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)

The existence of a protein approximately 48% identical with mast cell tryptases was predicted previously from a dog mastocytoma cDNA. Antibodies raised against a peptide based on the deduced sequence suggested that the protein (dog mast cell protease-3, dMCP-3) is expressed in mast cells. In this report, characterization of the protein purified from mastocytomas reveals an N-glycosylated, high molecular weight, tryptic serine protease, which appears to be a tetramer of catalytic subunits, approximately half of which are linked by disulfide bonds. The oligomeric complex yields a single NH2-terminal sequence, which is identical with that predicted by dMCP-3 cDNA. This finding, and the lack of closely related genes on blots of genomic DNA, predict that each subunit is the product of one gene. Although dMCP-3 binds to heparin, it is active and stable at low ionic strength in heparin's absence. It resists inactivation by inhibitors in plasma but is sensitive to small inhibitors, e.g. leupeptin and bis(5-amidino-2-benzimidazolyl)methane (BABIM). dMCP-3 hydrolyzes extended peptidyl p-nitroanilides ending in basic residues, with P1 arginine preferred to lysine; it hydrolyzes the Arg18-Ser19 bond of calcitonin gene-related peptide but cleaves neither vasoactive intestinal peptide nor casein. These data suggest that dMCP-3 is a unique serine protease whose stability, formation of intersubunit disulfide bonds, inhibitor susceptibilities and substrate preferences differ from those of its closest relatives, the mast cell tryptases.
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PMID:Purification and characterization of dog mast cell protease-3, an oligomeric relative of tryptases. 776 12

In this paper, data are presented on purification and properties of a new serine endopeptidase (duodenase) isolated from bovine duodenum mucosa. The enzyme has been purified to homogeneity by combinations of ammonium sulphate fractionation, carboxymethyl-cellulose 52 chromatography, and affinity chromatography on Sepharose 4B with Kunitz soybean trypsin inhibitor as a ligand. Some physicochemical properties of this protease have been investigated. The molecular mass of the purified duodenase was determined to be 29 +/- 0.5 kDa by SDS/PAGE and G-2000 SW column chromatography. The enzyme molecule is a single chain and the native enzyme is a monomeric protein. Its isoelectric point was estimated to be 10 +/- 0.2. Duodenase has two forms (I and II) which possess similar properties but differ in their amino acid composition. The new protease is a glycoprotein and contains approximately 3.5% sugars. The enzyme displays trypsin-like and chymotrypsin-like activities and hydrolyzes the amide bonds of substrates having Lys, Arg, Tyr, Phe and Leu residues at the P1 position. Duodenase is most active at pH 7.9-8.2. Duodenase was irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, indicative of an active-site serine in this protease. alpha-N-Tosyl-L-lysine chloromethane and alpha-N-tosyl-L-phenylalanine chloromethane, which react with an active His, caused marked inhibition of trypsin-like and chymotrypsin-like activities of duodenase. The enzyme activity was strongly suppressed by trypsin inhibitors from different sources (soybeans, bovine lungs and Lima beans). Chicken egg white ovomucoid had no effect on the duodenase activity. The N-terminal sequence of the native duodenase (24 amino acid residues) shows high similarity with those of human and murine cytotoxic T-lymphocyte granzymes, human leukocyte cathepsin G and rat mast cell chymases. The biological role of duodenase is discussed.
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PMID:Duodenase, a new serine protease of unusual specificity from bovine duodenal mucosa. Purification and properties. 786 48

