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Sample records for chymotrypsin

  1. Trypsin and Chymotrypsin Test

    Science.gov (United States)

    ... as persistent diarrhea, foul-smelling bulky greasy stools, malnutrition , and vitamin deficiency. Chymotrypsin may be ordered when ... relief and nutritional improvement by taking doctor-prescribed oral enzymes and vitamin supplements. ^ Back to top ... Health Professionals Get the Mobile App iTunes | Android | Kindle ...

  2. Purification and characterization of Locusta migratoria chymotrypsin.

    Science.gov (United States)

    Sakal, E; Applebaum, S W; Birk, Y

    1988-12-01

    A chymotrypsin-like enzyme (CTLE) was isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides by ion-exchange chromatography on diethylaminoethyl (DEAE) cellulose followed by affinity chromatography on phenylbutylamine (PBA) Sepharose. The purity and homogeneity of CTLE have been shown by SDS-PAGE and on cellulose acetate strips. The enzyme has a molecular weight of 24,000, determined by SDS-PAGE and on a Sephadex G-75 calibrated column. It has an isoelectric point of 10.1 and contains 0-1 half cystine residues. Sequence analysis of the first 20 N-terminal amino acids has shown 25% homology with bovine chymotrypsin and 40% homology with Vespa crabo and Vespa orientalis chymotrypsins and with Hypoderma lineatum trypsin. The optimal pH for enzyme activity and stability was in the range of 8.5-9.0. The Km and kcat values, determined on substrates for proteolytic, esterolytic and amidolytic activity, similar to those for bovine chymotrypsin. CTLE was inactivated by PMSF and TPCK indicating the involvement of serine and histidine in its active site. The enzyme was fully inhibited by the proteinaceous, double-headed, chymotrypsin-trypsin inhibitors BBI from soybeans and CI from chickpeas, by chicken ovomucoid (COM) and turkey ovomucoid (TOM), as well as by the Kunitz soybean trypsin inhibitor (STI) which hardly inhibits bovine chymotrypsin. Inhibition studies of CTLE with amino acid and peptide-chloromethylketones point towards the existence of an extended binding site. PMID:3246483

  3. The structure of an insect chymotrypsin.

    Science.gov (United States)

    Botos, I; Meyer, E; Nguyen, M; Swanson, S M; Koomen, J M; Russell, D H; Meyer, E F

    2000-05-19

    The South American imported fire ant (Solenopsis invicta), without natural enemies in the United States, widely infests the southern United States, causing more than a half billion dollars in health and agriculture-related damage annually in Texas alone. Fire ants are resistant to most insecticides, so control will require a more fundamental understanding of their biochemistry and metabolism leading to the design of selective, ecologically safe insecticides. The 4th instar larvae play a crucial role in the nutrition of the colony by secreting proteinases (especially chymotrypsin) which digest food products for the entire colony. The first structure of an ant proteolytic enzyme, fire ant chymotrypsin, was determined to atomic resolution (1.7 A). A structural comparison of the ant and mammalian structures confirms the "universality" of the serine proteinase motif and reveals a difference at residues 147-148, which are proteolytically removed in the bovine enzyme but are firmly intact in the ant chymotrypsin, suggesting a different activation mechanism for the latter. Likewise, the absence of the covalently attached propeptide domain (1-15) further suggests an uncharacteristic activation mechanism. The presence of Gly189 in the S1 site is an atypical feature of this chymotrypsin and is comparable only to human leukocyte elastase, hornet chymotrypsin and fiddler crab collagenase. Binding studies confirm the chymotrypsin nature of this novel enzyme. PMID:10801356

  4. 21 CFR 862.1180 - Chymotrypsin test system.

    Science.gov (United States)

    2010-04-01

    ... measure the activity of the enzyme chymotrypsin in blood and other body fluids and in feces. Chymotrypsin measurements are used in the diagnosis and treatment of pancreatic exocrine insufficiency. (b)...

  5. [Extraction and properties of microcapsulated alpha-chymotrypsin].

    Science.gov (United States)

    Aĭsina, R B; Kazanskaia, N F; Lukasheva, E V; Berezin, I V

    1976-09-01

    A method of microencapsulating of the proteolytic enzyme alpha-chymotrypsin into semi-permeable nylon membranes is worked out. The membrane is a polimer of 1,6-hexamethylenediamine and sebacoyl chloride. alpha-Chymotrypsin is enclosed into the capsule together with polyethyleneimine, capable of joining the walls of microcapsules and making the membrane more stable. The optimal concentrations of polyenthyleneimine and alpha-chymotrypsin are 5% and 1% correspondingly. The highest yield of microencapsulated enzyme was obtained for completely acetylated delta-chymotrypsin. The kinetic properties of microencapsulated alpha-chymotrypsin change very slightly as compared to those of the native one.

  6. Extraction, Purification and Characterization of Fish Chymotrypsin: A Review

    Directory of Open Access Journals (Sweden)

    Liang Zhou

    2011-01-01

    Full Text Available Problem statement: Solid fish waste is generated from the unwanted parts of fish including heads, tails, fins, frames, offal (guts, kidney and liver and skin. It accounts for up to 80% of material from production of surimi, 66% from production of fillet and 27% from production of headed and gutted fish. Currently, fish wastes are disposed off in land-based waste disposal systems or at sea generating toxic by-products during the decomposition process. However, fish processing waste can be used to produce commercially valuable by-products, such as chymotrypsin. Approach: A comperehensive review of the literature on the extraction, purification and characeterization of fish chymotrypsin was performed. Results: Chymotrypsin is an endopeptidase secreted by the pancreatic tissues of vertebrates and invertebrates. It has 3 different structures (chymotrypsin A, B and C varying slightly in solubility, electrophoretic mobility, isoelectric point and cleavage specificity. Only chymotrypsin A and B are found in fish. Compared with mammal chymotrypsin, fish chymotrypsins have similar amino acid composition and molecular weights. Fish chymotrypsins have higher specific activity, especially those from cold-water fish, and low pH and temperature tolerance. The factors affecting the concentration and activity of chymotrypsin in fish are water temperature, fish species, fish age, fish weight and starvation. Chymotrypsin has application in various industries including the food industry, leather production industry chemical industry and medical industry. Conclusion/Recommendations: Extraction techniques for chymotrypsin include: ultra-filtration, ammonium sulphate fractionation precipitation or water-in-oil microemulsions. Purification can be carried out using re-crystallization and gel-filtration, ion-exchange and hydrophobic interaction chromatography. Further studies should focus on the optimization of purifiying chymotrypsin from fish processing wastes.

  7. Structure of chymotrypsin variant B from Atlantic cod, Gadus morhua

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Asgeirsson, B; Thórólfsson, M;

    1996-01-01

    The amino-acid sequence of chymotrypsin variant B isolated from the pyloric caeca of Atlantic cod has been elucidated. The characterization of the primary structure is based on N-terminal Edman degradation and mass spectrometry of the native protein and enzymatically derived peptides. Chymotrypsi...

  8. A Chemical and Crystallographic Study of Carbamyl-Chymotrypsin A

    NARCIS (Netherlands)

    Robillard, George T.; Powers, James C.; Wilcox, Philip E.

    1972-01-01

    The reaction of p-nitrophenyl cyanate with chymotrypsinogen A and chymotrypsin A has been studied to determine the potential of this reagent in the field of enzyme modifications. These experiments have shown that p-nitrophenyl [14C]cyanate can react at specific loci on the enzyme under mild conditio

  9. Trypsin-chymotrypsin inhibitors from Vigna mungo seeds.

    Science.gov (United States)

    Cheung, Allen H K; Wong, Jack H; Ng, T B

    2009-01-01

    Three trypsin-chymotrypsin inhibitors were isolated from seeds of the black gram (Vigna mungo) with a procedure that entailed cation exchange chromatography on SP-Sepharose, anion exchange chromatography on Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q and Mono S, and gel filtration by FPLC on Superdex 75. Two of the trypsin-chymotrypsin inhibitors were adsorbed on the first four types of chromatographic media. All three inhibitors have a molecular mass of 16 kDa as judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The trypsin inhibitory activity of the inhibitors was attenuated in the presence of the reducing agent dithiothreitol. The remaining inhibitor was unadsorbed on SP-Sepharose but adsorbed on Q-Sepharose, Mono Q and Mono S. The protease inhibitors did not exert any inhibitory effect on hepatoma (Hep G2) and breast cancer (MCF 7) cells or antifungal action toward Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. Two of the inhibitors slightly inhibited the activity of HIV-1 reverse transcriptase, with an IC50 in the millimolar range.

  10. Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas

    Institute of Scientific and Technical Information of China (English)

    Haibo Wang; Duoduo Yuan; Rong Xu; Cheng-Wu Chi

    2011-01-01

    Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum, In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structurefunction studies.

  11. CARS and Raman spectroscopy of function-related conformational changes of chymotrypsin

    NARCIS (Netherlands)

    Brandt, N.N.; Chikishev, A.Yu.; Greve, J.; Koroteev, N.I.; Otto, C.; Sakodynskaya, I.K.

    2000-01-01

    We report on the comparative analysis of the conformation-sensitive bands of free enzyme (chymotrypsin), liganded enzyme (chymotrypsin anthranilate) and enzyme complex with 18-crown-6. The studies were carried out by Raman scattering spectroscopy and polarization-sensitive coherent anti-Stokes Raman

  12. Purification and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout oncorhynchus-mykiss

    DEFF Research Database (Denmark)

    Kristjansson, Magnus M.; Nielsen, Henrik Hauch

    1992-01-01

    Two chymotrypsins, called chymotrypsin I and II, were purified from the pyloric caeca of rainbow trout, by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The approximate molecular weights of chymotrypsin I and I...

  13. Effect of chymotrypsin C and related proteins on pancreatic cancer cell migration

    Institute of Scientific and Technical Information of China (English)

    Haibo Wang; Wei Sha; Zhixue Liu; Cheng-Wu Chi

    2011-01-01

    Pancreatic cancer is a malignant cancer with a bigh mortality rate. The amount of chymotrypsin C in pancreatic cancer cells is only 20% of that found in normal cells.Chymotrypsin C has been reported to be involved in cancer cell apoptosis, but its effect on pancreatic cancer cell migration is unclear. We performed cell migration scratch assays and Transwell experiments, and found that cell migration ability was downregulated in pancreatic cancer Aspc-1 cells that overexpressed chymotrypsin C, whereas the cell migration ability was upregulated in Aspc-1 cells in which chymotrypsin C was suppressed. Two-dimensional fluorescence differential in gel electrophoresis/mass spectrometry method was used to identify the proteins that were differentially expressed in Aspc-1 cells that were transfected with plasmids to induce either overexpression or suppressed expression of chymotrypsin C. Among 26 identified differential proteins, cytokeratin 18 was most obviously correlated with chymotrypsin C expression. Cytokeratin 18 is expressed in developmental tissues in early stages of cancer,and is highly expressed in most carcinomas. We speculated that chymotrypsin C might regulate pancreatic cancer cell migration in relation to cytokeratin 18 expression.

  14. Protection of chymotrypsin from inactivation by a N-mustard analog.

    Science.gov (United States)

    Brecher, A S; Koenig, M J

    1995-02-01

    Chymotrypsin activity is rapidly inactivated by the N-mustard anti-tumor drug, chlorambucil. Since mustards react with thiols, amines, carboxyls, imidazoles, and sulfide sites on proteins, N-acetylcysteine, 2 proprietary protein hydrolyzates, beta-mercaptoethanol, ethanolamine, and sodium lactate were tested for their capacity to protect chymotrypsin from inactivation by the mustard. In each instance, protection was afforded to chymotrypsin. In as much as N-acetylcysteine protected chymotrypsin from inactivation by chlorambucil, it is suggested that this thiol compound may serve as a detoxication agent and may not require prior transformation into glutathione by cells in order to reduce mustard levels within the cells, as suggested by Smith and Gross (Proceedings of the NATO Panel VIII meeting, Grenoble, France, 1991.) It is further suggested that amino acids present as biosynthetic and degradative components of cells may detoxify mustards. PMID:7701511

  15. Effect of Weaning Ages on Pancreatic and Intestinal Chymotrypsin Activity in Piglet

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hong-fu; LI Chang-zhong; GU Xian-hong; FANG Lu; MA Yong-xi; GONG Li-ming; FENG Guang-ming

    2002-01-01

    Twelve litters of new born piglets were divided randomly into groups Ⅰ, Ⅱ, Ⅲ and Ⅳ, and weaned at 17, 21, 28 and 35 days of age, respectively, to determine the effect of weaning age on pancreatic and intestinal chymotrypsin activity in piglets. The results showed that the relative and specific activity of pancreatic chymotrypsin increased significantly 12h after weaning compared with that of sucking piglets at the same age and then decreased and remained low for 2 - 3 weeks after weaning. Total pancreatic chymotrypsin activity had no change with the increase of pancreas weight during day 18 - 50 regardless of weaning (P >0.05). Chymotrypsin activity of jejunum had the same change as that of pancreas during sulking stage.Jejunum chymotrypin activity decreased in the first week post-weaning in the piglets weaned before day 28, but had no change in groups weaned at 28 or 35 days. The earlier the weaning age, the longer the restoring time.Chymotrypsin activity in jejunum is more sensitive to the effects of weaning age than in duodenum and ileum.

  16. Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom.

    Science.gov (United States)

    Zhou, Xing-Ding; Jin, Yang; Lu, Qiu-Min; Li, Dong-Sheng; Zhu, Shao-Wen; Wang, Wan-Yu; Xiong, Yu-Liang

    2004-02-01

    A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine alpha-chymotrypsin with a Ki of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities=89.5%) and other snake venom protease inhibitors. PMID:14990218

  17. Crystal structure of bovine duodenase, a serine protease, with dual trypsin and chymotrypsin-like specificities.

    Science.gov (United States)

    Pletnev, V Z; Zamolodchikova, T S; Pangborn, W A; Duax, W L

    2000-10-01

    The three-dimensional structure of duodenase, a serine protease from bovine duodenum mucosa, has been determined at 2.4A resolution. The enzyme, which has both trypsin-like and chymotrypsin-like activities, most closely resembles human cathepsin G with which it shares 57% sequence identity and similar specificity. The catalytic Ser195 in duodenase adopts the energetically favored conformation typical of serine proteinases and unlike the strained state typical of lipase/esterases. Of several waters in the active site of duodenase, the one associated with Ser214 is found in all serine proteinases and most lipase/esterases. The conservation of the Ser214 residue in serine proteinase, its presence in the active site, and participation in a hydrogen water network involving the catalytic triad (His57, Asp107, and Ser195) argues for its having an important role in the mechanism of action. It may be referred to as a fourth member of the catalytic triad. Duodenase is one of a growing family of enzymes that possesses trypsin-like and chymotrypsin-like activity. Not long ago, these activities were considered to be mutually exclusive. Computer modeling reveals that the S1 subsite of duodenase has structural features compatible with effective accommodation of P1 residues typical of trypsin (Arg/Lys) and chymotrypsin (Tyr/Phe) substrates. The determination of structural features associated with functional variation in the enzyme family may permit design of enzymes with a specific ratio of trypsin and chymotrypsin activities. PMID:10944388

  18. Large acceleration of α-chymotrypsin-catalyzed dipeptide formation by 18-crown-6 in organic solvents

    NARCIS (Netherlands)

    Unen, van Dirk-Jan; Engbersen, Johan F.J.; Reinhoudt, David N.

    1998-01-01

    The effects of 18-crown-6 on the synthesis of peptides catalyzed by α-chymotrypsin are reported. Lyophilization of the enzyme in the presence of 50 equivalents of 18-crown-6 results in a 425-fold enhanced activity when the reaction between the 2-chloroethylester of N-acetyl-L-phenylalanine and L-phe

  19. Alpha chymotrypsin coated clusters of Fe3O4 nanoparticles for biocatalysis in low water media

    Directory of Open Access Journals (Sweden)

    Mukherjee Joyeeta

    2012-11-01

    Full Text Available Abstract Background Enzymes in low water containing non aqueous media are useful for organic synthesis. For example, hydrolases in such media can be used for synthetic purposes. Initial work in this area was carried out with lyophilized powders of enzymes. These were found to have poor activity. Drying (removing bulk water by precipitation turned out to be a better approach. As enzymes in such media are heterogeneous catalysts, spreading these precipitates over a large surface gave even better results. In this context, nanoparticles with their better surface to volume ratio provide obvious advantage. Magnetic nanoparticles have an added advantage of easy separation after the reaction. Keeping this in view, alpha chymotrypsin solution in water was precipitated over a stirred population of Fe3O4 nanoparticles in n-propanol. This led to alpha chymotrypsin activity coated over clusters of Fe3O4 nanoparticles. These preparations were found to have quite high transesterification activity in low water containing n-octane. Results Precipitation of alpha chymotrypsin over a stirred suspension of Fe3O4 nanoparticles (3.6 nm diameter led to the formation of enzyme coated clusters of nanoparticles (ECCNs. These clusters were also magnetic and their hydrodynamic diameter ranged from 1.2- 2.6 microns (as measured by dynamic light scattering. Transmission electron microscopy (TEM, showed that these clusters had highly irregular shapes. Transesterification assay of various clusters in anhydrous n-octane led to optimization of concentration of nanoparticles in suspension during precipitation. Optimized design of enzyme coated magnetic clusters of nanoparticles (ECCN 3 showed the highest initial rate of 465 nmol min-1 mg-1protein which was about 9 times higher as compared to the simple precipitates with an initial rate of 52 nmol min-1 mg-1 protein. Circular Dichroism (CD(with a spinning cell accessory showed that secondary structure content of the alpha

  20. [Heterologous expression, purification, and properties of a chymotrypsin inhibitor isolated from potatoes].

