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Trypsin Inhibitor (trypsin + inhibitor)
Kinds of Trypsin Inhibitor Terms modified by Trypsin Inhibitor Selected AbstractsISOLATION AND CHARACTERIZATION OF TRYPSIN INHIBITORS FROM SOME THAI LEGUME SEEDSJOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2000SOOTTAWAT BENJAKUL ABSTRACT Trypsin inhibitors from cultivars of cowpea (Vigna unguiculata (L.) Wasp.), pigeon pea (Cajanus cajan (L.) Millsp.) and bambara groundnuts (Voandzeia subterranea (L.) Thou) grown in Thailand were isolated and characterized. Extraction of seeds with NaCl rendered a higher recovery of trypsin inhibitor than other solvents tested (P<0.05). The extraction time affected the inhibitor recovery (P<0.05). The extraction time of 3 h was optimum for the recovery of trypsin inhibitor from pigeon and bambara groundnuts, whereas 1 h was optimum for cowpea. Based cn inhibitor activity of zones separated by electrophoresis, the molecular mass of the inhibitor from bambara groundnuts was 13 kDa. Two inhibitory bands were observed for cowpea (10 and 18 kDa) and pigeon pea (15 and 25 kDa). Partial purification of inhibitors was achieved by heat-treatment at 90C for 10 min, followed by ammonium sulfate precipitation with 30,65% saturation. The partially purified inhibitors from four seeds were heat stable up to 30 min at 90C at pH 7.0. The activities were also retained over a wide pH range at 25C but were lost when samples were treated with ,-mercaptoethanol prior to electrophoresis. [source] Combining a polarizable force-field and a coarse-grained polarizable solvent model: Application to long dynamics simulations of bovine pancreatic trypsin inhibitorJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 11 2008Michel Masella Abstract The dynamic coupling between a polarizable protein force field and a particle-based implicit solvent model is described. The polarizable force field, TCPEp, developed recently to simulate protein systems, is characterized by a reduced number of polarizable sites, with a substantial gain in efficiency for an equal chemical accuracy. The Polarizable Pseudo-Particle (PPP) solvent model represents the macroscopic solvent polarization by induced dipoles placed on mobile Lennard-Jones pseudo-particles. The solvent-induced dipoles are sensitive to the solute electric field, but not to each other, so that the computational cost of solvent,solvent interactions is basically negligible. The solute and solvent induced dipoles are determined self-consistently and the equations of motion are solved using an efficient iterative multiple time step procedure. The solvation cost with respect to vacuum simulations is shown to decrease with solute size: the estimated multiplicative factor is 2.5 for a protein containing about 1000 atoms, and as low as 1.15 for 8000 atoms. The model is tested for six 20 ns molecular dynamics trajectories of a traditional benchmark system: the hydrated Bovine Pancreatic Trypsin Inhibitor (BPTI). Even though the TCPEp parameters have not been refined to be used with the solvent PPP model, we observe a good conservation of the BPTI structure along the trajectories. Moreover, our approach is able to provide a description of the protein solvation thermodynamic at the same accuracy as the standard Poisson-Boltzman continuum methods. It provides in addition a good description of the microscopic structural aspects concerning the solute/solvent interaction. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2008 [source] EFFECT of EXTRUSION ON TRYPSIN INHIBITOR CONTENTS of SOY-SWEET POTATO MIXTURESJOURNAL OF FOOD PROCESSING AND PRESERVATION, Issue 6 2000M.O. IWE Mixtures of soy and sweet potato (Ipomoea batatas) (L.) Lam), flours containing 18%, 25% and 30% moisture, respectively, were extruded in a single screw extruder. Results showed that inactivation of trypsin inhibitor was enhanced by both reductions in feed moisture and soy flour contents of sample mixtures. Hence subsequent extrusion was carried at 18% feed moisture with variable feed ratio, screw rotation speed and die diameter, using a central composite rotatable, near orthogonal experimental design. Results further showed that the effect of increasing the ratio of soy in the mixture was linearly significant (p > 0.05). Optimum Trypsin Inhibitor (TI) inactivation value of 3.40 mg/g was predicted at a feed composition of 80% sweet potato, 9 mm die diameter and 154 rpm, respectively. [source] Antifungal Activity of a Bowman,Birk-type Trypsin Inhibitor from Wheat KernelJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2000G. Chilosi A trypsin inhibitor from wheat kernel (WTI) was found to have a strong antifungal activity against a number of pathogenic fungi and to inhibit fungal trypsin-like activity. WTI inhibited in vitro spore germination and hyphal growth of pathogens, with protein concentration required for 50% growth inhibition (IC50) values ranging from 111.7 to above 500 ,g/ml. As observed by electron microscopy, WTI determined morphological alterations represented by hyphal growth inhibition and branching. One of the fungal species tested, Botrytis cinerea produced a trypsin-like protease, which was inhibited by the trypsin inhibitor. WTI, as well as other seed defence proteins, appear to be an important resistance factor in wheat kernels during rest and early germination when plants are particularly exposed to attack by potential soil-borne pathogens. Zusammenfassung Ein Trypsinhemmer aus Weizenkörnern (WTI) zeigte eine starke antifungale Aktivität gegenüber verschiedenen pathogenen Pilzen und hemmte deren trypsinähnliche Aktivität. WTI hemmte in vitro die Sporenkeimung und das Hyphenwachstum der Pathogene, wobei die IC50 -Werte zwischen 111,7 und mehr als 500 ,g/ml lagen. Elektronenmikroskopische Untersuchungen zeigten, dai WTI morphologische Veränderungen bewirkte, die aus einer Hemmung des Hyphenwachstums und einer veränderten Verzweigung bestanden. Eine der untersuchten Pilzarten, Botrytis cinerea, bildete eine trypsinähnliche Protease, die durch den Trypsininhibitor gehemmt wurde. Ebenso wie andere sameneigene Abwehrproteine scheint WTI während der Keimruhe und in den frühen Stadien der Keimung, wenn die Pflanzen gegenüber möglichen bodenbürtigen Pathogenen besonders exponiert sind, ein wichtiger Resistenzfaktor in Weizenkörnern zu sein. [source] Immobilized Metal Affinity Chromatography without Chelating Ligands: Purification of Soybean Trypsin Inhibitor on Zinc Alginate BeadsBIOTECHNOLOGY PROGRESS, Issue 1 2002Munishwar N. Gupta Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu2+, Zn2+, and Ni2+, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn2+ directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL -1, as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity. [source] A heat-stable trypsin inhibitor in adzuki bean (Vigna angularis): effect of extraction media, purification and biochemical characteristicsINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 1 2010Sappasith Klomklao Summary Trypsin inhibitor from adzuki bean (Vigna angularis) seed was isolated and characterised. Extraction of seed with NaCl at the concentration of 0.15 m showed a higher recovery of trypsin inhibitor than other solvents tested (P < 0.05). Optimal extraction time for the recovery trypsin inhibitor from adzuki bean seed was 30 min (P < 0.05). Purification of inhibitor was achieved by heat-treatment at 90 °C for 10 min, followed by ammonium sulphate precipitation with 30,65% saturation and size exclusion chromatography on Sephacryl S-200, presenting a yield and purification of 53.9% and 10.91-fold, respectively. The apparent molecular weight of trypsin inhibitor was estimated to be 14 kDa based on SDS-PAGE and inhibitor activity of zones separated by electrophoresis. The purified inhibitor was stable over a broad pH range and retained high inhibitory activity toward trypsin after incubation at 90 °C for 60 min. NaCl, at 0,3% concentration, did not affect the inhibitory activity of purified trypsin inhibitor, however, the activity was lost when sample was treated with ,-mercaptoethanol prior to electrophoresis. [source] Effect of cooking time on some nutrient and antinutrient components of bambaragroundnut seedsANIMAL SCIENCE JOURNAL, Issue 1 2009Stanley Omoh OMOIKHOJE ABSTRACT The proximate composition, gross energy, mineral composition, percentage sugar, oligosaccharides and antinutrient substances of bambaragroundnut seeds subjected to different cooking times were determined. The seeds were cooked for 30, 60, 90 and 120 min. Results of the proximate analysis showed that only the ether extract and ash were significantly (P < 0.05) reduced as the cooking time increased. In contrast, gross energy values significantly (P < 0.05) increased with increased cooking time. Amongst, the mineral elements assayed, calcium, magnesium and iron were significantly (P < 0.05) increased, while phosphorous, potassium, sodium and copper were reduced significantly (P > 0.05) with inreased cooking time. Percentage sucrose and glucose of bambaragroundnut seeds were significantly (P < 0.05) lowest in the raw form, but increased progressively with increased of cooking time. Raffinose and stachyose levels were reduced significantly by increased cookinf time (P < 0.05) with the least value in seeds cooked for 120 min. Trypsin inhibitor, hemagglutinin and tannin were completely eliminated in seeds cooked for 60 min or longer, but the phytin level was reduced significantly (P < 0.05) by cooking. For a significant detoxification of antinutrient substances and for optimal bioavailability of the component nutrients of bambaragroundnut seeds, an optimum cooking time of 60 min at 100°C is therefore recommended. [source] ISOLATION AND CHARACTERIZATION OF TRYPSIN INHIBITORS FROM SOME THAI LEGUME SEEDSJOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2000SOOTTAWAT BENJAKUL ABSTRACT Trypsin inhibitors from cultivars of cowpea (Vigna unguiculata (L.) Wasp.), pigeon pea (Cajanus cajan (L.) Millsp.) and bambara groundnuts (Voandzeia subterranea (L.) Thou) grown in Thailand were isolated and characterized. Extraction of seeds with NaCl rendered a higher recovery of trypsin inhibitor than other solvents tested (P<0.05). The extraction time affected the inhibitor recovery (P<0.05). The extraction time of 3 h was optimum for the recovery of trypsin inhibitor from pigeon and bambara groundnuts, whereas 1 h was optimum for cowpea. Based cn inhibitor activity of zones separated by electrophoresis, the molecular mass of the inhibitor from bambara groundnuts was 13 kDa. Two inhibitory bands were observed for cowpea (10 and 18 kDa) and pigeon pea (15 and 25 kDa). Partial purification of inhibitors was achieved by heat-treatment at 90C for 10 min, followed by ammonium sulfate precipitation with 30,65% saturation. The partially purified inhibitors from four seeds were heat stable up to 30 min at 90C at pH 7.0. The activities were also retained over a wide pH range at 25C but were lost when samples were treated with ,-mercaptoethanol prior to electrophoresis. [source] Preparation of trypsin-immobilised chitosan beads and their application to the purification of soybean trypsin inhibitorJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 13 2008Li Zhang Abstract BACKGROUND: Trypsin inhibitors are among the most important antinutritional factors in legumes. Recent research has shown that soybean trypsin inhibitor (SBTI) exhibits multiple bioactivities, but very few studies on the purification of SBTI are available. Enzymes are commonly used as biospecific ligands in affinity purification of their substrates or inhibitors. The aim of the present study was to prepare trypsin (EC 3.4.21.4)-immobilised chitosan beads and use them to purify trypsin inhibitor from soybean whey. RESULTS: Compared with free trypsin, the immobilised trypsin had higher thermal and pH stability. The adsorption ratio of SBTI from crude SBTI aqueous solution by trypsin-immobilised chitosan beads was 33.3%. The purified SBTI obtained by affinity chromatography was characterised by sodium dodecyl sulfate polyacrylamide gel electrophoresis as a single polypeptide band with an Mr of 8.3 kDa belonging to the Bowman,Birk family. CONCLUSION: Trypsin-immobilised chitosan beads were effectively used in the affinity separation of trypsin inhibitor from soybean seeds, thus indicating that immobilised trypsin may have practical application in the soybean-processing industry. The results of this study provide a background for further investigation of potential applications of soybean bioactive constituents in the areas of agriculture and food. Copyright © 2008 Society of Chemical Industry [source] Defense mechanisms against grazing: a study of trypsin inhibitor responses to simulated grazing in the sedge Carex bigelowiiOIKOS, Issue 9 2007Åsa Lindgren Trypsin inhibitors have been suggested to constitute an inducible defense in the sedge Carex bigelowii, and some former studies suggest that this might be a cause for the cyclic population dynamics in many alpine and arctic small mammals, for example lemmings (Lemmus lemmus). We investigated this further by using a method of simulated grazing (clipping) at different intensities, in three different habitats with varying resource availability, with different harvest times (hours after clipping), and two different stages of ramets (reproductive/vegetative) in a study from the Swedish mountain range. Our results do not indicate that C. bigelowii has an inducible defense constituted by an increase in trypsin inhibitor activity (TIA), but rather that the amount of soluble plant proteins (SPP) is lowered in wounded plants. The responses were somewhat different in the three habitats, with ramets growing in the marsh showing the highest ratio of TIA to SPP, due to low amounts of SPP. We did not find any significant effects of harvest time, or of the stage of the ramet that could support the hypothesis of an inducible defense. To conclude, we could not find any evidence for an inducible defense consisting of trypsin inhibitors in Carex bigelowii ramets, but we did find variations in the amount of SPP that may have nutritional consequences for herbivores. [source] NtKTI1, a Kunitz trypsin inhibitor with antifungal activity from Nicotiana tabacum, plays an important role in tobacco's defense responseFEBS JOURNAL, Issue 19 2010Hao Huang A cDNA library from tobacco inoculated with Rhizoctonia solani was constructed, and several cDNA fragments were identified by differential hybridization screening. One cDNA clone that was dramatically repressed, NtKTI1, was confirmed as a member of the Kunitz plant proteinase inhibitor family. RT-PCR analysis revealed that NtKTI1 was constitutively expressed throughout the whole plant and preferentially expressed in the roots and stems. Furthermore, RT-PCR analysis showed that NtKTI1 expression was repressed after R. solani inoculation, mechanical wounding and salicylic acid treatment, but was unaffected by methyl jasmonate, abscisic acid and NaCl treatment. In vitro assays showed that NtKTI1 exerted prominent antifungal activity towards R. solani and moderate antifungal activity against Rhizopus nigricans and Phytophthora parasitica var. nicotianae. Bioassays of transgenic tobacco demonstrated that overexpression of NtKTI1 enhanced significantly the resistance of tobacco against R. solani, and the antisense lines exhibited higher susceptibility than control lines towards the phytopathogen. Taken together, these studies suggest that NtKTI1 may be a functional Kunitz trypsin inhibitor with antifungal activity against several important phytopathogens in the tobacco defense response. [source] Thermodynamic analysis of binding of p -substituted benzamidines to trypsinFEBS JOURNAL, Issue 6 2001Reinskje Talhout Understanding the structural basis of inhibitor,enzyme interactions, important for the design of new drugs, requires a complete thermodynamic characterization of the binding process as well as a description of the structure of the complex. In this paper, the binding of p -substituted benzamidinium derivatives to the structurally well-characterized serine proteinase bovine pancreatic trypsin has been studied using isothermal titration calorimetry. These experiments have permitted a complete characterization of the temperature dependence of the inhibitor-binding thermodynamics. At 25 °C, both the enthalpy and entropy of binding are favourable for all studied derivatives, but this is only true for a relatively narrow temperature range. As binding is characterized by a negative change in heat capacity, the process is characterized by enthalpy,entropy compensation, resulting in a change of the net thermodynamic driving force for association from entropic to enthalpic with increasing temperature. These phenomena are not unusual when hydrophobic forces play an important role. The trend in the relative binding potencies can, to a significant extent, be attributed to the electron-donating/withdrawing character of the substituent at the para position, as shown by the Hammett plot for the different inhibitors; the more polar the p -substituted