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)

Rabbits were immunized with rat peritoneal mast cells (RMC) in complete Freund's adjuvant. The antisera (anti-RMC) were checked for their reactivity with RMC by intradermal skin tests in rats. The best serum was selected and absorbed with rat liver cells and rat immunoglobulins, including IgE. The absorbed serum (anti-RMCabs), as well as the anti-RMC serum, were then tested for their reactivity with RMC. Both sera were cytotoxic to RMC but only anti-RMC was cytotoxic for rat lymph node cells. Both sera gave positive reactions in rat skin, as seen by the permeability to Evan's blue dye. The binding of rat IgE to RMC was also inhibited by both sera. A control rabbit anti-rat sarcoma serum absorbed with liver cells did not show any interaction with RMC. When 125I-labeled RMC surface antigens were precipitated with anti-RMCabs and analyzed by SDS polyacrylamide gel electrophoresis, several components were observed. Among these was one with a mobility identical to that of a mast cell surface component that had previously been identified as the receptor for IgE or at least a component thereof.
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PMID:Characterization of an antiserum specific for cell surface antigens of rat mast cells. 5 33

The rat mast cell granule chymotrypsinlike enzyme was purified to homogeneity from 1 M NaCl solubilized membrane and granule-rich fractions of concentrated rat peritoneal mast cells by a preparative technique utilizing chromatography on Dowex 1, filtration on Sephadex G-75, and affinity chromatography with D-tryptophan methyl ester. Acid disk gel electrophoresis of the purified chymase disclosed a single stained band with activity being eluted from a replicate sliced gel in the same region. SDS-polyacrylamide gel electrophoresis of purified protein gave a single stained band that did not change in position with reduction and alkylation. Mast cell chymase is thus a cationic protein of 25,000 mol wt composed of a single polypeptide chain. The apparent K(m) of the chymase for BTEE was 1.5 x 10(-3) M and the V(max) was 67.8 mumol/min per mg. The enzyme was inhibited by TPCK and not by TLCK. The chymase complexed with native macromolecular rat mast cell heparin in molar ratios of 12:1 and 16:1, and complete heparin uptake occurred at a 40:1 ratio of chymase to heparin. Chymase activity was partially masked by combination with heparin in the isolated granule or experimental chymase-heparin complex, and soluble purified chymase was inhibited by concentrations of 5-HT comparable to those present in mast cells. It is therefore possible that the active site of chymase in the mast cell granule is largely masked by the combined effects of macromolecular heparin and 5-HT.
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PMID:Preparative purification of the rat mast cell chymase: characterization and interaction with granule components. 33 34

Nucleoside diphosphate (NDP) kinases have been found to be involved in a wide range of fundamental biological processes ranging from developmental control to signal transduction and metastasis. We have recently cloned and sequenced a cDNA encoding an NDP-kinase of the rat mucosal mast cell line RBL-2H3 [Hemmerich, S., Yarden, Y., & Pecht, I. (1992) Biochemistry (preceding paper in this issue)]. The enzyme itself has been isolated by means of its affinity to the bischromone cromoglycate. Here we report several of its biochemical characteristics: A structural model for the native protein is proposed in which two disulfide-linked pairs of similar 18-kDa subunits (p18) associate to form a 72-kDa tetramer (p72). This is based on the migration properties of the purified enzyme on gel filtration columns, sodium dodecylsulfate gel electrophoresis, and two-dimensional electrophoresis, together with peptide mapping data. In the absence of NDP, both intact p72 and the dissociated 18-kDa subunits (p18) were shown to undergo Mg(2+)-dependent stoichiometric autophosphorylation utilizing adenosine and guanosine triphosphate or gamma-thiotriphosphate as phosphate donor. This autophosphorylation activity was found to be retained by the 18-kDa subunits even following fractionation by SDS-PAGE and electrophoretic transfer to nitrocellulose. The Michaelis constant of this autophosphorylation reaction with either ATP, ATP gamma S, GTP, or GTP gamma S was determined to be 6.5 +/- 1 microM, and maximally 2 mol of phosphate were found to be incorporated per p72 molecule, thus indicating that phosphorylation occurs at a single site on only two of the four 18-kDa subunits of the holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Oligomeric structure and autophosphorylation of nucleoside diphosphate kinase from rat mucosal mast cells. 131 52

