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)

When a membrane suspension prepared from isolated rat brown fat mitochondria was incubated at 37 degrees C for 4 h, a loss of uncoupling protein (UCP) immunoreactivity was observed on Western blots. Analysis of [3H]GDP-binding characteristics to UCP in isolated membranes also showed a significant reduction in Bmax without significant effect on Kd. The loss of UCP was not due to protease contamination from lysosomes or mast cell granules, since loss of UCP was still observed when mitochondria were treated with digitonin to lyse lysosomes prior to membrane preparation and when mitochondria were isolated from rats injected with compound 48/80 to degranulate mast cells. Furthermore, loss of UCP was observed at alkaline pH and was not affected by inhibitors of lysosomal enzymes. Loss of UCP immunoreactivity was markedly reduced when membranes were incubated at 4 degrees C or in the presence of phenylmethylsulfonyl fluoride, but was not influenced by the addition of GDP. Overall, these results indicate the presence of a serine protease within brown fat mitochondrial membranes that may be involved in the breakdown of UCP.
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PMID:Temperature and phenylmethylsulfonyl fluoride sensitive loss of uncoupling protein in isolated brown adipose tissue mitochondrial membranes. 806 40

Mouse mast cells differentially express at least four chymases (mouse mast cell protease (mMCP) 1, mMCP-2, mMCP-4, and mMCP-5), a tryptase (mMCP-6), and an exopeptidase (mouse mast cell carboxypeptidase A (mMC-CPA)). The previously uncharacterized 2.5-kilobase mMCP-2 gene was isolated and found to consist of 5 exons. The 5'-flanking region of this gene is 89, 93, and 42% similar to that of the mMCP-1, mMCP-4, and mMCP-5 genes, respectively. Inheritance patterns of restriction-enzyme fragment length polymorphisms of these six mast cell protease genes in recombinant inbred mouse strains and interspecific backcrosses were used to determine their chromosomal locations. The mMCP-6 and mMC-CPA genes are located on chromosomes 17 and 3, respectively, whereas the four mast cell chymase genes all reside on chromosome 14 linked to a gene complex that encodes four cytotoxic T lymphocyte granzymes. Pulsed-field gel electrophoresis of genomic DNA digests demonstrated that the mMCP-1, mMCP-2, and mMCP-5 genes are within 850 kilobases of each other. Although clustering of the serine protease genes on chromosome 14 may be important at a higher level of genomic organization, the ability to independently induce or suppress the steady-state levels of the four chymase transcripts by treatment of mast cells with cytokines suggests that gene clustering is not the most critical factor for coordinate expression of these proteases. Because of the unique features of their tertiary structures, the substrate specificities of the serine proteases encoded by genes at the chromosome 14 complex are predicted to be more limited than those of pancreatic chymotrypsin and pancreatic trypsin, whose genes reside on chromosomes 8 and 6, respectively. Based on present day genomic distribution and sequence similarities, we propose that a primordial gene that encoded a serine protease with restricted substrate specificity underwent extensive duplication and divergence to form a family of cytokine-regulated transcripts from genes on chromosome 14.
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PMID:A closely linked complex of mouse mast cell-specific chymase genes on chromosome 14. 809 10

Heparin is a sulfated glycosaminoglycan, synthesized by connective tissue-type mast cells. Rat mast cell protease 1 (RMCP-1), a chymotrypsin-like serine protease expressed specifically by connective tissue-type mast cells, is recovered in a macromolecular complex with heparin proteoglycan. The heparin.RMCP-1 complexes are stored in the secretory granules of the cells and are released following mast cell activation. We showed previously that dissociation of RMCP-1 from heparin resulted in loss of protease activity, as measured by its ability to inactivate thrombin. In the present report the binding of heparin to RMCP-1 was characterized. Affinity chromatography on heparin-Sepharose showed that RMCP-1 displayed high affinity for heparin, with approximately 1.2 M NaCl being required for elution of RMCP-1 from the affinity matrix. The structural requirements for the binding of heparin to RMCP-1 were investigated. Heparan sulfate, chondroitin sulfate, and dermatan sulfate, three glycosaminoglycans structurally related to heparin, were > or = 80-fold less effective in binding to RMCP-1 than heparin. The 2-O-sulfate, 6-O-sulfate, and N-sulfate groups in heparin were all shown to contribute in the binding. The minimal heparin sequence required for binding to RMCP-1 was found in a 14-saccharide fraction. 14-Saccharide species, obtained after separation by anion exchange chromatography, showed continuously increased binding with increasing anionic charge densities. The 16-18-saccharides were the smallest heparin oligosaccharides capable of accelerating the inactivation of thrombin by RMCP-1.
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PMID:Interaction of heparin with rat mast cell protease 1. 818 50

