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Trypsin
Kinds of Trypsin Terms modified by Trypsin Selected AbstractsEFFECT OF SALTS AND POLYETHYLENE GLYCOLS ON THE PARTITIONING AND RECOVERY OF TRYPSIN FROM HYBRID CATFISH VISCERA IN AQUEOUS TWO-PHASE SYSTEMSJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2010SAPPASITH KLOMKLAO ABSTRACT The partitioning behavior of trypsin from hybrid catfish viscera in aqueous two-phase systems (ATPS) was studied. Factors such as polyethylene glycol (PEG) molecular mass and concentration, as well as types and concentration of salts, affected protein separation. Trypsin partitioned mainly in the top PEG-rich phase. ATPS formed by PEG of molecular weight 4,000 (20%, w/w) and NaH2PO4 (20%, w/w) showed the best capability for trypsin purification from hybrid catfish viscera. Under such conditions, the highest specific activity (30.05 units/µg protein) and purification (27.3-fold), were obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the enzyme after ATPS separation was near homogeneity and based on the activity staining, the band intensity of enzyme in ATPS fraction increased, indicating the greater specific activity of the viscera extract. The partitioned enzyme displayed optimal activity at pH 9.0 and 50C, respectively. The enzyme was stable up to 40C and within the pH range of 8,12. The enzyme exhibited a progressive decrease in activity with increasing NaCl concentration. PRACTICAL APPLICATIONS This paper describes the separation and recovery of trypsin from hybrid catfish viscera in ATPS and its properties. ATPS provides an efficient and attractive method for partitioning and recovery of trypsin from hybrid catfish viscera. Trypsins from various sources catalyze the hydrolysis of peptide bonds on the carboxyl sides of arginine and lysine. Therefore, it is expected that like other trypsins, trypsin after ATPS separation from hybrid catfish viscera could be useful in the biomedical, food and beverage industries. [source] PARTIAL PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE TRYPSIN FROM PYLORIC CAECA OF TAMBAQUI (COLOSSOMA MACROPOMUM)JOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2001RANILSON S. BEZERRA ABSTRACT A 38.5 kDa alkaline protease from pyloric caeca of tambaqui (Colossoma macropomumj, a tropical freshwater fish, was partially purified in three steps: thermal treatment (45Cfor 30 min), salting-out (ammonium sulfate at 40,80% of saturation) and gel filtration (Sephadex G-75), The purification and yield were 51.2-fold and 40%, respectively. The effects of pH, temperature, inhibitors, and substrates on proteolytic activities of partially purified enzyme were investigated. The optimum pH was 9.5, while the optimum temperature was 60C. This alkaline proteolytic activity remained unaltered after 30 min incubation at 55C. Active site inhibition provided additional evidence that this activity is attributed to a trypsin-like enzyme. [source] Synthesis of Core/Shell Colloidal Magnetic Zeolite Microspheres for the Immobilization of TrypsinADVANCED MATERIALS, Issue 13 2009Yonghui Deng Magnetic zeolite microspheres are synthesized by combining sol-gel synthesis and vapor-phase transport. The microspheres, which have magnetite cores and crystalline zeolite shells (see figure), exhibit super-paramagnetism and a high adsorption capacity for trypsin. Trypsin-adsorbed microspheres digest proteins very efficiently (in only 15,s) in the presence of microwave radiation. [source] EFFECT OF SALTS AND POLYETHYLENE GLYCOLS ON THE PARTITIONING AND RECOVERY OF TRYPSIN FROM HYBRID CATFISH VISCERA IN AQUEOUS TWO-PHASE SYSTEMSJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2010SAPPASITH KLOMKLAO ABSTRACT The partitioning behavior of trypsin from hybrid catfish viscera in aqueous two-phase systems (ATPS) was studied. Factors such as polyethylene glycol (PEG) molecular mass and concentration, as well as types and concentration of salts, affected protein separation. Trypsin partitioned mainly in the top PEG-rich phase. ATPS formed by PEG of molecular weight 4,000 (20%, w/w) and NaH2PO4 (20%, w/w) showed the best capability for trypsin purification from hybrid catfish viscera. Under such conditions, the highest specific activity (30.05 units/µg protein) and purification (27.3-fold), were obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the enzyme after ATPS separation was near homogeneity and based on the activity staining, the band intensity of enzyme in ATPS fraction increased, indicating the greater specific activity of the viscera extract. The partitioned enzyme displayed optimal activity at pH 9.0 and 50C, respectively. The enzyme was stable up to 40C and within the pH range of 8,12. The enzyme exhibited a progressive decrease in activity with increasing NaCl concentration. PRACTICAL APPLICATIONS This paper describes the separation and recovery of trypsin from hybrid catfish viscera in ATPS and its properties. ATPS provides an efficient and attractive method for partitioning and recovery of