Cleavage after Met596 of the beta-amyloid precursor protein to generate the N-terminus of beta-protein indicates the activity of a protease having chymotrypsin-like specificity. A chymotrypsin-like protease is further implicated in Alzheimer's disease by the increased synthesis of the protease inhibitor alpha 1-antichymotrypsin in pathologically affected brain regions and by the presence in the amyloid deposits of inactivated forms of alpha 1-antichymotrypsin (indicating irreversible binding to a target chymotrypsin-like protease). In the present report, we have purified from rat brain a chymotrypsin-like protease that (a) binds with high affinity to human alpha 1-antichymotrypsin, (b) proteolytically generates a beta-protein-containing C-terminal fragment from full-length recombinant human beta-amyloid precursor protein, and (c) selectively cleaves methoxysucinyl-Glu-Val-Lys-Met- p-nitroanilide (a substrate modeling the protease recognition domain for the beta-protein N-terminal cleavage site). Amino acid sequences of tryptic fragments of the purified rat brain chymotrypsin-like protease indicate an identity with rat mast cell protease I. Moreover, the ontogeny and compartmentalization of rat brain chymotrypsin-like protease are consistent with those of connective tissue-type mast cells in the meningeal and intracortical perivasculature. Because these areas in human brain form extensive beta-amyloid deposits in Alzheimer's disease, Down's syndrome, and hereditary cerebral hemorrhage with amyloidosis of Dutch origin, the present findings suggest that a brain mast cell chymotrypsin-like protease may participate in generating perivascular beta-protein, which ultimately aggregates into beta-amyloid deposits.
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PMID:Identification of a chymotrypsin-like mast cell protease in rat brain capable of generating the N-terminus of the Alzheimer amyloid beta-protein. 833 43

In order to examine the hypothesis that in aspirin-induced asthma (AIA) cyclooxygenase inhibition is associated with enhanced release of leukotrienes (LTs), we measured urinary leukotriene E4 (LTE4) and 11-dehydro-thromboxane B2 (TXB2) (as a measure of cyclooxygenase production) following challenge with oral aspirin or inhaled methacholine, in 10 AIA patients. We also determined serum tryptase and eosinophilic catonic protein (ECP) levels, in order to evaluate mast cell and eosinophil activation. Urinary LTE4 excretion was increased sevenfold 4-6 h after aspirin challenge, while 11-dehydro-TXB2 decreased gradually reaching 50% baseline levels 24 h after challenge (p < 0.05). This was accompanied by a significant fall in blood eosinophil count at 6 h, and a tendency to a rise in ECP. The intensity of both LTE4 and 11-dehydro-TXB2 responses depended on the dose of aspirin used (p < 0.001, analysis of variance (ANOVA)). The accompanying maximum fall in forced expiratory volume in one second (FEV1) was not correlated with peak LTE4 levels. In contrast to aspirin, methacholine challenge producing comparable bronchial obstruction, did not alter eicosanoid excretion or serum tryptase or ECP levels. In a separate study, lysine-aspirin inhalation challenge was performed in seven AIA patients, four of whom had responded with a rise in serum tryptase to oral aspirin challenge. Challenge with inhaled aspirin led to similar bronchoconstriction as with oral challenge, but non-respiratory symptoms such as scarlet flush or rhinorrhea were absent, and serum tryptase levels remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cysteinyl leukotrienes overproduction and mast cell activation in aspirin-provoked bronchospasm in asthma. 838 6

Transmembrane signaling initiated by the receptor with high affinity for Fc stem of IgE(Fc epsilon RI) requires the diffusion-dependent cross-linkage and persistent aggregation of the Fc epsilon RI. Disruption or prevention of receptor cross-links at any time during the secretory response quickly terminates secretion. We found that in the rat basophilic leukemia mast cell line, addition of wheat germ agglutinin, a lectin that binds to the Fc epsilon RI alpha subunit, caused a precipitous decline in the lateral diffusional and electrokinetic mobilities of the Fc epsilon RI. Both the unoccupied Fc epsilon RI and IgE-Fc epsilon RI complexes became immobilized, as determined from in situ electromigration and postelectric field relaxation. Immobilization of the Fc epsilon RI by wheat germ agglutinin was accompanied by a ligand-reversible association of 125I-IgE-Fc epsilon RI complexes with the Triton X-100-insoluble cytoskeletal fraction. Wheat germ agglutinin rapidly inhibited Fc epsilon RI-mediated signal transduction and secretion, whether cross-linkage was initiated by multivalent antigen, covalent IgE oligomers, anti-IgE, or anti-Fc epsilon RI antibody. Inhibition of signaling and secretion occurred on simultaneous addition of wheat germ agglutinin and antigen, and also when wheat germ agglutinin was added at increasing times after induction of Fc epsilon RI cross-linkage. Wheat germ agglutinin neither reduced the affinity of anti-DNP IgE for haptenic DNP-lysine nor reversed the binding of IgE to the Fc epsilon RI. Although wheat germ agglutinin caused internalization of the Fc epsilon RI, the onset of inhibition preceded and its extent exceeded that of internalization. Wheat germ agglutinin did not interfere with the secretory apparatus, as indicated by its lack of inhibition of secretion elicited by calcium ionophores. These findings suggest that inhibition of signal transduction is secondary to an initial event linked to immobilization of the Fc epsilon RI. Implications of these results are discussed with respect to the dynamics of Fc epsilon RI aggregation on rat basophilic leukemia cells.
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PMID:Immobilization of Fc epsilon receptors by wheat germ agglutinin. Receptor dynamics in IgE-mediated signal transduction. 839 54