benzamidine, the less potent it will be as a trypsin inhibitor. This behaviour might result from a bulk solvation effect, meaning that the more polar, lower potency inhibitors will be more stabilized in water than the less polar, higher potency inhibitors. [source] A heat-stable trypsin inhibitor in adzuki bean (Vigna angularis): effect of extraction media, purification and biochemical characteristicsINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 1 2010Sappasith Klomklao Summary Trypsin inhibitor from adzuki bean (Vigna angularis) seed was isolated and characterised. Extraction of seed with NaCl at the concentration of 0.15 m showed a higher recovery of trypsin inhibitor than other solvents tested (P < 0.05). Optimal extraction time for the recovery trypsin inhibitor from adzuki bean seed was 30 min (P < 0.05). Purification of inhibitor was achieved by heat-treatment at 90 °C for 10 min, followed by ammonium sulphate precipitation with 30,65% saturation and size exclusion chromatography on Sephacryl S-200, presenting a yield and purification of 53.9% and 10.91-fold, respectively. The apparent molecular weight of trypsin inhibitor was estimated to be 14 kDa based on SDS-PAGE and inhibitor activity of zones separated by electrophoresis. The purified inhibitor was stable over a broad pH range and retained high inhibitory activity toward trypsin after incubation at 90 °C for 60 min. NaCl, at 0,3% concentration, did not affect the inhibitory activity of purified trypsin inhibitor, however, the activity was lost when sample was treated with ,-mercaptoethanol prior to electrophoresis. [source] Chemical composition and toxic trace element composition of some Nigerian edible wild mushroomsINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 1 2008Olumuyiwa S. Falade Summary Two essential amino acids (methionine and tryptophan); anti-nutritional factors (tannin and trypsin inhibitor) and toxic elements (Pb, Cd, Ni, As, Hg and Cr) were determined spectrophotometrically from five edible wild mushrooms. The tryptophan content was between 1.00 and 1.82 g (100 g),1 but methionine was low at 0.26,1.38 g (100 g),1. Tannin content was high (30.3,40.0 mg g,1) but trypsin inhibitor was low (22.0,39.5 TIU g,1). Trace elements analysis reviled Pb (0.34,5.06 mg kg,1) to be the highest of all the trace elements. Cd was (0.06,1.70 mg kg,1), Ni (0.26,2.08 mg kg,1), As (0.17,0.92 mg kg,1), Hg (0.01,0.05 mg kg,1) and Cr (0.04,0.22 mg kg,1). These mushrooms are nutritious but must be well processed to eliminate or at least reduce the levels of tannin and Pb to improve their nutritional values. [source] Influence of the surface on thrombin generationINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 4 2008T. W. STIEF Summary Thrombin generation depends on the surface of the blood vessel or container. With a new ultra-sensitive and -specific thrombin assay the surface-dependent thrombin generation was quantified. Citrated blood or plasma was preincubated for 1 h (37 °C). Citrated blood, plasma, or plasma with 0,10 g/l hemoglobin,erythrocyte microparticles (Hb,MP) were preincubated at 23 °C or at 37 °C. Plasma samples (50 ,l) were recalcified in polystyrol (PS) wells and incubated for different coagulation reaction times (CRT). Final supramolar arginine concentrations, 0.1% Triton X 100, and chromogenic thrombin substrate concentrations in the onefold km,range were added and the linear ,A/t was measured in the recalcified coagulation activity assay (RECA). Aprotinin or corn trypsin inhibitor were added. (i) Recalcification of plasma (in different monovettes) pre-incubated for 1 h (37 °C) generated the following thrombin activities after 7 min (37 °C): 0.74 IU/ml (polypropylene (PP)-citrate), 0.39 IU/ml (PP-EDTA), 0.06 IU/ml (PP-heparin), 1.38 IU/ml (PS), 0.63 IU/ml (1 ml volume PP), 0.13 IU/ml (15 ml volume PP), and 3.62 IU/ml (glass). (ii) Recalcification of preincubated whole blood generated up to about fivefold more thrombin. (iii) Thrombin generation is proportional to the plasmatic concentration of Hb,MP, 10 g/l Hb,MP generating about 4 IU/ml thrombin within 20 min CRT. (iv) The IC50 of aprotinin and corn typsin inhibitor on thrombin generation in RECA are about 2 KIU/ml and about 1 U/ml, respectively. The reaction wall, the preincubation temperature, and hemolysis influences thrombin generation. The RECA allows to diagnose the prothrombotic capacity of any material. [source] Effect of addition of soybean trypsin inhibitor to colostrum on immunological status in goat kidsJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 1 2010J. J. Ramos Summary The aim of the study was to evaluate the influence of soybean trypsin inhibitor (TI) on immunoglobulin G (IgG) serum levels and growth in neonatal goat kids. Twenty-four newborn kids were fed with natural colostrum (group A), and 24 kids received the same colostrum with 1 g of TI per litre (group B). Blood samples were obtained at birth and on days 1, 2 and 4 of life to analyze serum proteins, IgG and haematological parameters. There were no clinical signs of disease and no significant differences in body weight between the groups. Haematological parameters were not affected by treatment. The peak of serum IgG was reached at 24 h of life, but no effects of soybean TI was observed on serum IgG levels. The apparent efficiency of absorption of IgG was similar in both groups (group A 24.5% vs. group B 25.2%, p > 0.05). The addition of TI to colostrum did not change the concentration of serum proteins and their fractions in goat kids. The correlation between serum IgG and ,-globulin was positive and significant (p < 0.01, r = 0.64) in group A, but not in group B (p > 0.05, r = 0.08), suggesting a negative influence of