trypsin from hybrid catfish viscera. Trypsins from various sources catalyze the hydrolysis of peptide bonds on the carboxyl sides of arginine and lysine. Therefore, it is expected that like other trypsins, trypsin after ATPS separation from hybrid catfish viscera could be useful in the biomedical, food and beverage industries. [source] ESR SPECTROSCOPY INVESTIGATION OF ANTIOXIDANT ACTIVITY AND PROTECTIVE EFFECT ON HYDROXYL RADICAL-INDUCED DNA DAMAGE OF ENZYMATIC EXTRACTS FROM PICRORRHIZA KURROAJOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2008SOUNG-HEE CHOI ABSTRACT The potential antioxidant activity of enzymatic extracts from Picrorrhiza kurroa was evaluated on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, hydroxyl radical and alkyl radical-scavenging activities using an electron spin resonance spectrometer (JEOL Ltd., Tokyo, Japan). P. kurroa was enzymatically hydrolyzed by seven carbohydrases and five proteases to prepare water-soluble extracts. The DPPH radical-scavenging activities of the pancreatic trypsin and Amyloglucosidase (AMG) (artificial carbohydrase by Novozyme Nordisk, Bagsvaerd, Denmark) extracts from P. kurroa were the highest among protease and carbohydrase extracts, and 50% inhibitory concentration (IC50) values were 35.58 and 29.03 µg/mL, respectively. The hydroxyl radical-scavenging activity of the Protamex and Viscozyme extracts from P. kurroa were the highest scavenging activities, and the IC50 values were 0.46 and 1.89 mg/mL, respectively. In addition, the Protamex and Maltogenase extracts from P. kurroa showed the highest alkyl radical-scavenging activities, and the IC50 values were 18.03 and 10.66 µg/mL, respectively. The protective effect of the Protamex extracts from P. kurroa on DNA damage which was free radical-induced was 92% at 3 mg/mL. These results indicate that enzymatic extracts of P. kurroa show potent antioxidant activity. PRACTICAL APPLICATIONS Picrorrhiza kurroa could be used to produce protein and carbohydrate extracts with antioxidative activity. Many industrial commercial enzymes such as Promozyme, Celluclast 1.5 L FG, Maltogenase L, Viscozyme L, Termamyl SC, Dextrozyme E, AMG 300 L, Protamex, Flavourzyme 500 MG, Neutrase 0.8 L, Pancreatic Trypsin and Alcalase 2.4 L could be also used to attain the extracts processing the high antioxidative activity. The extracts can be used as natural antioxidants. [source] Mass spectrometric identification of the trypsin cleavage pathway in lysyl-proline containing oligotuftsin peptidesJOURNAL OF PEPTIDE SCIENCE, Issue 4 2007Marilena Manea Abstract Trypsin cleaves specifically peptide bonds at the C -terminal side of lysine and arginine residues, except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis. Here we report evidence for a -Lys-Pro- tryptic cleavage in modified oligotuftsin derivatives, Ac-[TKPKG]4 -NH2) (1), using high-resolution mass spectrometry and HPLC as primary methods for analysis of proteolytic reactions. The proteolytic susceptibility of -Lys-Pro- bonds was strongly dependent on flanking residues, and the flexibility of the peptide backbone might be a prerequisite for this unusual cleavage. While -Lys-Gly- bonds in 1 were rapidly cleaved, the modification of these Lys residues by the attachment of a ß-amyloid(4,10) epitope to yield -Lys(X)-Gly derivatives prevented cleavage of this bond, and provided trypsin cleavage of -Lys-Pro- bonds, the pathway of this degradation being independent on the type of Lys- N, -side chains (acetyl group, amino acid, peptide). Substitution of the Lys residues by Ala at the P,2 positions decreased the tryptic cleavage, while replacement of the bulky side chain of Thr at the P2 positions strongly increased the cleavage of -Lys-Pro- bonds. Circular dichroism (CD) data of the modified oligotuftsin derivatives are in accord with enhanced flexibility of the peptide backbone, as a prerequisite for increased susceptibility to cleavage of -Lys-Pro- bonds. These results obtained of oligotuftsin derivatives might have implications for the proteolytic degradation of target peptides that require specific conformational preconditions. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Effect of Polycarboxylate Blocks on the Amidase Activity of Trypsin through Complexation with PEG/Polycarboxylate Block IonomersMACROMOLECULAR BIOSCIENCE, Issue 3 2007Atsushi Harada Abstract The amidase reaction of trypsin, which is a member of the serine proteinase family, is accelerated by its complexation with block ionomers containing a polycarboxylate block, such as PEG-PAA, PEG-PGA, or PEG-PMA. PEG-PAA and PEG-PGA had similar effects, causing an increase in the kcat value and a shift in the pH profile to a lower pH region. On the other hand, PEG-PMA showed not only an increase in the kcat value, but also a decrease in the activation energy; however, there was no shift in the pH dependence of the initial reaction rate. Such differences might be induced by the difference in pKa values of the polycarboxylate block in block