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)

Alkylation of DNA by xenobiotic agents, or their electrophilic metabolites, is believed to be the major initiating process that may result ultimately in carcinogenesis. The study of hemoglobin alkylated in vivo by chemical carcinogens has previously been proposed as an indicator for DNA alkylation. Xenobiotically modified proteins, however, are not readily amenable to conventional methods for amino acid sequencing. Tandem mass spectrometry allows unambiguous structural elucidation of chemically modified proteins. Styrene is a widely used chemical in the plastics industry and its major metabolite, styrene 7,8-oxide, is both mutagenic and carcinogenic in rodents. Human hemoglobin was modified in vitro with styrene 7,8-oxide and digested with trypsin. Tryptic peptides from unmodified hemoglobin were isolated by high performance liquid chromatography, and their molecular weights were determined by liquid secondary ion mass spectrometry. This allowed confirmation of the known sequence of the protein and provided a reference for the identification of modified peptides. High performance tandem mass spectrometry of modified peptides allowed unambiguous assignment of specific residues modified. The externally accessible histidines were found to be the dominant sites for alkylation at high modification levels of the protein.
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PMID:Characterization of structural xenobiotic modifications in proteins by high sensitivity tandem mass spectrometry. Human hemoglobin treated in vitro with styrene 7,8-oxide. 279 39

A fresh-water mussel Anodonta cygnea, an aquatic invertebrate resistant to pollution, possesses an inherent high potential to bind 2-acetylaminofluorene onto membrane vesicles. This binding is saturable and trypsin- and verapamil-sensitive. Simultaneously, this mussel reveals a relatively high inherent activity of glutathione-dependent enzyme activities with a distinct spectrum of substrate affinities. Both these activities are similar to the elements of the molecular mechanism involved in the acquired multi-drug resistance phenomenon described in tumor cell-lines. The recognition that in organisms exposed to polluted waters a multi-xenobiotic resistance mechanism may be involved is essential for understanding both the biological impact of pollution and the development of methods for rational risk assessment in regulatory policy.
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PMID:Distinct glutathione-dependent enzyme activities and a verapamil-sensitive binding of xenobiotics in a fresh-water mussel Anodonta cygnea. 281 96

A method for isolating mouse skin cells by enzymatic digestion with trypsin was developed. Cell populations of 33% viability could be further separated by metrizamide and Percoll gradient centrifugations into three fractions enriched in different cell types. in one fraction 80% of the cells were sebaceous, in the second fraction 50% of the cells were basal and the third fraction consisted predominantly of differentiated keratinocytes. Different cell types were characterized by electron microscopy, light microscopy, staining and enzyme activities. Measurement of benzo(a)pyrene hydroxylase, 7-ethoxycoumarin O-deethylase, UDP-glucuronosyltransferase and GSH-S-transferase activities in different cell types from control mice and mice topically treated with beta-naphthoflavone showed that different cell populations metabolized foreign compounds at different rates. The sebaceous cells were the most active xenobiotic-metabolizing cells. beta-Naphthoflavone increased relative enzyme activities of the original cell population and basal cell-enriched fraction more than that of the already highly active sebaceous cell population.
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PMID:Foreign compound metabolism by isolated skin cells from the hairless mouse. 640 43

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) functions in the packaging of nascent RNA polymerase II transcripts and participates in a variety of nuclear and cytoplasmic processes that modulate gene expression. The RNA binding characteristics of hnRNP A1 suggest that it can modulate the expression of specific genes, but little is known about its possible targets in vivo. In this article, we show that hnRNP A1 interacts with the transcript of a cytochrome P450 gene, Cyp2a5, induced by xenobiotics and during liver damage. Binding of the hnRNP A1 to CYP2A5 mRNA was demonstrated by immunoprecipitation of the xenobiotic-stimulated (37/39 kDa) CYP2A5 mRNA-protein complex with a monoclonal anti-hnRNP A1 antibody, by partial trypsin digestion of the complex, and by showing that the RNA-protein complex is not formed with protein extracts from cells lacking the hnRNP A1. We also show that a specific hepatotoxic inducer of the Cyp2a5 gene, pyrazole, increases the cytoplasmic levels of hnRNP A1 in vivo. Finally, we show that hnRNP A1 can be overexpressed in mouse primary hepatocytes, leading to an accumulation of the CYP2A5 mRNA. Collectively, these results indicate that the hnRNP A1 is an important regulator of the Cyp2a5 gene.
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PMID:Heterogeneous nuclear ribonucleoprotein A1 and regulation of the xenobiotic-inducible gene Cyp2a5. 1190 Dec 18

