Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Carboxypeptidase A beta and carboxypeptidase A tau-type from the pancreas of the ostrich were purified by water extraction of acetone powder, aminobenzylsuccinic acid affinity and hydroxylapatite chromatography. 2. The final preparations were homogeneous when subjected to SDS-PAGE and PAGE. The M(r) values obtained from SDS-PAGE for CPA beta and CPA tau-type were 34,600 and 34,400, respectively. 3. The effects of inhibitors (1,10 phenanthroline and indole-3-acetic acid), pH and temperature on CPA activity were examined. Ki-values for CPI, PPA, D-phe, D-trp and aminobenzylsuccinic acid were determined. 4. Km, kcat and kcat/Km values were determined for hipp-phe, cbz-gly-phe, cbz-(gly)2-phe, cbz-gly-leu, cbz-(gly)2-leu and cbz-(gly)2-val. 5. N-terminal sequencing and amino acid analysis were performed for CPA beta and CPA tau-type.
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PMID:Ostrich (Struthio camelus) carboxypeptidase A: purification, kinetic properties and characterization of the pancreatic enzyme. 801 41

Although the steady-state level of the mouse mast cell protease (mMCP) 7 transcript is below detection in the serosal and mucosal mast cells of the BALB/cJ mouse, the IL-3-dependent, bone marrow-derived mast cells (mBMMC) of this strain and four other strains contain a high steady-state level of the mMCP-7 transcript. To further analyze the expression of this mast cell tryptase, a mMCP-7-specific IgG was obtained by immunizing a rabbit with a 19-residue synthetic peptide that corresponds to its unique amino acid sequence at residues 160 to 178 (anti-mMCP-7(160-178). In a SDS-PAGE/immunoblot analysis of lysates of BALB/cJ mBMMC, anti-mMCP-7(160-178) IgG recognized a diffuse 31- to 36-kDa protein, which shifted to a sharp 27-kDa protein after treatment with N-glycanase. As assessed immunohistochemically, mMCP-7 protein is present not only in the secretory granules of BALB/cJ mBMMC, but also in the ear mast cells of this strain. In contrast, the ear mast cells of the C57BL/6J mouse do not contain detectable levels of mMCP-7 protein, although the ear mast cells of both mouse strains contain mMCP-5 protein. Because mMCP-7 mRNA and protein also were not detected in mBMMC from the C57BL/6J mouse, the failure of the ear mast cells of this strain to express mMCP-7 is most likely a consequence of an intrinsic abnormality in the mast cell-committed progenitor cells themselves, or in the bone marrow microenvironment that induces its mast cell progenitor cells to express this tryptase.
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PMID:Lack of expression of the tryptase mouse mast cell protease 7 in mast cells of the C57BL/6J mouse. 807 72

As previously reported, protoporphyrin plus long-wavelength UV light (PP/UVA) inhibits IgE-mediated degranulation of mouse bone marrow-derived mast cells, as assessed by measurement of the release of beta-hexosaminidase. This inhibitory effect was seen with cells sensitized with IgE either before or after PP/UVA treatment (57.8 and 55.3% inhibition, respectively). PP/UVA did not dissociate IgE already bound to cells as assessed either by measuring release of bound 125I-IgE or by flow cytometric analysis. Results from immunoadsorption followed by SDS-PAGE analysis suggested that PP/UVA treatment may cause stable conjugation of IgE to its receptor. In unsensitized cells, PP/UVA did not cause conjugation of the unoccupied Fc epsilon RI to other proteins in the plasma membrane. Nevertheless, Scatchard analysis revealed that PP/UVA decreased the number of Fc epsilon RI per cell by 37% (0.95 x 10(5) vs 1.51 x 10(5)/cell), whereas affinity of the receptor for IgE was comparable between PP/UVA-treated and untreated cells (3.40 nM vs 3.27 nM). Flow cytometric analysis also confirmed the decrease in Fc epsilon RI number in PP/UVA-treated unsensitized mouse bone marrow-derived mast cells. Although 84% of PP/UVA-treated and 82% of untreated cells expressed positive fluorescence when stained with FITC-conjugated IgE, fluorescence intensity was reduced by 40% after PP/UVA treatment. We conclude that PP/UVA alters the conformational structure and/or number of Fc epsilon RI expressed on the mast cell surface. This effect could potentially explain the ability of PP/UVA to inhibit mast cell secretory function and may be related to an ability of PP/UVA to alter the properties of the plasma membrane.
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PMID:Alterations in Fc epsilon RI induced by protoporphyrin plus long-wavelength ultraviolet light in mouse bone marrow-derived mast cells. 833 89