ionomers. [source] The Properties of Covalently Immobilized Trypsin on Soap-Free P(MMA-EA-AA) Latex ParticlesMACROMOLECULAR BIOSCIENCE, Issue 4 2005Kai Kang Abstract Summary: The covalent immobilization of trypsin onto poly[(methyl methacrylate)- co -(ethyl acrylate)- co -(acrylic acid)] latex particles, produced by a soap-free emulsion polymerization technique, was carried out using the carbodiimide method. The catalytic properties and kinetic parameters, as well as the stability of the immobilized enzyme were compared to those of the free enzyme. Results showed that the optimum temperature and pH for the immobilized trypsin in the hydrolysis of casein were 55,°C and 8.5, both of which were higher than that of the free form. It was found that Km (Michaelis constant) was 45.7 mg,·,ml,1 and Vmax (maximal reaction rate) was 793.0 ,g,·,min,1 for immobilized trypsin, compared to a Km of 30.0 mg,·,ml,1 and a Vmax of 5,467.5 ,g,·,min,1 for free trypsin. The immobilized trypsin exhibited much better thermal and chemical stabilities than its free counterpart and maintained over 63% of its initial activity after reusing ten times. TEM photograph of latex particles after trypsin immobilization. [source] Functional protease-activated receptors in the dorsal motor nucleus of the vagusNEUROGASTROENTEROLOGY & MOTILITY, Issue 4 2010H. Wang Abstract Background, Protease-activated receptors (PARs), a family member of G-protein coupled receptors, are present and functionally active in a wide variety of cells. The object of this study was to demonstrate the presence and function of PAR-1 and PAR-2 in the dorsal motor nucleus of the vagus (DMV). Methods, DMNV neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion. Neurons were cultured in Neurobasal medium A containing 2% B27 supplement. Intracellular calcium concentration ([Ca2 + ]i) was measured using fura-2 based microspectrometry. Expression of PARs was detected by RT-PCR and immunofluorescent staining. Key Result, Thrombin and PAR-1 agonist peptide activate PAR-1 with a maximum change in [Ca2 + ]i expressed as ,F/F0 of 229 ± 14% and 137 ± 7%, respectively. Trypsin and PAR-2 agonist peptide activate PAR-2 with a maximum ,F/F0 change of 258 ± 12% and 242 ± 10%, respectively. Inhibition of phospholipase C (PLC) by U73312 (1 ,m) decreased the maximal change in ,F/F0 induced by PAR-1 activation from 140 ± 17% to 21 ± 3%, while the PAR-2-mediated maximal change in ,F/F0 decreased from 185 ± 21% to 19 ± 6%. Blockade of IP3 receptor with 2APB inhibited the maximal change in ,F/F0 due to PAR-1 and PAR-2 activation by 72 ± 13% and 71 ± 20% respectively. PAR-1 immnuoreactivity was present in DMV neurons. Increase in transcripts for PAR-1 and PAR-2 were detected in DMV tissues derived from IBD rats relative to control animals. Conclusions & Inferences, Our results indicate that PAR-1 and PAR-2 are present in the DMV neurons, and their activation leads to increases in intracellular calcium via signal transduction mechanism that involves activation of PLC and the production of IP3. [source] Specificity and reactive loop length requirements for crmA inhibition of serine proteasesPROTEIN SCIENCE, Issue 2 2005Lisa D. Tesch Abstract The viral serpin, crmA, is distinguished by its small size and ability to inhibit both serine and cysteine proteases utilizing a reactive loop shorter than most other serpins. Here, we characterize the mechanism of crmA inhibition of serine proteases and probe the reactive loop length requirements for inhibition with two crmA reactive loop variants. P1 Arg crmA inhibited the trypsin-like proteases, thrombin, and factor Xa, with moderate efficiencies (,102,104 M,1sec,1), near equimolar inhibition stoichiometries, and formation of SDS-stable complexes which were resistant to dissociation (kdiss ,10,7 sec,1), consistent with a serpin-type inhibition mechanism. Trypsin was not inhibited, but efficiently cleaved the variant crmA as a substrate (kcat/KM of ,106 M,1 sec,1). N-terminal sequencing confirmed that the P1 Arg,P1,Cys bond was the site of cleavage. Altering the placement of the Arg in a double mutant P1 Gly-P1,Arg crmA resulted in minimal ability to inhibit any of the trypsin family proteases. This variant was cleaved by the proteases ,10-fold less efficiently than P1 Arg crmA. Surprisingly, pancreatic elastase was rapidly inhibited by wild-type and P1 Arg crmAs (105,106 M,1sec,1), although with elevated inhibition stoichiometries and higher rates of complex dissociation. N-terminal sequencing showed that elastase attacked the P1,Cys,P2,Ala bond, indicating that crmA can inhibit proteases using a reactive loop length similar to that used by other serpins, but with variations in this inhibition arising from different effective P2 residues. These results indicate that crmA inhibits serine proteases by the established serpin conformational trapping mechanism, but is unusual in inhibiting through either of two adjacent reactive sites. [source] Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false-positive controlPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2010Nestor Solis Abstract Proteolytic treatment of intact bacterial cells is an ideal means for identifying surface-exposed peptide epitopes and has potential for the discovery of novel vaccine targets. Cell stability during such treatment, however, may become compromised and result in the release of intracellular proteins that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital-acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface "shaving" technique with either trypsin or proteinase- K combined with LC-MS/MS. Trypsin-derived data were controlled using a "false-positive" strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell lysis and were removed from the trypsin-shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface-exposed peptides. Trypsin and proteinase- K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase- K treatment, 13 specific to trypsin and three identified in the control. The use of a subtracted false-positive strategy improved enrichment of surface-exposed peptides in the trypsin data set to approximately 80% (124/155 peptides). Predominant surface proteins were those associated with methicillin resistance,surface protein SACOL0050 (pls) and penicillin-binding protein 2, (mecA), as well as bifunctional autolysin and the extracellular matrix-binding protein Ebh. The cell shaving strategy is a rapid method for identifying surface-exposed peptide epitopes that may be useful in the design of novel vaccines against S. aureus. [source] Efficient on-chip proteolysis system based on functionalized magnetic silica microspheresPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2007Yan Li Abstract An easily replaceable enzymatic microreactor has been fabricated based on the glass microchip with trypsin-immobilized magnetic silica microspheres (MS microspheres). Magnetic microspheres with small size (,300,nm in diameter) and high magnetic responsivity to magnetic field (68.2,emu/g) were synthesized and modified with tetraethyl orthosilicate (TEOS). Aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were then introduced to functionalize the MS microspheres for enzyme immobilization. Trypsin was stably immobilized onto the MS microspheres through the reaction of primary amines of the proteins with aldehyde groups on the MS microspheres. The trypsin-immobilized MS microspheres were then locally packed into the microchannel by the application of a strong field magnet to form an on-chip enzymatic microreactor. The digestion efficiency and reproducibility of the microreactor were demonstrated by using cytochrome c (Cyt-C) as a model protein. When compared with an incubation time of 12,h by free trypsin in the conventional digestion approach, proteins can be digested by the on-chip microreactor in several minutes. This microreactor was also successfully applied to the analysis of an RPLC fraction of the rat liver extract. This opens a route for its further application in top-down proteomic analysis. [source] Backbone Dynamics of Cyclotide MCoTI-I Free and Complexed with Trypsin,ANGEWANDTE CHEMIE, Issue 39 2010Shadakshara S. Puttamadappa Freies Protein mit kräftigerem Rückgrat: {15N,1H}-NMR-spektroskopischen Studien zufolge sind die meisten NH-Gruppen im Rückgrat des Cyclotids MCoTI-I im freien Zustand fixiert, was für eine kompakte gefaltete Struktur spricht (siehe Bild). Wie der Ordnungsparameter S2 anzeigt, präsentiert sich das Rückgrat in Trypsin-gebundenem MCoTI-I deutlich mobiler. [source] Digestive proteinases and peptidases in the hepatopancreas of the southern brown shrimp (Farfantepenaeus subtilis) in two sub-adult stagesAQUACULTURE NUTRITION, Issue 4 2010D.S. BUARQUE Abstract The aim of this study was to examine proteinases and peptidases from the hepatopancreas of two sub-adult stages of Farfantepenaeus subtilis: SAS6 (5.93 ± 0.69 g wet weight) and SAS13 (13.26 ± 0.60 g wet weight). Trypsin and chymotrypsin activity was higher in the extract from the SAS6 individuals (P < 0.05). The highest activity among aminoacyl-,-naphthylamide substrates was found using alanine-, arginine-, leucine- and lysine-,-naphthylamide. There was a positive correlation between the recommended concentration of essential amino acids in penaeid shrimp feed and aminopeptidase activity in both sub-adult stages. Proteolytic activity of F. subtilis was strongly inhibited by specific trypsin inhibitors. The optimal temperature for trypsin, chymotrypsin and leucine aminopeptidase activity was between 45 and 55 °C. Six and seven bands were found in caseinolytic zymograms for SAS6 and SAS13 respectively. All bands were inhibited by phenylmethylsulfonyl fluoride in both sub-adult stages. The use of tosyl-lysine-chloromethyl-ketone and benzamidine caused strong inhibition of the proteolytic bands. Trypsin and chymotrypsin activity was the main difference observed between the protease pattern of SAS6 and SAS13F. subtilis. [source] Effects of dietary protein levels on the growth