Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The involvement of mast cells in the pathogenesis of aspirin (ASA)-induced respiratory reactions was investigated by measuring serum levels of tryptase, a neutral protease that is a specific marker of mast cell activation. ASA challenges were performed in 17 ASA-sensitive patients with asthma and rhinosinusitis, and tryptase and histamine levels were measured in their venous blood samples. In three subjects who experienced moderate to severe respiratory reactions extending to the skin and/or gastrointestinal tract, marked elevations of tryptase levels in postreaction serum samples (peak levels, 51.9 and 40.0 ng/ml) were discovered in two of these three subjects, and a small elevation of tryptase occurred in the serum of the third subject (3.1 ng/ml peak). Plasma histamine levels in postreaction samples were significantly elevated over baseline values in all three subjects (delta mean plasma histamine, 238 pg/ml versus 56 pg/ml for the remaining 14 subjects; p less than 0.04). In the remaining 14 subjects, who experienced similar respiratory reactions without extrapulmonary symptoms during aspirin challenge, changes in tryptase and histamine levels were not observed.
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PMID:Tryptase and histamine release during aspirin-induced respiratory reactions. 172 Jul 95

We have investigated the domain of the bindin polypeptide that selectively associates with gel-phase phospholipid vesicles. We found that small trypsin fragments of bindin retain the ability to selectively associate with gel-phase vesicles. The primary amino acid sequence of bindin suggests that these peptides are derived from the central portion of the polypeptide between residues 77 and 126, which is the most hydrophobic region of bindin. We have also employed 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID) and novel, radioiodinated, photoactivatable derivatives of the polar head group of phosphatidylethanolamine (ASD-PE and ASA-PE) to identify membrane-associated polypeptide segments after the transfer of radiolabel from the probe to the bindin polypeptide. After photolysis, bindin was selectively labeled only from probes incorporated in gel-phase vesicles. The labeling of bindin was much more efficient from the head group probes ASA-PE and ASD-PE (8 and 2% of the total label, respectively) in comparison to the hydrophobic probe TID (less than 0.02% of the total label), suggesting that bindin is localized within the polar part of the bilayer. Protease mapping experiments with V8 protease, trypsin, and endoprotease Lys-C suggest that some of the probe label is distributed along the amino-terminal portion of bindin between residues 1 and 76 and the rest of the label is restricted to the segments between residues 77 and 126 which also selectively bind to gel-phase vesicles. The carboxyl-terminal portion of bindin between residues 127 and 236 is not labeled.
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PMID:Analysis of the membrane-interacting domain of the sea urchin sperm adhesive protein bindin. 260 49

The 4-azidosalicylate derivative of 1,3-bis(D-mannos-4'-yloxy)-2-[2-3H]propylamine (ASA-[2-3H]BMPA) has been tested as a photoaffinity label for the sugar transporter in human erythrocytes. When photolysed in the presence of intact erythrocytes, ASA-[2-3H]BMPA covalently binds to the exofacial surface of the transporter. This labelled protein appears as a broad band in the 4.5 region in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The peak of radiolabel incorporation gives an apparent Mr of approx. 50 000 on 5-20% acrylamide gels. The binding is 80% inhibitable by 320 mM 4,6-O-ethylidene-D-glucose, by 320 mM D-glucose and by 50 microM cytochalasin B. Photoirradiation of a saturating concentration of ASA-BMPA in the presence of erythrocytes results in a 25-30% loss of D-galactose transport activity. From transport inactivation data and estimations of the amount of ASA-[2-3H]BMPA binding to the transporter it is calculated that there are approx. 220 000 exofacial hexose-transport binding sites per erythrocyte. The labelling of the transporter has been carried out using freshly drawn blood and 4-weeks-old transfusion blood. No change in the binding profile on SDS-polyacrylamide gel electrophoresis was observed. Proteolytic digestion of the ASA-[2-3H]BMPA-labelled transporter with either trypsin or alpha-chymotrypsin results in the appearance of a labelled 19 kDa fragment on SDS-polyacrylamide gel electrophoresis.
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PMID:Exofacial photoaffinity labelling of the human erythrocyte sugar transporter. 375 52

In an attempt to define glycolipid functions we have prepared photoactivatable, iodinatable derivative of globoside and used it for photoaffinity labeling of human erythrocyte membranes. Lysogloboside (Gb4Sph) was prepared from globoside through deacylation in methanolic KOH followed by re-N-acetylation of galactosaminyl residue. The NH2 group of sphingosine residue in Gb4 Sph reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid resulting in the formation of Gb4Sph-ASA which was purified by preparative tlc and column chromatography. It migrated on tlc as a single spot in two solvent systems, was susceptible to leech ceramide glycanase and could be radioiodinated to a specific radioactivity of about 200 Ci/mmol. Gb4Sph-[125I]ASA was incorporated into human erythrocytes in a time and concentration-dependent manner. Before photolysis 96% of the Gb4Sph-ASA could be removed with albumin but not with trypsin. After photolysis about 50% of the label was firmly bound to erythrocytes being resistant to albumin and trypsin treatment. The label was distributed between membrane proteins and lipids in about 1:2.3 ratio. Photolabeled proteins were analyzed by SDS-PAGE followed by autoradiography and immunostaining. Most of the radioactivity was detected in band 3 and its proteolytic fragments irrespective of the duration of photolysis. Photolabeling of erythrocyte lipids was demonstrated by Sephadex LH-20 column chromatography.
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PMID:Photochemical labeling of human erythrocyte membranes with radioiodinatable azidosalicylic acid derivative of globoside. 764 2

