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 interaction of serine protease (esterases) with 6-chloro-2-pyrones was investigated. Time-dependent inactivation of chymotrypsin, alpha-lytic protease, pig liver elastase, and cholinesterase was found with 3- and 5-benzyl-6-chloro-2-pyrone, as well as 3- and 5-methyl-6-chloro-2-pyrone. No inactivation was observed with the unsubstituted 6-chloro-2-pyrone. The substituted pyrones did not inactivate papain or carboxypeptidase A, as well as a number of other nonproteolytic enzymes. The substituted chloropyrones, therefore, show considerable selectivity toward serine proteases. Analogues in which the 6-chloro substituent is replaced by H or OH do not inactivate. The presence of the halogen is, therefore, essential for inactivation. Chymotrypsin catalyzes the hydrolysis of 3-benzyl-6-chloro-2-pyrone. At pH 7.5, (E)-4-benzyl-2-pentenedioic acid is the major product, and 2-benzyl-2-pentenedioic anhydride is a minor product. The ration of hydrolysis product found to the number of enzyme molecules inactivated varies from 14 to 40. The enzyme inactivated with the 3-benzyl compound does not show a spectrum characteristic of the pyrone ring. This suggests that inactivation by 3-benzyl-6-chloro-2-pyrone occurs in a mechanism-based fashion after enzymatic lactone hydrolysis. When the enzyme is inactivated with the 5-benzyl compound, absorbance due to the pyrone ring is observed. We suggest that inactivation occurs through an active site directed mechanism involving a 1,6-conjugate addition of an active site nucleophile to the pyrone ring.
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PMID:Novel inactivators of serine proteases based on 6-chloro-2-pyrone. 641 Nov 20

Carboxypeptidase A (EC 3.4.17.1) has been purified 44 000-fold in 33% yield from rat skeletal muscle by a four-step procedure. Purification in the presence of dichlorovinyl dimethyl phosphate conveniently inactivates an accompanying chymotrypsin-like enzyme and other serine protease(s) to ensure isolation of pure carboxypeptidase A free of polypeptide contaminants. The enzyme preparation consists of two components with molecular weights of approximately 39 300 and 37 800. The rat muscle carboxypeptidase is very similar to bovine pancreatic carboxypeptidase A in terms of (1) substrate specificity, (2) kinetics and molecular activity, (3) influence of metal ions on catalysis, (4) interaction with inhibitors, (5) effects of ionic strength on activity, and (6) stability and activity as functions of pH. Both muscle and pancreatic carboxypeptidases exhibit enhanced esterolytic activity when assayed in the presence of a variety of indoles and imidazoles or after incubation at relatively high concentrations of MnSO4. The muscle enzyme is substantially less stable than its pancreatic homologue, and in impure preparations is very much less soluble. The latter property is attributable to a binding substance present in such preparations which renders muscle but not pancreatic carboxypeptidase A insoluble until ionic strength is increased to values near 2 M.
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PMID:Purification and characterization of carboxypeptidase A from rat skeletal muscle. 701 67

Proteolytic enzyme activities were measured in skeletal muscle of Sprague-Dawley rats with streptozotocin-induced diabetes [tail vein injection of streptozotocin (100 mg/kg), under ether anesthesia]. Assay of rat muscle homogenates from diabetic rats revealed a significant increase in alkaline serine protease activity as compared to untreated control rats and diabetic rats given insulin. There were no significant changes in lysosomal cathepsin activities in diabetic muscle as compared to controls. Gel studies of myofibrils isolated from the three groups of rats, subjected to autolysis, revealed that the serine protease had copurified with the myofibrils. Treatment of rats with compound 48/80, which degranulates mast cells, abolished the alkaline protease activity. There was no serine protease activity associated with the myofibrils isolated from compound 48/80-treated rats. Results from this study indicate that serine proteases are not involved in muscle protein breakdown in diabetes and are of mast cell origin.
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PMID:Muscle proteolytic enzyme activities in diabetic rats. 703 84

