Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Pen a 1, the major shrimp allergen from brown shrimp Penaeus aztecus has been identified as the muscle protein tropomyosin. To identify Pen a 1 IgE binding sites, the reactivities of Pen a 1-specific monoclonal antibodies (mAbs) and shrimp-allergic subjects' IgE to shrimp and homologous mammalian tropomyosins were analyzed. Pen a 1, purified by preparative SDS-PAGE and commercially obtained porcine, bovine and rabbit tropomyosin were cleaved by CNBr or digested by endoproteinases Lys-C, Glu-C, trypsin, Arg-C and chymotrypsin. Reactivities of Pen a 1-specific mAbs and IgE to the resulting peptides were analyzed by dot blot and immunoblotting. The dot blot analysis showed that mAbs and IgE antibodies did not react with any of the mammalian tropomyosins. The immunoblot analysis showed that all Pen a 1 digests bound IgE or mAbs. However, not all peptides in each digest possessed an IgE binding site. IgE binding intensity and frequency varied by subject and peptide digest. IgE and mAb reactivity patterns were similar but no mAb reproduced the IgE binding patterns indicating that subject' IgE bound some epitopes that were not recognized by the Pen a 1-specific mAbs. These studies suggest that IgE-binding epitopes are restricted to certain parts of the Pen a 1 molecule, Pen a 1 may have several similar epitopes, and that Pen a 1 epitopes do not appear to be located in the highly homologous parts of the tropomyosin molecule.
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PMID:IgE and monoclonal antibody reactivities to the major shrimp allergen Pen a 1 (tropomyosin) and vertebrate tropomyosins. 909 46

We have isolated a full-length chitinase complementary DNA from the tiger shrimp Penaeus monodon that encodes a 621 amino acid protein possessing the functional domains of the chitinase protein family. The Penaeus monodon chitinase 1 (PmChi-1) gene product is 81.8% identical to a chitinase 1 protein expressed in the hepatopancreas of Penaeus japonicus. Analysis by reverse transcription-polymerase chain reaction (RT-PCR) indicates that PmChi-1 messenger RNA is detectable in the hepatopancreas and the gut. PmChi-1 expression during the molt cycle fluctuates markedly, with lowest mRNA levels at stages A(1), C, and D(3); there is a dramatic increase in transcript abundance at the D(2) stage. Using the same tissues and molt stages, RT-PCR analyses of genes encoding other digestive enzymes (trypsin, chymotrypsin, and cathepsin L), a muscle structural protein (tropomyosin II), and housekeeping proteins (elongation factor II and GTP-binding protein) indicate that PmChi-1 is expressed in a distinct tissue-specific and stage-specific manner. The other digestive enzyme genes are expressed in a similar spatiotemporal pattern, but none exhibited a dramatic increase in transcript abundance at stage D(2). Increased expression of PmChi-1 at D(2) suggests that hepatopancreas-expressed chitinase is involved in the degradation of endogenous chitin in the gut peritrophic membrane prior to molting.
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PMID:The Penaeus monodon Chitinase 1 Gene Is Differentially Expressed in the Hepatopancreas During the Molt Cycle. 1081 51

A single administration of monocrotaline to rats results in pathologic alterations in the lung and heart similar to human pulmonary hypertension. In order to produce these lesions, monocrotaline is oxidized to monocrotaline pyrrole in the liver followed by hematogenous transport to the lung where it injures pulmonary endothelium. In this study, we determined specific endothelial targets for (14)C-monocrotaline pyrrole using two-dimensional gel electrophoresis and autoradiographic detection of protein metabolite adducts. Selective labeling of specific proteins was observed. Labeled proteins were digested with trypsin, and the resulting peptides were analyzed using matrix-assisted laser desorption ionization mass spectrometry. The results were searched against sequence data bases to identify the adducted proteins. Five abundant adducted proteins were identified as galectin-1, protein-disulfide isomerase, probable protein-disulfide isomerase (ER60), beta- or gamma-cytoplasmic actin, and cytoskeletal tropomyosin (TM30-NM). With the exception of actin, the proteins identified in this study have never been identified as potential targets for pyrroles, and the majority of these proteins have either received no or minimal attention as targets for other electrophilic compounds. The known functions of these proteins are discussed in terms of their potential for explaining the pulmonary toxicity of monocrotaline.
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PMID:Protein targets of monocrotaline pyrrole in pulmonary artery endothelial cells. 1087 30

Two isoforms of lobster muscle tropomyosin, a fast muscle type, fTm, and a slow muscle type, sTm1, are identical except for 15 residues within the region of amino acids 39-80, which corresponds to exon 2 of the tropomyosin genes of many phyla. Although the difference in the sequence does not include the terminal regions, the two isoforms are extremely different in viscosity, which is a good measure of the head-to-tail interaction strength and should be dependent on the conformation of the terminal 7-9 residues. To determine the influence of amino-acid replacements in the internal region on the overall conformation and the functional properties of the molecule, we compared the physical properties of the two isoforms and their interactions with other proteins, such as actin and myosin subfragment 1 (S1). Limited proteolysis by trypsin and chymotrypsin showed that sTm1 is more susceptible than fTm at the sites outside the region with the replaced residues. Compared with fTm, sTm1 showed higher viscosity, had a higher actin affinity, and inhibited acto-S1 ATPase to a greater extent. Finally, the binding isotherm of S1-ADP to actin-sTm1 is less sigmoidal than that to actin-fTm. These results indicate that the amino-acid replacements in the internal region alter the conformation and the physical properties of the entire molecule as well as its interactions with actin and myosin.
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PMID:Amino-acid replacements in an internal region of tropomyosin alter the properties of the entire molecule. 1090 22