In a search for an invertebrate muscle from which the muscle regulatory proteins could be obtained in a great quantity and at high homogeneity, the regulatory proteins, tropomyosin (Tm) and three subunits of troponin (Tn), have been isolated from the lobster tail muscle, purified and partially characterized. The calcium-sensitive ATPase of lobster myofibril was restored when purified lobster Tm and lobster Tn were added to actin. Quantitative SDS-polyacrylamide gel electrophoresis showed that the lobster muscle contains actin, Tm, Tn with a molar ratio 7:1:1 and that lobster Tn consists of three subunits, one of each I, C and T. Each subunit was identified according to its effect on the acto-S1 ATPase rate. The isomer composition in each fraction of purified Tn subunit and in Tm are different from the rabbit skeletal muscle proteins; Tm consists of a single species of polypeptide of M(r) 38,000; the TnT fraction appears to be homogeneous with M(r) 43,000; the TnI fraction contains five isomers, all showing similar isoelectric pH, differing in M(r) in the range from 28,000 to 31,000; two TnC fractions contain three isomers in total with a range of M(r) from 18,500 to 19,000. Further study of the lobster Tm elucidated that digestion by carboxypeptidase A gave rise to a homogeneous preparation of truncated and non-polymerizable Tm which is devoid of 11 residues at the C-terminus of the molecule. The C-terminal amino acid sequence of 11 residues is homologous to the thoracic isomer generated from Drosophila melanogaster Tm-I gene. The present study indicated that, despite heterogeneities owing to the occurrence of isomers, the lobster regulatory proteins serve as an invertebrate source of the proteins for structural and biophysical studies, alternative to vertebrate counterparts.
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PMID:Isolation, purification and partial characterization of tropomyosin and troponin subunits from the lobster tail muscle. 149 Oct 69

The expression and function of Fc gamma RII and Fc gamma RIII on three mouse mast cell populations that differ in maturity as assessed by secretory granule constituents were analyzed by cellular and immunochemical approaches. As quantified by flow cytometric analysis of the binding of the rat 2.4G2 anti-Fc gamma RII/III mAb, mouse serosal mast cells (SMC) purified from the peritoneal cavity expressed more receptors per cell than did mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC), which are progenitors of SMC. Coculture of BMMC with mouse 3T3 fibroblasts for 2 wk, which alters the secretory granule composition toward that of SMC, also increased receptor epitope expression to a level equivalent to that of SMC. As assessed by rosette assays with mouse mAb to SRBC, all three mast cell populations bound IgG1, IgG2a, and IgG2b, essentially all binding was inhibited by 2.4G2 antibody, and greater quantities of the antibody were required to block immune adherence by cocultured mast cells and SMC as compared with BMMC. Immunoprecipitation and SDS-PAGE analysis of Fc gamma RII and Fc gamma RIII from BMMC, cocultured mast cells, and SMC that were surface radiolabeled with Na125I revealed predominant native forms of 62, 57, and 56 kDa, respectively, and an additional surface form of 43 kDa in SMC. Removal of N-linked carbohydrate from immunoprecipitates demonstrated that BMMC expressed peptide cores of 38 kDa (Fc gamma RII-1 gene product) and 31 kDa (Fc gamma RII-2 gene product), and barely detectable amounts of a 28-kDa (Fc gamma RIII gene product) core. The expression of all three was increased by coculture with 3T3 fibroblasts, consistent with the increased expression of their common epitope by cytofluorographic analysis. SMC expressed primarily the Fc gamma RII-1 and some Fc gamma RIII gene product. Thus, the three populations of mast cells express different amounts and ratios of the Fc gamma RII and Fc gamma RIII gene products, and maturation of BMMC during coculture with fibroblasts in vitro and in the peritoneal cavity in vivo augments cell-surface expression of the receptors and immune adherence function.
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PMID:Maturation-related changes in the expression of Fc gamma RII and Fc gamma RIII on mouse mast cells derived in vitro and in vivo. 170 9