Proteins of mast cells purified from human foreskin were separated by 2-D polyacrylamide gel electrophoresis using either nonequilibrium pH gradient electrophoresis or isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. Silver staining showed that a major feature of skin mast cell 2-D protein maps was a variety of relatively abundant proteins in the m.w. range of 29 to 37 kDa and covering a broad pH range from 5.0 to 8.5. Tryptase was identified on Western blots of 2-D-separated proteins by its binding of mAb and of 3H-diisopropylfluorophosphate. The precise distribution of tryptase varied among individuals but this protein generally occupied a continuum of molecular weights between 28 and 37 kDa and ranged in isoelectric point between 5.0 and 6.5. Tryptase was one of a number of mast cell proteins that bound the lectin concanavalin A as well as lectins specific for sialic acid, demonstrating that this enzyme is a sialylated glycoprotein. The diffuse m.w. distribution of skin mast cell tryptase (31 to 36 kDa) observed after SDS-PAGE was reduced to a single band of 30 kDa after treatment with protein-N-glycosidase F to remove asparagine-linked oligosaccharides. This finding suggests that intrinsic m.w. heterogeneity of tryptase in skin mast cells is largely a result of the addition of variable amounts of oligosaccharide to the tryptase polypeptide.
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PMID:Analysis of human skin mast cell proteins by two-dimensional gel electrophoresis. Identification of tryptase as a sialylated glycoprotein. 836 Apr 86

Although mouse interleukin-3-dependent, bone marrow culture-derived progenitor mast cells (BMMC) and a Kirsten sarcoma virus (KiSV)-immortalized mouse mast cell line (MC4w) both express on their surfaces receptors for the Fc portion of IgG (Fc gamma R), only MC4w degranulate upon Fc gamma R perturbation. As shown by surface iodination and SDS-polyacrylamide gel electrophoresis analysis of deglycosylated proteins immunoprecipitated with the Fc gamma R-specific monoclonal antibody 2.4G2, a 26-kDa protein, identified as Fc gamma RIII by immunoblotting with antibody to Fc gamma RIII, was predominantly expressed on the surface of MC4w but minimally on BMMC. However, both BMMC and MC4w expressed mRNA for Fc gamma RIII as determined by RNA blot analysis, and both translated Fc gamma RIII as assessed by intrinsic radiolabeling and SDS-polyacrylamide gel electrophoresis analysis of deglycosylated monoclonal antibody 2.4G2 immunoprecipitates. Pulse-chase analysis showed that intrinsically radiolabeled Fc gamma RIII was stable in MC4w cells but was degraded rapidly in BMMC and that newly synthesized Fc gamma RIII remained sensitive to digestion by endoglycosidase H in BMMC but rapidly became resistant in MC4w. These data suggest that the deficiency in surface Fc gamma RIII expression on BMMC is due to the degradation of Fc gamma RIII in the endoplasmic reticulum. Immunoprecipitation of surface Fc gamma R and Fc receptors for IgE (Fc epsilon RI) from digitonin-extracted cells followed by immunoblotting with antibody to Fc epsilon RI gamma-chain showed that gamma-chain is associated with surface Fc epsilon RI and Fc gamma R in MC4w, but only with Fc epsilon RI in BMMC, which lack surface Fc gamma RIII. Inasmuch as BMMC are progenitors of serosal mast cells, which, like MC4w, express surface Fc gamma RIII and undergo Fc gamma R-mediated activation, the data suggest that maturation of BMMC enables Fc gamma RIII to bypass degradation in the endoplasmic reticulum, resulting in the acquisition of functional Fc gamma RIII/gamma-chain complexes on the cell surface.
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PMID:Intracellular degradation of Fc gamma RIII in mouse bone marrow culture-derived progenitor mast cells prevents its surface expression and associated function. 841 24