soybean TI on ,-globulin absorption. These results show that addition of soybean TI to colostrum did not improve the performance or immunological status in goat kids. [source] Combining a polarizable force-field and a coarse-grained polarizable solvent model: Application to long dynamics simulations of bovine pancreatic trypsin inhibitorJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 11 2008Michel Masella Abstract The dynamic coupling between a polarizable protein force field and a particle-based implicit solvent model is described. The polarizable force field, TCPEp, developed recently to simulate protein systems, is characterized by a reduced number of polarizable sites, with a substantial gain in efficiency for an equal chemical accuracy. The Polarizable Pseudo-Particle (PPP) solvent model represents the macroscopic solvent polarization by induced dipoles placed on mobile Lennard-Jones pseudo-particles. The solvent-induced dipoles are sensitive to the solute electric field, but not to each other, so that the computational cost of solvent,solvent interactions is basically negligible. The solute and solvent induced dipoles are determined self-consistently and the equations of motion are solved using an efficient iterative multiple time step procedure. The solvation cost with respect to vacuum simulations is shown to decrease with solute size: the estimated multiplicative factor is 2.5 for a protein containing about 1000 atoms, and as low as 1.15 for 8000 atoms. The model is tested for six 20 ns molecular dynamics trajectories of a traditional benchmark system: the hydrated Bovine Pancreatic Trypsin Inhibitor (BPTI). Even though the TCPEp parameters have not been refined to be used with the solvent PPP model, we observe a good conservation of the BPTI structure along the trajectories. Moreover, our approach is able to provide a description of the protein solvation thermodynamic at the same accuracy as the standard Poisson-Boltzman continuum methods. It provides in addition a good description of the microscopic structural aspects concerning the solute/solvent interaction. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2008 [source] PROTEINASES IN HYBRID CATFISH VISCERA: CHARACTERIZATION AND EFFECT OF EXTRACTION MEDIAJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2010SAPPASITH KLOMKLAO ABSTRACT Proteolytic activity from viscera extract of hybrid catfish (Clarias macrocephalus × Clarias gariepinus) was investigated. Optimal pH and temperature for casein hydrolysis were 9.0 and 50C, respectively. The enzyme was stable to heat treatment up to 40C and over a pH range of 7,11 for 30,120 min. The proteolytic activity was effectively inhibited by soybean trypsin inhibitor, benzamidine, phenylmethylsulfonyl fluoride and N -p-tosyl-L-lysine chloromethyl ketone. Activities of the viscera extract continuously decreased as NaCl concentration increased, while activities increased as CaCl2 concentration increased. Based on the proteinase activity of zones separated by electrophoresis, the molecular mass of the major proteinases in hybrid catfish viscera was 23 and 20 kDa. The effect of extraction media on recovery of proteinases was also studied. Extraction of the viscera powder with 50 mM Tris-HCl, pH 7.0 containing 0.5 M NaCl and 0.2% (v/v) Brij 35 rendered a higher recovery of proteinase activity than other extractants tested (P < 0.05). The results suggested that major proteinases in hybrid catfish viscera were heat-activated alkaline proteinases, most likely trypsin-like serine proteinases. PRACTICAL APPLICATIONS Hybrid catfish viscera is an abundant and underutilized resource that can be used as a unique proteinase source. Proteinase from various sources catalyzes the hydrolysis of peptide bonds. Thus, it is expected that like other proteinases, hybrid catfish proteinase would be useful in biomedical, food and beverage application. Moreover, the presented extraction media could be adopted to recover the trypsin-like serine proteinase from hybrid catfish viscera, which is currently a solid waste of Pa-duk-ra industry. [source] COMPARATIVE STUDIES ON PROTEOLYTIC ACTIVITY OF SPLENIC EXTRACT FROM THREE TUNA SPECIES COMMONLY USED IN THAILANDJOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2004SUPPASITH KLOMKLAO ABSTRACT Proteolytic activities of splenic extract from three tuna species including skipjack tuna (Katsuwonus pelamis), yellowfin tuna (Thunnus albacores) and tongol tuna (Thunnus tonggol) were studied. Optimal activity of splenic extract from all tuna species was at pH 9.0 and 55C when casein was used as a substrate. Among all species tested, yellowfin tuna showed the highest activity, followed by skipjack tuna and tongol tuna. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid. E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibition. The effect of NaCl and CaCl2 on proteolytic activity was also investigated. Activities continuously decreased as NaCl concentration increased, and no activity remained in the presence of 30% NaCl. On the other hand, activities increased as CaCl2 concentration increased. The highest activity was obtained in the presence of 1 mM CaCl2. SDS-substrate gel electrophoresis revealed that major proteinases in splenic extract from different tuna species were different in apparent molecular weights and sensitivity to TLCK. Although the major activity bands of all species were strongly inhibited by soybean trypsin inhibitor, varying sensitivity to TLCK probably implied the differences in binding characteristic of enzyme to substrate and/or inhibitors. The results suggest that major proteinases in spleen of all tuna species were trypsin-like serine