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: UNIPROT:P15088 (mast cell)
14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Incubation of oat root plasma membrane vesicles in the presence of ATP with trypsin or chymotrypsin increased the rate of ATP hydrolysis and ATP-dependent proton pumping by the plasma membrane H(+)-ATPase. Proton pumping was stimulated more than 200%, whereas ATP hydrolytic activity was stimulated about 30%. The Km (ATP) for both proton pumping and ATP hydrolysis was lowered from about 0.3 mM to below 0.1 mM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trypsin-treated plasma membranes revealed a decrease in a 100-kDa band and the appearance of a 93-kDa band. Western blot analysis using antibodies against the H(+)-ATPase showed that both of these bands represented the H(+)-ATPase and suggested that a 7-kDa segment was released. Extensive treatment with carboxypeptidase A also activated the H(+)-ATPase indicating that the 7-kDa segment originated from the C terminus.
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PMID:Proteolytic activation of the plant plasma membrane H(+)-ATPase by removal of a terminal segment. 214 84

Peritoneal mast cells (PMC) and intestinal mucosal mast cells (IMMC) were purified from rats infected with the nematode Nippostrongylus brasiliensis. Overall protein constituents of both mast cell subtypes were analyzed by two-dimensional gel electrophoresis using either nonequilibrium pH gradient electrophoresis (NEPHGE) or isoelectric focusing (IEF) in the first dimension and SDS-PAGE (10%) in the second dimension followed by silver staining. PMC had seven dominant basic proteins (PB2-8; pI 9-9.5) with estimated molecular masses of 26 to 37 kDa, as well as 80 to 90 neutral or acidic proteins, most of which had pI 6 to 7.5 and estimated molecular masses of 20 to 100 kDa. All the basic proteins were granule-associated. Three basic proteins, PB6 (29 kDa), PB7 (28 kDa) and PB8 (RMCP I, 26 kDa), bound [3H]diisopropyl fluorophosphate (DFP), suggesting that they are serine proteases. However, only PB8 was reactive with antibodies to RMCP I. Another basic component (less than 14 kDa), perhaps a degradation product of PB6, PB7 or PB8, also bound [3H]DFP. By comparison, IMMC possessed nine basic proteins (IB1-9) and, in general, they were more acidic (pI about 8.5-9) than those of PMC. Four major basic proteins (IB6-9) were all 24 kDa but were slightly different in isoelectric points. These and another 46-kDa basic component (IB2) were reactive with antibodies to RMCP II and bound [3H]DFP. There were no other DFP-binding proteins in IMMC. In spite of remarkable differences between basic granule-associated proteins in PMC and basic proteins in IMMC, spots in the neutral-acidic range were for the most part similar in the two mast cell subsets, although quantitative differences were evident for some spots. Thus, rat mast cell populations from the peritoneal cavity and intestinal mucosa exhibit marked heterogeneity in their protein constituents with basic pI, including in their granule-associated proteins with serine protease activity.
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PMID:Mast cell heterogeneity: two-dimensional gel electrophoretic analyses of rat peritoneal and intestinal mucosal mast cells. 220

Human mast cells can be divided into two subsets based on serine proteinase composition: a subset that contains the serine proteinases tryptase and chymase (MCTC), and a subset that contains only tryptase (MCT). In this study we examined both types of mast cells for two additional proteinases, cathepsin G and elastase, which are the major serine proteinases of neutrophils. Because human mast cell chymase and cathepsin G are both chymotrypsin-like proteinases, the properties of these enzymes were further defined to confirm their distinctiveness. Comparison of their N-terminal sequences showed 30% nonidentity over the first 35 amino acids, and comparison of their amino acid compositions demonstrated a marked difference in their Arg/Lys ratios, which was approximately 1 for chymase and 10 for cathepsin G. Endoglycosidase F treatment increased the electrophoretic mobility of chymase on SDS gels, indicating significant N-linked carbohydrate on chymase; no effect was observed on cathepsin G. Immunoprecipitation and immunoblotting with specific antisera to each proteinase revealed little, if any, detectable cross-reactivity. Immunocytochemical studies showed selective labelling of MCTC type mast cells by cathepsin G antiserum in sections of human skin, lung, and bowel. No labeling of mast cells by elastase antiserum was detected in the same tissues, or in dispersed mast cells from lung and skin. A protein cross-reactive with cathepsin G was identified in extracts of human skin mast cells by immunoblot analysis. This protein had a slightly higher Mr (30,000) than the predominant form of neutrophil cathepsin G (Mr 28,000), and could not be separated from chymase (Mr 30,000) by SDS gel electrophoresis because of the size similarity. Using casein, a protein substrate hydrolyzed at comparable rates by chymase and cathepsin G, it was shown that about 30% of the caseinolytic activity in mast cell extracts was sensitive to inhibitors of cathepsin G that had no effect on chymase. Hydrolytic activity characteristic of elastase was not detected in these extracts. These studies indicate that human MCTC mast cells may contain two different chymotrypsin-like proteinases: chymase and a proteinase more closely related to cathepsin G, both of which are undetectable in MCT mast cells. Neutrophil elastase, on the other hand, was not detected in human mast cells by our procedures.
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PMID:Identification of a cathepsin G-like proteinase in the MCTC type of human mast cell. 221 56