performance, digestive capacity and amino acid metabolism of juvenile Jian carp (Cyprinus carpio var. Jian)AQUACULTURE RESEARCH, Issue 9 2009Yong Liu Abstract This experiment was conducted to evaluate the effects of protein levels on the growth performance, digestive capacity and amino acid metabolism of juvenile Jian carp. Brown fish meal was used as the sole protein source in the present study. Six isoenergetic experimental diets containing 14.4 MJ kg,1 of digestible energy and 220,495 g crude protein kg,1 diets were fed to triplicate groups of 50 fish with a mean initial weight of 16.67 ± 0.01 g for 45 days. Per cent weight gain (PWG) and feed efficiency ratio (FER) improved with an increase in the dietary protein levels up to 330 g kg,1 diet. The condition factor, relative gut length, intestinal folds height, hepatopancreas and intestine protein content improved with an increase in the protein levels up to 330,385 g kg,1 diet. Trypsin, creatinkinase, Na+, K+ -ATPase and alkaline phosphatase activities generally followed the same tendency as that of growth parameters. Amylase and ,-glutamyl transpeptidase (,-GT) activities were negatively correlated with increasing protein levels from 220 to 330 g kg,1 diet, and no differences were found thereafter. Lipase activity was unaffected by protein levels. Lactobacillus amount was increased with protein levels up to 275 g kg,1 diet, while Aeromonas amount followed the opposite pattern. Escherichia coli amount was not influenced by dietary protein levels. Glutamate,oxaloacetate transaminase (GOT) activities in the hepatopancreas and plasma ammonia concentration (PAC) were not influenced by protein levels between 220 and 275 g kg,1 diet, but significantly increased with increasing protein levels from 275 to 440 g kg,1 diet, and remained similar thereafter. Glutamate,pyruvate transaminase (GPT) activities significantly increased with protein levels >275 g kg,1 diet. Based on the broken-line model, the dietary protein requirement for PWG of Jian carp (16.7,55.0 g) was estimated to be 341 g kg,1 diet with a digestible energy of 14.4 MJ kg,1 diet. [source] Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae)AQUACULTURE RESEARCH, Issue 7 2009Diego Souza Buarque Abstract Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 °C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2,9.0) and 55 °C. CaCl2 did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 °C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated (P<0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols. [source] Effect of PbII on the Secondary Structure and Biological Activity of TrypsinCHEMBIOCHEM, Issue 7 2005Lin Yang Prof. Abstract The effects of PbIIon the secondary structure and biological activity of trypsin have been examined by monitoring changes in its conductivity and IR and circular dichroism (CD) spectra. The results show that PbIIreacts with trypsin, and that the binding sites might be OH and NH groups in pepsin. The CD spectra indicate that interaction with PbIIsignificantly affects the secondary structure of trypsin, the ,-sheet-structure content being increased by about 42,%, whilst those of ,-helix and ,-turn structures are decreased by 13,% and 21,%, respectively. The results clearly demonstrate that PbIIaffects the biological activity of trypsin by modifying its secondary structure. Most interesting is that PbIIup-regulates the activity of trypsin at low concentrations while down-regulating it at high concentrations. [source] Inhibition of Trypsin and Urokinase by Cbz-Amino(4-guanidinophenyl)methanephosphonate Aromatic Ester Derivatives: The Influence of the Ester Group on Their Biological Activity.CHEMINFORM, Issue 34 2006Marcin Sienczyk Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Overexpression of profilin reduces the migration of invasive breast cancer cellsCYTOSKELETON, Issue 2 2004Partha Roy Abstract The exact role profilin plays in cell migration is not clear. In this study, we have evaluated the effect of overexpression of profilin on the migration of breast cancer cells. Overexpression was carried out by stably expressing GFP-profilin in BT474 cells. It was observed that even a moderate level of overexpression of profilin significantly impaired the ability of BT474 cells to spread on fibronectin-coated substrate and migrate in response to EGF. GFP-profilin expressing cells also showed increased resistance to detachment in response to trypsin and increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin compared to the parental and GFP-expressing (control) cell lines. These results suggest that perturbation of profilin levels may offer a good strategy for controlling the metastatic potential of breast cancer cells. Cell Motil. Cytoskeleton 57:84,95, 2004. © 2004 Wiley-Liss, Inc. [source] Capillary sieving electrophoresis and micellar electrokinetic capillary chromatography produce highly correlated separation of tryptic digestsELECTROPHORESIS, Issue 14 2010Jane A. Dickerson Abstract We perform 2-D capillary