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)

Endometrial proteins showing cyclic expression during the normal menstrual cycle were localized on two-dimensional (2-D) electrophoresis gels separating proteins with isoelectric points (pl) ranging from 3.5 to 7 and relative molecular weights ranging from 10 to 300 kDa. Menstrual cycle-related proteins were excised from several 2-D gels, concentrated by one-dimensional (1-D) sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, and cleaved in situ by trypsin. The tryptic fragments were extracted and separated by reverse phase high performance liquid chromatography (RP-HPLC). Finally, the partial amino-terminal amino acid sequence of selected tryptic fragments were determined for each protein. We aimed at characterizing the 21 menstrual cycle-related proteins that were visible on silver-stained 2-D electrophoresis gels. Of the proteins being maximally synthesized in the proliferative phase endometrium, we identified proteins associated mainly with the cytoskeleton: vimentins, keratin, tropomyosin and tubulin, but also proteins such as proliferating cell nuclear antigen and beta-galactoside binding lectin. The partial amino acid sequences for another two proteins did not match any protein sequence in the Protein Identification Resource (PIR) and Swissprot databases. In the group of proteins having maximal synthesis in the secretory phase endometrium, we identified creatine kinase chain B and an isocitrate dehydrogenase-homologous protein, both of which are involved in energy metabolism. However, we also identified the annexin IV precursor, the 14-3-3 protein homologue also called stratifin or the epithelial cell marker protein 1 and the 21K tumour protein. Finally, four of the proteins were present in too low amounts to allow characterization. Interestingly, most of the identified proteins have not previously been described as having a menstrual cycle-related synthesis in the human endometrium. It may be considered that the concentration of some of the cycle-related proteins may be used in clinical situations to reflect specific endometrial phases.
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PMID:Human endometrial proteins with cyclic changes in the expression during the normal menstrual cycle: characterization by protein sequence analysis. 856 10

Photoaffinity labeling with [2'-32P]2N3NADP+ and [32P]2N3NAD+ was used to identify two overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of NADP(+)-dependent isocitrate dehydrogenase (IDH). Photolysis was required for insertion of radiolabel, and prior photolysis of photoprobes before addition of IDH prevented insertion. Photoincorportion of 2N3NAD+ inhibited the enzymatic activity of IDH. Photolabeling of IDH with both [32P]2N3NAD+ and [2'-32P]2N3-NADP+ showed saturation effects with apparent Kds of 20 and 14 microM (+/-12%), respectively. The efficiency of photoincorporation at saturation of binding sites was determined to be about 50%. Also, photolabeling was observed with [32P]8N3ATP and [32P]2N3ATP but with saturation effects observed at lower affinity. With all radiolabeled probes reduction of photoinsertion was effected best by the addition of NADP+ followed by NAD+ and then ATP, indicating that photoinsertion with all the probes was within the NADP+ binding site. Isolation of [32P]2N3NAD+ and [2'-32P]2N3NADP+ photolabeled peptides by use of immobilized boronate and immobilized Al3+ chromatography, respectively, followed by HPLC purification resulted in the identification of overlapping peptides corresponding to Ile244-Arg249 and Leu121-Arg133 (tryptic fragments) and Lys243-His248 and Leu121-His135 (chymotryptic fragments). Trp125 and Trp245 were identified as the sites of photoinsertion based on these residues not being detectable on sequencing, the lack of chymotryptic cleavage at these residues, and the decreased rate of trypsin digestion at nearby Lys243 and Lys127. Sequence analysis of [32P]8N3ATP and [32P]2N3ATP photolabeled peptides gave essentially the same peptide regions being photolabeled but at much lower efficiency, indicating that the effects of ATP on IDH activity are dependent on competition for the same site.
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PMID:Identification of adenine binding domain peptides of the NADP+ active site within porcine heart NADP(+)-dependent isocitrate dehydrogenase. 888 29

A multivariate model is proposed relating short-term biomarker measurements in Daphnia magna to chronic effects (21-d exposure) occurring at the population level (time to death, mean brood size, mean total young per female, intrinsic rate of natural increase, net reproductive rate, and growth). The results of the short-term exposure (48 h-96 h) to eight model toxicants (cadmium, chromium, mercury, tributyl tin, linear alkylsulfonic acid, sodium pentachlorophenolate, lindane, and 2,4-dichlorophenoxyacetic acid) on the following biomarkers were used for the multivariate model: digestive enzymes (amylase, cellulase, beta-galactosidase, trypsin, and esterase), enzymes of the intermediary metabolism (glycogen phosphorylase, glucose-6-phosphate dehydrogenase, pyruvate kinase, lactate dehydrogenase, and isocitrate dehydrogenase), cellular energy allocation (CEA) (protein, carbohydrate, and lipid content and electron transport activity), and DNA damage and antioxidative stress activity. Using partial least squares to latent structures (PLS), a two-component model was obtained with R2 of 0.68 and a Q2 value of 0.60 based on the combined analysis of a limited number of the 48- and 96-h biomarker responses. For the individual population-level responses, the R2 values varied from 0.66 to 0.77 and the Q2 values from 0.52 to 0.69. Energy-related biomarkers (cellular energy allocation, lipid contents, anaerobic metabolic activity--pyruvate kinase, and lactate dehydrogenase), combined with parameters related to oxidative stress (catalase) and DNA damage measured after 48 and 96 h of exposure, were able to predict long-term effects at higher levels of biological organization.
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PMID:A multivariate biomarker-based model predicting population-level responses of Daphnia magna. 1295 51