Exogenous addition of purified chymase, a rat serosal mast cell (RSMC) chymotryptic enzyme, results in RSMC degranulation at 37 degrees, but not at 1 degree. Chymase can cause an active site-dependent inducing event at 1 degree such that RSMC degranulation occurs if the cells are later incubated at 37 degrees. RSMC exposed to chymase or other stimuli were surface radiolabelled using 125I and Iodo-Gen, solubilized with 1% Nonidet-40, and the resulting 25,000 g supernatants analysed by SDS-PAGE and autoradiography. A 125I-labelled RSMC membrane protein of approximate 90,000 MW decreased upon exposure to either chymase or alpha-chymotrypsin (alpha-CT) for 5 min at 37 degrees or to chymase for 60 min at 1 degree. Exposure of RSMC to the secretagogues ionophore A23187, compound 48/80, and anti-IgE for 5 min at 37 degrees resulted in beta-hexosaminidase (a secretory granule enzyme) release, but did not cause a detectable change in the 90,000 MW surface-labelled protein. Lima bean trypsin inhibitor, which inhibits both the esterase and RSMC degranulation activities of chymase and alpha-CT, prevented the disappearance of the 125I-labelled 90,000 MW band when added with chymase or alpha-CT. Exposure of RSMC to chymase at 1 degree for 0-10 min, prior to addition of LBTI, led to a progressive disappearance of the 90,000 MW band, which corresponded to the kinetics of priming for subsequent RSMC degranulation at 37 degrees. When RSMC were exposed to trypsin (2.5 micrograms/ml) for 0-120 min at 1 degree, a progressive disappearance of the 90,000 MW band occurred, in association with a loss of sensitivity to subsequent activation by chymase at 37 degrees. The disappearance of the 90,000 MW determinant in association with chymase-mediated priming for degranulation and the inability of chymase to mediate degranulation of trypsin-treated RSMC, which lack this membrane protein, suggests that it is involved in chymase-mediated RSMC degranulation.
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PMID:Cleavage of a rat serosal mast cell membrane component during degranulation mediated by chymase, a secretory granule protease. 231 65

The expression of the antigenic determinant identified by the B54.2 rat monoclonal antibody on four populations of mouse mast cells has been quantified, and the epitope-bearing surface antigen and its biosynthesis have been characterized. As assessed by indirect immunofluorescence staining and flow cytometric analysis, B54.2 antibody bound to serosal mast cells (S-MC), bone marrow culture-derived mast cells (BM-MC), fetal liver culture-derived mast cells (FTL-MC), and Abelson murine leukemia virus-transformed FTL-MC (ABFTL-MC). However, the intensity of cell surface fluorescence exhibited by ABFTL-MC was approximately eightfold less per cell compared with nontransformed, culture-derived mast cells. Immunoprecipitation of B54.2 antibody-binding molecules from each population of mast cells labeled intrinsically with [35S]methionine and analysis by SDS-PAGE demonstrated that the B54.2 epitope was expressed in each case on two noncovalently associated proteins of 110,000 Mr and approximately 130,000 Mr, but that the percentage of radiolabel in the latter species was approximately threefold less in ABFTL-MC than in BM-MC. As assessed by pulse-chase analysis with [35S]methionine, the 110,000 Mr protein was a precursor of the 130,000 Mr molecule ("B54.2 antigen") synthesized by BM-MC. Labeling of BM-MC with [35S]methionine in the presence of tunicamycin followed by immunoprecipitation and SDS-PAGE of B54.2 antibody-binding material revealed a single species of 93,000 Mr, indicating that the native molecules contained N-linked carbohydrate. Endoglycosidase H treatment of the glycoproteins precipitated by B54.2 antibody from BM-MC reduced the Mr of the 110,000-Mr molecule to 93,000 Mr without an appreciable change in the 130,000-Mr species. These data indicate that the 110,000-Mr precursor form is a "high mannose" type glycoprotein and the 130,000-Mr membrane surface B54.2 antigen is a "complex" type glycoprotein, and that the epitope recognized by the B54.2 antibody on the surface of the mouse mast cell populations is located on the 93,000-Mr peptide core.
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PMID:Biochemical characterization of a mast cell plasma membrane antigen shared by mouse serosal, culture-derived, and virally transformed mast cells. 243 44