The cell line HMC-1, derived from a patient with mast cell leukaemia, is the only established cell line exhibiting a phenotype similar to that of human mast cells. This paper reports on a detailed characterization of the expression of a panel of markers for various types of immature and mature haematopoietic cells in the HMC-1. We also studied the potential of HMC-1 to differentiate upon treatment with conditioned media from the human T-cell line Mo, retinoic acid or DMSO. HMC-1 was found to express several mast cell-related markers. A high expression of Kit, the receptor for stem-cell factor, was detected. The majority of the cells were stained with a MoAb against the mast cell-specific serine protease tryptase. Of particular interest was the finding that beta-tryptase mRNA, but not alpha-tryptase mRNA, was expressed in HMC-1. Using enzyme-histochemistry we were able to show that the beta-tryptase was enzymatically active, indicating that tryptase can form active homotetramers. Both heparin and chondroitin sulfate were found to be present in approximately equal amounts. HMC-1 lacked surface expression of the high-affinity IgE receptor, which was confirmed by the absence of mRNA of the alpha- and beta-chains of the IgE-receptor complex. However, a strong expression of the gamma-chain of the IgE-receptor complex was detected. A positive staining of the monocyte/macrophage marker CD68 was obtained, as well as a strong hybridization signal for the eosinophilic/basophilic-related differentiation marker the Charcot-Leyden crystal. Treatment of HMC-1 with conditioned media from the human T-cell line Mo, retinoic acid or DMSO induced only moderate changes in the surface or intracellular expression of the studied markers. The agents tested neither induced any of the monocyte/granulocyte markers examined, nor expression of the Fc epsilon RI alpha-chain.
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PMID:Phenotypic characterization of the human mast-cell line HMC-1. 819 Dec 24

The expression of a tryptic serine protease was detected in the cell line KU812 by Northern blot analysis with an oligonucleotide probe directed against a conserved region present in all of the five presently cloned human mast cell tryptases. PCR primers designed for the amplification of a nearly full-length copy of tryptase mRNAs were used to study the identity of the KU812 tryptase. Ten clones were characterized and all were found to be identical to one of the tryptases previously cloned from a human skin cDNA library. This tryptase has been thought to originate from mast cells of the skin. Two possible explanations may account for the observed identity between the presumed mast cell tryptase and the KU812 tryptase. Firstly, it is possible that the KU812 tryptase is a basophil-specific tryptase which has previously been cloned from a human skin cDNA library containing low levels of cDNA copies derived from basophils in the starting material. Secondly, the KU812 cell line, and possibly normal basophils, express a tryptase which is identical to one of the tryptases expressed in normal skin mast cells. We cannot at present rule out any of the two possibilities, but we favour the second explanation as being the most likely.
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PMID:Characterization of a tryptase mRNA expressed in the human basophil cell line KU812. 843 31

Tryptase, a mast cell serine protease, has been implicated in the pathophysiology of allergic asthma, but formal evidence to support this hypothesis has been limited by the lack of specific inhibitors for use in vivo. Therefore, in this study we examined the effects of two inhibitors of tryptase, APC 366 [N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolinamide hydrochloride] and BABIM [bis(5-amidino-2-benzimidazolyl)methane] on antigen-induced early and late responses, airway responsiveness as measured by carbachol provocation, microvascular permeability as measured by bronchoalveolar lavage (BAL) albumin concentrations, and tissue eosinophilia from biopsies in allergic sheep. APC 366 and BABIM were administered by aerosol in all experiments. In vehicle control trials, antigen challenge resulted in peak early and late increases in specific lung resistance (SRL) of (mean +/- SE, n = 6) 259 +/- 30% and 183 +/- 27% over baseline, respectively. Treatment with APC 366 (9 mg/3 ml H2O given 0.5 h before, 4 h after, and 24 h after antigen challenge) slightly reduced the peak early response (194 +/- 41%), but significantly inhibited the late response (38 +/- 6%, p < 0.05 versus control trials). Twenty-four hours after challenge, APC 366 also completely blocked the antigen-induced airway hyperresponsiveness to inhaled carbachol observed in the control trial.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tryptase inhibitors block allergen-induced airway and inflammatory responses in allergic sheep. 852 Jul 78