Monoclonal antibody 4E3 directed against a glycosylated surface protein on human ovarian teratocarcinoma cells (CRL-1572 cell line) was conjugated to bovine carboxypeptidase A (CPA) using a 3400 Da polyethylene glycol chain bearing an N-hydroxysuccinimide group at both ends. The conjugate preparation was purified by fast protein liquid chromatography on a Superose 12/30 HR column. The 4E3-CPA conjugate was recovered in the third fraction by SDS-PAGE analysis. The specific binding of the 4E3-CPA conjugate to CRL-1572 cells was confirmed by a FACS analysis and the enzymatic activity of the conjugate remained while tested with hippuryl-L-phenylalanine. In vitro cytotoxic assays on CRL-1572 cells showed that the prodrug methotrexate-phenylalanine (MTX-Phe) alone was non-toxic (ID50 > 1000 ng ml-1) but was selectively converted to MTX when the cells were pretreated with 50 micrograms ml-1 4E3-CPA conjugate, which enhanced considerably the pharmacological activity of the prodrug with an ID50 of 70 ng ml-1. The co-culture assays with CRL-1572 and MRC-5 cells (human normal lung diploid fibroblast cell lines) demonstrated the specificity of the 4E3-CPA conjugate for the CRL-1572 cells since no cytotoxicity was observed on the MRC-5 cells. When both cell lines were mixed in ratios ranging from 1:10,000 to 1:5 (CRL-1572:MRC-5), the significant increase in the ID25 was correlated with the proportion of tumoral cells present in the cell inoculum. These results suggest that MTX-Phe combined with 4E3-CPA conjugate is a promising model for a more selective and localised anti-cancer chemotherapy based on the ADEPT concept.
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PMID:Activation of methotrexate-phenylalanine by monoclonal antibody--carboxypeptidase A conjugate for the specific treatment of ovarian cancer in vitro. 856 31

Two monoclonal antibodies (mAbs), XB6 and YD3, which recognise ovine immunoglobulin E (IgE) were produced. Mast cells isolated from ovine intestinal mucosa were used as a source of IgE to immunize mice. Culture supernatants of hybridomas were screened by immunoassays on small-intestine tissue sections, isolated mucosal cells, and dot blots of lysed mast cell homogenate. Two mAbs were chosen for their specific binding to mast cells. Antigen bound by these mAbs was purified by immunoaffinity chromatography using XB6 mAb, and this produced two bands consistent with IgE heavy chain (86,000 Daltons) and immunoglobulin light chain (28,000 Daltons) when run under reducing conditions on SDS-PAGE gels. Purified IgE was shown on dot blots to react weakly with mAb to chimeric ovine IgE and strongly to polyclonal anti-sheep antibodies. The two mAbs induced an immediate hypersensitivity-like reaction when injected into the skin of sheep. The mAbs bound to mast cells and other mononuclear cells, presumably IgE-secreting B-cells in mesenteric lymph node sections. These mAbs proved useful for detecting IgE-bearing cells in various ovine tissues, for purifying mast cells from cell isolates by panning and immunomagnetic bead separation, for purifying serum IgE using immunoaffinity chromatography and for detecting IgE in an ELISA. Competitive binding assays showed that the two mAbs bind to different epitopes on IgE. These mAbs will be useful in research applications and in diagnostic assays.
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PMID:Production and characterisation of monoclonal antibodies recognising ovine IgE. 879 63