The rapid development and integration of liquid chromatography-tandem mass spectrometry (LC-MS-MS) has enabled the high-throughput identification of proteins and driven the expanding field of proteomics. LC-MS-MS also offers an attractive general approach to the analysis of xenobiotic adducts on proteins. The aim of this study was to examine the combined use of LC-MS-MS and the SALSA algorithm as a general approach to map xenobiotic adducts on proteins at the level of amino acid sequence. Hemoglobin (Hb) adducts are commonly used as biomarkers for exposure to environmental toxicants. Human Hb was incubated with styrene oxide, ethylene oxide, and butadiene dioxide (40 mM) to form adducts, digested with trypsin and analyzed by LC-MS-MS on a ThermoFinnigan LCQ ion trap MS instrument. Data-dependent scanning was used for acquisition of MS-MS spectra. The SALSA algorithm was used to detect MS-MS spectra of native and modified Hb peptides. The adducted sites identified are the N-terminal valines of both Hbalpha and Hbbeta, glutamic acid 7, cysteine 93, and histidines 77, 97, and 143 of the beta chain and histidine 45 of the alpha chain. Specific shifts in the b- and y-ion series in MS-MS spectra confirmed the locations of each adduct. This approach offers a means to simultaneously identify multiple Hb adducts resulting from exposures to known or unknown toxicants. Combined application of LC-MS-MS and SALSA thus provides a general means of mapping protein modifications at the level of amino acid sequence.
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PMID:Sequence mapping of epoxide adducts in human hemoglobin with LC-tandem MS and the SALSA algorithm. 1206 47

Peroxisomes increase in size and number in responsive animals ranging from mammals to marine mussels and fish species when treated with certain compounds named peroxisome proliferators. This phenomenon, known as peroxisome proliferation, is mediated by nuclear receptors termed peroxisome proliferator-activated receptors (PPARs). Three PPAR subtypes have been described (alpha, beta, and gamma) and in mammals PPARalpha is mainly expressed in tissues that catabolize fatty acids, PPARbeta is ubiquitously distributed, and PPARgamma is mainly expressed in the adipose tissue and immune system. The aim of this study was to analyze the tissue distribution of different PPAR subtypes in zebrafish Danio rerio using commercially available antibodies against PPARalpha, PPARbeta, and PPARgamma. In western blots, specific bands were detected at about 58 kDa for PPARalpha and PPARbeta. For PPARgamma the band was detected at 56 kDa. Similar results were obtained in mouse liver homogenates used as positive control, indicating the specificity of the antibodies. Immunohistochemistry was performed in paraformaldehyde-fixed tissue using either microwave or microwave plus trypsin pretreatment for antigen retrieval. In zebrafish, PPARalpha was expressed mainly in liver parenchymal cells, proximal tubules of kidney, enterocytes, and pancreas. PPARbeta showed a widespread distribution and was expressed in the liver, proximal and distal tubules and glomeruli of the kidney, pancreas, enterocytes and smooth muscle of the intestine, skin epithelium, lymphocytes, and male and female gonads. PPARgamma expression was weak in pancreatic cells, intestine, and gonads for both pretreatments. Most of the signal detected was cytoplasmic; only in the cases of PPARalpha and PPARbeta was some nuclear labeling detected in the liver. In mouse tissues, the distribution of PPAR subtypes was similar to that described previously for rats. Our results demonstrate that all three distinct PPAR subtypes are present in zebrafish. The tissue and cellular distribution of PPAR subtypes in zebrafish resembled partly that described before in mammals. Further studies are needed to decipher the functions of PPAR subtypes in zebrafish and other aquatic organisms and particularly their role in regulation of metabolic responses to xenobiotic exposure.
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PMID:Expression of peroxisome proliferator-activated receptors in zebrafish (Danio rerio). 1227 59

The current knowledge on biological protein acetylation is confined to acetyl CoA-dependent acetylation of protein catalyzed by specific acetyl transferases and the non-enzymatic acetylation of protein by acetylated xenobiotics such as aspirin. We have discovered a membrane-bound enzyme catalyzing the transfer of acetyl groups from the acetyl donor 7,8-diacetoxy-4-methyl coumarin (DAMC) to glutathione S-transferase 3-3 (GST3-3), termed DAMC:protein transacetylase (TAase). The purified enzyme was incubated with recombinant GST3-3 subunit and DAMC, the modified protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gel digested with trypsin and the tryptic digest was analyzed by mass spectrometry. The N-terminus and six lysines, Lys-51, -82, -124, -181, -191 and -210, were found to be acetylated. The acetylation of GST3-3 described above was not observed in the absence of either DAMC or TAase. These results clearly establish the phenomenon of protein acetylation independent of acetyl CoA catalyzed by a hitherto unknown enzyme (TAase) utilizing a certain xenobiotic acetate (DAMC) as the active acetyl donor.
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PMID:Establishment of the enzymatic protein acetylation independent of acetyl CoA: recombinant glutathione S-transferase 3-3 is acetylated by a novel membrane-bound transacetylase using 7,8-diacetoxy-4-methyl coumarin as the acetyl donor. 1238 81