    Science.gov (United States)

    2013-01-01

    The PKPIJ-B gene encoding a chymotrypsin inhibitor from a subfamily of potato Kunitz-type proteinase inhibitors (PKPI) in potatoes (Solanum tuberosum L. cv. Yubilei Zhukova) was cloned into a pET23a vector and then expressed in Escherichia coli. The recombinant PKPIJ-B protein obtained in the inclusion bodies was denatured, purified by high-performance liquid chromatography (HPLC) on Mono Q under denaturing conditions, and renaturated. The renaturated protein was additionally purified using HPLC on DEAE-ToyoPearl. The PKPIJ-B protein efficiently suppressed chymotrypsin activity, had a weaker effect on trypsin, and inhibited the growth and development of phytopathogenic microorganisms affecting potato plants. PMID:23662448

  1. N-Acetylbenzotriazole as a protein reagent. Specific behaviour towards delta-chymotrypsin.

    Science.gov (United States)

    Reboud-Ravaux, M

    1976-05-17

    When N-[14C] acetylbenzotriazole, presented here as a new agent for the acetylation of proteins, reacted at pH 8 and 25 degrees C with delta-chymotrypsin, 15 amino groups (the epsilon-amino groups of lysing residues and the alpha-amino terminus of half-cystine-1) and two phenolic groups (those of the two exposed tyrosine residues) were acetylated with respective pseudo first-order constants of 0.056 +/- 0.003 and 0.15 +/- 0.03 min(-1). Surprisingly, in contrast with the acetic anhydride reaction, the alpha-amino group of Ile-16 was found to be not acetylated as shown by N-terminus determination and activity measurements: the modified delta-chymotrypsin (or acetylated delta-chymotrypsin) was fully active after neutral dialysis. Only a transient inactivation due to the incorporation of one [14C] acetyl group per mole of catalytic site was observed. The kinetic constant found for reactivation at pH 8.5 was 0.315 +/- 0.005 min(-1) at 25 degrees C. The enzyme-catalyzed hydrolysis of N-acetylbenzotriazole was described by a k(cat) value of 0.093 +/- 0.005 min(-1) at pH 7 and 25 degrees C. Circular dichroism changes observed at 230 nm during the reaction at pH 8.5, of acetylated delta-chymotrypsin with N-acetylbenzotriazole indicated a total conversion of the amount of enzyme molecules which were in the 'inactive' or 'alkaline' conformation at this pH, into the 'active' or 'neutral' one. Benzotriazole alone was unable to induce such a conformational change. The rate constant of the reverse structural process from the 'neutral' to the 'alkaline' conformation was 0.32 +/- 0.02 min(-1): identical to that of the deacetylation of the catalytic site. Thus, the unusual lack of acetylation of Ile-16 alpha-amino group during delta-chymotrypsin treatment with N-acetylbenzotriazole is interpreted as a stabilization of the enzyme 'neutral' conformation where the Ile-16 alpha-amino group is buried, thus inaccessible to the reagent. The properties of the delta-chymotrypsin modification

  2. Expression and characterization of honeybee, Apis mellifera, larva chymotrypsin-like protease

    OpenAIRE

    Matsuoka, Takuma; Takasaki, Akihiko; Mishima, Tomoyuki; Kawashima, Takuji; Kanamaru, Yoshihiro; Nakamura, Tadasi; Yabe, Tomio

    2015-01-01

    International audience Previously, we found three enzyme fractions containing activities for the hydrolysis of royal jelly proteins from honeybee queen larvae. In this study, we identified a honeybee chymotrypsin-like protease (HCLPase) by LC-MS/MS and expressed it as a recombinant protein in Escherichia coli. The protease had an estimated molecular weight of around 26 kDa and showed high specificity for succinyl-Ala-Ala-Pro-Phe p-nitroanilide as a proteolytic substrate. Furthermore, the p...

  3. Selective Inhibition of the Synthesis of Sindbis Virion Proteins by an Inhibitor of Chymotrypsin

    Science.gov (United States)

    Pfefferkorn, E. R.; Boyle, Mary K.

    1972-01-01

    Treatment of chick embryo fibroblasts infected with Sindbis virus with TPCK, the choloromethyl ketone derivative of tosyl-phenylalanine and an inhibitor of chymotrypsin, resulted in reduced synthesis of viral structural proteins and the accumulation of a high-molecular-weight polypeptide, thought to be a precursor. The analogous inhibitor of trypsin, TLCK, the chloromethyl ketone derivative of tosyllysine, had no such effect. PMID:5061988

  4. Ultrasonic Cole-Cole diagram for solutions and application to alpha-chymotrypsin.

    OpenAIRE

    Cerf, R.; Salehi, S T; Rogez, D

    1989-01-01

    Deconvolution of ultrasonic data into single relaxations is rarely feasible when only the absorption or the velocity of the waves is measured. Here we use both series of data to construct a Cole-Cole diagram for a solution. When applied to alpha-chymotrypsin, this method shows two relaxations that are well separated on the time scale, a result that will help simplify analyses of the ultrasonic data for this enzyme.

  5. Ultrasonic Cole-Cole diagram for solutions and application to alpha-chymotrypsin.

    Science.gov (United States)

    Cerf, R; Salehi, S T; Rogez, D

    1989-04-01

    Deconvolution of ultrasonic data into single relaxations is rarely feasible when only the absorption or the velocity of the waves is measured. Here we use both series of data to construct a Cole-Cole diagram for a solution. When applied to alpha-chymotrypsin, this method shows two relaxations that are well separated on the time scale, a result that will help simplify analyses of the ultrasonic data for this enzyme.

  6. Purification and characterization of chymotrypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991.

    Science.gov (United States)

    Villalba-Villalba, Ana Gloria; Ramírez-Suárez, Juan Carlos; Pacheco-Aguilar, Ramón; Valenzuela-Soto, Elisa Miriam; Lugo-Sánchez, María Elena; Figueroa-Soto, Ciria Guadalupe

    2013-04-01

    Pterygoplichthys disjunctivus viscera chymotrypsin was purified by fractionation with ammonium sulfate (30-70 % saturation), gel filtration, affinity, and ion exchange chromatography. Chymotrypsin molecular weight was approximately 29 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), shown a single band in zymogram. Electrofocusing study suggested being an anionic enzyme (pI ≈ 3.9), exhibiting maximal activity at pH 9 and 50 °C, using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulfonyl fluoride (PMSF) (99 %), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (94 %). Enzyme activity was affected by the following ions in decreasing order: Hg(2+), Fe(2+), Cu(2+), Li(1+), Mg(2+), K(1+), Mn(2+), while Ca(2+) had no effect. Chymotrypsin activity decreased continuously as NaCl concentration increased (from 0 to 30 %). K m and V max values were 0.72 ± 1.4 mM and 1.15 ± 0.06 μmol/min/mg of protein, respectively (SAAPNA as substrate). Results suggest the enzyme has a potential application where low processing temperatures are needed, such as in fish sauce production.

  7. Modeling stability and flexibility of α-Chymotrypsin in room temperature ionic liquids.

    Science.gov (United States)

    Latif, Muhammad A M; Tejo, Bimo A; Abedikargiban, Roghayeh; Abdul Rahman, Mohd B; Micaêlo, Nuno M

    2014-01-01

    We investigate the structure and dynamics of α-Chymotrypsin in five room temperature ionic liquids (RTILs) sharing a common cation, hydrated with different water percentages (w/w) (weight of water over protein). Results from molecular dynamics simulations are correlated with experimental evidences from studies on the activity of enzymes in RTILs. α-Chymotrypsin protein structure is closer to its native crystallographic structure in RTILs than in aqueous environment. We show that the structural properties of α-Chymotrypsin were affected by the water concentration assayed in a typical bell-shaped profile, which is also frequently reported for organic solvents. The protein structure was more native like at 10-20% of water (w/w) for all RTILs except for [BMIM][Cl]. We found that the fluctuations of the main chain in [BMIM][BF4] and [BMIM][TfO] were not significantly affected by the increasing amount of water. However, we were able to show that the flexible regions were the ones more hydrated, indicating that water is responsible for the flexibility of the protein. The solvation of the enzyme in water-immiscible RTILs, such as [BMIM][PF6] and [BMIM][Tf2N] lead to higher enzyme flexibility at increased water content. Enzyme solvation by [BMIM][Cl] resulted in ion penetration in the core enzyme structure, causing incremented flexibility and destabilization at low water percentages. All RTILs stripped water molecules from the protein surface, following a similar behavior also found in organic solvents. Anions formed structured arrangements around the protein, which allowed non-stripped water molecules to localize on the protein surface. PMID:23844874

  8. Application of Divide and Conquer Extended Genetic Algorithm to Tertiary Protein Structure of Chymotrypsin Inhibitor-2

    Directory of Open Access Journals (Sweden)

    A. Alfaro

    2006-01-01

    Full Text Available Determining the method by which a protein thermodynamically folds and unfolds in three-dimension is one of the most complex and least understood problems in modern biochemistry. Misfolded proteins have been recently linked to diseases including Amyotrophic Lateral Sclerosis and Alzheimer's disease. Because of the large number of parameters involved in defining the tertiary structure of proteins, based on free energy global minimisation, we have developed a new Divide and Conquer (DAC Extended Genetic Algorithm. The approach was applied to explore and verify the energy landscape of protein chymotrypsin inhibitor-2.

  9. Effect of polyols on the native structure of {alpha}-chymotrypsin: A comparable study

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Awanish; Attri, Pankaj [Department of Chemistry, University of Delhi, Delhi - 110 007 (India); Venkatesu, Pannuru, E-mail: venkatesup@hotmail.com [Department of Chemistry, University of Delhi, Delhi - 110 007 (India)

    2012-05-20

    Highlights: Black-Right-Pointing-Pointer We have studied stability of {alpha}-chymotrypsin in polyols. Black-Right-Pointing-Pointer We have performed DSC, CD and fluorescence spectroscopy. Black-Right-Pointing-Pointer Our {Delta}G{sub u} of CT in polyol increase as polyol concentration increases. Black-Right-Pointing-Pointer All polyols acted as enhancers for CT stability. Black-Right-Pointing-Pointer Our results show that trehalose is strong stabilizer. - The influence of polyols on the structure and stability of {alpha}-chymotrypsin (CT) have been explored by using differential scanning calorimeter (DSC), circular dichroism (CD) and fluorescence spectroscopy. We have predicted the thermodynamic folding properties (transition temperature (T{sub m}), enthalpy change ({Delta}H), heat capacity change ({Delta}C{sub p}) and Gibbs free energy change ({Delta}G{sub u}) from DSC to understand the clear picture of folding studies of CT. All polyols (trehalose, sucrose, sorbitol, and glycerol) acted as enhancers for CT stability, with varying efficacies and efficiencies. The DSC, CD and fluorescence spectral analysis clearly showed the ability of polyols to protect the native structural conformation of enzyme and preventing the unfolding which occurs in the aqueous media. These results explicitly explain that stabilizing polyols are preferentially excluded from the surface of CT, since water has a higher tendency toward favourable interactions with functional groups of the CT than with polyols.

  10. Chemical constituents of Stereospermum acuminatissimum and their urease and α-chymotrypsin inhibitions.

    Science.gov (United States)

    Ramsay, Kamdem Soup T; Wafo, Pascal; Ali, Zulfiqar; Khan, Ajmal; Oluyemisi, Ogbole O; Marasini, Bishnu P; Khan, Ikhlas A; Bonaventure, Ngadjui T; Choudhary, M Iqbal; Atta-ur-Rahman

    2012-01-01

    Phytochemical investigation of the stem bark of Stereospermum acuminatissimum K. Schum. resulted in the isolation of 21 compounds, including two new guanine derivatives, 1,3,7-trimethylguanin-1/3-ium (1) and 3,7-dimethylguanin-1/3-ium (2), and one new phenolic long chain ester, 2-(4-hydroxyphenyl)ethyl hentriacontanoate (3). The known compounds were identified as sterequinones A, F, and H (4, 5, and 6), zenkequinones A-B (7-8), p-coumaric acid (9), methyl caffeate (10), caffeic acid (11), psilalic acid (12), syringaldehyde (13), norviburtinal (14), specioside (15), verminoside (16), tyrosol (17), eutigoside A (18), ellagic acid (19), atranorin (20), and ursolic acid (21). The metabolites were screened for their potential against urease and α-chymotrypsin enzymes, as urease is targeted in peptic ulcer while α-chymotrypsin is used to remove protein debris in ulcer. Compound 20 was found to be excellent urease inhibitor with IC(50) value of 18.2 ± 0.03 μM. Compounds 13 and 18-20 are reported for the first time from the genus Stereospermum. The chemotaxonomic significance of the isolated compounds was also described. PMID:22062354

  11. The third serine proteinase with chymotrypsin specificity isolated from Atlantic cod (Gadus morhua) is a type-II elastase

    DEFF Research Database (Denmark)

    Asgeirsson, B; Leth-Larsen, Rikke; Thórólfsson, M;

    1998-01-01

    efficiency of elastase C. The effects of several inhibitors on cod elastase C were identical to effects on chymotrypsins variants A and B, but dissimilar when compared with porcine pancreatic elastase. On the basis of the specificity and amino acid sequence, we conclude that the enzyme under study is most...

  12. A Jonah-like chymotrypsin from the therapeutic maggot Lucilia sericata plays a role in wound debridement and coagulation.

    Science.gov (United States)

    Pöppel, Anne-Kathrin; Kahl, Mareike; Baumann, Andre; Wiesner, Jochen; Gökçen, Anke; Beckert, Annika; Preissner, Klaus T; Vilcinskas, Andreas; Franta, Zdeněk

    2016-03-01

    Lucilia sericata larvae are used in maggot debridement therapy, a traditional wound healing approach that has recently been approved for the treatment of chronic wounds. Maggot excretion products (MEP) contain many different proteases that promote disinfection, debridement and the acceleration of wound healing, e.g. by activating the host contact phase/intrinsic pathway of coagulation. In order to characterise relevant procoagulant proteases, we analysed MEP and identified a chymotrypsin-like serine protease with similarities to Jonah proteases from Drosophila melanogaster and a chymotrypsin from Lucilia cuprina. A recombinant form of the L. sericata Jonah chymotrypsin was produced in Escherichia coli. The activated enzyme (Jonahm) had a pH optimum of 8.0 and a temperature optimum of 37 °C, based on the cleavage of the chromogenic peptide s-7388 and casein. Jonahm reduced the clotting time of human plasma even in the absence of the endogenous protease kallikrein, factor XI or factor XII and digested the extracellular matrix proteins fibronectin, laminin and collagen IV, suggesting a potential mechanism of wound debridement. Based on these characteristics, the novel L. sericata chymotrypsin-like serine protease appears to be an ideal candidate for the development of topical drugs for wound healing applications.

  13. Covalent immobilization of mixed proteases, trypsin and chymotrypsin, onto modified polyvinyl chloride microspheres.

    Science.gov (United States)

    Li, Dong-Fang; Ding, Hao-Chen; Zhou, Tao

    2013-11-01

    A commercially available trypsin-chymotrypsin mixture was covalently immobilized onto modified polyvinyl chloride (PVC) microspheres, which were activated by the subsequent treatment of PVC microspheres with ethylenediamine and glutaraldehyde. The immobilized mixed protease was characterized by FT-IR and SEM analyses. Immobilization conditions were optimized by Box-Behnken design and the response surface method. The activity of the immobilized mixed protease prepared under optimal conditions (pH 6.6, 23 °C, 2 h) reached 1341 U/g. Compared with the free form, the immobilized enzyme possesses a slightly higher optimal pH value and a wider pH-activity profile, superior thermal stability, and a higher Km value. Reusability of the immobilized mixed protease indicated that >70% of the original activity was retained after having been recycled six times.

  14. A new chymotrypsin-like serine protease involved in dietary protein digestion in a primitive animal, Scorpio maurus: purification and biochemical characterization

    Directory of Open Access Journals (Sweden)

    Gargouri Youssef

    2011-07-01

    Full Text Available Abstract Background Most recent works on chymotrypsins have been focused on marine animals and insects. However, no study was reported in chelicerate. Results Scorpion chymotrypsin-like protease (SCP was purified to homogeneity from delipidated hepatopancreases. The protease NH2-terminal sequence exhibited more than 60% monoacids identity with those of insect putative peptidases. The protease displayed no sequence homology with classical proteases. From this point of view, the protease recalls the case of the scorpion lipase which displayed no sequence homology with known lipases. The scorpion amylase purified and characterized by our time, has an amino-acids sequence similar to those of mammalian amylases. The enzyme was characterized with respect its biochemical properties: it was active on a chymotrypsin substrate and had an apparent molecular mass of 25 kDa, like the classically known chymotrypsins. The dependence of the SCP activity and stability on pH and temperature was similar to that of mammalian chymotrypsin proteases. However, the SCP displayed a lower specific activity and a boarder pH activity range (from 6 to 9. Conclusion lower animal have a less evaluated digestive organ: a hepatopancreas, whereas, higher ones possess individualized pancreas and liver. A new chymotrypsin-like protease was purified for the first time from the scorpion hepatopancreas. Its biochemical characterization showed new features as compared to classical chymotrypsin-higher-animals proteases.

  15. The effect of trypsin and chymotrypsin on the bactericidal activity and specific antibody activity of bovine colostrum.

    Science.gov (United States)

    Brock, J H; Arzabe, R; Piñeiro, A; Olivito, A M

    1977-01-01

    Digestion of bovine colostral whey with trypsin or chymotrypsin caused a progressive loss of the complement-mediated bactericidal activity of naturally-occurring colostral antibodies of E. coli 0111. Bactericidal activity was associated primarily with IgG1 immunoglobulin and to a lesser extent with IgM. Chymotrypsin preferentially attacked IgM, destroying its antibacterial activity and producing an apparent decrease in its mol wt. Trypsin preferentially attacked IgG1, but loss of antibacterial activity was in this case not accompanied by a decrease in molecular weight. Using colostral whey with antiperoxidase activity it was shown that the kinetics of loss of specific antibody activity were similar to those of loss of bactericidal activity. It is therefore suggested that trypsin may cause a loss of specific antibody activity of colostral IgG1 without cleaving the immunoglobulin molecule. PMID:321342

  16. pH-responsive polymer-assisted refolding of urea- and organic solvent-denatured alpha-chymotrypsin.

    Science.gov (United States)

    Roy, I; Gupta, M N

    2003-12-01

    A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of alpha-chymotrypsin denatured with 8 M urea and 100 mM dithiothreitol at pH 8.2. The complete activity could be regained within 10 min during refolding. Both native and refolded enzymes showed emission of intrinsic fluorescence with lambda(max) of 342 nm. Gel electrophoresis showed that the presence of Eudragit S-100 led to dissociation of multimers followed by the appearance of a band at the monomer position. The unfolding (by 8 M urea) and folding (assisted by the polymer) also led to complete renaturation of alpha-chymotrypsin initially denatured by 90% dioxane. The implications of the data in recovery of enzyme activity from inclusion bodies and the interesting possibility in the in vivo context of reversing protein aggregation in amyloid-based diseases have been discussed.