The complex temporal evolution of on-column exocytotic release of serotonin and proteins from individual rat peritoneal mast cells (RPMCs) was monitored by using capillary electrophoresis. Laser-induced native fluorescence detection with 275-nm excitation was used, and a detection limit of 1.7 amol (S/N = 3; rms) was obtained for serotonin. A physiological running buffer was used to ensure that the cell remained viable throughout. The secretagogue was polymyxin B sulfate (Pmx). Following the injection of a single mast cell into the capillary, electromigration of Pmx toward and past the cell induced degranulation and release of serotonin. The time course of release was registered in the electropherograms with subsecond resolution. Subsequent introduction of SDS caused the cell to lyse completely and allowed the residual serotonin to be quantified. The average amount of serotonin observed per RPMC was 1.6 +/- 0.6 fmol; the average percentage of serotonin released was 28 +/- 14%. Events that are consistent with released serotonin from single submicrometer granules (250 aL each) were evident, each of which contained an average amount of 5.9 +/- 3 amol.
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PMID:Monitoring exocytosis and release from individual mast cells by capillary electrophoresis with laser-induced native fluorescence detection. 879 24

Sheep mast cell proteinase-1 (sMCP-1), a serine proteinase with dual chymase/tryptase activity, is expressed in gastrointestinal mast cells, and released systemically and on to the mucosal surface during gastrointestinal nematode infection. The potential for native plasma proteinase inhibitors to control sMCP-1 activity was investigated. Sheep alpha1-proteinase inhibitor (alpha1PI) inhibited sMCP-1 slowly, with second-order association rate constant (kass) 1. 1x10(3) M-1.s-1, whereas sheep contrapsin inhibited trypsin (kass 2.2x10(6) M-1.s-1) but not sMCP-1. Western-blot analysis and gel filtration showed that when added to serum or plasma, sMCP-1 was partitioned between alpha1PI and alpha2-macroglobulin. The possibility that significant cleavage of plasma proteins could occur before sMCP-1 was inhibited was investigated using gel filtration and SDS/PAGE after adding sMCP-1 to plasma. Cleavage of ovine fibrinogen occurred in the presence of excess alpha1PI and alpha2-macroglobulin, the alpha-chain being cleaved C-terminally and the beta-chain at the putative Lys-27. In addition, sMCP-1 was found to be mitogenic for bovine pulmonary artery fibroblasts, but was not mitogenic in the presence of soya-bean trypsin inhibitor. In terms of fibrinogen cleavage and fibroblast stimulation, sMCP-1 shows functional similarities to mast cell tryptase.
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PMID:Sheep mast cell proteinase-1, a serine proteinase with both tryptase- and chymase-like properties, is inhibited by plasma proteinase inhibitors and is mitogenic for bovine pulmonary artery fibroblasts. 916 5

Soybean Bowman-Birk protease inhibitor (BBI) is an inhibitor of serine proteases with two functional inhibitory domains of different specificities: one is specific for chymotrypsin-like proteases, the other for trypsin-like proteases. Chymase and tryptase are serine proteases which are stored in mast cell granules and released upon degranulation. This work investigated the inhibition of human chymase and tryptase by BBI. Active-site titration of human skin chymase by BBI demonstrated that BBI was a highly effective inhibitor of human chymase. Virtually stoichiometric inhibition of chymase by BBI was observed at 10 nM chymase. Kinetic studies of the inhibition reaction yielded an association rate constant of 4.0 x 10(5) M(-1) s(-1) and a dissociation rate constant of 1.7 x 10(-5) s(-1). From these two constants we estimate a K(i) of 50 pM. Chymase/BBI complexes did not dissociate in SDS-PAGE analyses under nonreducing conditions, consistent with the formation of a very tight complex with little tendency to dissociate. In contrast to chymase, human tryptase was not inhibited by BBI. These studies demonstrate that BBI is a good inhibitor of human chymase, exhibiting reaction properties better than physiological inhibitors described to date.
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PMID:Soybean Bowman-Birk protease inhibitor is a highly effective inhibitor of human mast cell chymase. 924 90


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