proteinases. [source] IDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARPJOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2004MIN-JIE CAO ABSTRACT Myofibril-bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55,60C as shown by SDS-PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as ,-actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril-bound serine proteinase. [source] ISOLATION AND CHARACTERIZATION OF TRYPSIN INHIBITORS FROM SOME THAI LEGUME SEEDSJOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2000SOOTTAWAT BENJAKUL ABSTRACT Trypsin inhibitors from cultivars of cowpea (Vigna unguiculata (L.) Wasp.), pigeon pea (Cajanus cajan (L.) Millsp.) and bambara groundnuts (Voandzeia subterranea (L.) Thou) grown in Thailand were isolated and characterized. Extraction of seeds with NaCl rendered a higher recovery of trypsin inhibitor than other solvents tested (P<0.05). The extraction time affected the inhibitor recovery (P<0.05). The extraction time of 3 h was optimum for the recovery of trypsin inhibitor from pigeon and bambara groundnuts, whereas 1 h was optimum for cowpea. Based cn inhibitor activity of zones separated by electrophoresis, the molecular mass of the inhibitor from bambara groundnuts was 13 kDa. Two inhibitory bands were observed for cowpea (10 and 18 kDa) and pigeon pea (15 and 25 kDa). Partial purification of inhibitors was achieved by heat-treatment at 90C for 10 min, followed by ammonium sulfate precipitation with 30,65% saturation. The partially purified inhibitors from four seeds were heat stable up to 30 min at 90C at pH 7.0. The activities were also retained over a wide pH range at 25C but were lost when samples were treated with ,-mercaptoethanol prior to electrophoresis. [source] EFFECT of EXTRUSION ON TRYPSIN INHIBITOR CONTENTS of SOY-SWEET POTATO MIXTURESJOURNAL OF FOOD PROCESSING AND PRESERVATION, Issue 6 2000M.O. IWE Mixtures of soy and sweet potato (Ipomoea batatas) (L.) Lam), flours containing 18%, 25% and 30% moisture, respectively, were extruded in a single screw extruder. Results showed that inactivation of trypsin inhibitor was enhanced by both reductions in feed moisture and soy flour contents of sample mixtures. Hence subsequent extrusion was carried at 18% feed moisture with variable feed ratio, screw rotation speed and die diameter, using a central composite rotatable, near orthogonal experimental design. Results further showed that the effect of increasing the ratio of soy in the mixture was linearly significant (p > 0.05). Optimum Trypsin Inhibitor (TI) inactivation value of 3.40 mg/g was predicted at a feed composition of 80% sweet potato, 9 mm die diameter and 154 rpm, respectively. [source] Tuna Pepsin: Characteristics and Its Use for Collagen Extraction from the Skin of Threadfin Bream (Nemipterus spp.)JOURNAL OF FOOD SCIENCE, Issue 5 2008S. Nalinanon ABSTRACT:, Pepsin from the stomach of albacore tuna, skipjack tuna, and tongol tuna was characterized. Pepsin from all tuna species showed maximal activity at pH 2.0 and 50 °C when hemoglobin was used as a substrate. Among the stomach extract of all species tested, that of albacore tuna showed the highest activity (40.55 units/g tissue) (P < 0.05). Substrate-Native-PAGE revealed that pepsin from albacore tuna and tongol tuna consisted of 2 isoforms, whereas pepsin from skipjack tuna had only 1 form. The activity was completely inhibited by pepstatin A, while EDTA (ethylenediaminetetraacetic acid), SBTI (soybean trypsin inhibitor), and E-64 (1-(L -trans-epoxysuccinyl-leucylamino)-4-guanidinobutane) exhibited negligible effect. The activity was strongly inhibited by SDS (sodium dodecyl sulfate) (0.05% to 0.1%, w/v). Cysteine (5 to 50 mM) also showed an inhibitory effect in a concentration dependent manner. ATP, molybdate, NaCl, MgCl2, and CaCl2 had no impact on the activity. When tuna pepsin (10 units/g defatted skin) was used for collagen extraction from the skin of threadfin bream for 12 h, the yield of collagen increased by 1.84- to 2.32-fold and albacore pepsin showed the comparable extraction efficacy to porcine pepsin. The yield generally increased with increasing extraction time (P < 0.05). All collagen obtained with the aid of tuna pepsin showed similar protein patterns compared with those found in acid-solubilized collagen. Nevertheless, pepsin from skipjack tuna caused the degradation of , and , components. All collagens were classified as type I with large portion of ,-chain. However, proteins with molecular weight (MW) greater than 200 kDa were abundant in acid-solubilized collagen. [source] Soy-Derived Immunoglobulin Production Stimulating Factor Enhances IgM Production of Mouse Spleen LymphocytesJOURNAL OF FOOD SCIENCE, Issue 7 2006N. Maeda ABSTRACT:, We have reported previously that some proteins such as lactoferrin and lysozyme control the immune system via immunoglobulin (Ig) production. In the course of the study about the function of dietary proteins and peptides, Ig production-stimulating activity of an unknown protein contained in soybean trypsin inhibitor (STI) preparation was found. Thus, we examined the activity of the unknown activator and the mechanism of the activity. The factor significantly elevated IgM production by mouse spleen lymphocytes in a dose-dependent manner. In addition, the unknown activator up-regulated the expression of interleukin (IL)-6 and IL-10 mRNA. And IgM production enhancing activity of STI was significantly suppressed by neutralization of IL-6. Then, to clarify whether STI itself is the active compound or not, STI was fractionized by gel filtration chromatography. We found that the activity the content of STI did not correlate with and the active fractions contained some proteins whose molecular weight is more than 20 kDa. These suggest