, F(ab') fragments of polyclonal antibodies against peptide Leu155-Val260 interfered with TGF-beta binding to peptide Asp45-Lys359 in a dose-dependent manner. Peptide Leu155-Val260, however, is only a weak competitor of the binding of wild-type decorin to reconstituted type I collagen fibrils. Therefore, independent binding sites of decorin for TGF-beta and type I collagen should exist. In support of this hypothesis saturable binding of TGF-beta1 and TGF-beta2 to collagen-bound native decorin could be demonstrated. The bound cytokine could be released in a biologically active form by collagenase treatment. Thus, decorin may play a biological role in storing this cytokine temporarily in the extracellular matrix and in thereby modulating an interaction of TGF-beta with its signaling receptors.
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PMID:Decorin core protein fragment Leu155-Val260 interacts with TGF-beta but does not compete for decorin binding to type I collagen. 967 33

The prtV gene, encoding a collagenase of Vibrio parahaemolyticus, was expressed in Escherichia coli and purified by affinity chromatography. The transformant E. coli BL21(DE3)(pPRT2) secreted the recombinant PrtV, and the highest enzyme activity was detected in the culture supernatant after 5 h IPTG induction. The molecular mass of purified PrtV was 62 kDa as determined by gel filtration, which was similar to that obtained by SDS-PAGE (64 kDa). This suggested that PrtV was a monomer protein having no subunit structure. The isoelectric point of PrtV was 8.52. In addition, PrtV contained a 27 amino acid signal peptide, and the amino acid composition of the PrtV showed satisfactory agreement with that predicted from the DNA sequence. The optimum temperature and pH of PrtV were 40 degrees C and pH 7.5, respectively. The activity of PrtV was inhibited by chelators such as EDTA, EGTA and 1,10-phenanthroline; however, its activity was restored by the addition of various metal ions (Co2+, Mn2+, Ca2+, Cu2+, Ni2+ and Zn2+), indicating that PrtV is a metalloprotease. PrtV degraded both type I collagen and synthetic substrate FALGPA well, showing that PrtV is indeed a collagenase.
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PMID:Expression and characterization of the prtV gene encoding a collagenase from Vibrio parahaemolyticus in Escherichia coli. 1020 92

Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research. Equine recombinant interleukin-1beta was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized. Reverse transcriptase polymerase chain reaction methods were used to amplify the entire coding region of the equine IL-1beta mRNA, which was cloned into an expression vector, expressed in E. coli, and purified using a Ni2+ chromatographic method. The effects of the recombinant peptide on chondrocyte gene expression were determined by Northern blotting using RNA from equine chondrocyte cultures hybridized to probes for matrix metalloproteinases (MMP 1, MMP 3, MMP 13), tissue inhibitor of matrix metalloproteinases 1 (TIMP 1) and cyclooxygenase 2 (COX 2). Effects on selected mediators of cartilage degradation (nitrite concentrations and MMP activity) were determined using conditioned medium from reIL-1beta-treated equine cartilage explant cultures. A recombinant peptide of approximately 21 kd was obtained. Northern blotting analyses revealed a marked up-regulation of expression of all MMPs, TIMP 1, and COX 2 in mRNA from treated chondrocytes. Furthermore, cartilage explants exposed to reIL-1beta had augmented collagenase/gelatinase and stromelysin activities as well as increased concentration of nitrite in conditioned media. The development of a biologically active, species-specific IL-1beta provides a valuable tool in the study of osteoarthritis pathophysiology and its treatment in horses.
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PMID:Recombinant equine interleukin-1beta induces putative mediators of articular cartilage degradation in equine chondrocytes. 1185 44


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