The goal of this study was the development of a method for quantitative expression proteomics on the limited sample amounts obtained through laser capture microdissection (LCM) of tissues, e.g., approximately 10 000 cells, which typically contain roughly 1-4 microg protein. The 16O/18O labeling method was selected as an approach to measure differential expression. A sample preparation protocol including lysis, digestion and 16O/18O labeling was first developed for LCM cell samples. The selected protocol was examined using two LCM caps of 10 000 cells from invasive ductal carcinoma of the breast and shown to be repeatable. A further test of LC-IT-MS/MS in combination with the 16O/18O post-digestion labeling method for studying low level samples was conducted first on a single protein (BSA) and then on a 5-standard protein mixture digest of different protein amounts, each with a total content approximately 1 microg. Next, protein expression was compared between 10 000 cells, each of microdissected normal ductal epithelium and metastatic ductal carcinoma, using the developed method. The proteins from the microdissected cells were extracted, precipitated, digested with trypsin and then 16O/18O labeled. The normal and metastatic cell samples were analyzed using reversed phase LC-ESI-MS/MS on the ion trap mass spectrometer. A total of 76 proteins were identified. Some, such as mitochondrial isocitrate dehydrogenase, actin and 14-3-3 protein xi/delta were found to be significantly up-regulated in the breast tumor cells.
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PMID:Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 16O/18O isotopic labeling. 1525 14

Using mouse hearts from Swiss Webster mice, calcineurin was immunoprecipitated using commercially available anti-calcineurin antibody and the resulting complex analyzed by using sodium dodecyl sulfate-gel electrophoresis with silver staining. Distinct proteins were observed and subjected to in situ trypsin digestion followed by extraction of the resulting peptides. Peptides from each protein band were loaded onto a target for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and analyzed. The resulting peptide mass spectrum was compared with the Mascot and Protein Prospector databases and resulted in the specific identification of heart mitochondrial proteins, specifically Mn-superoxide dismutase (SOD), aconitase (ACN), and malate dehydrogenase (MDH). Each of the three mitochondrial enzymes was identified with approximately 15-25% sequence coverage and all with statistical significance (P < 0.05) according to the Mascot database search engine. Tandem mass spectrometry analysis of the peptide fragmentation spectra confirmed the identification of these protein partners and also yielded the identification of mitochondrial isocitrate dehydrogenase (ICDH) as another protein in the immunoprecipitated complex. Using antibody preparations against Mn-SOD, ACN, and ICDH showed the presence of calcineurin and each of the three proteins in the immunoprecipitated complex by Western slot blotting. The activity of ACN, but not MDH or ICDH, was enhanced after incubation with calcineurin indicating one possible regulatory function for the complex. The mitochondrial forms of Mn-SOD, ACN, MDH, and ICDH were identified as partner proteins of calcineurin with all the proteins present in a single multiprotein complex.
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PMID:Association of calcineurin with mitochondrial proteins. 1663 48

Heterologous expression of Vitreoscilla hemoglobin (VHb) has been reported to improve cell growth, protein synthesis, metabolite productivity and nitric oxide detoxification. Although it has been proposed that such phenomenon is attributed to the enhancement of respiration and energy metabolism by facilitating oxygen delivery, the mechanism of VHb action remains to be elucidated. In the present study, changes of protein expression profile in Escherichia coli as a consequence of VHb production was investigated by two-dimensional gel electrophoresis (2-DE) in conjunction with peptide mass fingerprinting. Total protein extracts derived from cells expressing native green fluorescent protein (GFPuv) and chimeric VHbGFPuv grown in Luria-Bertani broth were prepared by sonic disintegration. One hundred microgram of proteins was individually electrophoresed in IEF-agarose rod gels followed by gradient SDS-PAGE gels. Protein spots were excised from the gels, digested to peptide fragments by trypsin, and analyzed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Results revealed that expression of VHbGFPuv caused an entire disappearance of tryptophanase as well as down-regulated proteins involved in various metabolic pathways, e.g. glycerol kinase, isocitrate dehydrogenase, aldehyde dehydrogenase, and D-glucose-D-galactose binding protein. Phenotypic assay of cellular indole production confirmed the differentially expressed tryptophanase enzymes in which cells expressing chimeric VHbGFP demonstrated a complete indole-negative reaction. Supplementation of delta-aminolevulinic acid (ALA) to the culture medium enhanced expression of glyceraldehyde-3-phosphate dehydrogenase and glycerol kinase. Our findings herein shed light on the functional roles of VHb on cellular carbon and nitrogen consumptions as well as regulation of other metabolic pathway intermediates, possibly by autoregulation of the catabolite repressor regulons.
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PMID:Shedding light on the role of Vitreoscilla hemoglobin on cellular catabolic regulation by proteomic analysis. 1834 84