that an unknown activator exists in STI preparation. [source] Plasma and urine levels of urinary trypsin inhibitor in patients with acute and fulminant hepatitisJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 2 2002SHI DE LIN Abstract Background and Aim Urinary trypsin inhibitor (UTI) is synthesized by hepatocytes and excreted into urine. Plasma and urine UTI levels have been measured to evaluate whether these levels may be useful markers in various pathological conditions. However, there has been no study on plasma and urine UTI levels in patients with acute liver diseases. The aim of the present study was to evaluate plasma and urine UTI levels and their relationship with the severity of hepatic damage in patients with acute liver diseases. Methods Plasma and urine UTI levels were measured by newly developed enzyme-linked immunosorbent assay in 15 patients with acute hepatitis (AH), 12 patients with acute severe hepatitis (ASH) and 10 patients with fulminant hepatitis (FH), as assessed on admission. The serial changes in plasma and urine UTI were also observed in some patients with AH and ASH. Results Plasma UTI levels (U/mL, median [25,75th percentile]) were: 11.0, (9.5,16.1) in patients with AH; 7.8 (5.6,11.5) in those with ASH; 6.5 (4.0,9.5) in patients with FH; and 9.7 (7.3,11.0) in normal controls. Plasma UTI levels in patients with FH were significantly lower than in those with AH. Plasma UTI levels showed significant positive correlations with the levels of prothrombin time (PT), hepaplastin test, antithrombin III, ,2-plasmin inhibitor, plasminogen (Plg) and fibrinogen. After the recovery of liver dysfunction, increased plasma UTI levels in patients with AH were decreased, whereas previously decreased plasma UTI levels in patients with ASH were increased. Urine UTI levels were significantly increased in patients with AH compared with those of normal controls. In patients with ASH and FH, urine UTI levels were increased but not significantly. Urine UTI levels significantly positively correlated with PT and Plg. After the recovery of liver dysfunction, previously increased urine UTI levels in patients with AH were decreased. The correlation between plasma UTI and urine UTI levels was not significant. Conclusions The findings of the present study suggested that the levels of plasma and urine UTI changed in patients with AH and were closely related to the abnormalities of coagulo-fibrinolysis, including PT. Further studies are needed to clarify whether these levels may be useful markers to predict the prognosis of acute hepatitis. [source] Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 8 2001Victor J. Nesatyy Abstract The potential of electrospray ionization (ESI) mass spectrometry (MS) to detect non-covalent protein complexes has been demonstrated repeteadly. However, questions about correlation of the solution and gas-phase structures of these complexes still produce vigorous scientific discussion. Here, we demonstrate the evaluation of the gas-phase binding of non-covalent protein complexes formed between bovine pancreatic trypsin inhibitor (BPTI) and its target enzymes over a wide range of dissociation constants. Non-covalent protein complexes were detected by ESI-MS. The abundance of the complex ions in the mass spectra is less than expected from the values of the dissociation constants of the complexes in solution. Collisionally activated dissociation (CAD) tandem mass spectrometry (MS/MS) and a collision model for ion activation were used to evaluate the binding of non-covalent complexes in the gas phase. The internal energy required to induce dissociation was calculated for three collision gases (Ne, Ar, Kr) over a wide range of collision gas pressures and energies using an electrospray ionization source. The order of binding energies of the gas-phase ions for non-covalent protein complexes formed by the ESI source and assessed using CAD-MS/MS appears to differ from that of the solution complexes. The implication is that solution structure of these complexes was not preserved in the gas phase. Copyright © 2001 John Wiley & Sons, Ltd. [source] Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activityJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2005Yasuhiro Nishiyama Abstract Covalent nucleophile,electrophile interactions have been established to be important for recognition of substrates by several enzymes. Here, we employed an electrophilic amidino phosphonate ester (EP1) to study the nucleophilic reactivity of the following proteins: albumin, soluble epidermal growth factor receptor (sEGFR), soluble CD4 (sCD4), calmodulin, casein, ,-lactalbumin, ovalbumin, soybean trypsin inhibitor and HIV-1 gp120. Except for soybean trypsin inhibitor and ,-lactalbumin, these proteins formed adducts with EP1 that were not dissociated by denaturing treatments. Despite their negligible proteolytic activity, gp120, sEGFR and albumin reacted irreversibly with EP1 at rates comparable to the serine protease trypsin. The neutral counterpart of EP1 reacted marginally with the proteins, indicating the requirement for a positive charge close to the electrophilic group. Prior heating resulted in altered rates of formation of the EP1,protein adducts accompanied by discrete changes in the fluorescence emission spectra of the proteins, suggesting that the three-dimensional protein structure governs the nucleophilic reactivity. sCD4 and vasoactive intestinal peptide (VIP) containing phosphonate groups (EP3 and EP4, respectively) reacted with their cognate high-affinity binding proteins gp120 and calmodulin, respectively, at rates exceeding the corresponding reactions with EP1. Reduced formation of EP3,gp120 adducts and EP4,calmodulin adducts in