In order to elucidate the mechanism responsible for infiltration of nasal mucosa by granulocytes, we tested neutrophil chemotactic activity (NCA) in nasal lavages, by the modified Boyden chamber method, in 16 patients with perennial allergic rhinitis (AR), six ASA-sensitive patients with chronic rhinosinusitis (CRS), and seven normal, nonatopic control subjects (NC). Nasal secretions from all three groups showed significant NCA (mean 157.1 +/- 54.0, 62.2 +/- 20.7, and 39.4 +/- 11.4% of FMLP chemotactic activity for AR, CRS, and NC subjects, respectively). Nasal secretions from patients with AR expressed significantly higher NCA (P < 0.02) than did secretions from NA patients. NCA was unchanged by heating at 56 degrees C for 60 min and was not susceptible to degradation by trypsin. Nasal challenge with Dermatophagoides pteronyssinus antigen induced clinical symptoms and resulted in significant increases in total protein and albumin concentrations in nasal lavages in AR patients, but failed to change the mean NCA activity for up to 40 min after the challenge. These results indicate that nasal secretions from both atopic and nonatopic patients express NCA, but its relation to allergic inflammation remains to be established.
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PMID:Neutrophil chemotactic activity (NCA) in nasal secretions from atopic and nonatopic subjects. Effect of antigen challenge. 823 96

We have photolabeled the inositol 1,4,5-trisphosphate (IP3) receptor and probed the IP3 ligand binding site using two novel photoaffinity ligands, [125I] (azidosalicyl)aminopropyl-IP3 ([125I]ASA-IP3) and [3H] (benzoyldihydrocinnamyl)aminopropyl-IP3 ([3H]BZDC-IP3). Both ligands have high affinity for the IP3 receptor and, when photoactivated, label the IP3 receptor protein with appropriate inositol phosphate selectivity. The high specific activity of [125I]ASA-IP3 allowed identification of a single photolabeling site within the IP3R by two-dimensional peptide analysis. Substantially higher levels of incorporation into the receptor are achieved with [3H]BZDC-IP3 (50-60% efficiency) than with [125I]ASA-IP3 (3%), facilitating the use of [3H]BZDC-IP3 as a better ligand for the high-efficiency labeling and purification of IP3R-labeled peptides. Peptides were generated from photolabeled IP3 receptor by trypsin digestion and purified by high-pressure liquid chromatography (HPLC). A single purified [3H]BZDC-IP3-labeled peptide, corresponding to IP3R amino acids 476-501, was sequenced and shown to match specific sequences in the N-terminal 20% of the IP3 receptor, an area suggested on the basis of mutagenesis studies to contain the IP3 recognition site.
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PMID:Inositol 1,4,5-trisphosphate receptors: labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands. 838 18

Benzimidazoles (BZ) are broad spectrum anthelmintics thought to exert their effects by interacting with and disrupting the functions of microtubules. However, direct biochemical evidence for binding between BZ and tubulin has not been shown nor is it known what sequences in tubulin interact with BZ. In this study, a photoactive analogue of 2-acetamido-5-(3-aminophenoxy)benzimidaz ole that has biological activity similar to other benzimidazoles was synthesized and used to photoaffinity label cell lysates from the parasitic nematode of sheep Haemonchus contortus. The photoactive analogue, 2-acetamido-5-[3-(4-azido-3-125I-salicyl amido)phenoxy]benzimida zol e or 125I-ASA-BZ, was shown to photolabel a 54-kDa protein that was specifically immunoprecipitated with anti-tubulin monoclonal antibodies. Tubulin photoaffinity labeling by 125I-ASA-BZ was also inhibited with molar excess of various BZ analogues and colchicine. Interestingly, 125I-ASA-BZ photoaffinity-labeled the beta- and not the alpha-subunits of tubulin. Proteolytic digestion of 125I-ASA-BZ-labeled tubulin with Staphylococcus aureus V8 proteinase revealed one major peptide with an apparent molecular mass of 3.5 kDa. Exhaustive digestion of 125I-ASA-BZ-labeled beta-tubulin with trypsin resulted in two fractions containing radioactive peptides. Protein sequencing of the high performance liquid chromatography-purified tryptic ASA-BZ-photolabeled peptides identified the N-terminal 63-77 and 78-103 sequences as the BZ binding domain.
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PMID:p-Azidosalicyl-5-amino-6-phenoxybenzimidazole photolabels the N-terminal 63-103 amino acids of Haemonchus contortus beta-tubulin 1. 862 85