Protein from hog which is recognized by human monoclonal antibody (HB4C5), generated from a patient with large cell lung carcinoma, was identified as carboxypeptidase A by comparison of the protein with carboxypeptidase A in enzymatic activity, immunologic reactivity, and amino acid sequence. Carboxypeptidase A activity was also found in human cancer tissue, and purified antigen from cancer tissue recognized by the antibody HB4C5 was reacted with rabbit anti-carboxypeptidase A serum, indicating that carboxypeptidase A is an antigen of HB4C5. Since large amounts of carboxypeptidase A can be obtained from porcine sources, a simple method for its purification was established. The fraction which was most reactive with HB4C5 was obtained from acetone powder of porcine pancreas by successive applications of water extraction, ammonium sulfate precipitation, trypsin treatment, and Mono Q column chromatography. Its apparent molecular weight was 40,000, according to SDS polyacrylamide gel electrophoresis. When the reactivity of IgG in sera with the purified carboxypeptidase A was measured, the detection rates for lung, ovary, larynx, uterus, and liver cancer were more than 50%, while the rates for stomach and breast cancer were around 30%, and pancreatic cancer, benign diseases, and normal controls were minimally detected.
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PMID:Serodiagnosis of cancer using porcine carboxypeptidase A as an animal antigen recognized by human monoclonal antibody HB4C5. 187 99

A juvenile-hormone-binding protein (juvenile-hormone carrier), isolated from Galleria mellonella haemolymph, was treated with trypsin, chymotrypsin, carboxypeptidase A and subtilisin. Among these enzymes, only subtilisin was able to affect juvenile-hormone-binding activity of this protein. With SDS/PAGE it was shown that juvenile-hormone-binding protein, a 32-kDa peptide, is first slowly converted into a 30-kDa molecule, then into two or three smaller-molecular-mass species (20-25 kDa), which in turn were further digested to small peptides undetectable in PAGE. The 30-kDa peptide has a 2.4-times-higher dissociation constant for juvenile hormone than the native protein. No binding activity was detected for 20-25-kDa peptides. The rate of proteolysis of juvenile-hormone-binding protein was decreased by more than twofold in the presence of hormone, however, the overall cleavage pattern was unchanged. Under non-denaturing conditions, free binding-protein molecules could be separated from juvenile-hormone-binding-protein complex due to a slower electrophoretic mobility of the complex. As judged from ultracentrifugation and cross-linking experiments, binding of the hormone to its haemolymph carrier does not induce formation of oligomers, but shifts the sedimentation coefficient from 2.30S to 2.71S. It is concluded that juvenile-hormone binding induces a conformational transition of its carrier protein. This hormone-induced change might have a physiological significance for signal transmission.
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PMID:Conformational change of the haemolymph juvenile-hormone-binding protein from Galleria mellonella (L). 193 32

The neutral protease tryptase has been isolated from a human mast cell line, HMC-1. The HMC-1 line was established from the peripheral blood of a patient with mast cell leukemia and maintained as continuously proliferating clones in vitro and as solid mast cell tumors in nude mice. HMC-1-derived tryptase was purified by sequential chromatography on Dowex 1, DEAE 5 PW, and heparin-agarose. Purified tryptase has an apparent molecular weight of 150,000, as determined by molecular sieve HPLC, but migrates as a doublet of bands of 32/35,000 on SDS-PAGE gels. Maximal enzymatic activity was observed at pH 8.5. Cleavage of tosyl-L-arginine methyl ester by purified tryptase was inhibited by dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone 2 HCl, HgCl2, tosyl-L-lysine chloromethyl ketone, leupeptin, and PMSF but not by benzamidine, aprotinin, tosyl-L-phenyl-alanine chloromethyl ketone, soybean trypsin inhibitor, human plasma, ovomucoid inhibitor, or lima bean trypsin inhibitor. Microsequencing of purified tryptase yielded an amino terminal sequence that was identical to that previously reported for human pituitary-derived tryptase.
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PMID:Purification of tryptase from a human mast cell line. 211 May 91

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


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