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)

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.
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PMID:Gamete interactions in Xenopus laevis: identification of sperm binding glycoproteins in the egg vitelline envelope. 906 Apr 74

Cholestatic liver injury induces an inflammatory response that follows the activation of hepatic macrophages. Constitutive activation of the transcription factor, NF-kappaB, was found in these macrophages over the course of hepatic injury. Since NF-kappaB activation has been shown to have a key role in the inflammatory process, the modulatory effects of the antioxidant, alpha-tocopherol succinate, and the glucocorticoid, dexamethasone, on NF-kappaB activation were examined in this study. Male Sprague Dawley rats underwent 2-7 days of common bile duct division and ligation (CBDL) or sham laparotomy. Hepatic macrophages were isolated by collagenase Pronase perfusion and purified by centrifugal elutriation. Activation was determined by electrophoretic mobility shift assay and ELISA. We determined that NF-kappaB activation in injured hepatic macrophages could only be inhibited by dexamethasone. Dexamethasone-mediated inhibition of NF-kappaB activation required the synthesis of a regulatory protein since cycloheximide-treated cells were resistant to its effects. Furthermore, dexamethasone-treated hepatic macrophages showed elevated steady-state levels of IkappaB-alpha mRNA, suggesting the role of IkappaB-alpha as a potential regulatory mediator. Consistent with constitutive transcriptional activation we showed constitutive secretion of TNF-alpha from injured hepatic macrophages which could be inhibited by dexamethasone. These data show for the first time, in a biologically significant model of hepatic injury, constitutive activation of the key inflammatory transcription factor NF-kappaB and cytokine TNF-alpha. These results support an approach focused on the NF-kappaB/IkappaB-alpha pathway as a critical target for therapeutic intervention during hepatic injury, and the consideration of possible steroid-based therapies.
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PMID:NF-kappaB activation and modulation in hepatic macrophages during cholestatic injury. 935 33

1. Hepatic stellate cells are key mediators of hepatic fibrosis. We have studied hepatic stellate cell expression of the collagenase and general protease inhibitor alpha2-macroglobulin after activation in tissue culture and in response to certain cytokines. 2. Hepatic stellate cells isolated by Pronase-collagenase digestion were activated by culture on uncoated plastic. By Northern analysis hepatic stellate cells undergoing activation (5 days) expressed alpha2-macroglobulin mRNA and alpha2-macroglobulin could be immunolocalized to hepatic stellate cells from 5 to 15 days of culture. 3. By ELISA of cell culture supernatants hepatic stellate cell secretion of alpha2-macroglobulin was found to increase from 2. 78+/-1.13 ng x ml-1 x microgram-1 DNA per 24 h at 5 days of culture (n=8) to 13.55+/-4.64 ng x ml-1 x microgram-1 DNA per 24 h at 15 days of culture (n=7). Stimulation of hepatic stellate cells with interleukin-6 at 5 days caused a significant increase in alpha2-macroglobulin expression as did exposure to Kupffer-cell conditioned medium. However, exposure of hepatic stellate cells to interleukin-1, transforming growth factor-beta1 and tumour necrosis factor-alpha had no significant effect. 4. During profibrotic liver injury plasma alpha2-macroglobulin levels were found to increase to between 850% and 250% of the control value (100%) after bile duct ligation (72 h to 13 days respectively), and to 1166% and 1106% of the control value during progressive CCl4-induced fibrosis (24 h to 4 weeks respectively). 5. These data suggest that hepatic stellate cells are a potential source of the potent protease inhibitor alpha2-macroglobulin, expression of which may inhibit matrix remodelling during progressive fibrosis.
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PMID:Rat hepatic stellate cell expression of alpha2-macroglobulin is a feature of cellular activation: implications for matrix remodelling in hepatic fibrosis. 968 May

CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPS-induced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-alpha (TNF-alpha) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-alpha mRNA in cells isolated via collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently.
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PMID:Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells. 1007 34

The aim of this study was to investigate the individual capabilities of the proteolytic enzyme preparation Pronase, the enzyme collagenase and sodium hypochlorite to disintegrate and solubilize carious dentin. Samples of carious dentin, and samples of sound dentin for comparison, were extracted 4 times in succession for 24 h with buffered solutions of Pronase. Separate carious dentin samples were extracted in the same manner with buffered solutions of collagenase or with aqueous sodium hypochlorite. The extracts, the solid residues left over after the extractions and untreated samples of carious and sound dentin were digested with sulfuric acid-H(2)O(2) and then analyzed for nitrogen content by a special adaptation of the Berthelot color reaction. Although Pronase did not attack sound dentin, it solubilized more than 90% of the nitrogen present in carious dentin. Collagenase solubilized approximately 66% of the nitrogen, whereas sodium hypochlorite released only 12-20% of the nitrogen of carious dentin. In clinical dentistry, chemical disintegration of carious dentin may reduce the need for mechanical removal of sound tooth structure.
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PMID:Pronase digestion of carious dentin. 1052 33

A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited. The comet assay is a simple technique for determining levels of DNA damage in individual cells. In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content. A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied. Cells were incubated with H(2)O(2) and DNA damage was assessed. Pronase and collagenase provided optimum digestion conditions, releasing 1. 12x10(5) cells per biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had high inter-observer (6.1%) and inter-assay (4.5%) reproducibility. Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migration. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005). Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt. Levels of DNA migration were significantly lower with each digestion (r=-0.94, p<0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt. The comet assay is a reproducible measure of DNA damage in gastric epithelial cells. Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.
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PMID:In vivo DNA damage in gastric epithelial cells. 1086 59