  17. Creation of an artificial metalloprotein with a Hoveyda-Grubbs catalyst moiety through the intrinsic inhibition mechanism of α-chymotrypsin.

    Science.gov (United States)

    Matsuo, Takashi; Imai, Chie; Yoshida, Takefumi; Saito, Takashi; Hayashi, Takashi; Hirota, Shun

    2012-02-01

    An L-phenylalanyl chloromethylketone-based inhibitor equipped with a Hoveyda-Grubbs catalyst moiety was regioselectively incorporated into the cleft of α-chymotrypsin through the intrinsic inhibition mechanism of the protein to construct an artificial organometallic protein.

  18. Studies on interaction between CdTe quantum dots and -chymotrypsin by molecular spectroscopy

    Indian Academy of Sciences (India)

    Jianniao tian; Shengzhi Wei; Yanchun Zhao; Rongjun Liu; Shulin Zhao

    2010-05-01

    In this article, the interaction between -Chymotrypsin and CdTe QDs was investigated by fluorescence, synchronous fluorescence, and circular dichroism (CD) spectroscopic methods at pH 7.20 and pH 9.05. The intrinsic fluorescence of -Chy is quenched by CdTe QDs. Under different pH conditions, the level of binding constants is determined to be 103 from fluorescence data. The hydrogen bond or van der Waals force is involved in the binding process when pH is 9.05, while the hydrophobic and electrostatic interactions play main role in the binding process when pH is 7.20. The red-shift of synchronous fluorescence spectral peak of protein after the addition of CdTe QDs reveals that the microenvironments around tryptophan residues are disturbed by CdTe QDs. The secondary structure of -Chy undergoes slight changes as similar by far-UV CD data. The activity and stability of -Chy in the presence of CdTe QDs were also studied. -Chy can maintain its high activity and stability under different pH conditions for 24 h in the presence of CdTe QDs.

  19. Nazumazoles D-F, Cyclic Pentapeptides That Inhibit Chymotrypsin, from the Marine Sponge Theonella swinhoei.

    Science.gov (United States)

    Fukuhara, Kazuya; Takada, Kentaro; Okada, Shigeru; Matsunaga, Shigeki

    2016-06-24

    Nazumazoles D-F (1-3) were isolated from the marine sponge Theonella swinhoei. The compounds gave extremely broad peaks by reversed-phase HPLC using an ODS column. HPLC using a gel permeation column was instrumental for the separation of the three compounds. Their planar structures were determined by interpretation of NMR data to be cyclic pentapeptides. Nazumazoles D-F contained one residue each of α-keto-l-norvaline (l-Knv) {or α-keto-d-leucine (l-Kle)}, l-alanyloxazole (l-Aox), d-Abu (or d-Ser), N-α-CHO-β-l-Dpr, and cis-4-methyl-l-proline. The absolute configuration of each amino acid residue was determined by Marfey's method in combination with conversion of the α-keto-β-amino acid to the α-amino acid by oxidation. Nazumazoles D-F are not cytotoxic against P388 cells at 50 μM, but inhibit chymotrypsin. PMID:27213234

  20. Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin.

    Science.gov (United States)

    Meller, Kinga; Pomastowski, Paweł; Grzywiński, Damian; Szumski, Michał; Buszewski, Bogusław

    2016-04-01

    The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (μ-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (Nα-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research. PMID:26947160

  1. Stretch regulates expression and binding of chymotrypsin-like elastase 1 in the postnatal lung.

    Science.gov (United States)

    Joshi, Rashika; Liu, Sheng; Brown, Montell D; Young, Sarah M; Batie, Matthew; Kofron, J Matthew; Xu, Yan; Weaver, Timmothy E; Apsley, Karen; Varisco, Brian M

    2016-02-01

    Lung stretch is critical for normal lung development and for compensatory lung growth after pneumonectomy (PNX), but the mechanisms by which strain induces matrix remodeling are unclear. Our prior work demonstrated an association of chymotrypsin-like elastase 1 (Cela1) with lung elastin remodeling, and that strain triggered a near-instantaneous elastin-remodeling response. We sought to determine whether stretch regulates Cela1 expression and Cela1 binding to lung elastin. In C57BL/6J mice, Cela1 protein increased 176-fold during lung morphogenesis. Cela1 was covalently bound to serpin peptidase inhibitor, clade A, member 1, resulting in a higher molecular mass in lung homogenate compared to pancreas homogenate. Post-PNX, Cela1 mRNA increased 6-fold, protein 3-fold, and Cela1-positive cells 2-fold. Cela1 was expressed predominantly in alveolar type II cells in the embryonic lung and predominantly in CD90-positive lung fibroblasts postnatally. During compensatory lung growth, Cela1 expression was induced in nonproliferative mesenchymal cells. In ex vivo mouse lung sections, stretch increased Cela1 binding to lung tissue by 46%. Competitive inhibition with soluble elastin completely abrogated this increase. Areas of stretch-induced elastase activity and Cela1 binding colocalized. The stretch-dependent expression and binding kinetics of Cela1 indicate an important role in stretch-dependent remodeling of the peripheral lung during development and regeneration. PMID:26443822

  2. Intracellular localization of Treponema denticola chymotrypsin-like proteinase in chronic periodontitis

    Directory of Open Access Journals (Sweden)

    Emilia Marttila

    2014-07-01

    Full Text Available Treponema denticola is an important periodontal pathogen capable of tissue invasion. Its chymotrypsin-like proteinase (CTLP can degrade a number of basement membrane components in vitro, thus suggesting a contribution to tissue invasion by the spirochete. The aim of this study was to analyze the localization of CTLP in chronic periodontitis tissues ex vivo. A polyclonal antibody specific to T. denticola cell-bound CTLP was used to detect the spirochetes in the gingival tissues of patients with moderate to severe chronic periodontitis (n=25 by immunohistochemistry and periodic acid-Schiff staining (PAS. The presence of T. denticola in the periodontal tissue samples was analyzed by PCR. Periodontal tissue samples of 12 of the 25 patients were found to be positive for T. denticola by PCR. Moreover, CTLP could be detected in the periodontal tissues of all these patients by immunohistochemistry. In the epithelium, the CTLP was mostly intracellular. Typically, the positive staining could be seen throughout the whole depth of the epithelium. When detected extracellularly, CTLP was localized mainly as granular deposits. The connective tissue stained diffusely positive in four cases. The positive staining co-localized with the PAS stain in nine cases. T. denticola and its CTLP could be detected in diseased human periodontium both intra- and extracellularly. The granular staining pattern was suggestive of the presence of T. denticola bacteria, whereas the more diffused staining pattern was indicative of the recent presence of the bacterium and shedding of the cell-bound proteinase.

  3. High Temperature Unfolding and Low Temperature Refolding Pathway of Chymotrypsin Inhibitor 2 Using Molecular Dynamics Simulation

    Science.gov (United States)

    Malau, N. D.; Sumaryada, T.

    2016-01-01

    The mechanism that explains the unfolding/refolding process of the protein is still a major problem that has not been fully understood. In this paper we present our study on the unfolding and refolding pathway of Chymotrypsin Inhibitor 2 (CI2) protein through a molecular dynamics simulation technique. The high temperature unfolding simulation were performed at 500 K for 35 ns. While the low temperature refolding simulation performed at 200 K for 35 ns. The unfolding and refolding pathway of protein were analysed by looking at the dynamics of root mean squared deviation (RMSD) and secondary structure profiles. The signatures of unfolding were observed from significant increase of RMSD within the time span of 10 ns to 35 ns. For the refolding process, the initial structure was prepared from the structure of unfolding protein at t=15 ns and T=500 K. Analysis have shown that some of the secondary structures of CI2 protein that have been damaged at high temperature can be refolded back to its initial structure at low temperature simulation. Our results suggest that most of α-helix structure of CI2 protein can be refolded back to its initial state, while only half beta-sheet structure can be reformed.

  4. Immunomodulatory Effect of Chymotrypsin in CNS Is Sex-independent: Evidence of Anti-inflammatory Role for IL-17 in EAE.

    Science.gov (United States)

    Ghaffarinia, Ameneh; Parvaneh, Shahram; Jalili, Cyrus; Riazi-Rad, Farhad; Yaslianifard, Somayeh; Pakravan, Nafiseh

    2016-04-01

    Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory autoimmune diseases of the central nervous system. Chymotrypsin is a serine protease with immunomodulatory effect in the peripheral organs. We previously demonstrated the immunomodulatory effect of chymotrypsin in ameliorating the EAE in female Lewis rats. However, there are sex-based differences in the immune system, drug activity, and CNS structure and composition. In addition, female gender is a better prognostic indicator of MS and males are more severely affected by EAE than females. Consequently, gender may have an important impact on therapeutic effect. Therefore, in this study we investigated the anti-inflammatory effect of chymotrypsin in male Lewis rat model of EAE. The disease was induced in male Lewis rats and the animals were evaluated for weight loss and clinical signs for 14 days. Intra-CSF injection of chymotrypsin was done on day 7 and expression of mRNA for IFN-γ, IL-4, IL-17, and FoxP3 in brain, spinal cord and deep cervical lymph node were determined using a two-step real-time PCR. Administration of 0.2mg/ml chymotrypsin ameliorated the disease by decreasing IFN-γ and increasing expression of IL-4 and IL-17 at the inflammatory foci. This is consistent with anti-inflammatory effect of IL-4 and IL-17 at high concentrations. We conclude that Immunomodulatory affect of chymotrypsin in CNS is sex-independent. Our result also provides more evidence on the anti-inflammatory role of IL-17. However more research is needed to elucidate the underlying immunomodulatory role of chymotrypsin and how to increase its beneficial effect by modification of dosage and/or regimen of administration. PMID:27090368

  5. Activity of α-Chymotrypsin Enhanced in the Presence of Iron Oxide Nanoparticles in Organic Solvent: Application to Peptide Synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Cheolwoo; Kim, Mahnjoo [Pohang Univ. of Science and Technology, Pohang (Korea, Republic of)

    2013-03-15

    We have demonstrated that α-CT displays a significantly enhanced activity in the presence of IONs relative to its IONs-free counterparts in organic solvent. IONs-activated α-CT catalyzed efficiently the synthesis of peptides without the formation of hydrolyzed byproducts. Enzymes are a useful class of catalysts for the preparation of enantiomeric compounds. The applications of enzymes in synthetic transformations, however, are limited by their reduced activities in organic solvent. Particularly, proteases such as subtilisin and α-chymotrypsin display several orders of magnitude lower activities in organic solvent than their aqueous counterparts.

  6. Comparison of the Contributions of Tetrahydrofurfuryl Alcohol and PEG to α-Chymotrypsin Renaturation with High Performance Hydrophobic Interaction Chromatography

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The contributions of tetrahydrofurfuryl alcohol (THFA) and polyethylene glycol (PEG) to the renatured efficiency of α-chymotrypsin were investigated and compared with each other. The maximum increments of bioactivity recovery of α-Chy were found to be 25.1% for THFA, 10.4% for PEG, respectively. The experimental results indicated that the denaturant solution containing THFA contributed more to the renaturation of α-Chy in high performance hydrophobic interaction chromatography (HPHIC) than that containing PEG, when the concentration of THFA was 3.2%, the bioactivity recovery of α-Chy is the highest.

  7. Moisture-induced solid state instabilities in α-chymotrypsin and their reduction through chemical glycosylation

    Directory of Open Access Journals (Sweden)

    Solá Ricardo J

    2010-08-01

    Full Text Available Abstract Background Protein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state. Results First, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme α-chymotrypsin (α-CT under controlled humidity conditions. Results showed that α-CT aggregates and inactivates as a function of increased relative humidity (RH. Furthermore, α-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that α-CT structural dynamics increased at increasing RH. The magnitude of the structural changes in tendency parallels the solid-state instability data (i.e., formation of buffer-insoluble aggregates, inactivation, and loss of native conformation upon reconstitution. To determine if these moisture-induced instability issues could be ameliorated by chemical glycosylation we proceeded to modify our model protein with chemically activated glycans of differing lengths (lactose and dextran (10 kDa. The various glycoconjugates showed a marked decrease in aggregation and an increase in residual activity after incubation. These stabilization effects were found to be independent of the glycan size. Conclusion Water sorption leads to

  8. Lung injury-dependent oxidative status and chymotrypsin-like activity of skeletal muscles in hamsters with experimental emphysema

    Directory of Open Access Journals (Sweden)

    Tonon Jair

    2013-01-01

    Full Text Available Abstract Background Peripheral skeletal muscle is altered in patients suffering from emphysema and chronic obstructive pulmonary disease (COPD. Oxidative stress have been demonstrated to participate on skeletal muscle loss of several states, including disuse atrophy, mechanical ventilation, and chronic diseases. No evidences have demonstrated the occurance in a severity manner. Methods We evaluated body weight, muscle loss, oxidative stress, and chymotrypsin-like proteolytic activity in the gastrocnemius muscle of emphysemic hamsters. The experimental animals had 2 different severities of lung damage from experimental emphysema induced by 20 mg/mL (E20 and 40 mg/mL (E40 papain. Results The severity of emphysema increased significantly in E20 (60.52 ± 2.8, p Conclusions Taken together, the results of the present study suggest that muscle atrophy observed in this model of emphysema is mediated by increased muscle chymotrypsin-like activity, with possible involvement of oxidative stress in a severity-dependent manner.

  9. 13C High-Resolution Nuclear Magnetic Resonance Studies of Enzyme-Substrate Reactions at Equilibrium. Substrate Strain Studies of Chymotrypsin-N-Acetyltyrosine Semicarbazide Complexes

    NARCIS (Netherlands)

    Robillard, George; Shaw, Elliott; Shulman, R.G.

    1974-01-01

    N-Acetyl-L-tyrosine semicarbazide is hydrolyzed by chymotrypsin (EC 3.4.21.1) to N-acetyl-L-tyrosine and semicarbazide. If a high concentration of semicarbazide is present, the equilibrium for the reaction can be shifted from hydrolysis to synthesis. Using N-acetyl-L-[13C]tyrosine enriched at the ca

  10. High Resolution Nuclear Magnetic Resonance Studies of the Active Site of Chymotrypsin. I. The Hydrogen Bonded Protons of the “Charge Relay” System

    NARCIS (Netherlands)

    Robillard, G.; Shulman, R.G.

    1974-01-01

    High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin Aδ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. Th

  11. Effects of crown ethers and small amounts of cosolvent on the activity and enantioselectivity of α-chymotrypsin in organic solvents

    NARCIS (Netherlands)

    Engbersen, Johan F.J.; Broos, Jaap; Verboom, Willem; Reinhoudt, David N.

    1996-01-01

    Addition of crown ethers to α-chymotrypsin, subtilisin, and other proteases considerably enhances the activity of these enzymes in transesterification reactions of N-acetyl-alanine and -phenylalanine esters in organic solvents. Even much higher enhancements of activity (up to 640 ×) are obtained by

  12. Digestion of atopic allergens with trypsin α-chymotrypsin and pancreatic kallikrein, and influence of the allergens upon the proteolytic and esterolytic activity of these enzymes

    NARCIS (Netherlands)

    Berrens, L.

    1968-01-01

    The action of bovine trypsin, α-chymotrypsin and pancreatic kallikrein upon a number of atopic allergens has been studied by pH-stat measurements during short-term incubation. Most atopic allergens proved chemically resistant towards these enzymes. Graphs of enzyme susceptibility vs. the ratio of ex

  13. High Resolution Nuclear Magnetic Resonance Studies of the Active Site of Chymotrypsin. II. Polarization of Histidine 57 by Substrate Analogues and Competitive Inhibitors

    NARCIS (Netherlands)

    Robillard, G.; Shulman, R.G.

    1974-01-01

    The proton nuclear magnetic resonance signal of the His57-Asp102 hydrogen bonded proton in the charge relay system of chymotrypsinogen A and chymotrypsin Aδ has been monitored to determine the influence of substrate analogues and competitive inhibitors on the electronic state of the active site regi

  14. Nutritive value and effect of blanching on the trypsin and chymotrypsin inhibitor activities of selected leafy vegetables.