electrophoresis on fluorescently labeled proteins and peptides. Capillary sieving electrophoresis (CSE) was performed in the first dimension and MEKC was performed in the second. A cellular homogenate was labeled with the fluorogenic reagent FQ and separated using the system. This homogenate generated a pair of ridges; the first had essentially constant migration time in the CSE dimension, while the second had essentially constant migration time in the MEKC dimension. In addition, a few spots were scattered through the electropherogram. The same homogenate was digested using trypsin, and then labeled and subjected to the 2-D separation. In this case, the two ridges observed from the original 2-D separation disappeared and were replaced by a set of spots that fell along the diagonal. Those spots were identified using a local-maximum algorithm and each was fit using a 2-D Gaussian surface by an unsupervised nonlinear least squares regression algorithm. The migration times of the tryptic digest components were highly correlated (r=0.862). When the slowest migrating components were eliminated from the analysis, the correlation coefficient improved to r=0.956. [source] Mass spectrometrical analysis of the mitochondrial carrier Aralar1 from mouse hippocampusELECTROPHORESIS, Issue 11 2010Seok Heo Abstract Aralar1 is a mitochondrial aspartate/glutamate carrier and a key component of the malate,aspartate NADH shuttle system. An analytical approach to obtain high sequence coverage is important to predict conformation, identify splice variants and binding partners or generate specific antibodies. Moreover, a method allowing determination of Aralar1 from brain samples is a prerequisite for evaluating a biological role. Sucrose gradient ultracentrifugation was applied to enrich native membrane protein fractions and these were run on blue-native PAGE, followed by multidimensional gel electrophoresis. Spots from the third-dimensional gel electrophoresis were in-gel digested with trypsin, chymotrypsin and subtilisin. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using collision-induced dissociation and electron transfer dissociation modes. ModiroŌ v1.1 along with Mascot v2.2 software was used for data handling. Aralar1 could be clearly separated, unambiguously identified and characterized from protein extracts of mouse hippocampus by the use of the multidimensional gel electrophoretic steps. The combined sequence coverage of Aralar1 from trypsin, chymotrypsin and subtilisin digestions was 99.85%. The results provide the basis for future studies of Aralar1 at the protein chemical rather than at the immunochemical level in the brain and thus challenge and enable determination of Aralar1 levels required for understanding biological functions in health and disease. [source] Highly efficient capture and enumeration of low abundance prostate cancer cells using prostate-specific membrane antigen aptamers immobilized to a polymeric microfluidic deviceELECTROPHORESIS, Issue 18 2009Udara Dharmasiri Abstract Prostate tumor cells over-express a prostate-specific membrane antigen (PSMA) that can be used as a marker to select these cells from highly heterogeneous clinical samples, even when found in low abundance. Antibodies and aptamers have been developed that specifically bind to PSMA. In this study, anti-PSMA aptamers were immobilized onto the surface of a capture bed poised within a PMMA, microchip, which was fabricated into a high-throughput micro-sampling unit (HTMSU) used for the selective isolation of rare circulating prostate tumor cells resident in a peripheral blood matrix. The HTMSU capture bed consisted of 51 ultra-high-aspect ratio parallel curvilinear channels with a width similar to the prostate cancer cell dimensions. The surface density of the PSMA-specific aptamers on an ultraviolet-modified PMMA microfluidic capture bed surface was determined to be 8.4×1012,molecules/cm2. Using a linear velocity for optimal cell capture in the aptamer-tethered HTMSU (2.5,mm/s), a recovery of 90% of LNCaP cells (prostate cancer cell line; used as a model in this example) was found. Due to the low abundance of these cells, the input volume required was 1,mL and this could be processed in ,29,min using an optimized linear flow rate of 2.5,mm/s. Captured cells were subsequently released intact from the affinity surface using 0.25%,w/w trypsin followed by counting individual cells using a contact conductivity sensor integrated into the HTMSU that provided high detection and sampling efficiency (,100%) and did not require staining of the cells for enumeration. [source] Sensitive, label-free protein assay using 1-ethyl-3-methylimidazolium tetrafluoroborate-supported microchip electrophoresis with laser-induced fluorescence detectionELECTROPHORESIS, Issue 9 2008Yuanhong Xu Abstract Based on the dimer,monomer equilibrium movement of the fluorescent dye Pyronin Y (PY), a rapid, simple, highly sensitive, label-free method for protein detection was developed by microchip electrophoresis