Tryptase is a trypsin-type serine protease that is released from mast cells. Bradykinin (BK) is released directly from kininogens or through activation of either Hageman factor or subsequent plasma prekallikrein. Its nasal administration or inhalation induces allergy-like symptoms. Although elevated levels of tryptase and BK in allergic fluids have been detected, the role of this proteinase and the mechanism of BK production at allergic reaction sites are still unknown. To investigate the pathologic functions of tryptase, the enzyme, purified from human lung, was incubated with normal human plasma, deficient plasmas, kininogens, or prekallikrein. High molecular weight kininogen was then added, and the mixtures were examined for vascular permeability enhancement (VPE) activity, a representative function of bradykinin, using guinea pig skin. Tryptase-treated plasma induced VPE in a dose-dependent manner; activity was lost in the absence of a kininase inhibitor but not an antihistamine drug. Tryptase produced VPE activity from normal or Hageman factor-deficient plasma, but only 30% of this activity was produced from prekallikrein-deficient plasma. Significantly, no activity was obtained from kininogen-deficient plasma. Deficient plasma that were reconstituted with each missing factor resulted in VPE-inducing capacity by tryptase, equivalent to that found with normal plasma. Incubation of tryptase with high or low molecular weight kininogen induced VPE activity in a dose- and incubation time-dependent manner. Prekallikrein incubated with tryptase also generated a soybean trypsin inhibitor-sensitive VPE-inducing activity from high molecular weight kininogen. The loss of tryptase VPE-producing activity as a function of incubation time was found to be a result of spontaneous inactivation of the enzyme and not of the degradation of high molecular weight kininogen by the enzyme. We conclude that tryptase induces VPE by releasing BK, primarily through prekallikrein activation, but also through direct release from kininogens. This indicates that this mast cell-derived proteinase contributes to kinin production in allergic diseases.
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PMID:Induction of vascular permeability enhancement by human tryptase: dependence on activation of prekallikrein and direct release of bradykinin from kininogens. 864 82

Tryptase (EC 3.4.21.59), the major secretory product of human mast cells, has become widely used as a biochemical marker for mast cells and mast cell activation, and is attracting attention as a mediator of allergic disease. However, there is little information available on the properties, or even the presence, of this protease in commonly used species of laboratory animals. We, here, report the demonstration and characterisation of this enzyme in the guinea pig lung. Tryptic activity resistant to alpha 1-proteinase inhibitor and soybean trypsin inhibitor was detected in sections of guinea pig lung tissue with the histochemical substrate Z-Gly-Pro-Arg-MNA. It was localised to mast cells and appeared to be present in all mast cells staining with Alcian Blue. A tryptic protease was purified 2400-fold from whole lung tissue by high salt extraction, cetylpyridinium chloride precipitation, heparin agarose chromatography, and gel filtration. This enzyme was found to be multimeric with a subunit of 38 kDa and a native molecular mass of 860 +/- 100 kDa. Inhibitor studies identified it as a serine protease. Like human tryptase, it was inhibited by leupeptin, benzamidine, and APC 366 (N-(1-hydroxy-2- naphthoyl)-L-arginyl(-L-prolinamide hydrochloride), but not by alpha 1-proteinase inhibitor, soybean trypsin inhibitor, or antithrombin III. Its response to changes in pH and ionic strength was similar to that of human tryptase. Differences between the guinea pig and human enzymes were seen in activity toward a panel fo 10 tryptic p_nitroanilide peptide substrates. Kinetic constants were determined for two of these: with L-Pyr-Pro-Arg-pNA the guinea pig tryptase had a similar Km but a 5-fold lower kcat than human tryptase, and with L-Pyr-Gly-Arg-pNA the guinea pig enzyme had a 10-fold lower Km and a 30% greater kcat than human counterpart. Heparin stabilised guinea pig tryptase, but did not alter its kinetic parameters as it did with human tryptase, decreasing the Km towards both substrates. The presence of a protease with similarities to human tryptase in the mast cells of guinea pigs suggests that this species may be an appropriate model to investigate the actions to tryptase in vivo, provided cognizance is taken of the differences that do exist.
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PMID:Guinea pig lung tryptase. Localisation to mast cells and characterisation of the partially purified enzyme. 869 58