The functional role of mast cells in rheumatoid synovium was investigated by assessing the ability of mast cell tryptase to activate latent collagenase derived from rheumatoid synoviocytes. Tryptase, a mast cell neutral protease, was demonstrated in situ to reside in rheumatoid synovial mast cells, by an immunoperoxidase technique using a mouse mAb against tryptase, and in vitro to be released by dispersed synovial mast cells after both immunologic and nonimmunologic challenge. Each rheumatoid synovial mast cell contains an average of 6.2 pg of immunoreactive tryptase and the percent release values of this protease correlated with those of histamine (r = 0.58, p less than 0.01). The ability of purified tryptase to promote collagenolysis was demonstrated in a dose-dependent fashion using latent collagenase derived from rheumatoid synovium, synovial fluid, IL-1-stimulated cultured synoviocytes, and partially purified latent collagenase derived from conditioned media, with between 10 and 92% of the collagen substrate degraded. [3H] Collagen, treated with tryptase-activated latent collagenase, was subjected to electrophoresis on SDS polyacrylamide gels and autoradiography showed the collagen degradation pattern (A, B) characteristically produced by collagenase. Mast cell lysates also activated synovial latent collagenase yielding 24% digestion of collagen substrate. This activator in mast cell lysates could be inhibited by diisopropylflurophosphate or by immunoadsorption of tryptase. Thus, mast cells may activate metalloproteinases and play a role in the catabolism of collagen that occurs in rheumatoid synovium.
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PMID:Activation of latent rheumatoid synovial collagenase by human mast cell tryptase. 245 61

Derivatives of the antiallergic drug cromolyn [disodium 5,5'-[(2-hydroxy-1,3-propanediyl)-bis(oxy)]bis [4-oxo-(4H-1-benzopyran)-2- carboxylate]], which can be conjugated covalently at the propane 2-position to macromolecules and to insoluble matrices, were synthesized. Conjugates of these derivatives with macromolecules were examined for their binding to cells of the rat basophilic leukemia line RBL-2H3, which is widely employed as a model for immunologically induced mast cell degranulation. Only those drug-protein conjugates in which the cromolyn analogue with an amino group at the propane 2-carbon instead of the hydroxyl was linked to the carrier by glutaraldehyde were found to exhibit specific and saturable binding to these cells. Analysis of the binding data for these conjugates yielded an apparent binding constant of 3.8 +/- 0.2 X 10(8) M-1 and an apparent number of binding sites for the probe of 4000-8000 per cell. The conjugates found to bind specifically to the cells were also immobilized on agarose matrices and employed in an affinity-based isolation of the membrane component responsible for the observed binding. A single labeled polypeptide was eluted from these columns, onto which either whole cell lysates or solubilized purified plasma membranes of surface-radioiodinated RBL-2H3 cells had been adsorbed. This membrane protein appears on autoradiograms of nonreducing SDS-PAGE as a single broad band of approximately 110,000 daltons (Da) apparent molecular mass. On autoradiograms of reducing gels, the only band detected has an apparent mass of approximately 50,000 Da and appears narrower. Elution of the columns with the drug and disulfide-reducing agents or with the latter alone resulted in significantly higher yields of the 50-kDa polypeptide. Both the intact and reduced proteins bind strongly to immobilized concanavalin A and less so to immobilized wheat germ agglutinin, suggesting that the isolated intact protein is probably a dimer of two glycosylated subunits of similar molecular mass. Treatment of the reduced protein with endoglycosidase F leads to a decrease in its apparent molecular mass by approximately 12 kDa, suggesting that the extent of glycosylation of this polypeptide is approximately 25%. As shown in the following paper, the intact protein constitutes a Ca2+ channel that is activated upon IgE-Fc epsilon receptor aggregation.
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PMID:Isolation and purification of an Fc epsilon receptor activated ion channel from the rat mast cell line RBL-2H3. 246 4