with LIF detection. PY formed a nonfluorescent dimer induced by the premicellar aggregation of an anionic surfactant, SDS, however, the fluorescence intensity of the system increased dramatically when proteins such as BSA, bovine hemoglobin, cytochrome c, and trypsin were added to the solution due to the transition of dimer to fluorescent monomer. Furthermore, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF4) instead of PBS was applied as running buffers in microchip electrophoresis. Due to the excellent properties of EMImBF4, not only nonspecific protein adsorption was more efficiently suppressed, but also approximately ten-fold higher fluorescence intensity enhancement was obtained than that using PBS. Under the optimal conditions, detection limits for BSA, bovine hemoglobin, cytochrome c, and trypsin were 1.00×10,6, 2×10,6, 7×10,7, and 5×10,7 mg/mL, respectively. Thus, without covalent modification of the protein, a protein assay method with high sensitivity was achieved on microchips. [source] The resistance of metallothionein to proteolytic digestion: An LC-MS/MS analysisELECTROPHORESIS, Issue 16 2007Rongying Wang Abstract Metallothioneins (MTs) are a family of cysteine-rich metalloproteins which strongly bind to heavy metals, such as Cd(II), Zn(II), and Cu(I). Previous works by other group using gel electrophoresis and fluorescence showed MTs were resistant to proteolytic digestion by a variety of enzymes, raising the difficulties in proteomic identification of MTs. The present work was attempted to analyze the resistance of MTs to trypsin using LC with MS/MS (LC-MS/MS), which was able to determine the sequences of the produced peptides and thus precisely characterize the cleavages. The results showed that metal-saturated MTs were completely resistant to trypsin. This resistance problem could be overcome by the addition of EDTA to MT samples, which rendered MTs readily digested into peptides and identified by MS/MS. Interestingly, the partially metal binding MTs were digested into peptides predominantly with miss cleavages which were well dependent on the amount of heavy metals bound to MTs. An explanation for these observations was proposed. The potential applications of the MT's resistance to trypsin in isolation and identification of MTs in complex mixtures such as cultured cells was demonstrated. The preliminary data also showed the same proteomic approach of proteolytic digestion followed by MS/MS analysis may provide information on metal binding status of MTs, along with the identification of MTs in a mixture. [source] Toward an integrated microchip sized 2-D polyacrylamide slab gel electrophoresis device for proteomic analysisELECTROPHORESIS, Issue 3 2007Zuzana Demianovį Abstract We describe a miniaturized instrument capable of performing 2-DE. Our miniaturized device is able to perform IEF and polyacrylamide slab gel electrophoresis (PASGE) in the same unit. It consists of a compartment for a first-dimensional IEF gel, which is connected to a second-dimensional PASGE gel. The focused samples are automatically transferred from the IEF gel to the PASGE gel by electromigration. Our preliminary experiments show that the device is able to focus and separate a mixture of proteins in approximately 1,h, excluding the time required for the staining procedure. On average, the gel-to-gel retardation factor,(Rf) variation was 6.2% (±0.9%) and pI variation was 2.5% (±0.6%). Separated protein spots were excised from stained gels, digested with trypsin, and further identified by MS, thus enabling direct proteomic analysis of the separated proteins. [source] Insoluble eggshell matrix proteins , their peptide mapping and partial characterization by capillary electrophoresis and high-performance liquid chromatographyELECTROPHORESIS, Issue 5 2003Ivan Mik Abstract Avian eggshell matrix proteins were studied by two analytical approaches. Peptide mapping was done by trypsin and pepsin followed by collagenase cleavage; analyses were carried out by capillary electrophoresis and reversed-phase high-performance liquid chromatography (HPLC). Comparison of peptide maps obtained by both methods revealed a complex mixture of peptides in the insoluble layers of the eggshell; it was concluded that there are at least three different insoluble protein/peptide layers in the avian eggshell (cuticle, palisade, and mammillary layer). Partial characterization of peptides in each layer was made by HPLC-mass spectrometry analysis. There is an evidence that the eggshell insoluble proteins contain species susceptible to collagenase cleavage, however, the sequences split by this enzyme probably are not those typical for the main triple-helical core of collagenous proteins. It is proposed that the action of collagenase upon eggshell proteins is caused by the side effect of collagenase described previously with synthetic peptides. Some of the proteins present are probably glycosylated. Fatty acid content in the insoluble eggshell layers (after decalcification) was in the range