Allergen-induced bronchoconstriction involves mast cell activation. Tryptase is a mast cell serine protease that is released during this process, but little is known about the action of tryptase in the airway. The purpose of this study was to determine: (1) if aerosolized tryptase causes bronchoconstriction, and (2) the mechanism by which this occurs. We measured mean pulmonary flow resistance (RL) in five allergic sheep before and after consecutive inhalations of 100 and 500 ng tryptase (in 2 ml total volume). Inhaled tryptase at 100 and 500 ng increased RL (mean +/- SE) by 33 +/- 12 and 122 +/- 8% (p < 0.05) over baseline. The response was reproducible upon repeat challenges. These studies were repeated in the same animals after pretreatment with aerosolized APC 366 (9 mg/3 ml), a specific tryptase inhibitor. In APC-366-treated sheep, tryptase increased RL by 10 +/- 3 and 6 +/- 2% (p < 0.05 versus control values) at 100 and 500 ng, respectively. The response to tryptase was also blocked by pretreating the sheep intravenously with the histamine H1-antagonist chlorpheniramine (2 mg/kg), in which RL increased only 5 +/- 4 and 7 +/- 6% after 100 and 500 ng tryptase. APC 366, however, did not block histamine-induced bronchoconstriction. Consistent with these findings was the observation that segmental bronchial challenge with tryptase (1 microgram) resulted in a significant increase in histamine levels in bronchoalveolar lavage. Inhaled tryptase (500 ng) also caused airway hyperresponsiveness to aerosolized carbachol 2 h after tryptase challenge. This tryptase-induced airway hyperresponsiveness could be blocked either by pretreating the sheep with APC 366 (30 min before challenge) or by treating the sheep 30 min after challenge. These results indicate that inhaled tryptase causes bronchoconstriction and airway hyperresponsiveness in allergic sheep by an event that may involve mast cell activation.
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PMID:Inhaled tryptase causes bronchoconstriction in sheep via histamine release. 881 Jun

Proteinase 3 (PR3) is a neutrophil serine protease stored in the azurophil granules of the promyelocyte and its successors. The protease has been identified as an autoantigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) occurring in patients with Wegener's granulomatosis. To characterize the biosynthesis and processing of human PR3 in a transgenic cellular model, cDNA encoding human pre-proproteinase 3 cloned from U-937 cells was transfected to the rat basophilic/mast cell line RBL-1 and the murine myeloblast-like cell line 32D c13. The stable expression of transgenic proteinase 3 was characterized by biosynthetic labeling, followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. After pulse labeling for 30 min two proforms of PR3 (32 and 35 kDa), differing in carbohydrate content but with protein cores of identical size, were demonstrated. Chase of the label resulted in a processed 32-kDa form clearly visible in RBL, but only faintly in 32D cells, probably indicating delayed intracellular transfer in the latter cell line. Partial digestion with N-glycosidase F showed that both potential N-glycosylation sites on PR3 were occupied and conversion of the oligosaccharide side chains into complex forms was demonstrated by acquisition of resistance to endoglycosidase H. Translocation of PR3 to granules was shown by subcellular fractionation and immunocytochemistry. Enzymatic activation of PR3 was suggested by affinity to diisopropylfluorophosphate and removal of an amino-terminal propeptide. Cells transfected with PR3 showed positive immunofluorescence for ANCA-containing sera from patients with Wegener's granulomatosis. Our results show that human PR3 transfected to RBL or 32D cells is synthesized as a 29-kDa protein core glycosylated on two distinct sites. Oligosaccharide trimming and proteolytic processing occur and the protein is targeted for granular storage in a form antigenic for ANCA.
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PMID:Characterization of the processing and granular targeting of human proteinase 3 after transfection to the rat RBL or the murine 32D leukemic cell lines. 900 May 44


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