As assessed by immunoprecipitation analyses, expression of the epitope recognized by the rat mAb B23.1 is approximately sevenfold greater on the surface of mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) than on serosal mast cells (SMC) obtained directly from the peritoneal cavity. Immunoprecipitation of B23.1 antibody-binding molecules from Na[125I] surface-labeled BMMC and SMC followed by sizing on SDS-polyacrylamide gels under reducing conditions demonstrated that the epitope is located on molecules of 49,000 and 47,500 Mr, respectively. An additional immunoprecipitated molecule of 42,000 Mr was detected from BMMC intrinsically radiolabeled with [35S]methionine, and pulse-chase analyses revealed that this species was a biosynthetic precursor of the 49,000 Mr cell surface form of the Ag. Treatment of the immunoprecipitated 42,000 and 49,000 Mr forms with endoglycosidase F reduced the Mr of both to 37,000, as did intrinsic radiolabeling of BMMC in the presence of tunicamycin, indicating that both the 42,000 Mr precursor form and the 49,000 Mr cell surface molecule (gp49) contained N-linked carbohydrate. Activation of [32P]orthophosphate-labeled BMMC by sensitization with mouse monoclonal IgE anti-TNP and challenge with TNP-BSA or by exposure to the calcium ionophore A23187 elicited the rapid phosphorylation of gp49 but not of its precursor forms, as did treatment of the cells with PMA. Elution of phosphorylated and immunoprecipitated gp49 from SDS-polyacrylamide gels followed by partial acid hydrolysis of the protein and phosphoamino acid analysis by high voltage thin-layer electrophoresis on cellulose plates indicated that serine, but not threonine or tyrosine, was phosphorylated upon stimulation of BMMC with IgE/Ag, calcium ionophore, or PMA. Cholera toxin did not elicit phosphorylation of gp49. These data suggest that gp49, a plasma membrane glycoprotein preferentially expressed by mouse BMMC, may be either directly or indirectly phosphorylated via protein kinase C during mast cell activation-secretion.
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PMID:Activation- and phorbol ester-stimulated phosphorylation of a plasma membrane glycoprotein antigen expressed on mouse IL-3-dependent mast cells and serosal mast cells. 246 32

High- (alpha chain) and low-affinity IgE receptors from purified populations of rat intestinal mucosal (IMMC) and peritoneal mast cells (PMC) were characterized by SDS-PAGE. Receptor expression and molecular weight were compared. IMMC yielded 59-kilodalton (kDa) alpha chains of the high-affinity receptors and two forms (58, 50 kDa) of low-affinity receptors, whereas PMC possessed only 51-kDa alpha chains and 56-kDa low-affinity receptors. These differences extend the evidence for functional diversity between mast cell subtypes.
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PMID:IgE receptors from rat intestinal mucosal and peritoneal mast cells show mast cell subtype-specific differences. 252 56

Beta-adrenergic agonists can prevent mediator release from guinea pig pulmonary mast cells. By pharmacologic characterization, this response is mediated through a beta-2 receptor. Structural characterization of this receptor on the lung mast cell, however, has been limited by methods for isolation of this pulmonary cell. In this study, the guinea pig lung mast cell was isolated to greater than 90% purity, and its beta-adrenergic receptor identified by photoaffinity labeling with [125I]iodoazidobenzylpindolol (125IABP) and separation of membrane proteins by SDS-PAGE. We found the guinea pig pulmonary mast cell beta-adrenergic receptor to electrophorese as a heterogeneous protein between 68 and 116 kD. Photoaffinity labeling with 125IABP was protectable by alprenolol and isoproterenol but not by phentolamine and norepinephrine. Using subtype-selective compounds, the pulmonary mast cell receptor was established to be of a beta-2 subtype. This is the first report of the structural identification of a lung mast cell beta-adrenergic receptor and the first report of a beta-adrenergic receptor of approximately 100 kD in mass. This mast cell receptor is considerably larger than the 65 kD beta-adrenergic receptors that have been identified in whole lung and other tissues. Data we have obtained using Northern blot analysis of mast cell RNA suggest a protein message of 45 kD for this beta-adrenergic receptor and a high degree of glycosylation most likely accounts for the large molecular size observed.
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PMID:Photoaffinity labeling of the guinea pig pulmonary mast cell beta-adrenergic receptor. 256 88


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