    Science.gov (United States)

    Mosha, T C; Gaga, H E

    1999-01-01

    Proximate composition, energy, mineral and vitamin contents and the effect of blanching methods and times on the trypsin and chymotrypsin inhibitor activities were studied using cabbage, collard, turnip, peanut, and sweet potato leaves. Results of this study indicated that, crude protein, crude fat, carbohydrate and ash contents were in the range of 15.5-25.6%, 1.4-6.5%, 60.4-73.1% and 6.8-7.5%, respectively. Total dietary fiber was lowest in cabbage (28.2 g/100 g) and highest in the collard leaves (43.1%) while energy content per 100 g of vegetables was highest in sweet potato leaves (402 kcal) and lowest in cabbage (379 kcal). The mineral content per 100 g of vegetables were in the range of 33.4-249.8 mg, 241.2-471.2 mg, 12.1-75.1 mg, 14.9-98.9 mg, 0.5-3.5 mg and 0.9-3.1 mg for Ca, K, Na, Mg, Fe and Zn, respectively. For ascorbic acid, riboflavin, thiamin and total carotenoids, concentrations in 100 g of vegetables were in the range of 45.1-112.7 mg, 0.2-0.3 mg, 0.3-0.8 mg and 2.0-7.3 mg, respectively. The trypsin inhibitory activity per gram of the vegetables was highest in collard (60.1 TIU/g) and lowest in peanut leaves (41.0 TIU/g). Chymotrypsin inhibitor activity was highest in the peanut (69.6 CIU/g) but lowest in the collard leaves (48.0 CIU/g). Both trypsin and chymotrypsin inhibitor activities were significantly (p blanching methods. In the conventional blanching method, trypsin inhibitor activity was reduced by 0.5, 6.8, 11.9, 9.0 and 19.3 percent in cabbage, collard, turnip, sweet potato and peanut leaves, respectively, when the vegetables were blanched for 2.5 minutes but after blanching for 10 minutes, the trypsin inhibitor activity was reduced by 29.7, 34.9, 54.3, 52.3 and 65.6 percent in cabbage, collard, turnip, sweet potato and peanut greens, respectively. For the microwave oven blanching, trypsin inhibitor activity was reduced by 3.8, 3.3, 32.7, 5.0 and 9.5 percent in cabbage, collard, turnip, sweet potato and peanut leaves, respectively when

  15. Elastase-like Activity Is Dominant to Chymotrypsin-like Activity in 20S Proteasome's β5 Catalytic Subunit.

    Science.gov (United States)

    Bensinger, Dennis; Neumann, Theresa; Scholz, Christoph; Voss, Constantin; Knorr, Sabine; Kuckelkorn, Ulrike; Hamacher, Kay; Kloetzel, Peter-Michael; Schmidt, Boris

    2016-07-15

    The ubiquitin/proteasome system is the major protein degradation pathway in eukaryotes with several key catalytic cores. Targeting the β5 subunit with small-molecule inhibitors is an established therapeutic strategy for hematologic cancers. Herein, we report a mouse-trap-like conformational change that influences molecular recognition depending on the substitution pattern of a bound ligand. Variation of the size of P1 residues from the highly β5-selective proteasome inhibitor BSc2118 allows for discrimination between inhibitory strength and substrate conversion. We found that increasing molecular size strengthens inhibition, whereas decreasing P1 size accelerates substrate conversion. Evaluation of substrate hydrolysis after silencing of β5 activity reveals significant residual activity for large residues exclusively. Thus, classification of the β5 subunit as chymotrypsin-like and the use of the standard tyrosine-containing substrate should be reconsidered. PMID:27111844

  16. Synthesis of SP II albumin, β-amylase and chymotrypsin inhibitor CI-1 on polysomes from the endoplasmic reticulum of barley endosperm

    International Nuclear Information System (INIS)

    Free and initially membrane bound polysomes were isolated from 20 day old endosperms of Bomi and Hiproly barley and used as templates in an in vitro protein synthesizing system based on wheat germ extract. Three 35S--labelled translation products were identified as SP II albumin, β-amylase and chymotrypsin inhibitor CI-1 among the polypeptides synthesized by the polysomes of the endoplasmic reticulum. Identification employed immunoaffinity isolation. Protein Z was not detectable among the in vitro translation products of either initially membrane bound or free endosperm polysomes. Compared to Bomi barley the Hiproly endosperm is enriched for translatable mRNA coding SP II albumin, β-amylase and chymotrypsin inhibitor CI-1. In vitro and in vivo synthesized β-amylases have identical molecular weights. Only one form of SP II albumin is produced in vitro, confirming that the lower molecular weight SP II B albumin is a proteolytic cleavage product of the SP II A albumin. (author)

  17. Allosteric Inhibitory Molecular Recognition of a Photochromic Dye by a Digestive Enzyme: Dihydroindolizine makes α-chymotrypsin Photo-responsive

    Science.gov (United States)

    Bagchi, Damayanti; Ghosh, Abhijit; Singh, Priya; Dutta, Shreyasi; Polley, Nabarun; Althagafi, Ismail. I.; Jassas, Rabab S.; Ahmed, Saleh A.; Pal, Samir Kumar

    2016-09-01

    The structural-functional regulation of enzymes by the administration of an external stimulus such as light could create photo-switches that exhibit unique biotechnological applications. However, molecular recognition of small ligands is a central phenomenon involved in all biological processes. We demonstrate herein that the molecular recognition of a photochromic ligand, dihydroindolizine (DHI), by serine protease α-chymotrypsin (CHT) leads to the photo-control of enzymatic activity. We synthesized and optically characterized the photochromic DHI. Light-induced reversible pyrroline ring opening and a consequent thermal back reaction via 1,5-electrocyclization are responsible for the photochromic behavior. Furthermore, DHI inhibits the enzymatic activity of CHT in a photo-controlled manner. Simultaneous binding of the well-known inhibitors 4-nitrophenyl anthranilate (NPA) or proflavin (PF) in the presence of DHI displays spectral overlap between the emission of CHT-NPA or CHT-PF with the respective absorption of cis or trans DHI. The results suggest an opportunity to explore the binding site of DHI using Förster resonance energy transfer (FRET). Moreover, to more specifically evaluate the DHI binding interactions, we employed molecular docking calculations, which suggested binding near the hydrophobic site of Cys-1-Cys-122 residues. Variations in the electrostatic interactions of the two conformers of DHI adopt unfavorable conformations, leading to the allosteric inhibition of enzymatic activity.

  18. Adsorption isotherms, kinetics and thermodynamic studies towards understanding the interaction between cross-linked alginate-guar gum matrix and chymotrypsin.

    Science.gov (United States)

    Woitovich Valetti, Nadia; Picó, Guillermo

    2016-02-15

    The adsorption kinetics of chymotrypsin, a pancreatic serine protease, onto an alginate-gum guar matrix cross-linked with epichlorohydrin has been performed using a batch-adsorption technique. The effect of various experimental parameters such as pH, salt presence, contact time and temperature were investigated. The pseudo-first-order and pseudo-second-order kinetic models were used to describe the kinetic data which shows that the adsorption of the enzyme followed the pseudo-second-order rate expression. The Langmuir, Freundlich and Hill adsorption isotherm models were applied to describe the equilibrium isotherms, and the isotherm constants were determined. It was found that Hill model was more suitable for our data because the isotherm data showed a sigmoidal behavior with the free enzyme concentration increasing in equilibrium. At 8°C and at pH 5.0, 1g hydrate matrix adsorbed about 7mg of chymotrypsin. In the desorption process 80% of the biological activity of chymotrypsin was recovered under the condition of 50mM phosphate buffer, pH 7.00-500mM NaCl. When successive cycles of adsorption/washing/desorption were performed, it was observed that the matrix remained functional until the fourth cycle of repeated batch enzyme adsorption. These results are important in terms of diminishing of cost and waste generation.

  19. Adsorption isotherms, kinetics and thermodynamic studies towards understanding the interaction between cross-linked alginate-guar gum matrix and chymotrypsin.

    Science.gov (United States)

    Woitovich Valetti, Nadia; Picó, Guillermo

    2016-02-15

    The adsorption kinetics of chymotrypsin, a pancreatic serine protease, onto an alginate-gum guar matrix cross-linked with epichlorohydrin has been performed using a batch-adsorption technique. The effect of various experimental parameters such as pH, salt presence, contact time and temperature were investigated. The pseudo-first-order and pseudo-second-order kinetic models were used to describe the kinetic data which shows that the adsorption of the enzyme followed the pseudo-second-order rate expression. The Langmuir, Freundlich and Hill adsorption isotherm models were applied to describe the equilibrium isotherms, and the isotherm constants were determined. It was found that Hill model was more suitable for our data because the isotherm data showed a sigmoidal behavior with the free enzyme concentration increasing in equilibrium. At 8°C and at pH 5.0, 1g hydrate matrix adsorbed about 7mg of chymotrypsin. In the desorption process 80% of the biological activity of chymotrypsin was recovered under the condition of 50mM phosphate buffer, pH 7.00-500mM NaCl. When successive cycles of adsorption/washing/desorption were performed, it was observed that the matrix remained functional until the fourth cycle of repeated batch enzyme adsorption. These results are important in terms of diminishing of cost and waste generation. PMID:26849187

  20. Effect of ligand structure of stationary phase of high per-formance hydrophobic interaction chromatography on re-naturation efficiency of GuHCl-denatured α-chymotrypsin

    Institute of Scientific and Technical Information of China (English)

    SHEN Yehua; WANG Haibo; BAI Quan; GENG Xindu

    2005-01-01

    The renaturation of the denatured α-chymotrypsin (α-Chy) with 1.7 mol · L-1 guanidine hydrochloride (GuHCI) by three kinds of stationary phase of high performance hydrophobic interaction chromatography (STHIC) with a comparable hydrophobicity but different ligand structures was investigated. The obtained result indicates that the ligand structures of the three STHIC contribute to the renaturation efficiency of α-Chy in the order of the end ligands PEG-600< phenyl group < tetrahydrofurfuryl alcohol (THFA).

  1. Radiation-induced heterogeneity of chymotrypsin of mus musculus. On the characterization of structurally and functionally in vitro modified enzyme forms

    International Nuclear Information System (INIS)

    The distribution of in vitro induced 60Co-γ (structural heterogeneity of mouse chymotrypsin has been studied in terms of molecular weight, catalytic activity and net charge distribution. It was found that the enzyme stucture, with retained molecular weight, could partly accumulate structural changes subsequently not leading to modification of catalytic properties. Loss of petide fragments (0 < Mw (lt 6000) the enzyme showed native function but also modified as well as total loss of function. Further loss of peptide fragments results in modified function and total loss of function. These results indicate the capability of the enzyme to accumulate in vitro changes partly without a total loss of function. (author)

  2. The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex

    Science.gov (United States)

    Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

    2016-02-01

    Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases.

  3. Purification of a chymotrypsin-like enzyme present on adult Schistosoma mansoni worms from infected mice and its characterization as a host carboxylesterase.

    Science.gov (United States)

    Igetei, Joseph E; Liddell, Susan; El-Faham, Marwa; Doenhoff, Michael J

    2016-04-01

    A serine protease-like enzyme found in detergent extracts of Schistosoma mansoni adult worms perfused from infected mice has been purified from mouse blood and further characterized. The enzyme is approximately 85 kDa and hydrolyses N-acetyl-DL-phenylalanine β-naphthyl-ester, a chromogenic substrate for chymotrypsin-like enzymes. The enzyme from S. mansoni worms appears to be antigenically and enzymatically similar to a molecule that is present in normal mouse blood and so is seemingly host-derived. The enzyme was partially purified by depleting normal mouse serum of albumin using sodium chloride and cold ethanol, followed by repeated rounds of purification by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified material was subjected to tandem mass spectrometry and its derived peptides found to belong to mouse carboxylesterase 1C. Its ability to hydrolyse α- or β-naphthyl acetates, which are general esterase substrates, has been confirmed. A similar carboxylesterase was purified and characterized from rat blood. Additional evidence to support identification of the enzyme as a carboxylesterase has been provided. Possible roles of the enzyme in the mouse host-parasite relationship could be to ease the passage of worms through the host's blood vessels and/or in immune evasion. PMID:26924446

  4. Purification of a chymotrypsin-like enzyme present on adult Schistosoma mansoni worms from infected mice and its characterization as a host carboxylesterase.

    Science.gov (United States)

    Igetei, Joseph E; Liddell, Susan; El-Faham, Marwa; Doenhoff, Michael J

    2016-04-01

    A serine protease-like enzyme found in detergent extracts of Schistosoma mansoni adult worms perfused from infected mice has been purified from mouse blood and further characterized. The enzyme is approximately 85 kDa and hydrolyses N-acetyl-DL-phenylalanine β-naphthyl-ester, a chromogenic substrate for chymotrypsin-like enzymes. The enzyme from S. mansoni worms appears to be antigenically and enzymatically similar to a molecule that is present in normal mouse blood and so is seemingly host-derived. The enzyme was partially purified by depleting normal mouse serum of albumin using sodium chloride and cold ethanol, followed by repeated rounds of purification by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified material was subjected to tandem mass spectrometry and its derived peptides found to belong to mouse carboxylesterase 1C. Its ability to hydrolyse α- or β-naphthyl acetates, which are general esterase substrates, has been confirmed. A similar carboxylesterase was purified and characterized from rat blood. Additional evidence to support identification of the enzyme as a carboxylesterase has been provided. Possible roles of the enzyme in the mouse host-parasite relationship could be to ease the passage of worms through the host's blood vessels and/or in immune evasion.

  5. Relative importance of phytohemagglutinin (lectin) and trypsin-chymotrypsin inhibitor on bean (Phaseolus vulgaris L) protein absorption and utilization by the rat.

    Science.gov (United States)

    Carvalho, M R; Sgarbieri, V C

    1998-10-01

    The main objective of this work was to perform a comparative study of the antinutritional and/or toxic properties of phytohemagglutinin and trypsin-chymotrypsin inhibitor extracted from the seed of a commercial cultivar of edible bean used in Brazil. Bean proteins were extracted in acidic salt solution and fractionated by dialysis and centrifugation, then freeze-dried. The total freeze-dried bean extract and the globulin or albumin protein fraction were resuspended in distilled water and heated (100 degrees C, 30 min) for inactivation of hemagglutinin. Diets were prepared with unheated bean protein fractions and heated ones (100% trypsin inhibitor activity, but 0% phytohemagglutinin activity). As a result, the inhibition of growth and poor dietary protein utilization were observed in rats fed diets containing unheated bean protein fractions, but not in rats fed diets containing heated fractions. It was thus assumed that phytohemagglutinin is the main antinutritional and toxic factor that in dry bean (Phaseolus) protein and that trypsin inhibitor (Bowman-Birk type) did not interfere with rat growth. PMID:9919488

  6. Notable Stabilization of α-Chymotrypsin by the Protic Ionic Additive, [ch][dhp]: Calorimetric Evidence for a Fine Enthalpy/Entropy Balance.

    Science.gov (United States)

    Uchaneishvili, Sophio; Makharadze, Maya; Shushanyan, Mikhael; van Eldik, Rudi; Khoshtariya, Dimitri E

    2014-01-01

    An impact of 0.5 to 3 M choline dihydrogen phosphate, [ch][dhp], the biotechnologically relevant ionic substance, on the thermal stability of a model globular protein, α-chymotrypsin (α-CT), has been studied exploiting the highly sensitive differential scanning calorimetry (DSC) technique. The notable overall stabilizing effect of 11 ± 2 K regarding the thermal transition (melting) temperature, T m , has been detected. For this kind of series, for the first time, the calorimetric melting enthalpy (ΔH cal) and transition entropy (ΔS m ) parameters have been determined simultaneously throughout. The first analysis indicated a two-phase impact implying (a) the initial, dramatic drop in both ΔH cal and ΔS m , obviously connected to specific, direct interaction between the [ch][dhp] components and α-CT's charged groups (within 0 to 1 mol/L [ch][dhp]), leading to the essential rearrangement of the interfacial hydrogen-bonded (HB) network; and (b) the follow-up (within 1 to 3.0 mol/L [ch][dhp]), modest changes in ΔH cal and lack of changes in ΔS m , seemingly connected with a subsequent steady strengthening of already reformed HB network, respectively. These changes, presumably, are primarily facilitated by Coulombic interactions between the [dhp] anions and solvent-exposed positively charged amino groups of α-CT.

  7. Activity, stability and kinetic parameters for -chymotrypsin catalysed reactions in AOT/isooctane reverse micelles with nonionic and zwitterionic mixed surfactants

    Indian Academy of Sciences (India)

    Santosh Kumar Verma; Kallol K Ghosh

    2013-07-01

    Reverse micelles (RMs) of sodium 1,4-bis(2-ethylhexyl)sulphosuccinate (AOT) in nonpolar organic solvents are widely known to have very high solubilization power for water. The method is applied to the hydrolysis of -nitrophenyl acetate (PNPA) catalysed by -chymotrypsin (-CT) in AOT/isooctane/buffer RMs. The increase in -CT activity and stability was an optimum at wo ([H2O]/[AOT]) = 10, z [Isooctane]/[AOT]) = 5. Three typical surfactants were selected based on their head group charges: a non-ionic surfactant Triton-X 100 and two zwitterionic sulphobetaine surfactants of the type CH2+1N+Me2 (CH2)3 SO$^{−}_{3}$ (n = 10; SB3-10, n = 16; SB3-16). The kinetic parameters (such as cat and M) of the -CT at 27°C were determined and compared in the absence and presence of three surfactants. The effect of chain length of zwitterionic surfactant (SB3-10 and SB3-16) on the enzymatic efficacy of -CT as a function of mixed surfactant addition has been investigated in AOT/isooctane RMs at pH 7.75.

  8. Urease and α-chymotrypsin inhibitory activities of transition metal complexes of new Schiff base ligand: Kinetic and thermodynamic studies of the synthesized complexes using TG–DTA pyrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Ikram, Muhammad, E-mail: ikram.chemistry@suit.edu.pk [Department of Chemistry, Sarhad University of Science and Information Technology, Peshawar (Pakistan); Rehman, Sadia [Department of Chemistry, Sarhad University of Science and Information Technology, Peshawar (Pakistan); Institute of Chemical Sciences, University of Peshawar (Pakistan); Ali, Muhammad [Department of Biotechnology, Bacha Khan University Charsadda (Pakistan); Faridoon [Institute of Chemical Sciences, University of Peshawar (Pakistan); Schulzke, Carola [Institut für Biochemie, Ernst-Moritz-Arndt, Universität Greifswald, Felix-Hausdorff-Straße 4, 17487, Greifswald (Germany); Baker, Robert J. [School of Chemistry, University of Dublin, Trinity College, Dublin 2 (Ireland); Blake, Alexander J. [School of Chemistry, The University of Nottingham, Nottingham NG7 2RD (United Kingdom); Malook, Khan [Centralized Resource Laboratory, University of Peshawar (Pakistan); Wong, Henry [School of Chemistry, The University of Nottingham, Nottingham NG7 2RD (United Kingdom); Saeed-Ur-Rehman [Institute of Chemical Sciences, University of Peshawar (Pakistan)

    2013-06-20

    Graphical abstract: - Highlights: • Single crystal structural analysis of co-crystallized Schiff base ligand. • Urease and α-chymotrypsin inhibition activities of all the synthesized compounds. • TG–DTA studies of all the synthesized compounds under static air. • Kinetic and thermodynamic parameters evaluation using Horrowitz–Mettzger method. - Abstract: The Schiff base 2-[(E)-(quinolin-3-ylimino)methyl]phenol (H-QMP) was crystallized in Pc space group and complexed with Ni(II) and Co(II) in [M(QMP){sub 2}] and Cu(II) and Zn(II) in [M(QMP)(CH{sub 3}COO)]H{sub 2}O compositions. Elemental analyses, mass spectrometry, IR, UV–vis spectroscopy, conductance study and magnetic susceptibilities were used to characterize the complexes. The thermograms obtained in the range of 30–1000 °C were used for kinetic and thermodynamic calculations. The activation energies and order of pyrolysis were calculated using Horowitz–Metzger method. The calculated activation energies were subsequently used for the calculations of thermodynamic parameters including ΔS*, ΔH* and ΔG*. It was found that the thermal stability and activation energy follow the order Cu(II) > Ni(II) > Co(II) > Zn(II) and E{sub Ni}{sup *}>E{sub Cu}{sup *}>E{sub Co}{sup *}>E{sub Zn}{sup *}, respectively. All the compounds were also studied for their urease and α-chymotrypsin inhibition, showing medium to moderate activities for both the enzymes except nickel complex. Nickel complex shows IC{sub 50} = 9.9 ± 0.124 μM ± SEM, and the activity was rationalized by carrying out molecular modeling studies.