Bronchopulmonary dysplasia (BPD), the most common cause of chronic lung disease in prematurely born infants, is histologically characterized by various degrees of airway and alveolar septal fibrosis. Tryptase, a serine protease specific to mast cells, has been shown to have potent fibroblast mitogenic properties and in addition has been shown to be increased in adult fibrotic lung disorders. Based on this analogy, the distribution of pulmonary mast cells exhibiting tryptase immunoreactivity was investigated by immunoperoxidase staining in autopsy specimens of infants dying with BPD. Morphologically normal lung specimens from similarly aged infants dying of sudden infant death syndrome (SIDS) served as controls. Tryptase-positive mast cell counts were performed at 250x from at least 10 random fields in bronchial, peribronchiolar, and alveolar regions. Compared to controls, in lung sections exhibiting typical histologic features of long-standing BPD, tryptase positive cells were significantly increased in bronchial (23.9 +/- 3.6 vs 14.4 +/- 2.3) and peribronchiolar (15.3 +/- 3.2 vs 4.63 +/- 0.6) regions compared to controls (P < 0.05, Student's t test). In particular, alveolar regions exhibiting moderate to severe degrees of septal fibrosis exhibited a dramatic increase in the number of tryptase-positive cells (9.83 +/- 1.89 vs 0.34 +/- 0.18, P = 0.003). These findings of a tryptase-positive mast cell hyperplasia in BPD suggest potential roles of mast cells as well as tryptase in the pathogenesis of this disease.
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PMID:Tryptase immunoreactive mast cell hyperplasia in bronchopulmonary dysplasia. 756 12

Rat mast cell protease 1 (RMCP-1) is a chymotrypsin-like serine protease (chymase) that is specifically expressed by connective-tissue-type mast cells. It is stored in the secretory granules of the cells in a complex with heparin proteoglycan, and the chymase/heparin proteoglycan complexes are released following mast cell activation. The present study was undertaken to examine if the association with heparin proteoglycan influenced the regulation of RMCP-1 by various macromolecular protease inhibitors. Endogenous mast cell heparin proteoglycan was shown to significantly block the inhibition of RMCP-1 by the serpins alpha 1-protease inhibitor and alpha 1-antichymotrypsin, as well as the inhibition by alpha 2-macroglobulin, soybean trypsin inhibitor and plasma. The blocking of protease inhibition showed an optimum at a RMCP-1/proteoglycan ratio of 5:1 (by mass), corresponding to approximately 80 RMCP-1 molecules bound/proteoglycan molecule. Chymase activity present on intact peritoneal mast cells, i.e. present in its native complex with heparin proteoglycan, was also shown to be largely resistant to inhibition by alpha 1-antichymotrypsin and alpha 1-protease inhibitor. Heparin 10-saccharides and 20-saccharides were inefficient in preventing the interaction of RMCP-1 with alpha 1-antichymotrypsin, whereas pig mucosal heparin (approximately 50 monosaccharide units) blocked protease inhibition. We have previously shown that heparin potentiates the catalytic activity of RMCP-1 and, in the present study, we show that the mechanism for chymase activation involves a sixfold reduction of the Km,app value of RMCP-1 for the chromogenic substrate S-2586. Thus, the association of mast cell chymase with heparin proteoglycan may serve both to potentiate the catalytic activity of the enzyme and to increase the life-span of the chymases by preventing their inhibition after exocytosis.
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PMID:Regulation of rat mast cell protease 1 activity. Protease inhibition is prevented by heparin proteoglycan. 758 46

2H3 subline of rat basophilic leukemia (RBL-2H3) cells are mast cell analogs that lack responsiveness to nonimmunologic stimuli such as compound 48/80 and substance P. To determine if fibroblasts can influence this responsiveness, RBL-2H3 cells were cocultured with confluent monolayers of mouse 3T3 fibroblasts and assayed for secretagogue-induced histamine release. After 1 wk in coculture, RBL-2H3 cells began to respond to compound 48/80. Responsiveness reached a maximum at 2 wk in coculture and remained at this level for an additional 2 wk. Histamine release was specific, noncytotoxic, dose-dependent, and occurred even in the absence of extracellular Ca2+. No soluble factor from 3T3 cells was found that induced these alterations. Moreover, neither recombinant rat or mouse steel factor, at concentrations up to 250 ng/ml, was able to alter RBL-2H3 cell reactivity to compound 48/80. By 2 wk in coculture, RBL-2H3 cells also became responsive to substance P, although no changes in histamine content, Alcian blue+/safranin- staining or type of serine protease were detected. These results show that 3T3 fibroblasts cause an alteration in the functional repertoire of RBL-2H3 cells and that soluble steel factor cannot duplicate the effect.
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PMID:Mouse 3T3 fibroblasts induce rat basophilic leukemia (RBL-2H3) cells to acquire responsiveness to compound 48/80. 767 78