Crude extracts of Leishmania amazonensis, but not of L. guyanensis, are lytic to erythrocytes and nucleated cells, including macrophages. L. amazonensis-mediated lysis is caused by a membrane-associated pore-forming protein, named a-leishporin. Here we show that L. amazonensis, but not L. guyanensis, promastigote extracts increase their hemolytic activity when kept at 4 degrees C for a few days or at 37 degrees C for a few hours. We show that the activation in the extracts is mediated by a cytosolic serine-protease. Although L. guyanensis extracts are hemolytically inactive and unable to generate hemolytic activity, their membrane fraction becomes hemolytic in the presence of the cytosolic fraction of L. amazonensis, also by the action of a serine-protease. This suggests that L. guyanensis contains a potential lytic molecule, named here g-leishporin. The cytosolic fraction of L. guyanensis is unable to activate either a- or g-leishporin, indicating that this species does not possess the protease(s) that activate(s) the cytolysin. Trypsin, chymotrypsin, collagenase, Pronase and proteinase K, are also effective in activating a-leishporin but not g-leishporin. This suggests that the inactive forms of a-leishporin and g-leishporin are distinct in structure and/or are activated by different mechanisms. We are considering two hypotheses for the activation of leishporins: (1) proteolysis of an inactive precursor and (2) dissociation and/or proteolytic degradation of an inhibitory oligopeptide. The present data and preliminary results argue for the second hypothesis. We speculate that leishporin could be activated in the protease-rich, low pH, and dissociating environment of parasitophorous vacuole contributing for the release of the parasites from the macrophage.
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PMID:Proteolytic activation of leishporin: evidence that Leishmania amazonensis and Leishmania guyanensis have distinct inactive forms. 1116 43

Gelatin extracted from Alaska pollack skin was hydrolyzed with serial digestions in the order of Alcalase, Pronase E, and collagenase using a three-step recycling membrane reactor. The fraction from the second step, which was hydrolyzed with Pronase E, was composed of peptides ranging from 1.5 to 4.5 kDa and showed high antioxidative activity. Two different peptides showing strong antioxidative activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an ODS column. The isolated peptides, P1 and P2, were composed of 13 and 16 amino acid residues, respectively; and both peptides contained a Gly residue at the C-terminus and the repeating motif Gly-Pro-Hyp. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability was measured with MTT assay. The results showed that P2 had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by addition of the peptide. These results indicate that the purified peptide, P2, from gelatin hydrolysate of Alaska pollack skin is a natural antioxidant which has potent antioxidative activity.
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PMID:Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska pollack skin. 1130 57

Bovine skin gelatin was hydrolyzed with sequential protease treatments in the order of Alcalase, Pronase E, and collagenase using a three-step ultrafiltration membrane reactor. The molecular weight distributions of the first, second, and third hydrolysates were 4.8-6.6, 3.4-6.6, and 0.9-1.9 kDa, respectively. The angiotensin I converting enzyme (ACE) inhibitory activity of the third hydrolysate (IC(50) = 0.689 mg/mL) was higher than that of the first and second hydrolysates. Two different peptides showing strong ACE inhibitory activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration chromatography, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The isolated peptides were composed of Gly-Pro-Leu and Gly-Pro-Val and showed IC(50) values of 2.55 and 4.67 microM, respectively.
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PMID:Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate. 1140 99

Alpha-synuclein modulates dopamine homeostasis in dopamine-producing neurons of substantia nigra, partly through regulation of human dopamine transporter (hDAT) activity. To identify the underlying mechanisms, we disrupted the modulation of hDAT activity by wild-type (wt) alpha-synuclein, and its familial Parkinson's disease linked mutants A30P and A53T, by mild trypsinization (0.1%, 30 s) of Ltk(-) cotransfected cells. Trypsin completely reversed the attenuation of hDAT function mediated by wt and the A30P mutant. In A53T coexpressing cells, where DAT activity is not downregulated, trypsinization did not induce any changes. These effects of trypsin were mimicked by collagenase I and Dispase (0.1%, 1 min each) but not by chymotrypsin, Pronase, or papain (0.1%, up to 2 min each). Trypsin increased dopamine uptake in rat primary mesencephalic neurons, suggesting that DAT activity is also subjected to modulation by alpha-synuclein in these neurons that endogenously coexpress both proteins. In trypsinized cells, dopamine accelerated both production of reactive oxygen species and cell death in hDAT and wt or A30P, but not A53T, coexpressing cells, compared to nontrypsinized cells. Paradoxically, trypsin increased the protein-protein interactions between the synuclein variants and hDAT, without any noticeable proteolysis of these proteins. hDAT-alpha-synuclein protein-protein interactions occurred through residues 58-107 (NAC domain) of the alpha-synuclein variants and residues 598-620 of the carboxy-terminal tail of hDAT, in both trypsinized and nontrypsinized cells. Confocal microscopy and biotinylation studies show that, in cells expressing the wt or A30P variants, but not the A53T mutant, hDAT is sequestered away from the plasma membrane into the cytoplasm, an effect that is reversed by trypsin. These results show that alpha-synuclein modulates hDAT function through trafficking of the transporter in a process that can be disrupted by trypsin.
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PMID:Trypsin disrupts the trafficking of the human dopamine transporter by alpha-synuclein and its A30P mutant. 1475 60


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