  9. Observation of the efficacy of combination of chymotrypsin flush with debridement in the treatment of 70 cases of phase III decubitus%糜蛋白酶冲洗联合清创术治疗III期褥疮疗效70例观察

    Institute of Scientific and Technical Information of China (English)

    王建雄; 谢言秋; 高红云; 王芝珺; 陈艳清; 王胜

    2013-01-01

      目的:探讨糜蛋白酶联合清创术对促进III期褥疮愈合的治疗作用。方法:将近5年(2006-2011年)收集的70例III期褥疮的患者分为治疗组与对照组,在基本治疗方法不变的情况下,治疗组加用糜蛋白酶治疗,比较两组脓性分泌物坚守和消失时间。结果:治疗1周后,治疗组的效果明显高于对照组,两组差异有统计学意义。且治疗组的其他指标均有显著改善。结论:治疗III期褥疮在重视加强营养的同时,联合糜蛋白酶清创治疗褥疮明显缩短愈合时间,可推广应用。%  Objective:To investigate the therapeutic effect of chymotrypsin plus debridement on the phase III bedsore healing. Methods:Seventy patients with phase III bedsore collected in recent five years (2006-2011) were divided into either a treatment group or a control group. Patients in the treatment group were additionally treated with chymotrypsin besides the basic treatment. Purulent secretions stick and disappearance time between these two groups were compared. Results:After one week treatment, the efficacy in the treatment group was significantly higher than that in the control group and the difference between them was statistically significant. Other indicators in the treatment group were significantly improved as compared with the control group. Conclusion:Combination of chymotrypsin with debridement in the treatment of the phase III bedsore can significantly shorten the healing time at the same time to strengthen nutrition.

  10. Efficacy of Amoxicillin-Clavulanate Potassium Combined With Chymotrypsin for Pediatric Acute Suppurative Tonsillitis%阿莫西林克拉维酸钾联合糜蛋白酶治疗儿童急性化脓性扁桃体炎疗效观察

    Institute of Scientific and Technical Information of China (English)

    楚选云

    2016-01-01

    Objective The research aims to explore the clinical effect of amoxicilin-clavulanate potassium combined with chymotrypsin on pediatric acute suppurative tonsilitis.Methods The research chose 100 cases of children patients with suppurative tonsilitis from the hospital,and distributed them into the observation group and the control group randomly of which each had 50 cases. The control group received the treatment of amoxicilin-clavulanate potassium alone,while the observation group received the additional treatment of chymotrypsin on the basis. After the treatment,the research compared the two groups in terms of the improvement time of main symptoms and signs,total effective rate and untoward effect. Results The symptoms improve time of in the observation group were significantly better than that in the control group,the total efficiency in the observation group were significantly higher than that in the coTntrol group(P<0.05).Conclusion Treating pediatric acute suppurative tonsilitis with amoxicilin-clavulanate potassium combined with chymotrypsin can reduce the duration of symptoms and improve the effective rate of treatment,thus should be spread and applied clinicaly.%目的:探讨阿莫西林克拉维酸钾联合糜蛋白酶治疗儿童急性化脓性扁桃体炎的疗效。方法选取我院100例化脓性扁桃体炎患儿,随机分为观察组和对照组,每组患者均为50例,对照组单用阿莫西林克拉维酸钾,观察组在此基础上加用糜蛋白酶治疗,比较两组治疗后主要症状体征改善时间、总有效率和不良反应。结果观察组症状改善时间优于对照组,总有效率高于对照组,差异均有统计学意义(P<0.05)。结论阿莫西林克拉维酸钾联合糜蛋白酶治疗儿童急性化脓性扁桃体炎,能缩短症状持续时间,提高治疗有效率。

  11. Effects of amoxicillin and clavulanate potassium combined with chymotrypsin on children with acute suppurative tonsillitis%阿莫西林克拉维酸钾联合糜蛋白酶治疗儿童急性化脓性扁桃体炎疗效观察

    Institute of Scientific and Technical Information of China (English)

    乔勇; 余霞辉; 邓骥; 邱有波; 罗扬

    2015-01-01

    Objective To explore the effects of amoxicillin and clavulanate potassium combined with chymotrypsin on children with acute suppurative tonsillitis .Methods One hundred and twenty-two children with acute suppurative tonsillitis were randomly di-vided into treatment group ( n =62 ) and control group ( n =60 ) .The treatment group was administrated with amoxicillin and clavu-lanate potassium combined with chymotrypsin while the control group was administrated with amoxicillin and clavulanate potassium on -ly.Two groups were treated for 7 to 10 days.The improvement time of main symptom and the clinical effect were compared between the two groups.Results The times of temperature returning to normal ,angina relieve,pharyngeal hyperaemia fading ,swollen tonsils relieve and discharge extinction in the treatment group were significantly shorter than that in the control group ( P<0.05 ) .The total efficiency in the treatment group(96.77%)was significantly higher than that in the control group (85.0%)(P<0.05).There was no significant difference in incidence of adverse reactions between the two groups .Conclusion Amoxicillin and clavulanate potassium combined chy-motrypsin can shorten the duration of symptoms and improve the treatment efficiency in the treatment of children with acute suppurative tonsillitis.%目的:探讨阿莫西林克拉维酸钾联合糜蛋白酶治疗儿童急性化脓性扁桃体炎的疗效。方法采用随机数字表法将122例化脓性扁桃体炎患儿分为治疗组(62例)和对照组(60例),对照组单用阿莫西林克拉维酸钾,治疗组在此基础上加用糜蛋白酶治疗,疗程均为7~10 d。比较两组治疗后主要症状体征改善时间、总有效率和不良反应。结果治疗组体温恢复正常、咽痛缓解、咽部充血消退、扁桃体肿大减轻、分泌物消退等时间均明显优于对照组,总有效率(96.77%)高于对照组(85.0%),差异均有统计学意义( P<0

  12. Clinical Effect of Different Atomization Methods of Ambroxol Hydrochloride and Chymotrypsin in the Treatment of Neonatal Pneumonia%盐酸氨溴索和糜蛋白酶不同雾化方式治疗新生儿肺炎的临床疗效

    Institute of Scientific and Technical Information of China (English)

    许英

    2015-01-01

    Objective To study clinical effect of different atomization methods of Ambroxol hydrochloride and chymotrypsin in the treatment of neonatal pneumonia.Methods 90 cases of neonatal pneumonia were randomly divided into control group and research group ,45 cases each.Both groups were given ambroxol hydrochloride and chymotrypsin treatment.In control group, ultrasonic atomization method was taken to the team and observation group took oxygen drive atomization method.Results The team cough, lung sounds and asthma clinical symptoms disappear time was shorter than the control group; Group total effective rate 97.2% was significantly higher than the 85.4% in the control group (P< 0.05).Conclusion Newborn pneumonia of children take oxygen atomization method for drug treatment is better than ultrasonic atomization method.%目的:研究盐酸氨溴索(沐舒坦)和糜蛋白酶不同雾化方式对治疗新生肺炎患儿的临床效果。方法选取90例新生儿肺炎随机分成对照组与研究组各45例。两组都进行常规治疗,都给予沐舒坦和糜蛋白酶进行治疗,对照组采取超声雾化法进行给药,研究组采取氧驱动雾化法进行给药。结果研究组咳嗽、肺部啰音以及气喘等临床症状的完全消失时间都比对照组短;研究组总有效率高达97.2%,高于对照组的85.4%(P<0.05)。结论对于新生肺炎患儿采取氧驱动雾化法给药进行治疗比超声雾化法给药治疗效果更好。

  13. 煤工尘肺雾化吸入α-糜蛋白酶和可必特的临床疗效观察%Clinical observation of α-chymotrypsin and Combivent by aerosol inhalation in treatment of coal workers' pneumoconiosis

    Institute of Scientific and Technical Information of China (English)

    王建国; 闫香果; 黄晓罡; 多彩虹; 蔡淑琪

    2012-01-01

    [Objective]To study the therapeutic effect of a-chymotiypsin and Combivent (M-receptor blocker combined with p2 receptor agonist) by aerosol inhalation in treatment of coal workers'pneumoconiosis (CWP) patients who were complicated with pulmonary dysfunction and had stable clinical symptoms. [Methods] Among 123 patients with pulmonary dysfunction, 115 patients had completed the trial, and they were randomly divided into the Combivent group (35 cases) , a-chymotrypsin group (48 cases) and the control group (32 cases). Three groups were treated with aerosol inhalation, Combivent group was given Combivent 2.5 ml with injection water 3.5 ml, a-chymotrypsin group was given a-chymotrypsin 4 000 U with injection water 6 ml, and the control group was given injection water 6 ml, for 5 days, twice daily, 15 min for every time. The pulmonary function (FEV-1, FVC, EVC, FEF25% -75% , MEF75% and MEF 50% ) , clinical symptom scores and six minute walking distance of patients before and after treatment were observed, the adverse reactions were recorded, and the compliance was evaluated. [ Results ] Before treatment, there was no significant difference in clinical symptom scores, six minute walking distance and pulmonary function indexes among three groups. After treatment, the clinical symptom scores in Combivent group were lower than those before treatment, and six minute walking distance was higher than that before treatment ( both P<0. 01). FEV-1, MEF75% and EVC had improved significantly compared with before treatment (all P<0.05). In a-chymotrypsin group, six minute walking distance was higher than that before treatment (P<0. 05) , there was no significant difference in clinical symptom scores, and EVC had improved significantly compared with before treatment (P<0.01). In the control group, there was no significant difference in clinical symptom scores and six minute walking distance between before and after treatment (both P>0. 05). FEF25% -75% (P<0. 01) and MEF

  14. In situ activity of chymotrypsin in sugar-salted herring during cold storage

    DEFF Research Database (Denmark)

    Engvang, K.L.; Nielsen, Henrik Hauch

    2000-01-01

    % lower content of free amino acids. The relative amount of single free amino acids was unaffected by the presence of inhibitor, except for valine, leucine, isoleucine, methionine, phenylalanine and tyrosine, which were formed in lower relative amount. Removal of intestines (gutted herring) and thereby...... all intestinal proteolytic activity did not change the free amino acid profile, except for the concentration of histidine which decreased more when intestines were present. (C) 2000 Society of Chemical Industry....

  15. Introductory Bioinformatics Exercises Utilizing Hemoglobin and Chymotrypsin to Reinforce the Protein Sequence-Structure-Function Relationship

    Science.gov (United States)

    Inlow, Jennifer K.; Miller, Paige; Pittman, Bethany

    2007-01-01

    We describe two bioinformatics exercises intended for use in a computer laboratory setting in an upper-level undergraduate biochemistry course. To introduce students to bioinformatics, the exercises incorporate several commonly used bioinformatics tools, including BLAST, that are freely available online. The exercises build upon the students'…

  16. Chromosomal localization of silkworm (Bombyx mori) sericin gene 1 and chymotrypsin inhibitor 13 using fluorescence in situ hybridization

    Institute of Scientific and Technical Information of China (English)

    Yutaka; BANNO; Hiroshi; FUJII

    2008-01-01

    The chromosomal locations of two single-copy genes, Ser-1 and CI-13, in silkworm (Bombyx mori) were detected at the molecular cytogenetics level by fluorescence in situ hybridization in the study. The results showed that Ser-1 is located near the distal end of the 11th linkage group, relatively at the 12.5±1.4 position in pachytene; and that CI-13 has been mapped near the distal end of the 2nd linkage group, relatively at the 8.2±1.2 position in pachytene. Furthermore, their location model map-FISH map on silkworm chromosome was drawn. The FISH technique and its application to silkworm are also discussed in this paper.

  17. Bacillus subtillis RTSBA6 6.00, a new strain isolated from gut of Helicoverpa armigera (Lepidoptera: Noctuidae) produces chymotrypsin-like proteases

    OpenAIRE

    Shinde, Ashok A.; Shaikh, Faiyaz K.; Padul, Manohar V.; Kachole, Manvendra S.

    2012-01-01

    Exploring bacterial communities with proteolytic activity from the gut of the Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) insect pests was the purpose of this study. As initial efforts to achieve this goal here we report the isolation of new Bacillus subtillis RTSBA6 6.00 strain from the gut of H. armigera and demonstrated as proteases producer. Zymographic analysis revealed 12 proteolytic bands with apparent molecular weights ranging from 20 to 185 kDa. Although some activity was ...

  18. Observation on the Effect of Local Pericoronal Rinsing with Chymotrypsin on Acute Pericoronitis%糜蛋白酶冠周冲洗治疗急性冠周炎的疗效观察

    Institute of Scientific and Technical Information of China (English)

    廖益辉; 李夏缨; 刘晓兵

    2012-01-01

    目的 评价糜蛋白酶对急性冠周炎治疗的效果.方法 选择86 例急性智齿冠周炎的患者,分成2 组:试验组用糜蛋白酶冠周冲洗,盲袋导入碘甘油,临床观察5d 复诊;对照组用3% 过氧化氢、生理盐水,盲袋导入碘甘油,临床观察5d 复诊.结果 试验组与对照组总有效率分别为98% 和86%,差异有统计学意义.结论 糜蛋白酶促使冠周盲袋内脓性分泌物及坏死组织液化分解,使炎症得到改善,是一种适合冠周炎治疗的有效局部用药.

  19. Enzymatic Synthesis of a CCK-8 Tetrapeptide Fragment

    Institute of Scientific and Technical Information of China (English)

    Guang Ya XIANG; Heiner ECKSTEIN

    2004-01-01

    The enzymatic synthesis of a tetrapeptide Phac-Met-Gly-Trp-Met-OEt is reported. It was synthesized by coupling Phac-Met-OEt with Gly-OMe>·HCl, Trp-OMe and Met-OEt successively, catalyzed by α-chymotrypsin, papain and α-chymotrypsin respectively. The results of FAB-MS showed that the products had the correct molecular mass.

  20. Baltic cyanobacteria- A source of biologically active compounds

    Digital Repository Service at National Institute of Oceanography (India)

    Mazur-Marzec, H.; Błaszczyk, A.; Felczykowska, A.; Hohlfeld, N.; Kobos, J.; Toruńska-Sitarz, A.; PrabhaDevi; Montalva`o, S.; DeSouza, L.; Tammela, P.; Mikosik, A.; Bloch, S.; Nejman-Faleńczyk, B.; Węgrzyn, G.

    of proteolytic enzymes of medicinal relevance such as chymotrypsin, trypsin, elastase, plasmin and thrombin (Kodani et al., 1998; Ishida et al., 1999; Itou et al., 1999; Taori et al., 2007, Lifshits et al., 2011). Peptidic structures with protease inhibition...

  1. The effect of dietary fiber on human pancreatic enzyme activity in vitro.

    Science.gov (United States)

    Dunaif, G; Schneeman, B O

    1981-06-01

    Human pancreatic juice was used as a source of amylase, lipase, trypsin, and chymotrypsin. The human pancreatic juice was incubated with one of several dietary fibers, including alfalfa, oat bran, pectin. Solka Floc, wheat bran, and xylan. In addition, the human pancreatic juice was incubated without any fiber, which was used as the control. Incubation with Solka Floc (cellulose) and xylan (a hemicellulose) resulted in a substantial loss of activity in all enzymes assayed. Wheat bran and oat bran decreased amylase and chymotrypsin activity, while alfalfa decreased trypsin and chymotrypsin activity. Incubation with pectin significantly increased amylase and chymotrypsin activity. The mechanism by which sources of dietary fiber can alter enzyme activity is currently unknown. This effect of a dietary component on the activity of human pancreatic enzymes emphasizes the need to investigate further the effects of dietary fiber on digestion and absorption in the small intestine to understand fully its effects on metabolism. PMID:6165234

  2. Controlling the Covalent and Noncovalent Adsorption of Proteins on Polymeric Surfaces in Auqeous Liquids%水溶液中蛋白质在聚合物表面共价及非共价吸附的控制

    Institute of Scientific and Technical Information of China (English)

    E.P.Ivanova; D.; K.; Pham; Y.; V.; Alekseeva; G.; M.; Demyashev; D.; V.; Nicolau

    2005-01-01

    @@ The adsorption and covalent immobilization of human immunoglobulin (HIgG), lysozyme, α-chymotrypsin, and myoglobin have been compared using differentexperimental techniques: ellipsometry (ELM), X-ray photoelectron spectroscopy (XPS),optical fluorescence and atomic force microscopy (AFM).

  3. Silk Microgels Formed by Proteolytic Enzyme Activity

    OpenAIRE

    Samal, Sangram K.; Dash, Mamoni; Chiellini, Federica; Kaplan, David L; Chiellini, Emo

    2013-01-01

    The proteolytic enzyme α-chymotrypsin selectively cleaves the amorphous regions of silk fibroin protein (SFP) and allows the crystalline regions to self-assemble into silk microgels (SMG) at physiological temperature. These microgels consist of lamellar crystals in the micrometer scale, in contrast to the nanometer scaled crystals in native silkworm fibers. SDS-PAGE and zeta potential results demonstrated that α-chymotrypsin utilized only the nonamorphous domains or segments of the heavy chai...

  4. カエル舌水受容器に対するトリプトファナーゼおよびヒスチダーゼの作用

    OpenAIRE

    野村, 浩道; 浅沼, 直和

    1993-01-01

    N-bromosuccinimide, which is known to act on histidine and tryptophan residues of polypeptides and proteins, has been shown to inhibit Ca response specifically without inhibiting Na response in the water receptor of the frog. In order to ascertain the involvement of histidine and tryptophan residues in the receptor site responsible for Ca response, effects of histidase, tryptophanase and α-chymotrypsin on Ca and Na responses were examined. Histidase , tryptophanase and α-chymotrypsin inhibite...