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

The complete amino acid sequence of duodenase, a new serine endopeptidase from bovine duodenal mucosa, has been determined. The sequence was reconstructed by the automated sequence analysis of the peptides obtained after cleavage with trypsin, Staphylococcus aureus V8 protease, cyanogen bromide and duodenase. The enzyme is composed of 226 amino acid residues yielding a molecular mass of 29.06 kDa. The presence of six cysteine residues and one potential sugar-chain-binding site at Asn50 was revealed. A predicted catalytic triade characteristic of the serine proteases was traced in the duodenase primary structure at the corresponding positions (His44, Asp87 and Ser181 in the sequence). Comparison of the sequence of duodenase with the other known primary structures of mammalian serine proteinases reveales the duodenase identity to granzymes from human and mice, human cathepsin G and mast cell chymases from rat, and gives an overall sequence identity of 47-55% with the mentioned enzymes. Alignment of the known serine protease and duodenase primary structures showed unique amino acid residues within the duodenase substrate-binding pocket at positions 189 (Asn) and 226 (Asp) (the bovine chymotrypsinogen A numbering). These results are discussed with respect to the relation between the duodenase unique residues within the primary specificity pocket S1 and the unusual dual specificity of the enzyme.
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PMID:Duodenase, a new serine protease of unusual specificity from bovine duodenal mucosa. Primary structure of the enzyme. 786 49

The azurophil granules of neutrophil granulocytes contain neutral proteases such as leukocyte elastase and cathepsin G. These are synthesized as inactive precursors, but following proteolytic processing, they are stored in granules as active enzymes. We describe the establishment of a transgenic cellular model for expression of the human myeloid serine protease cathepsin G. The cDNA for preprocathepsin G was stably expressed in the rat basophilic/mast cell line RBL-1 and the translation product was characterized by use of biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Conversion into complex form of an asparagine-linked carbohydrate unit of approximately 3.5 kDa was shown, as judged by the products obtained upon treatment with endoglycosidase H and N-glycanase. Proteolytic processing of 32.5-kDa procathepsin G into a 31-kDa form, within 1-2 h after synthesis, was demonstrated by pulse-chase experiments. Further processing into a 30-kDa form also occurred to a minor extent. The processed forms were enzymatically active, as judged by affinity for the serine protease inhibitors diisopropylfluorophosphate and aprotinin. Translocation of processed forms of cathepsin G to high density fractions, indicating targeting of the protease to granules, was demonstrated by subcellular fractionation. The weak base NH4Cl was shown to delay the processing and enzymatic activation of cathepsin G, whereas the monovalent ionophore monensin completely inhibited both events. Our data demonstrate that human cathepsin G transfected to rat RBL-1 cells, is proteolytically processed into enzymatically active forms and that subcellular transfer to granular organelles occurs. As the processing of transgenic human cathepsin G corresponds to that of endogenous protease of myeloid cells, the model should provide new unique possibilities to further characterize the activation and granular targeting of myeloid serine proteases.
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PMID:Processing of human cathepsin G after transfection to the rat basophilic/mast cell tumor line RBL. 792 11

Trypsin-related serine proteases are encoded by a very large gene family in mammals. We describe here a comparative analysis of the genomic DNA sequences of mouse, rat, and human mast-cell-specific serine protease genes. Strong evidence was found for multiple exchanges of genetic information between closely related members of this gene family. The 5' regulatory regions of MMCP-1 and MMCP-L share a remarkably high degree of sequence identity (98%), starting 10 base pairs downstream of exon 1 and extending to the end of the presently sequenced region at position -1347 of the MMCP-1 gene. The remaining parts of the two genes share approximately 80% sequence identity. Evidence for at least two additional, but not so recent, exchanges was found in the 3' regions of the MMCP-4 and MMCP-L genes and in the 5' regions of the genes for MMCP-1 and MMCP-2. The 5' regulatory regions of all presently characterized mouse mast-cell-specific chymotrypsin-like serine protease genes exhibit over 88% sequence identity in the region from the transcription initiation site to approximately position -600. An exception is MMCP-5 which is the most distantly related member of this subfamily. The high degree of sequence similarities indicates a strong evolutionary homogenization of the 5' regulatory region, possibly by several gene conversion events. In addition, several insertions of genetic information have been identified in genes for mast-cell chymases and genes for T-cell granzymes. A number of these have been found to represent repetitive sequences, such as L1. The previously characterized tissue-specific enhancer element of the RMCP II gene was identified as a member of a middle repetitive sequence. A cDNA for a newly discovered pseudogene, closely related to the mouse mast cell chymases was isolated by polymerase chain reaction amplification from a mouse connective tissue-like mast cell line. The structure of this cDNA is presented. We also present the characterization of a novel spliced variant of MMCP-6 that contains an alternative 3' terminal exon (exon 6). The function of this variant, if any, is still unknown. A comparative analysis of amino acid sequence identities between different hematopoietic serine proteases shows that a high degree of sequence similarity does not always correlate with relateness in cleavage specificity. This indicates that the substrate specificity evolved with a higher evolutionary rate than the degree of overall amino acid sequence identity of these proteases.
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PMID:Genes for mast-cell serine protease and their molecular evolution. 795 52


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