of 2,4% (which reflected both lipid and lipoproteins bound fatty acids). Porphyrin pigments are dominant in the cuticle layer. [source] Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac diseaseENTOMOLOGICAL RESEARCH, Issue 3 2007Elena N. ELPIDINA Abstract Digestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post-proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin-like and chymotrypsin-like, are compartmentalized to the PM. Trypsin-like activity is due to one cationic and three anionic proteinases. Chymotrypsin-like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N-like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM , cathepsin L and post-proline cleaving peptidase , in the treatment of celiac disease is discussed. [source] A multivariate biomarker-based model predicting population-level responses of Daphnia magnaENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2003Wim M. De Coen Abstract 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 (48h-96 h) to eight model toxicants (cadmium, chromium, mercury, tributyl tin, linear alkylsulfonic acid, sodium pentachlorophenolate, lindane, and 2,4-dichloro-phenoxyacetic acid) on the following biomarkers were used for the multivariate model: digestive enzymes (amylase, cellulase, ,-galactosidase, trypsin, and esterase), enzymes of the intermediary metabolism (glycogen phosphorylase, glucose-6-phosphate de-hydrogenase, 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. [source] Exocrine pancreatic dysfunction in sepsisEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 3 2003B. Tribl Abstract Background Sepsis in critical illness is associated with the progressive failure of multiple organs. This study aims to establish a correlation between the severity of sepsis and exocrine pancreatic dysfunction. Materials and methods In a prospective cohort study pancreatic exocrine function was tested by means of a secretin-cholecystokinin test in 21 critically ill, mechanically ventilated patients with sepsis according to criteria of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee (ACCP/SCCM): 11 patients with shock and 10 patients without shock. Data were compared with seven healthy controls. Results The volume of duodenal fluid was not statistically different in the three groups. Sepsis patients without shock had significantly reduced content of amylase and chymotrypsin in duodenal juice compared with healthy controls (P < 0·01). Secretion of amylase, chymotrypsin, trypsin (P < 0·01 each) and bicarbonate in duodenal fluid (P < 0·05) was impaired in the septic shock patients when compared with the healthy controls. The content of trypsin was different between sepsis patients and septic shock patients (P < 0·05). Spearman correlation analysis was significant between the amylase secretion and the APACHE III and SOFA scores (P < 0·01). The SOFA score was also related to secretion of trypsin (P < 0·05). In patients on pressor therapy, use of norepinephrine was associated with a significant decrease in bicarbonate secretion (P < 0·05). Conclusions Sepsis is associated with secretory pancreatic dysfunction that is worse in septic shock than in sepsis without shock. Impaired exocrine function was significantly correlated to the APACHE III and SOFA scores. [source] Expression of psoriasis-associated fatty acid-binding protein in senescent human dermal microvascular endothelial cellsEXPERIMENTAL DERMATOLOGY, Issue 9 2004Moon Kyung Ha Abstract:, Aging is associated with the progressive pathophysiologic modification of endothelial cells. In vitro endothelial cell senescence is accompanied by proliferative activity failure and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in aging organisms. In order to observe the changing patterns of protein expression in senescent human dermal microvascular endothelial cells (HDMECs), proteins obtained from both early- and late-passaged HDMECs were separated by two-dimensional electrophoresis, visualized by silver staining, and quantified by image processing. Proteins of interest were extracted by in-gel digestion with trypsin and quantified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), by searching the National Center for Biotechnology Information protein-sequence database. More than 2000 spots were detected by 2D electrophoresis within a linear pH range of 3,10. Twenty-two major differentially expressed spots were observed in serially passaged HDMECs and identified with high confidence by MALDI-TOF-MS. One of these spots was found to be a 14,15 kDa psoriasis-associated fatty acid-binding protein (PA-FABP) with high affinity for long-chain fatty acids. The expression of PA-FABP was confirmed to be elevated in senescent HDMECs (passage 20) by fluorescence-activated cell sorting (FACS), confocal laser microscopy, and by immunohistochemistry in aged human skin tissue. Our results suggest that the overexpression of FABP in cultured senescent HDMECs is closely related to skin aging. [source] | ||||||||||||||||||||||||||||||||||||||||