We updated the two-dimensional protein database for mouse liver. Microsomal and cytosolic fractions of the liver proteins from male mice were separated by two-dimensional electrophoresis. The proteins were identified by Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) on the basis of peptide mass fingerprinting, following in-gel digestion with trypsin and matching with the theoretical peptide masses of all known proteins from all species. Approximately 5800 spots, excised from 14 two-dimensional gels, were analyzed which resulted in the identification of about 2500 proteins that were the products of 328 different genes. The database includes 112 newly identified gene products. The fractionation prior to two-dimensional electrophoresis was essential for the detection of the new proteins, 55% of which were found in the microsomal and 35% in the cytosolic fraction. The more frequently identified proteins in the various gels were heat shock proteins, house-keeping enzymes, such as ATP synthase chains, disulfide isomerase, and structural proteins, such as tropomyosin. About 45% of the identified proteins were detected 1-3 times, 45% 4-9 times, and the rest 10 or more times. Most proteins were represented by many spots. In average, about 18-20 spots were detected per gene product.
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PMID:Two-dimensional database of mouse liver proteins. An update. 1142 30

The protein content of muscle is determined by the relative rates of synthesis and degradation. The balance between this process determines the number of functional contractile units within each muscle cell. Myofibril-bound protease, protease M previously reported in mouse skeletal muscle could be solubilized from the myofibrillar fraction by salt and acid treatment and partially purified by Mono Q and Superose 12 chromatography. Isolated protease M activity in vitro on whole myofibrils resulted in myosin, actin, troponin T, alpha-actinin and tropomyosin degradation. Protease M is serine type and was able to hydrolyze trypsin-type synthetic substrates but not those of chymotrypsin type. In gel filtration chromatography, protease M showed Mr 120.0 kDa. The endogenous inhibitor (MHPI) is a glycoprotein (110.0 kDa) that efficiently blocks the protease M-dependent proteolysis of myofibrillar proteins in a dose-dependent way, as shown by electrophoretic analysis and synthetic substrates assays. Protease M-Inhibitor system would be implicated in myofibrillar proteins turnover.
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PMID:Myofibril-bound serine protease and its endogenous inhibitor in mouse: extraction, partial characterization and effect on myofibrils. 1192 84

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.
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PMID:Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins. 1208 38

We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.
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PMID:Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosis. 1613 Jan 69

1. Tropomyosin preparations of the Bailey type, and those prepared in the presence of dithiothreitol to prevent oxidation of protein thiol groups, inhibit the Ca(2+)-activated adenosine triphosphatase (ATPase) of desensitized actomyosin by up to 60%. 2. The inhibitory activity of myofibrillar extracts and tropomyosin survives various agents known to denature proteins but to the action of which tropomyosin is unusually stable, namely heating at 100 degrees and mild tryptic digestion. It is destroyed by prolonged treatment with trypsin. 3. The ethylenedioxybis-(ethyleneamino)tetra-acetic acid (EGTA)-sensitizing factor present in extracts of natural actomyosin and myofibrils could be selectively destroyed, leaving unchanged the inhibitory effect on the Ca(2+)-activated ATPase. There was no correlation between the EGTA-sensitizing and the Ca(2+)-activated inhibitory activities of tropomyosin prepared under different conditions. 4. Optimum inhibition was achieved when tropomyosin and the myosin of desensitized actomyosin were present in approximately equimolar proportions. Tropomyosin had no effect on the Ca(2+)-activated ATPase of myosin measured under similar conditions. 5. Evidence is presented showing that the tropomyosin binds to desensitized actomyosin under the conditions in which the ATPase is inhibited.
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PMID:The effect of tropomyosin on the adenosine triphosphatase activity of desensitized actomyosin. 1674 51

Atrazine (ATRA) is the most commonly applied herbicide in the United States and is frequently detected in drinking water at significant levels. After oral exposure, ATRA metabolism yields diaminochlorotriazine (DACT), an electrophilic molecule that has been shown to form covalent protein adducts. This research was designed to identify ATRA-induced protein adducts formed in the pituitary gland of ATRA-exposed rats and in DACT-exposed LbetaT2 rat pituitary cells. Immunohistochemistry showed diffuse cytoplasmic and nuclear staining in both pituitary sections and LbetaT2 cells indicating the formation of DACT protein adducts. Protein targets from both rat pituitaries and LbetaT2 cell culture were identified following two-dimensional electrophoresis (2DE), immunodetection, and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Western blots from both exposed rats and LbetaT2 cells revealed over 30 DACT-modified spots that were not present in control animals. Protein spots were matched to concurrently run 2DE gels stained with Sypro Ruby, excised, and in-gel-digested with trypsin. Mass spectrometry analysis of digest peptides resulted in the identification of 19 spots and 8 unique proteins in the rats and 21 spots and 19 unique proteins in LbetaT2 cells. The identified proteins present in both sample types included proteasome activator complex subunit 1, ubiquitin carboxyl-terminal hydrolase isozyme L1, tropomyosin, ERp57, and RNA-binding proteins. Each of these proteins contains active-site or solvent-exposed cysteine residues, making them viable targets for covalent modification by DACT.
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PMID:Proteomic analysis of diaminochlorotriazine adducts in wistar rat pituitary glands and LbetaT2 rat pituitary cells. 1837 Apr 13


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