  5. Pregnancy zone protein, a proteinase-binding macroglobulin. Interactions with proteinases and methylamine.

    Science.gov (United States)

    Christensen, U; Simonsen, M; Harrit, N; Sottrup-Jensen, L

    1989-11-28

    Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein, strongly related to alpha 2-macroglobulin (alpha 2M). Its properties and its reactions with a number of enzymes, particularly chymotrypsin, and with methylamine have been investigated. It is concluded that native PZP molecules are dimers of disulfide-bridged 180-kDa subunits and that proteinase binding results in covalent 1:1 (tetrameric)PZP-enzyme complexes. Native PZP is unstable, and storage should be avoided, but when kept unfrozen at 0 degree C most PZP preparations stay native 1-3 months. The reaction of PZP with chymotrypsin involves (i) proteolysis of bait regions, (ii) cleavage of beta-cysteinyl-gamma-glutamyl thiol ester groups, (iii) some change of the conformation and quaternary structure of PZP, and (iv) the formation of covalent 1:1 chymotrypsin-PZP(tetramer) complexes in which chymotrypsin is active but shows less activity than free chymotrypsin. The emission spectra of intrinsic fluorescence show significant differences between the PZP-chymotrypsin complex and its native components, whereas no differences are observed between methylamine-reacted PZP and native PZP. Methylamine reacts with the beta-cysteinyl-gamma-glutamyl thiol ester groups of PZP in a second-order process with k = (13.6 +/- 0.5) M-1 s-1, pH 7.6, 25 degrees C. The reaction product is PZP(dimers); no PZP(tetramers) are formed. The proteinase-binding specificity of PZP is far more restricted than that of alpha 2M. Certain chymotrypsin-like and trypsin-like enzymes are bound much less efficiently than is chymotrypsin itself.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Crystallization, data collection and processing of the chymotrypsin–BTCI–trypsin ternary complex

    Energy Technology Data Exchange (ETDEWEB)

    Esteves, Gisele Ferreira; Teles, Rozeni Chagas Lima; Cavalcante, Nayara Silva; Neves, David; Ventura, Manuel Mateus [Laboratório de Biofísica, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília-DF (Brazil); Barbosa, João Alexandre Ribeiro Gonçalves, E-mail: joao@lnls.br [Center for Structural Molecular Biology (CeBiME), Brazilian Synchrotron Light Laboratory (LNLS), CP 6192, 13083-970 Campinas-SP (Brazil); Freitas, Sonia Maria de, E-mail: joao@lnls.br [Laboratório de Biofísica, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília-DF (Brazil)

    2007-12-01

    A ternary complex of the proteinase inhibitor (BTCI) with trypsin and chymotrypsin was crystallized and its crystal structure was solved by molecular replacement. A ternary complex of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) with trypsin and chymotrypsin was crystallized by the sitting-drop vapour-diffusion method with 0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 6000 and 5%(v/v) 2-methyl-2,4-pentanediol as precipitant. BTCI is a small protein with 83 amino-acid residues isolated from Vigna unguiculata seeds and is able to inhibit trypsin and chymotrypsin simultaneously by forming a stable ternary complex. X-ray data were collected from a single crystal of the trypsin–BTCI–chymotrypsin ternary complex to 2.7 Å resolution under cryogenic conditions. The structure of the ternary complex was solved by molecular replacement using the crystal structures of the BTCI–trypsin binary complex (PDB code) and chymotrypsin (PDB code) as search models.

  7. HYDROLYSIS OF CHEESEWHEY PROTEINSWITH TRYPSIN, CHYMOTRYPSINAND CARBOXYPEPTIDASEA

    Directory of Open Access Journals (Sweden)

    M. F. CUSTÓDIO

    2009-01-01

    Full Text Available

    This work presents a method for adding value to cheese whey residues by whey proteins hydrolysis, using trypsin, chymotrypsin and carboxypeptidase A as catalysts. Sweet cheese whey was dialyzed and filtered in kaolin. Lactose and protein contents were analyzed after each step. The activities of bovine pancreas trypsin and chymotrypsin were measured at different pHs and temperatures. The optimal pH for the hydrolysis of whey proteins was 9.0 for both enzymes. Optima temperatures were 60ºC for trypsin, and 50ºC for chymotrypsin. Trypsin exhibited typical Michaelis-Menten behavior, but chymotrypsin did not. Electrophoretic analysis showed that neither trypsin nor chymotrypsin alone hydrolyzed whey proteins in less than three hours. Hydrolysis rates of -lactalbumin by trypsin, and of bovine serum albumin by chymotrypsin were low. When these enzymes were combined, however, all protein fractions were attacked and rates of hydrolysis were enhanced by one order of magnitude. The addition of carboxypeptidase A to the others enzymes did not improve the process yield.

  8. Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae.

    Directory of Open Access Journals (Sweden)

    Jianxiu Yao

    Full Text Available Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and their homologs from the European corn borer (Ostrinia nubilalis larval gut. Our analyses of the cDNA-deduced amino acid sequences indicated that 12 were putative trypsins, 12 were putative chymotrypsins, and the remaining 10 were trypsin and chymotrypsin homologs that lack one or more conserved residues of typical trypsins and chymotrypsins. Reverse transcription PCR analysis indicated that all genes were highly expressed in gut tissues, but one group of phylogenetically-related trypsin genes, OnTry-G2, was highly expressed in larval foregut and midgut, whereas another group, OnTry-G3, was highly expressed in the midgut and hindgut. Real-time quantitative PCR analysis indicated that several trypsin genes (OnTry5 and OnTry6 were significantly up-regulated in the gut of third-instar larvae after feeding on Cry1Ab protoxin from 2 to 24 h, whereas one trypsin (OnTry2 was down-regulated at all time points. Four chymotrypsin and chymotrypsin homolog genes (OnCTP2, OnCTP5, OnCTP12 and OnCTP13 were up-regulated at least 2-fold in the gut of the larvae after feeding on Cry1Ab protoxin for 24 h. Our data represent the first in-depth study of gut transcripts encoding expanded families of protease genes in O. nubilalis larvae and demonstrate differential expression of protease genes that may be related to Cry1Ab intoxication and/or resistance.

  9. Probing different conformational states of pregnancy-zone protein. Fluorescence studies utilizing the binding of 4,4'-bis(8-anilino-1-naphthalenesulphonate).

    Science.gov (United States)

    Arbelaez, L F; Jensen, P E; Shanbhag, V P; Stigbrand, T

    1993-12-01

    The binding of the fluorescence probe 4,4'-bis(8-anilino-1-naphthalenesulphonate) (bis-ANS) to the human proteinase inhibitor pregnancy-zone protein (PZP) and its complexes with methylamine and chymotrypsin were investigated. The existence of dimeric PZP-chymotrypsin complex was demonstrated and both the dimeric and the tetrameric PZP-chymotrypsin complexes could be studied separately. The fluorescence data indicate that bis-ANS binds to two different sites on PZP and its complexes. The values of the dissociation constant, Kd1, for the binding to the high-affinity site were determined to be 231 +/- 14, 220 +/- 28, 114 +/- 15 and 49 +/- 1 nM, for the binding to native PZP, PZP-methylamine and dimeric and tetrameric PZP-chymotrypsin, respectively. An 11-30-fold decrease was observed in the affinity for the second site, the corresponding values of the dissociation constant, Kd2, being 1.5-2.8 +/- 1.0 microM, which are not significantly different for PZP and its derivatives. The results suggest that the probe bis-ANS discriminates between the different conformational states of PZP and that while the conformation of the complex with methylamine does not differ much from that of the native protein, there is a significant change in conformation when chymotrypsin cleaves the bait region. This is substantiated by a 30%-45% decrease in the maximum enhancement of fluorescence intensity when PZP is treated with chymotrypsin. Although the dimeric and tetrameric forms of PZP-chymotrypsin complexes differ in Kd1 values, the difference in the maximum enhancement of the fluorescence of bis-ANS by the two forms is not significant. This indicates that dimer-dimer interaction in the tetrameric form does not involve hydrophobic sites. The necessity of bait-region cleavage for extensive conformational changes in PZP distinguishes it from alpha 2-macroglobulin, the other alpha-macroglobulin in human plasma.

  10. Enzymatic spectrophotometric reaction rate determination of aspartame

    Directory of Open Access Journals (Sweden)

    Trifković Kata T.

    2015-01-01

    Full Text Available Aspartame is an artificial sweetener of low caloric value (approximately 200 times sweeter than sucrose. Aspartame is currently permitted for use in food and beverage production in more than 90 countries. The application of aspartame in food products requires development of rapid, inexpensive and accurate method for its determination. The new assay for determination of aspartame was based on set of reactions that are catalyzed by three different enzymes: α-chymotrypsin, alcohol oxidase and horseradish peroxidase. Optimization of the proposed method was carried out for: (i α-chymotrypsin activity; (ii time allowed for α-chymotrypsin action, (iii temperature. Evaluation of the developed method was done by determining aspartame content in “diet” drinks, as well as in artificial sweetener pills. [Projekat Ministarstva nauke Republike Srbije, br. III46010

  11. Erythrocyte invasions and receptor heterogeneity in field isolates of Nanay river basin Iquitos

    Institute of Scientific and Technical Information of China (English)

    Kuppusamy Chenniappan; Sarah H Johns

    2012-01-01

    Objective:To determine whether the requirements for sialic acid varies and whether several types of silaic acid independent receptors utilized for invasion mechanisms of fresh filed isolates collected aroundNanay river basin,Iquitos.Methods:The field isolates were cultured as described previously byJensen andTrager andMR4 protocol with little modifications.The erythrocytes preparation and subsequent enzyme treatment was done as described previously bySharma. with little modification.Invasion assay was performed as described previously by Sharmaet al with little modification.Results:TheNanay river basin isolates showed five types of invasion mechanisms or types of receptors-ligand interactions.Here we observed that an equal numbers of neuraminidase sensitive and resistant invasion receptor-ligand interaction profiles as the most common receptor-ligand invasion profiles.Neuraminidase resistance trypsin sensitive chymotrypsin sensitive(NMRTSCTS) invasion of receptor-ligand interaction profile was found in seven isolates,Five field isolates and one reference strain showed neuraminidase sensitive, trypsin sensitive and chymotrypsin resistant(NMSTSCTR) invasion of receptor-ligand interactions, six isolates including one reference strains dd2 showed neuraminidase sensitive, trypsin and chymotrypsin resistance(NMSTRCTR) indicating its dependence on sialic acids and independence of trypsin and chymotrypsin sensitive proteins.Four isolates showed neuraminidase sensitive, trypsin sensitive and chymotrypsin sensitive(NMSTSCTS) invasion of receptor-ligand interactions, seven isolates were neuraminidase resistant, trypsin sensitive and chymotrypsin resistance (NMRTSCTR) invasion of receptor-ligand interactions, indicating its dependence on trypsin sensitive proteins.Conclusions:TheNanay river basin isolates showed five types of invasion mechanisms or types of receptors-ligand interactions.A full understanding of theses invasion mechanisms may allow the development of novel

  12. Enzymatic Synthesis of a CCK-8 Tripeptide Derivative

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The enzymatic synthesis of CCK-8 tripeptide derivative Phac-Met-Asp(OMe)-Phe-NH2 is reported. Starting with Phac-Met-OCam, we have successfully synthesized the target tripeptide with three free or immobilized enzymes, α-chymotrypsin, papain and thermolysin in reasonable yields. The key steps in this synthesis were the coupling of Phac-Met-OCam and H-Asp(OMe)2 to form Met-Asp peptide bond catalyzed by α-chymotrypsin and the selective hydrolysis of α-ester of Phac-Met-Asp(OMe)2 catalyzed by papain.

  13. Structures and Activity of New Anabaenopeptins Produced by Baltic Sea Cyanobacteria.

    Science.gov (United States)

    Spoof, Lisa; Błaszczyk, Agata; Meriluoto, Jussi; Cegłowska, Marta; Mazur-Marzec, Hanna

    2015-12-30

    Anabaenopeptins, bioactive cyclic hexapeptides, were isolated by preparative reversed-phase high performance liquid chromatography from an extract of Baltic Sea cyanobacterial bloom material composed of Nodularia spumigena (50%), Aphanizomenon flos-aquae (40%) and Dolichospermum spp. (10%). Five new anabaenopeptins and nine previously known anabaenopeptins were isolated, and their putative structures were determined by tandem mass spectrometry. The activity of the peptides against carboxypeptidase A and protein phosphatase 1 as well as chymotrypsin, trypsin and thrombin was tested. All anabaenopeptins inhibited carboxypeptidase A (apart from one anabaenopeptin variant) and protein phosphatase 1 with varying potency, but no inhibition against chymotrypsin, trypsin and thrombin was observed.

  14. Immobilization-stabilization of proteases as a tool to improve the industrial design of peptide synthesis.

    Science.gov (United States)

    Blanco, R M; Bastida, A; Cuesta, C; Alvaro, G; Fernandez-Lafuente, R; Rosell, C M; Guisan, J M

    1991-01-01

    Synthesis of dipeptides benzoyl Arginine leucinamide and kyotorphin catalyzed by highly stabilized derivatives of trypsin and chymotrypsin have been performed. Extreme experimental conditions could be tested and parameters of industrial interest could be improved provided the high activity and stability of the derivatives in these unfavourable environments. Thermodynamically controlled synthesis catalyzed by trypsin could be optimized and 97% conversion was obtained in 90% organic cosolvents. 100% yields were achieved in kinetically controlled synthesis catalyzed by trypsin in aqueous medium in the presence of IM Ammonium Sulphate. Higher starting concentrations of poorly soluble substrates of chymotrypsin could be used in a reaction medium containing 50% DMF and 95% yield were obtained.

  15. Enzymatic treatment of peanut butter to reduce the concentration of major peanut allergens

    OpenAIRE

    Yu, Jianmei; Goktepe, Ipek; Ahmedna, Mohamed

    2013-01-01

    This study investigated the effects of enzymatic treatment of peanut butter on two-major peanut allergens (Ara h 1 and Ara h 2). Home-made and commercial peanut butter samples were treated with alpha-chymotrypsin, trypsin or the combination of these enzymes and incubated at room temperature for 24�h or at 37��C for 3�h. Treated peanut butter samples were sampled weekly for evaluation of total soluble proteins and extractable Ara h 1/Ara h 2. Data show that 1:1 alpha-chymotrypsin: trypsin at 0...

  16. Identification of strong aggregating regions in soy glycinin upon enzymatic hydrolysis

    NARCIS (Netherlands)

    Kuipers, B.J.H.; Gruppen, H.

    2008-01-01

    Upon hydrolysis with chymotrypsin, soy glycinin has a strong tendency to aggregate. The regions of glycinin from which the aggregating peptides originate were identified by accumulative-quantitative peptide mapping. To this end, the aggregating peptides were further hydrolyzed with trypsin to obtain

  17. Synthesis and biological evaluation of both enantiomers of p-boronophenylalaninol

    Energy Technology Data Exchange (ETDEWEB)

    Kumanishi, A.; Ozaki, N.; Tanimori, S.; Tsuda, T.; Kirihata, M. [Osaka Prefecture Univ., Graduate School of Agriculture and Life Sciences, Sakai, Osaka (Japan); Takagaki, M.; Ono, K. [Kyoto Univ., Kumatori, Osaka (Japan). Research Reactor Inst

    2000-10-01

    The both enantiomers of p-borono-phenylalaninol (BPA-ol) were synthesized from racemic p-boronophenylalanine (BPA) ethyl ester by a hybrid process involving enzymatic resolution using {alpha}-chymotrypsin. The relationships between absolute configuration and biological properties of BPA-ol were evaluated using C6 gliosarcoma cells. Clear differences of biological activities between the both enantiomers were not found. (author)

  18. Magnetic enzyme reactors for isolation and study of heterogeneous glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Korecka, Lucie [Department of Analytical Chemistry, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic)]. E-mail: lucie.korecka@upce.cz; Jezova, Jana [Department of Analytical Chemistry, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Bilkova, Zuzana [Department of Biological and Biochemical Sciences, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Benes, Milan [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Namesti 2, 162 06 Prague (Czech Republic); Horak, Daniel [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Namesti 2, 162 06 Prague (Czech Republic); Hradcova, Olga [Department of Biological and Biochemical Sciences, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Slovakova, Marcela [Department of Biological and Biochemical Sciences, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Laboratoire Physicochimie Curie, UMR 168 CNRS/Institute Curie, Paris Cedex 05 (France); Viovy, Jean-Louis [Laboratoire Physicochimie Curie, UMR 168 CNRS/Institute Curie, Paris Cedex 05 (France)

    2005-05-15

    The newly developed magnetic micro- and nanoparticles with defined hydrophobicity and porosity were used for the preparation of magnetic enzyme reactors. Magnetic particles with immobilized proteolytic enzymes trypsin, chymotrypsin and papain and with enzyme neuraminidase were used to study the structure of heterogeneous glycoproteins. Factors such as the type of carrier, immobilization procedure, operational and storage stability, and experimental conditions were optimized.

  19. Enzyme-polymer composites with high biocatalytic activity and stability

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jungbae; Kosto, Timothy J.; Manimala, Joseph C.; Nauman, E B.; Dordick, Jonathan S.

    2004-08-22

    We have applied vacuum-spraying and electrospinning to incorporate an enzyme into a polymer matrix, creating a novel and highly active biocatalytic composite. As a unique technical approach, enzymes were co-dissolved in toluene with polymers, and the solvent was then rapidly removed by injecting the mixture into a vacuum chamber or by electrospinning. Subsequent crosslinking of the enzyme with glutaraldehyde resulted in stable entrapped enzyme within the polymeric matrices. For example, an amorphous composite of alpha-chymotrypsin and polyethylene showed no significant loss of enzymatic activity in aqueous buffer for one month. Nanofibers of alpha-chymotrypsin and polystyrene also showed no decrease in activity for more than two weeks. The normalized activity of amorphous composite in organic solvents was 3-13 times higher than that of native alpha-chymotrypsin. The activity of nanofibers was 5-7 times higher than that of amorphous composite in aqueous buffer solution. The composites of alpha-chymotrypsin and polymers demonstrate the feasibility of obtaining a wide variety of active and stable biocatalytic materials with many combinations of enzymes and polymers.