Magnesium is crucial for baculovirus transmission in Culex nigripalpus (Theobald) and Culex quinquefasciatus (Say) larvae. However, the mechanistic role of magnesium in baculovirus transmission is unknown. To investigate the possible role of host response factors in baculovirus transmission, suppression subtractive hybridization was used to identify genes differentially transcribed after magnesium exposure in Cx. quinquefasciatus larvae. Suppression subtractive hybridization was performed in both directions enriching for cDNAs differentially transcribed between a nonmagnesium larval control and magnesium (15 mM MgSO4) treatment of Cx. quinquefasciatus larvae held for 1 h at 27 degrees C. Clones from differentially transcribed genes were evaluated by sequencing, and relative gene transcription levels were analyzed using quantitative real-time polymerase chain reaction quantitative real-time polymerase chain reaction. Target transcripts up/downregulated by magnesium included Cx. quinquefasciatus troponin C, isocitrate dehydrogenase, allergen, cytochrome b5, chymotrypsinogen, apolipophorins, tryptase gamma, carboxylesterase, prolylcarboxypeptidase, imaginal disc growth factor, aldehyde dehydrogenase, tropomyosin-1, chitotriosidase, heat shock protein 70 B2, inorganic phosphate cotransporter, and many other hypothetical protein genes. Magnesium can alter gene transcription in a vector mosquito population, and understanding this process can provide insight into the mechanistic role of magnesium in baculovirus transmission.
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PMID:Identification and expression profile of multiple genes in response to magnesium exposure in Culex quinquefasciatus larvae. 2117 53

Although a series of protein levels from several protein pathways have been shown to differ between white (WA) and brown (BA) adipocytes, proteomic work on this subject with the exception of mitochondrial protein differences is limited. It was, therefore, the aim of the study to compare WA with BA soluble protein levels. Proteins were extracted from WA and BA and the soluble fraction was run on two-dimensional gel electrophoresis. Quantification of spot volume was carried out and protein spots, statistically different between groups (P < 0.01), were in-gel digested with trypsin and peptides were identified using nano-LC-ESI-MS/MS in the CID and ETD mode. Differences between selected proteins were evaluated by immunoblotting. A network was generated using the ingenuity pathway analysis. Five proteins, protein DJ-1, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, isocitrate dehydrogenase subunit alpha, electron transfer flavoprotein subunit alpha and immunoglobulin-binding protein 1, were increased in BA based on a gel-based proteomic method and differential expression was verified by immunoblotting. These individual proteins were represented by one spot each and sequence coverages were between 28 and 65%. A network generated based on these results indicated a link to ubiquitination. Differential protein levels between WA and BA allow interpretation of previous work on adipocyte biochemistry and form the basis for future studies with genetic or pharmacological inhibition of these proteins accompanied by work on phenotype and adipocyte function.
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PMID:Proteomic differences between white and brown adipocytes. 2439 Apr 58

The present study was undertaken to identify potentially immunoreactive proteins of the muscle larvae (ML) and adult stage (Ad) of the nematode Trichinella spiralis Owen, 1835. To identify immunoreactive proteins that are specifically recognised by anti-Trichinella antibodies, ML and Ad crude extracts and their excretory-secretory (E-S) products were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot with serum samples from pigs experimentally infected with T. spiralis. A total of 18 bands were selected for final identification by liquid chromatography-tandem mass spectrometry. To further understand the functions of the proteins identified in this study, gene ontology terms were applied. Results showed that the specific antibodies against T. spiralis reacted with protein bands matching heat shock proteins, aminopeptidase, enolase, isocitrate dehydrogenase NADP-dependent, tropomyosin, P49 antigen, serine proteinase, secreted 5'-nucleotidase, antigen targeted by protective antibodies, 53 kDa E-S antigen, putative trypsin and paramyosin. Three proteins common for both adult stage and muscle larvae, including heat shock proteins, enolase and 5'-nucleotidase, might play important role during T. spiralis infection. These proteins are presumably presented to the host immune system and may induce humoral immune response. Thus, these proteins may be potential antigens for early diagnosis and the development of a vaccine against the parasite.
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PMID:Proteomic analysis of potential immunoreactive proteins from muscle larvae and adult worms of Trichinella spiralis in experimentally infected pigs. 2596 May 66


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