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.
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PMID:Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi. 1457 68

Recent advances in proteomics have provided an excellent opportunity to understand biological adaptation under complex environmental stress at the protein level. Gaobeidian Lake, located in Beijing, China, is characterized by complex environmental stresses by serving as both the effluent of a wastewater treatment plant and a coolant of a nearby thermal power plant. Liver is the primary organ of energy metabolism and xenobiotic detoxification. To further our understanding of how organisms that live in Gaobeidian Lake acclimatize themselves to these complex environmental stresses, hepatic protein expression patterns were examined in goldfish Carassius auratus that inhabit the lake. Huairou Reservoir, a drinking water source, was used as a reference site. Twenty four protein spots, which were differently expressed in the two sites, were further digested with trypsin and analyzed by matrix-assisted laser desorption/ionization (MALDI) tandem time of flight mass spectrometry (TOF/TOF). The expression of several energy metabolism and oxidative stress proteins, such as glutathione peroxidase (GPx), ferritin H3, and liver basic fatty acid-binding protein (Lb-FABP) were found to be altered in this stressful environment. In addition to the up-regulation of GPx translation, both the mRNA levels and enzymatic activity of GPx protein were elevated in goldfish living in Gaobeidian Lake. The expression of both peroxisome proliferator activated receptor (PPAR), one of the most important metabolism and stress regulation genes as well as cytochrome P450 1A1 (CYP1A1), a detoxification gene, was also detected by real-time PCR at the two sites. Increased expression levels of both PPAR-beta and CYP1A1 (P < 0.1) were observed in Gaobeidian Lake. Our study provides an integrative view of the expression levels of hepatic proteins and genes in goldfish under complex environmental stress that live in Gaobeidian Lake. Our results showed that anthropogenic environmental stresses in Gaobeidian Lake activated the regulation gene of lipid metabolism PPAR, elevated the lipid metabolism levels, and activated the anti-oxidative adaptation mechanism of organisms in the lake.
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PMID:Proteomic study of the effects of complex environmental stresses in the livers of goldfish (Carassius auratus) that inhabit Gaobeidian Lake in Beijing, China. 1808 Jul 50

Heterodera glycines, the soybean cyst nematode (SCN), causes the most damaging chronic disease of soybean (Glycine max). Host resistance requires the resistance allele at rhg1. Resistance destroys the giant cells created in the plant's roots by the nematodes about 24 to 48 h after commencement of feeding. In addition, 4 to 8 d later, a systemic acquired resistance develops that discourages later infestations. The molecular mechanisms that control the rhg1-mediated resistance response appear to be multigenic and complex, as judged by transcript abundance changes, even in near isogenic lines (NILs). This study aimed to focus on key posttranscriptional changes by identifying proteins and metabolites that were increased in abundance in both resistant and susceptible NILs. Comparisons were made among NILs 10 d after SCN infestation and without SCN infestation. Two-dimensional gel electrophoresis resolved more than 1,000 protein spots on each gel. Only 30 protein spots with a significant (P < 0.05) difference in abundance of 1.5-fold or more were found among the four treatments. The proteins in these spots were picked, trypsin digested, and analyzed using quadrupole time-of-flight tandem mass spectrometry. Protein identifications could be made for 24 of the 30 spots. Four spots contained two proteins, so that 28 distinct proteins were identified. The proteins were grouped into six functional categories. Metabolite analysis by gas chromatography-mass spectrometry identified 131 metabolites, among which 58 were altered by one or more treatment; 28 were involved in primary metabolism. Taken together, the data showed that 17 pathways were altered by the rhg1 alleles. Pathways altered were associated with systemic acquired resistance-like responses, including xenobiotic, phytoalexin, ascorbate, and inositol metabolism, as well as primary metabolisms like amino acid synthesis and glycolysis. The pathways impacted by the rhg1 allelic state and SCN infestation agreed with transcript abundance analyses but identified a smaller set of key proteins. Six of the proteins lay within the same small region of the interactome identifying a key set of 159 interacting proteins involved in transcriptional control, nuclear localization, and protein degradation. Finally, two proteins (glucose-6-phosphate isomerase [EC 5.3.1.9] and isoflavone reductase [EC 1.3.1.45]) and two metabolites (maltose and an unknown) differed in resistant and susceptible NILs without SCN infestation and may form the basis of a new assay for the selection of resistance to SCN in soybean.
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PMID:The nematode resistance allele at the rhg1 locus alters the proteome and primary metabolism of soybean roots. 1942 3


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