  20. Comparison of the aggregation behavior of soy and bovine whey protein hydrolysates

    NARCIS (Netherlands)

    Kuipers, B.J.H.; Alting, A.C.; Gruppen, H.

    2007-01-01

    Abstract Soy-derived proteins (soy protein isolate, glycinin, and ß-conglycinin) and bovine whey-derived proteins (whey protein isolate, ¿-lactalbumin, ß-lactoglobulin) were hydrolyzed using subtilisin Carlsberg, chymotrypsin, trypsin, bromelain, and papain. The (in)solubility of the hydrolysates ob

  1. Polyphosphorous acid catalyzed cyclization in the synthesis of cryptolepine derivatives

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    11-Oxo-10,11-dihydroxy-5H-indolo[3,2,b]quinoline7-carboxylic acid was obtained specifically by polyphosphorous acid catalyzed cyclization with optimal reaction conditions. Biological assays showed that it potentially inhibits the proteasomal chymotrypsin-like activity in vitro and suppresses breast cancer cell growth.

  2. Main: 1COA [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available are Chymotrypsin Inhibitor 2 (Ci2) Mutant With Ile 76 Replaced By Val (I76v) 1coaa 1 Serine Protease Inhibitor S.E.Jackson,... M.Moracci, N.Elmasry, C.M.Johnson, A.R.Fersht S.E.Jackson, M.Moracci, N.Elmasry, C.M.Johnson

  3. Isolation and removal of proteolytic enzymes with magnetic cross-linked erythrocytes

    Science.gov (United States)

    Šafařík, Ivo; Šafaříková, Mirka

    2001-01-01

    New magnetic adsorbents for batch isolation and removal of various proteolytic enzymes were prepared by glutaraldehyde cross-linking of bovine, porcine and human erythrocytes in the presence of fine magnetic particles. Trypsin, chymotrypsin, alkaline bacterial protease and proteases present in various commercial enzyme preparations were efficiently adsorbed on these adsorbents; on the contrary, proteins without proteolytic activity were not adsorbed.

  4. Main: 1YPB [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available placed By Ala (S31g,E33a,E34a) Proteinase Inhibitor(Chymotrypsin) Y.Harpaz, N.Elmasry, A.R.Fersht, K.Henrick... Y.Harpaz, N.Elmasry, A.R.Fersht, K.Henrick Direct Observation Of Better Hydratio

  5. Main: 1YPA [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available placed By Ala (S31a,E33a,E34a) Proteinase Inhibitor(Chymotrypsin) Y.Harpaz, N.Elmasry, A.R.Fersht, K.Henrick... Y.Harpaz, N.Elmasry, A.R.Fersht, K.Henrick Direct Observation Of Better Hydratio

  6. Role of proteases and antiprotease in the etiology of chronic pancreatitis

    Directory of Open Access Journals (Sweden)

    Srimanjari Kavutharapu

    2012-01-01

    Full Text Available Background/Aim: Chronic pancreatitis (CP is the progressive and irreversible destruction of the pancreas characterized by the permanent loss of endocrine and exocrine function. Trypsin, the most important digestive enzyme plays a central role in the regulation of all other digestive enzymes. Chymotrypsin, an endopeptidase hydrolyzes peptides at amino acids with aromatic side chains. Alpha-1-antitrypsin is a principal antiprotease which protects the mucosal tissue from the proteolytic effects of trypsin and chymotrypsin by the formation of molar complexes. The present study is aimed at examining the role of proteases (trypsin and chymotrypsin and anti-protease (α1-anti-trypsin in the etiopathogenesis of chronic pancreatitis. Patients and Methods: A total of 90 CP patients and 110 age and sex matched controls were considered for the study. Serum trypsin, chymotrypsin and α1-anti-trypsin levels were determined prospectively in CP patients and compared to healthy controls as described previously. Results: The mean activity of trypsin were found to be increased in CP patients (X ± SD = 0.82 ± 0.838 in comparison to normal control group (X ± SD = 0.55 ± 0.328, (P = 0.001. Chymotrypsin activity were also found to be elevated in CP patients (X ± SD = 0.63 ± 0.278 in comparison to control group (X ± SD = 0.39 ± 0.295, (P = 0.0001. The mean α-1-anti-trypsin activity were found to be lowered in CP patients (X ± SD = 0.42 ± 0.494 in comparison to control group (X ± SD = 0.67 ± 0.465, with the variation being significant (P = 0.0003. Conclusion: The findings suggest an imbalance in the synthesis and degradation of proteolytic enzymes and antiprotease indicating an altered aggressive and defensive role in the pathogenesis of chronic pancreatitis.

  7. Effect of Feed Additives on the Development of Proteolytic Enzymes of the Tropical Sport Fish Malaysian Mahseer (Tor tambroides-Bleeker Fry

    Directory of Open Access Journals (Sweden)

    K. C. Jalal

    2005-01-01

    Full Text Available Tor tambroides fry with a mean of 8.0±72 mm standard length (SL and weighing (W 0.06±0.01g were stocked at the rate of fifty (50 individuals in each of the fifteen 150l rectangular fibre glass tanks for a period of 5 weeks. The development of proteolytic enzymes (Trypsin and Chymotrypsin was studied during these feeding treatments. 45% protein diet without additives was treated as control diet. The gut of fish fry fed on control diet incorporated with 0.10% Spirulina, 0.10% enzyme and 0.10% vitamin additives were examined. Fish fry fed on diet with 0.10% Spirulina showed significantly higher enzymatic activity (PSpirulina to a diet could be a vital factor to activate the proteolytic enzymes such as trypsin and chymotrypsin of the Tor tambroides fry. This suggests a new approach in the use of feed additive Spirulina in fish feed.

  8. Cyanoacetamide based Barbiturates, Thiobarbiturates and their Biological Studies

    International Nuclear Information System (INIS)

    Various cyanoacetamide based Knoevenagel adducts were coupled with barbituric acid / thiobarbituric acid and triethylorthoformate via a one pot three component reaction in 2-butanol availing the desired compound in excellent yields. All the synthesized compounds (2-15) were extensively characterized by 1H-NMR, 13C-NMR, Mass spectrometry and elemental analysis and were screened for antibacterial, antiurease, antioxidant, cytotoxicity and chymotrypsin inhibition studies. In case of antibacterial studies 2 was found appreciably active against the six selected strains whereas the rest of the compounds were moderately active. The urease inhibition studies revealed compound 5 and 12 as potent whereas the rest were found inactive where thiourea was used as control. Antioxidant activity results exhibited with 2 as the most active and rest of compounds showed good activity. In case of chymotrypsin inhibition studies all the synthesized compounds were found inactive with the exception of 6 which was moderately active. (author)

  9. Functional analysis of the interactions between reovirus particles and various proteases in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Sargent, M.D.; Long, D.G.; Borsa, J.

    1977-01-01

    The digestion of purified reovirus particles by various proteases including chymotrypsin, trypsin, pronase, papain, bromelain, proteinase K, and fibrinolysin has been examined as it relates to virion transcriptase activation and alteration of infectivity. In every case uncoating to the level of active transcriptase proceeds via two mechanistically distinct steps. All the proteases tested serve to mediate only the first of the two steps, converting intact virions to intermediate subviral particles (ISVP) in which the transcriptase is retained in a latent state. The second step of the uncoating process is mediated by a K/sup +/ ion-triggered, endogenous mechanism and results in conversion of ISVP to cores, concomitant with transcriptase activation and loss of infectivity. All of the tested enzymes, except trypsin, reversibly block the second step of uncoating. These results indicate the generality, with respect to protease employed, of the two-step process for reovirus uncoating and transcriptase activation demonstrated previously with chymotrypsin.

  10. Solid and papillary neoplasm of the pancreas

    DEFF Research Database (Denmark)

    Jørgensen, L J; Hansen, A B; Burcharth, F;

    1992-01-01

    In two cases of solid and papillary neoplasm of the pancreas (SPN), positive staining for argyrophil granules, chromogranin-A, neuron-specific enolase, chymotrypsin, alpha 1-antitrypsin, vimentin, cytokeratin, and estrogen receptors was present. Ultrastructurally, neurosecretory as well as zymoge......In two cases of solid and papillary neoplasm of the pancreas (SPN), positive staining for argyrophil granules, chromogranin-A, neuron-specific enolase, chymotrypsin, alpha 1-antitrypsin, vimentin, cytokeratin, and estrogen receptors was present. Ultrastructurally, neurosecretory as well...... as zymogenlike granules were demonstrated. Measurements of mean nuclear volume and volume-corrected mitotic index discriminated between SPN and well-differentiated ductal adenocarcinoma of the pancreas, with notably lower values being seen in SPN. Silver-stained nucleolar organizer region counts showed wide...

  11. Comparison of the aggregation behavior of soy and bovine whey protein hydrolysates.

    Science.gov (United States)

    Kuipers, Bas J H; Alting, Arno C; Gruppen, Harry

    2007-01-01

    Soy-derived proteins (soy protein isolate, glycinin, and beta-conglycinin) and bovine whey-derived proteins (whey protein isolate, alpha-lactalbumin, beta-lactoglobulin) were hydrolyzed using subtilisin Carlsberg, chymotrypsin, trypsin, bromelain, and papain. The (in)solubility of the hydrolysates obtained was studied as a function of pH. At neutral pH, all soy-derived protein hydrolysates, particularly those from glycinin, obtained by hydrolysis with subtilisin Carlsberg, chymotrypsin, bromelain, and papain showed a stronger aggregation compared to the non-hydrolyzed ones. This increase in aggregation was not observed upon hydrolysis by trypsin. None of the whey-derived protein hydrolysates exhibited an increase in aggregation at neutral pH. The high abundance of theoretical cleavage sites in the hydrophobic regions of glycinin probably explains the stronger exposure of hydrophobic groups than for the other proteins, which is suggested to be the driving force in the aggregate formation.

  12. Main: 1CIR [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1CIR 大麦 Barley Hordeum vulgare l. Subtilisin-Chymotrypsin Inhibitor-2a Hordeum Vulg...are Molecule: Chymotrypsin Inhibitor 2; 1cir 5 Chain: A, B; 1cir 6 Domain: Residues 1 - 40, 41 - 64; 1cir 7 Synonym: Ci2; 1ci...r 8 Engineered: Yes; 1cir 9 Other_details: Cleaved Between Residues 40 And 41 1cir 10 Serin...e Protease Inhibitor B.J.Davis, A.R.Fersht 1cir 18 B.Davis, A.M.Buckle, G.De Prat Gay, A.R.Fersht 1ci...r 20 Towards The Complete Structural Characterization 1cir 21 Of A Protein Folding Pathw

  13. Main: 1CIQ [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available q 27 Cleaved Inhibitor 1ciq 17 ICI2_HORVU:57,21|ICI2_HORVU:83,60|EMBL; X05404; CAA28988....1CIQ 大麦 Barley Hordeum vulgare l. Subtilisin-Chymotrypsin Inhibitor-2a Hordeum Vulg...are Molecule: Chymotrypsin Inhibitor 2; 1ciq 5 Chain: A, B; 1ciq 6 Fragment: Residues 1 - 40, 41 - 64; 1ciq 7 Synonym: Ci2; 1ci...q 8 Engineered: Yes; 1ciq 9 Other_details: Cleaved Between 40 And 41 1ciq 10 Serine Prote...ase Inhibitor A.M.Buckle, A.R.Fersht 1ciq 19 B.Davis, A.M.Buckle, G.De Prat Gay, A.R.Fersht 1ci

  14. Effects of Mucuna pruriens protease inhibitors on Echis carinatus venom.

    Science.gov (United States)

    Hope-Onyekwere, Nnadozie Stanley; Ogueli, Godwin Ifeanyi; Cortelazzo, Alessio; Cerutti, Helena; Cito, Annarita; Aguiyi, John C; Guerranti, Roberto

    2012-12-01

    The medicinal plant Mucuna pruriens, with reputed anti-snake venom properties has been reported to contain a kunitz-type trypsin inhibitor. This study was undertaken to further evaluate the protease inhibitory potential of gpMuc, a multiform glycoprotein, and other protein fractions from M. pruriens seeds against trypsin, chymotrypsin, Echis carinatus snake venom, ecarin and thrombin. The results showed that gpMuc inhibited both trypsin and chymotrypsin activities and was thermally stable, maintaining its trypsin inhibitory activity at temperatures of up to 50°C. Its structural conformation was also maintained at pH ranges of 4-7. Immunoreactivity study confirms that it contains protease-recognizing epitope on one of its isoforms. The whole protein extract of M. pruriens seeds inhibited prothrombin activation by ecarin and whole E. carinatus venom, and also thrombin-like activity using chromogenic assay. PMID:22447581

  15. Inhibitory serpins from rye grain with glutamine as P-1 and P-2 residues in the reactive center

    DEFF Research Database (Denmark)

    Hejgaard, Jørn

    2001-01-01

    were determined for each serpin. Three of four serpins containing the unusual reactive center P-2-P-1' QQ down arrowS and one with P-2-P-1' PQ down arrowM were equally efficient inhibitors of chymotrypsin (k(a)'similar to 10(5) M-1 s(-1)). One serpin with P-2-P-1 PY down arrowM was a faster inhibitor...

  16. Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins

    OpenAIRE

    1982-01-01

    Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-proteins by pronase (1 mg/ml for 20 min at 37 degrees C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A, and thermolysin. Protein synthesis was required to r...

  17. Pyrrolidine dithiocarbamate-zinc(II) and -copper(II) complexes induce apoptosis in tumor cells by inhibiting the proteasomal activity☆

    OpenAIRE

    Milacic, Vesna; Chen, Di; Giovagnini, Lorena; Diez, Alejandro; Fregona, Dolores; Dou, Q. Ping

    2008-01-01

    Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for su...

  18. Multiple phases of protien gels

    Science.gov (United States)

    Annaka, Masahiko; Tanaka, Toyoichi

    1994-03-01

    A multiple phase transition was observed in gels made by covalently cross-linking proteins in either native or denatured state. The enzymatic activity of the gels prepared from native α-chymotrypsin was determined for each of the multiple phases. The reversibility of the swelling degrees and the enzymatic reaction rates upon phase transition suggests that the protein is at a free energy minimum and thus in a phase.

  19. [Stability of the structure and antigenic determinants of adenovirus type 1 native hexon to proteases].

    Science.gov (United States)

    Kiseleva, E K; Khil'ko, S N; Grigor'ev, V G; Diachenko, N S; Vantsak, N P

    1986-08-01

    Hexon capsomers of human adenovirus type 1 (h1) labeled by iodine 125 were digested in a native state (trimers) by trypsin, chymotrypsin or papain, and the resulting hydrolysates were analyzed by SDS-PAGE. In each case, a discrete and temporally stable pattern of relatively large fragments was revealed. The degree of hexon polypeptide hydrolysis was maximal for papain, intermediate for chymotrypsin and minimal for trypsin, the largest fragments in the digest being 32, 40 and 80 kD, respectively. At room temperature, all the electrophoretically discernible hexon proteolytical fragments were held together in structures resembling intact hexon trimers and could be regarded as "hexon cores", of which papain hexon cores were the most stable during SDS-PAGE. Radioimmunoprecipitation analysis revealed a complete absence of native hexon antigenicity in thermodenaturated fragments of hexon protease digests, while native trypsin, chymotrypsin and papain hexon cores could be precipitated by hexon-specific antibodies. The immunoprecipitated material contained all of the hexon fragments found in appropriate hexon cores and retained the structure of the original cores. Trypsin, chymotrypsin and papain hexon cores were shown to possess at least part of native Ad h1 hexon antigenic determinants of each of the following specificities: species-specific (epsilon), cross-reactive with hexon of human adenoviruses (h3 and h6), simian adenovirus (sim 16), bovine adenoviruses (bos 3 and bos 7) and avian adenovirus (Aviadenovirus gal 1 or CELO). Thus, the full spectrum of known hexon antigenic determinants (species-specific to intergenus-crossreactive) is at least portly stable against protease attack of native hexon capsomers.

  20. Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals

    OpenAIRE

    Mourão, Caroline B.F.; Elisabeth F Schwartz

    2013-01-01

    The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomo...

  1. Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification

    OpenAIRE

    Jager, M; Nir, E; Weiss, S

    2006-01-01

    An often limiting factor for studying protein folding by single-molecule fluorescence resonance energy transfer (FRET) is the ability to site-specifically introduce a photostable organic FRET donor (D) and a complementary acceptor (A) into a polypeptide chain. Using alternating-laser excitation and chymotrypsin inhibitor 2 as a model, we show that chemical labeling of a unique cysteine, followed by enzymatic modification of a reactive glutamine in an N-terminally appended substrate sequence r...

  2. Strategies in protein sequencing and characterization: Multi-enzyme digestion coupled with alternate CID/ETD tandem mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Nardiello, Donatella; Palermo, Carmen, E-mail: carmen.palermo@unifg.it; Natale, Anna; Quinto, Maurizio; Centonze, Diego

    2015-01-07

    Highlights: • Multi-enzyme digestion for protein sequencing and characterization by CID/ETD. • Simultaneous use of trypsin/chymotrypsin for the maximization of sequence. • Identification of PTMs, sequence variants and species-specific residues. • Increase of accuracy in sequence assignments by orthogonal fragmentation techniques. - Abstract: A strategy based on a simultaneous multi-enzyme digestion coupled with electron transfer dissociation (ETD) and collision-induced dissociation (CID) was developed for protein sequencing and characterization, as a valid alternative platform in ion-trap based proteomics. The effect of different proteolytic procedures using chymotrypsin, trypsin, a combination of both, and Lys-C, was carefully evaluated in terms of number of identified peptides, protein coverage, and score distribution. A systematic comparison between CID and ETD is shown for the analysis of peptides originating from the in-solution digestion of standard caseins. The best results were achieved with a trypsin/chymotrypsin mix combined with CID and ETD operating in alternating mode. A post-database search validation of MS/MS dataset was performed, then, the matched peptides were cross checked by the evaluation of ion scores, rank, number of experimental product ions, and their relative abundances in the MS/MS spectrum. By integrated CID/ETD experiments, high quality-spectra have been obtained, thus allowing a confirmation of spectral information and an increase of accuracy in peptide sequence assignments. Overlapping peptides, produced throughout the proteins, reduce the ambiguity in mapping modifications between natural variants and animal species, and allow the characterization of post translational modifications. The advantages of using the enzymatic mix trypsin/chymotrypsin were confirmed by the nanoLC and CID/ETD tandem mass spectrometry of goat milk proteins, previously separated by two-dimensional gel electrophoresis.