Tryptase is the major secretory protease of human mast cells and is proposed to be involved in neuropeptide processing and tissue inflammation. Exploration of the biology of tryptase has been hindered by the lack of potent, selective inhibitors. The current study explores the properties of aromatic diamidines as inhibitors of dog and human tryptase. The strongest inhibitors of tryptase in this series are bis(5-amidino-2-benzimidazolyl)methane (BABIM) and (5-amidino-2-benzimidazolyl)-(5-(N,N'-dimethylamidino)-2- benzimidazolyl)methane, which exhibit K(i) values of 1.8 and 1.4 nM, respectively, in blocking the hydrolysis of tosyl-L-Gly-Pro-Lys-4-nitroanilide by human tryptase. These compounds are approximately 10,000-fold more potent than benzamidine, and are the strongest reversible inhibitors of tryptase described to date. Other aromatic mono- and diamidines, including amiloride and pentamidine, are less potent. Nonetheless, they abolish tryptase activity at high inhibitor concentrations. The rank order of tryptase inhibitor potency parallels that of inhibitors tested against trypsin. BABIM, the only highly active member of this series whose potency against other targets has been examined previously, is a far stronger inhibitor of tryptase than of other trypsin-like serine proteases, including those involved with hemostasis, fibrinolysis and the complement system. Therefore, BABIM appears to have selective affinity for tryptase. In addition to inhibiting tryptase-induced hydrolysis of peptide-based chromogenic substrates, BABIM blocks completely the reversal of vasoactive intestinal peptide-induced relaxation of isolated trachea by dog tryptase. Thus, BABIM and related amidines are potent inhibitors of mast cell tryptases that may be useful in exploring mast cell protease biology.
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PMID:Bis(5-amidino-2-benzimidazolyl)methane and related amidines are potent, reversible inhibitors of mast cell tryptases. 843 15

A hapten (DNP) model of topically induced ocular anaphylaxis has been developed. Rats immunized with DNP-Ascaris were skin-tested with DNP-bovine serum albumin (DNP-BSA) and Evans blue and challenged topically with varying amounts of di-DNP-lysine. The degree of clinical conjunctival edema was assessed, and eye tissues were evaluated histologically. Clinical conjunctival edema and histologic mast cell degranulation increased with higher concentration of di-DNP-lysine. In general, rats with positive skin tests showed more clinical conjunctival edema and more mast cell degranulation than those with negative skin tests. Three other groups of rats with positive skin tests to the DNP-BSA were injected intravenously with 125I-BSA and challenged topically with di-DNP-lysine. Retention of 125I-BSA in ocular adnexa and in globes was higher in di-DNP-lysine- than in PBS-challenged eyes. The hapten model simulates the ocular component of human hay fever in that ocular anaphylaxis is induced in immunized rats by topical challenge with antigen alone.
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PMID:A hapten model of topically-induced ocular anaphylaxis in the rat. 859 6