the presence of sCD4 and VIP devoid of the phosphonate groups was evident, suggesting that the nucleophilic reactivity is expressed in coordination with non-covalent recognition of peptide determinants. These observations suggest the potential of EPs for specific and covalent targeting of proteins, and raise the possibility of nucleophile,electrophile pairing as a novel mechanism stabilizing protein,protein complexes. Copyright © 2005 John Wiley & Sons, Ltd. [source] Influence of thermal motion on 1H chemical shifts in proteins: the case of bovine pancreatic trypsin inhibitorJOURNAL OF PEPTIDE SCIENCE, Issue 3 2001Bernard Busetta Abstract The possible influence of thermal motion on 1H chemical shifts is discussed for a small stable protein, the bovine pancreatic Kunitz trypsin inhibitor (BPTI). The thermal effects on the aromatic side chains and on the backbone are treated separately. The thermal motion of the aromatic side chains is accounted for in terms of their rotation around the C,C, bond and the motion of each individual proton is interpreted as a ratio between the amount of ordered and quite disordered states. The influence of hydrogen bonds is introduced as an extra contribution to the chemical shifts of the bonded proton. Their contribution to the chemical shifts resulting from the polarization of the peptide bond is investigated, as is their influence on local flexibility. Finally, the relative importance of each contribution to the chemical shift information is compared. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd. [source] Antifungal Activity of a Bowman,Birk-type Trypsin Inhibitor from Wheat KernelJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2000G. Chilosi A trypsin inhibitor from wheat kernel (WTI) was found to have a strong antifungal activity against a number of pathogenic fungi and to inhibit fungal trypsin-like activity. WTI inhibited in vitro spore germination and hyphal growth of pathogens, with protein concentration required for 50% growth inhibition (IC50) values ranging from 111.7 to above 500 ,g/ml. As observed by electron microscopy, WTI determined morphological alterations represented by hyphal growth inhibition and branching. One of the fungal species tested, Botrytis cinerea produced a trypsin-like protease, which was inhibited by the trypsin inhibitor. WTI, as well as other seed defence proteins, appear to be an important resistance factor in wheat kernels during rest and early germination when plants are particularly exposed to attack by potential soil-borne pathogens. Zusammenfassung Ein Trypsinhemmer aus Weizenkörnern (WTI) zeigte eine starke antifungale Aktivität gegenüber verschiedenen pathogenen Pilzen und hemmte deren trypsinähnliche Aktivität. WTI hemmte in vitro die Sporenkeimung und das Hyphenwachstum der Pathogene, wobei die IC50 -Werte zwischen 111,7 und mehr als 500 ,g/ml lagen. Elektronenmikroskopische Untersuchungen zeigten, dai WTI morphologische Veränderungen bewirkte, die aus einer Hemmung des Hyphenwachstums und einer veränderten Verzweigung bestanden. Eine der untersuchten Pilzarten, Botrytis cinerea, bildete eine trypsinähnliche Protease, die durch den Trypsininhibitor gehemmt wurde. Ebenso wie andere sameneigene Abwehrproteine scheint WTI während der Keimruhe und in den frühen Stadien der Keimung, wenn die Pflanzen gegenüber möglichen bodenbürtigen Pathogenen besonders exponiert sind, ein wichtiger Resistenzfaktor in Weizenkörnern zu sein. [source] Association Analyses of Genetic Polymorphisms of GSTM1, GSTT1, NQO1, NAT2, LPL, PRSS1, PSTI, and CFTR With Chronic Alcoholic Pancreatitis in JapanALCOHOLISM, Issue 2010Katsuya Maruyama Background:, Excessive consumption of alcohol is involved in the onset of pancreatitis. However, most of heavy drinkers do not always develop chronic pancreatitis. Various genetic factors appear to be involved in these individual differences in onset of chronic alcoholic pancreatitis. Here we investigated a possible association of alcoholic pancreatitis with polymorphisms of the various genes belong to the phase II detoxification enzymes responsible for metabolism of the oxidative compounds, and the several genes that have relevance to inherited pancreatitis. Methods:, The subjects consisted of 53 patients with chronic alcoholic pancreatitis, 54 alcoholic patients without pancreatic dysfunction, and 42 healthy individuals. DNA was extracted from the peripheral nucleated blood cells of all subjects and genetic mutations and subtypes were analyzed by the PCR and RFLP methods. We examined the correlation between chronic alcoholic pancreatitis and variants of the phase II detoxification enzymes such as Glutathione S-transferase M1 (GSTM1), glutathione S-transferase theta 1 (GSTT1), NADPH-quinone oxidoreductase 1 (NQO1), and N-acetyl transferase (NAT2). In addition, genes of lipoprotein lipase (LPL), cationic trypsinogen (PRSS1), pancreatic secretory trypsin inhibitor (PSTI), and cystic fibrosis transmembrane conductance regulator (CFTR) were also analyzed. Results:, Frequencies of the gene deletion of GSTM1 and GSTT1 in addition to the C-allele frequency of NQO1 tended to be higher in the alcoholic patients with (AlCP) or without pancreatic dysfunction (Alc) than in the healthy controls although the difference was not significant. The NAT2 gene showed no relation with Alc and AlCP patients. PSTI, LPL, PRSS1, and CFTR genes presented no association with chronic alcoholic pancreatitis. Conclusions:, All genes analyzed in the present study lacked association with chronic alcoholic pancreatitis. However, the gene deletion of GSTM1 and GSTT1, and the C-allele of NQO1 cannot be ruled out for association with alcoholism. [source] | ||||||||||||||||||||||||||||||||||