We used photocross-linking of peptides to DnaK to identify elements of the peptide binding site of DnaK. We attached a photoactivatable group (N-hydroxysuccinimidyl-4-azido-salicylic acid (NHS-ASA) or N-iodoacetamidobutyl-4-azido-salicylic acid (I-ABASA)) to different positions on peptide C of the vesicular stomatitis virus glycoprotein, 125I-radiolabeled the cross-linker, cross-linked the peptide to DnaK by UV irradiation, and then determined the amino acid residues of DnaK that were cross-linked to the peptide. Limited trypsin digestion of the DnaK-peptide complex revealed that the derivatives modified with photoactivatable cross-linker peptide C cross-linked to a C-terminal fragment of DnaK and that the N-terminal 45-kDa fragment of DnaK was not cross-linked by these modified peptides. The attachment points of the three peptide C derivatives carrying photoactivatable cross-linkers at different locations on the peptide, PepC-ASA, PepC-S7C-ABASA, and PepC-S8C-ABASA, have been identified as Arg-536, Arg-527, and His-541 of DnaK, respectively. Thus all three peptides cross-linked to amino acids located close together in a sequence that includes one end of the long alpha-helix in the NMR-based secondary structure model of the peptide binding domain of Hsp70 family (Morshauser, R., Wang, H., Flynn, G., and Zuiderweg, E. (1995) Biochemistry 34, 6261-6266).
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PMID:Identification of elements of the peptide binding site of DnaK by peptide cross-linking. 870 69

A prospective, randomized, double-blind study was performed in 62 patients (ASA Classes I and II) treated with either 0.15 or 0.25 mg/kg cisatracurium or 0.15 mg/kg vecuronium administered as a rapid bolus. We wished to determine whether the muscle relaxants caused cutaneous, systemic, or chemical evidence of histamine release. Six minutes after induction of anesthesia with thiopental, patients received one of the muscle relaxants over 5 s. Plasma histamine levels were measured by radioimmunoassay after thiopental administration and 3 and 5 min after the administration of the relaxant. Additionally, plasma was assayed for tryptase, a marker of mast cell release. Cutaneous manifestations to both thiopental and the muscle relaxant were graded by an independent observer. Arterial blood pressure and heart rate were measured every minute. Although systolic and diastolic blood pressure decreased and heart rate increased significantly after thiopental administration (P < 0.0001), there were no further hemodynamic changes after either cisatracurium or vecuronium. One patient who received 0.25 mg/kg cisatracurium exhibited a slight elevation in plasma histamine level 5 min after hemodynamic changes. Cutaneous signs of histamine release were noted in five patients after thiopental administration (flush in four, erythema in one), but no further cutaneous reactions were observed after administration of either cisatracurium or vecuronium. We conclude that cisatracurium and vecuronium do not cause systemic or cutaneous histamine release. Tryptase levels showed no evidence of mast cell degranulation.
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PMID:The lack of histamine release with cisatracurium: a double-blind comparison with vecuronium. 905 14

A novel polyanhydride, poly[(5-carboxybutyl formamide)-2-acetyl salicylic anhydride] (P(CBFAS)), with 5-aminosalicylic acid (5-ASA) incorporated into the polymer backbone was synthesized and characterized by infrared, (1)H-nuclear magnetic resonance, differential scanning calorimetry, vapor pressure osmometry, etc. The polyanhydride was subjected to degradation and simultaneously released 5-ASA and its derivative 5-acetyl aminosalicylic acid (5-acetyl ASA) in vitro under various conditions. The factors influencing the release profiles of 5-ASA and 5-acetyl ASA, including polymer molecular weights, pH value, enzyme and rat gastrointestinal contents, were examined. The results showed that the release rate of 5-ASA and 5-acetyl ASA increases with increasing pH value and with decreasing molecular weights. In PBS (pH 8.0, 37 degrees C) total ASA released was 8.0% for P(CBFAS)(1) (Mn 10770) in 13 h, but only 1.1 and 2.6% at pH 2.0 and 6.5, respectively. Enzymes including pepsin and trypsin, as well as rat gastric and jejunum contents had little effect on the release rate of 5-ASA and 5-acetyl ASA at pH 2.0 and 6.5 (less than 4% in 13 h). However, the release rate of 5-ASA and 5-acetyl ASA was much fast in PBS(pH 8.0) containing 5% of cecal contents, the total ASA released was 13.6% for the polymer in 13 h. Considering the high drug loading of the polymer (50.2% of 5-ASA moieties in the backbones) and the degradation characters, it is possible to reach high local concentration of 5-ASA in the colon site via oral administration. Therefore, P(CBFAS) may be potentially useful in the colon specific delivery of 5-ASA.
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PMID:Synthesis, characterization and in vitro release of 5-aminosalicylic acid and 5-acetyl aminosalicylic acid of polyanhydride--P(CBFAS). 1263 98


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