A rapid, sensitive, semiautomated method to measure the activity of mast cell-derived tryptase has been developed. The assay is based on the tryptase-mediated hydrolysis of vasoactive intestinal peptide that was modified to include an N-terminal dansyl reporter group. Tryptase cleaves vasoactive intestinal peptide (VIP) producing two major and two minor products. Full-length VIP was separated from the proteolysis products by reverse-phase (C18) chromatography. The amount of each product as well as the amount of full-length substrate remaining was determined using an in-line fluorescence detector. As little as 0.5 pmol of peptide could be detected. Predominant cleavages were after Arg-14 and Lys-20 with minor cleavages after Lys-15 and Lys-21. The hydrolysis of VIP at Arg-14 was slightly affected by the presence of the dansyl group at the N-terminus. While the Km value was unaffected, the kcat value decreased, yielding a kcat/Km ratio that was eightfold lower. The addition of calcium chloride to the reaction mixture resulted in a slight decrease in the kcat/Km ratio. Cleavage of dansyl-VIP by tryptase was completely inhibited by DesPro2-Arg15-Aprotinin.
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PMID:High-performance liquid chromatographic assay for tryptase based on the hydrolysis of dansyl-vasoactive intestinal peptide. 861 98

The practical application of exopeptidase has been limited by the high cost of the enzymes resulting from the low content of individual exopeptidase in the raw material. This can be overcome by the use of a combination of all the exopeptidases in the same enzyme source, as well as the use of the enzyme immobilization technology. A porcine pancreatic exopeptidase mixture was prepared by the ammonium sulfate precipitation at 35% saturation of the autolyzed pancreatic juice. The enzyme preparation was immobilized on thin shrimp chitin film by crosslinking with glutaraldehyde. The immobilized porcine pancreatic exopeptidases (IPPE) was effective in releasing the free amino acids from peptides. Of these amino acids, the concentrations of arginine, lysine, histidine, tyrosine, phenylalanine, leucine, and glutamine were increased much more than those of other amino acids. This indicated that both the porcine pancreatic exopeptidases preparation and the IPPE contained carboxypeptidase A, B, and aminopeptidase. The IPPE was also efficient in the decrease of the hydrophobicity of protein hydrolysates demonstrated by hydrophobic chromatographic analysis. This led to the application of the immobilized exopeptidases in protein hydrolysate debittering. The IPPE was able to remove the bitterness of the tryptic/chymotryptic casein hydrolysates.
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PMID:The immobilized porcine pancreatic exopeptidases and its application in casein hydrolysates debittering. 867 84

Prostaglandin (PG)D2 is a major product of arachidonic acid metabolism in pulmonary mast cells. We therefore attempted to determine whether measurement of the stable urinary metabolite of PGD2, 9 alpha, 11 beta-PGF2, could serve as a marker of mast cell activation in the lungs. A commercially available enzyme immunoassay was validated and found to be specific and sensitive when applied to unpurified urine. There was no diurnal variation in the levels of 9 alpha, 11 beta-PGF2 in healthy volunteers. Morning baseline values of urinary 9 alpha, 11 beta-PGF2 were measured in three groups--healthy volunteers (n = 9), patients with atopic asthma (n = 14), and aspirin-intolerant patients with asthma (n = 12)--and found to be very similar, 54 +/- 9, 62 +/- 6, and 71 +/- 15 ng/mmol creatinine, respectively (means +/- SEM). Urinary excretion of 9 alpha, 11 beta-PGF2 was increased threefold immediately after allergen-induced bronchoconstriction in nine patients with atopic asthma. Bronchial challenge with inhaled lysine aspirin in eight aspirin-intolerant patients with asthma produced bronchoconstriction without extrapulmonary symptoms and was also followed by a significant increase in the urinary excretion of 9 alpha, 11 beta-PGF2. In addition, challenge with a higher dose of aspirin produced an even greater increase in urinary 9 alpha, 11 beta-PGF2, supporting dose-dependent release of PGD2 during aspirin-induced bronchoconstriction. In contrast, the postchallenge levels of urinary 9 alpha, 11 beta-PGF2 were not increased when bronchoconstriction was induced by histamine challenge in the aspirin-intolerant patients with asthma. The study confirms mast cell involvement in allergen-induced bronchoconstriction and provides novel data, which strongly support the hypothesis that pulmonary mast cells are activated during aspirin-induced airway obstruction. It is finally suggested that measurement of urinary 9 alpha, 11 beta-PGF2 with enzyme immunoassay may be used as a new noninvasive strategy to monitor mast cell activation in vivo.
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PMID:Increased urinary excretion of the prostaglandin D2 metabolite 9 alpha, 11 beta-prostaglandin F2 after aspirin challenge supports mast cell activation in aspirin-induced airway obstruction. 875 20


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