  3. A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro

    OpenAIRE

    Jorge E Suarez; Mauricio Urquiza; Hernando Curtidor; Rodriguez, Luis E.; Marisol Ocampo; Elizabeth Torres; Fanny Guzman; Manuel Elkin Patarroyo

    2000-01-01

    The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falcip...

  4. What determines the strength of noncovalent association of ligands to proteins in aqueous solution?

    OpenAIRE

    Miyamoto, S; Kollman, P A

    1993-01-01

    Free energy perturbation methods using molecular dynamics have been used to calculate the absolute free energy of association of two ligand-protein complexes. The calculations reproduce the significantly more negative free energy of association of biotin to streptavidin, compared to N-L-acetyltryptophanamide/alpha-chymotrypsin. This difference in free energy of association is due to van der Waals/dispersion effects in the nearly ideally performed cavity that streptavidin presents to biotin, w...

  5. Physical and chemical characterization of a Giardia lamblia-specific antigen useful in the coprodiagnosis of giardiasis.

    OpenAIRE

    Rosoff, J D; Stibbs, H H

    1986-01-01

    We recently reported the isolation and identification of a Giardia lamblia-specific antigen (GSA 65) that is shed in the stool of giardiasis patients. In the present study, this antigen was affinity purified from sonic extracts of axenically cultured G. lamblia trophozoites and characterized to better understand its biological function and its potential usefulness in the design of coprodiagnostic assays for giardiasis. GSA 65 was resistant to proteolytic digestion with trypsin, chymotrypsin, ...

  6. Proteolytic activity in the midgut of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae), Pomegranate carob moth

    OpenAIRE

    M. Ranjbar; JJ Sendi; A Zibaee

    2011-01-01

    In this study, the proteolytic activity in the midgut of Ectomyelois ceratoniae as the major pest of pomegranate was investigated to find nature of specific proteases and their properties for adopting possible pest management procedure. It was found that fourth and fifth instar larvae had the highest proteolytic activity as well as specific proteinases including, elastase, trypsin-like, chymotrypsin-like and two exopeptidases. The optimal pH of general protease was 10 and 9 for azocasein, cas...

  7. Transformation of Human Cathelicidin LL-37 into Selective, Stable, and Potent Antimicrobial Compounds

    OpenAIRE

    Wang, Guangshun; Hanke, Mark L.; Mishra, Biswajit; Lushnikova, Tamara; Heim, Cortney E.; Chittezham Thomas, Vinai; Bayles, Kenneth W; Kielian, Tammy

    2014-01-01

    This Letter reports a family of novel antimicrobial compounds obtained by combining peptide library screening with structure-based design. Library screening led to the identification of a human LL-37 peptide resistant to chymotrypsin. This d-amino-acid-containing peptide template was active against Escherichia coli but not methicillin-resistant Staphylococcus aureus (MRSA). It possesses a unique nonclassic amphipathic structure with hydrophobic defects. By repairing the hydrophobic defects, t...

  8. Isolated co-lipase deficiency in two brothers.

    OpenAIRE

    Hildebrand, H; Borgström, B.; Békássy, A; Erlanson-Albertsson, C; Helin, I

    1982-01-01

    Two normally developed Assyrian brothers with isolated pancreatic co-lipase deficiency are described. They presented at the age of 5-6 years with loose stools. They had steatorrhoea, and analysis of exocrine pancreatic enzymes in the small intestine showed co-lipase deficiency, while amylase, chymotrypsin, trypsin and lipase were normal. Intraduodenal infusion of purified co-lipase improved fat digestion measured by the triolein breath test. Their steatorrhoea diminished on treatment with ent...

  9. Prevalence of S and Z alpha 1-antitrypsin mutations in patients with pancreatic diseases in Serbian population

    OpenAIRE

    Nikolić Aleksandra; Divac Aleksandra; Stanković Marija; Dinić Jelena; Lukić Snežana; Anđelić-Jelić Marina; Popović Dragan; Radojković Dragica

    2010-01-01

    One of the key points in research of pancreatic disease pathology is further elucidation of the role of proteases and antiproteases, since their imbalance can lead to pancreatic injury. Alpha 1-antitrypsin (AAT) is one of the most important serum inhibitors of proteolytic enzymes, including pancreatic enzymes trypsin, chymotrypsin and elastase. It is speculated that mutations in the AAT gene may influence the onset and the development of pancreatic disease. The presence of the most common AAT...

  10. Main: 1CQ4 [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available es; Mutation: Yes; Other_details: Exists As A1b2/A2b1 Domain-Swapped Dimer; Molecule: Serine Proteinase Inhi...ion: Yes; Other_details: Exists As A1b2/A2b1 Domain-Swapped Dimer Hydrolase Inhibitor Y.W.Chen, K.R.Stott Y....W.Chen, K.Stott, M.F.Perutz Crystal Structure Of A Dimeric Chymotrypsin Inhibitor 2 Mutant Containing An Ins

  11. Strategies in protein sequencing and characterization: Multi-enzyme digestion coupled with alternate CID/ETD tandem mass spectrometry

    International Nuclear Information System (INIS)

    Highlights: • Multi-enzyme digestion for protein sequencing and characterization by CID/ETD. • Simultaneous use of trypsin/chymotrypsin for the maximization of sequence. • Identification of PTMs, sequence variants and species-specific residues. • Increase of accuracy in sequence assignments by orthogonal fragmentation techniques. - Abstract: A strategy based on a simultaneous multi-enzyme digestion coupled with electron transfer dissociation (ETD) and collision-induced dissociation (CID) was developed for protein sequencing and characterization, as a valid alternative platform in ion-trap based proteomics. The effect of different proteolytic procedures using chymotrypsin, trypsin, a combination of both, and Lys-C, was carefully evaluated in terms of number of identified peptides, protein coverage, and score distribution. A systematic comparison between CID and ETD is shown for the analysis of peptides originating from the in-solution digestion of standard caseins. The best results were achieved with a trypsin/chymotrypsin mix combined with CID and ETD operating in alternating mode. A post-database search validation of MS/MS dataset was performed, then, the matched peptides were cross checked by the evaluation of ion scores, rank, number of experimental product ions, and their relative abundances in the MS/MS spectrum. By integrated CID/ETD experiments, high quality-spectra have been obtained, thus allowing a confirmation of spectral information and an increase of accuracy in peptide sequence assignments. Overlapping peptides, produced throughout the proteins, reduce the ambiguity in mapping modifications between natural variants and animal species, and allow the characterization of post translational modifications. The advantages of using the enzymatic mix trypsin/chymotrypsin were confirmed by the nanoLC and CID/ETD tandem mass spectrometry of goat milk proteins, previously separated by two-dimensional gel electrophoresis

  12. A Novel Method for Diminishing Protein Aggregation during Denatuaration Process

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The addition of packing material for high performance hydrophobic interaction chrornatograghy (HPHIC) into the denaturant solution to prevent, or depress protein aggregation in the denatuaration process is presented. The renaturation of α-chymotrypsin (α-Chy)denatured with guanidine hydrochloride (GuHCl) solution indicated that renaturation efficiency can be enhanced from 36.1% to 59.0% by this new method. The structure of the ligand linking of HPHIC packings is also important for the protein renaturation.

  13. Improving the in vitro protein digestibility of sorghum with reducing agents

    OpenAIRE

    Hamaker, B. R.; Kirleis, A. W.; Butler, L G; Axtell, J. D.; Mertz, E T

    1987-01-01

    We have shown in previous reports that cooked sorghum protein is less digestible than other cooked cereal proteins. The pepsin-indigestible proteins in sorghum were found to be mainly prolamin proteins. Cooking sorghum in the presence of 2-mercaptoethanol increased protein digestibility (in vitro with pepsin or trypsin/chymotrypsin) to a level comparable with other cereals. At a concentration of 100 mM, other reducing agents (dithiothreitol, sodium bisulfite, and L-cysteine) were equally effe...

  14. Protease activation in glycerol-based deep eutectic solvents

    OpenAIRE

    Zhao, Hua; Baker, Gary A.; Holmes, Shaletha

    2011-01-01

    Deep eutectic solvents (DESs) consisting of mixtures of a choline salt (chloride or acetate form) and glycerol are prepared as easily accessible, biodegradable, and inexpensive alternatives to conventional aprotic cation-anion paired ionic liquids. These DES systems display excellent fluidity coupled with thermal stability to nearly 200 °C. In this work, the transesterification activities of cross-linked proteases (subtilisin and α-chymotrypsin), immobilized on chitosan, were individually exa...

  15. Some properties of the intestinal proteases of the rabbitfish, Siganus canaliculatus (Park).

    Science.gov (United States)

    Sabapathy, U; Teo, L H

    1995-06-01

    Some properties of the intestinal proteases of the rabbitfish were examined. At 25°C, both trypsin and chymotrypsin showed pH optima of 8.0. Leucine aminopeptidase, however, displayed maximum activity in the pH range, 7.0-9.0. Leucine aminopeptidase had the highest optimum temperature (60°C), and chymotrypsin, the lowest (30°C). The optimum temperature of trypsin was 55°C. The activation energy, Ea, was found to be 8.24 for trypsin and 8.50 kcal mol(-1) for chymotrypsin. The Ea for leucine aminopeptidase was 6.29 kcal mol(-1) above 40°C and 1.73 kcal mol(-1) below 40°C. Substrate concentration-velocity plots showed that all three enzymes followed Michaelis-Menten kinetics; the Km and Vmax were estimated for the three enzymes. The effects of various protease inhibitors on enzyme activity were also examined and confirmed the protease classes to which each enzyme belonged. The three proteases examined have similar properties to proteases in other fishes.

  16. Development of Enzymes and In Vitro Digestibility during Metamorphosis and Molting of Blue Swimming Crab (Portunus pelagicus

    Directory of Open Access Journals (Sweden)

    Phanu Chamchuen

    2014-01-01

    Full Text Available The work focuses on development of digestive enzymes (amylase, total protease, trypsin, and chymotrypsin and activity ratio of trypsin to chymotrypsin (T/C ratio for digestive efficiency and growth, in blue swimming crab (Portunus pelagicus during metamorphosis and molting. Specific activities of all enzyme parameters studied were associated with growth during metamorphosis, while only those of trypsin and T/C ratio were associated during molting cycle where trypsin and chymotrypsin specific activities associated with consumption rate with especially high levels during late intermolt and early premolt stages. About 50% increased weight gain was observed with at least double increased T/C ratio at the end of molting period, compared to the stages prior to molting. Growth of carapace would be more significant after finishing molting. Carapace width gain and T/C ratio were highest at the first crab stage. Studies of in vitro protein digestibility of different feed raw materials indicated that Artemia, Rotifer, and Moina are the best for larval stages. Otherwise, the use of shrimp feed and Artemia flake could be the alternatives. Incorporating of cassava meal into the feed formula for early adult stage (juvenile could be an advantage. The proteins from animals are more beneficial for adult crab culture than the proteins from plants and bacteria. The digestible quality of dietary protein is very important during larval stages, while the protein level of diet is more important during adult stages with fully developed digestive enzymes.

  17. Digestive enzyme activity during ontogenetic development and effect of live feed in green catfish larvae (Mystus nemurus Cuv. & Val.

    Directory of Open Access Journals (Sweden)

    Vichai Vatanakul

    2012-07-01

    Full Text Available Mystus nemurus is one of the economically important freshwater species fish for aquaculture in Thailand. The aim ofthis study was to describe the development of proteolytic enzymes (pepsin, trypsin and chymotrypsin, carbohydrase (-amylase and lipase, and the influence of live feed during early stage of larval development. Enzymatic assays were conductedfrom day 1 to day 45 after hatching in larvae fed the following feed sequence: Moina sp. during 3-15 day after hatching(DAH, co-feeding between Moina sp. and powder feed during 16-29 DAH and artificial diet during 30-45 DAH. Threesamples of larvae were collected before morning feeding on days 1, 3, 5, 7, 9, 12, 15, 20, 25, 30, 35, 40 and 45. All enzymeswere detected at low level as early as hatching and remained constant until 20 DAH. Significant increases of pepsin, trypsin,chymotrypsin and -amylase activity were detected during 25-45 DAH (p<0.05 whereas lipase activity showed an increaseduring 30-45 DAH, which was related to an increased growth of fish larvae at 30 DAH onward (p<0.05. Live feed, Moina sp.,significantly increased enzyme activity of trypsin, chymotrypsin and -amylase of fish larvae during 3-15 DAH in comparison with those of unfed fish larvae (p<0.05.

  18. Structure basis 1/2SLPI and porcine pancreas trypsin interaction

    Energy Technology Data Exchange (ETDEWEB)

    Fukushima, Kei; Kamimura, Takashi; Takimoto-Kamimura, Midori, E-mail: m.kamimura@teijin.co.jp [Teijin Institute for Bio-Medical Research, 4-3-2 Asahigaoka, Hino-shi, Tokyo 191-8512 (Japan)

    2013-11-01

    1/2SLPI is a C-terminal domain of SLPI (secretory leukocyte protease inhibitor) which inhibits various serine proteases broadly. The present study is the first X-ray structural report on how 1/2SLPI with P1 Leu strongly inhibits trypsin and how it can inhibit multiple serine proteases. SLPI (secretory leukocyte protease inhibitor) is a 107-residue protease inhibitor which inhibits various serine proteases, including elastase, cathepsin G, chymotrypsin and trypsin. SLPI is obtained as a multiple inhibitor in lung defense and in chronic airway infection. X-ray crystal structures have so far reported that they are full-length SLPIs with bovine α-chymotrypsin and 1/2SLPI (recombinant C-terminal domain of SLPI; Arg58–Ala107) with HNE (human neutrophil elastase). To understand the role of this multiple inhibitory mechanism, the crystal structure of 1/2SLPI with porcine pancreas trypsin was solved and the binding modes of two other complexes compared. The Leu residue surprisingly interacts with the S1 site of trypsin, as with chymotrypsin and elastase. The inhibitory mechanism of 1/2SLPI using the wide primary binding site contacts (from P2′ to P5) with various serine proteases is discussed. These inhibitory mechanisms have been acquired in the evolution of the protection system for acute inflammatory diseases.

  19. Enzymatic Hydrolysis Does Not Reduce the Biological Reactivity of Soybean Proteins for All Allergic Subjects.

    Science.gov (United States)

    Panda, Rakhi; Tetteh, Afua O; Pramod, Siddanakoppalu N; Goodman, Richard E

    2015-11-01

    Many soybean protein products are processed by enzymatic hydrolysis to attain desirable functional food properties or in some cases to reduce allergenicity. However, few studies have investigated the effects of enzymatic hydrolysis on the allergenicity of soybean products. In this study the allergenicity of soybean protein isolates (SPI) hydrolyzed by Alcalase, trypsin, chymotrypsin, bromelain, or papain was evaluated by IgE immunoblots using eight soybean-allergic patient sera. The biological relevance of IgE binding was evaluated by a functional assay using a humanized rat basophilic leukemia (hRBL) cell line and serum from one subject. Results indicated that hydrolysis of SPI by the enzymes did not reduce the allergenicity, and hydrolysis by chymotrypsin or bromelain has the potential to increase the allergenicity of SPI. Two-dimensional (2D) immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the chymotrypsin-hydrolyzed samples indicated fragments of β-conglycinin protein are responsible for the apparent higher allergenic potential of digested SPI. PMID:26447491

  20. Digestive peptidase evolution in holometabolous insects led to a divergent group of enzymes in Lepidoptera

    KAUST Repository

    Dias, Renata O.

    2015-03-01

    © 2015 Elsevier Ltd. Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

  1. Effects of dietary fish oil replacement by vegetable oil on the digestive enzymes activity and intestinal morphology in Meagre, Argyrosomus regius (Asso, 1801

    Directory of Open Access Journals (Sweden)

    Fernando Antunes Magalhães

    2014-07-01

    The results were analyzed by three way factorial. Amylase activity was bigger in FO when compared with VO (Table 1. The same result was observed in chymotrypsin activity. On the other hand, lipase activity was higher in VO. Regarding the levels of lipids, diets with 17% had higher amylase activity than diets with 12%. The inverse was observed in chymotrypsin activity. In relation to lipase activity, no differences were observed on the two levels of lipids studied. No differences in digestive enzymes activities were observed when diets were supplemented with selenium. Epithelium architecture of the posterior intestine was slightly affected by dietary treatments. Higher levels of lipids seem to induce enterocyte vacuolization, and vacuoles seem to be larger when a blend of vegetable oils was used instead of fish oil. No clear role can be attributed to selenium regarding intestinal morphology. In conclusion, our study showed that the source and levels of lipid in diets for meagre have influence in activity of digestible enzymes like amylase, lipase and chymotrypsin. Furthermore, levels of selenium do not cause an alteration in studied digestible enzymes.

  2. Extracellular proteolysis of apolipoprotein E (apoE by secreted serine neuronal protease.

    Directory of Open Access Journals (Sweden)

    Irfan Y Tamboli

    Full Text Available Under normal conditions, brain apolipoprotein E (apoE is secreted and lipidated by astrocytes, then taken up by neurons via receptor mediated endocytosis. Free apoE is either degraded in intraneuronal lysosomal compartments or released. Here we identified a novel way by which apoE undergoes proteolysis in the extracellular space via a secreted neuronal protease. We show that apoE is cleaved in neuronal conditioned media by a secreted serine protease. This apoE cleavage was inhibited by PMSF and α1-antichymotrypsin, but not neuroserpin-1 or inhibitors of thrombin and cathepsin G, supporting its identity as a chymotrypsin like protease. In addition, apoE incubation with purified chymotrypsin produced a similar pattern of apoE fragments. Analysis of apoE fragments by mass spectrometry showed cleavages occurring at the C-terminal side of apoE tryptophan residues, further supporting our identification of cleavage by chymotrypsin like protease. Hippocampal neurons were more efficient in mediating this apoE cleavage than cortical neurons. Proteolysis of apoE4 generated higher levels of low molecular weight fragments compared to apoE3. Primary glial cultures released an inhibitor of this proteolytic activity. Together, these studies reveal novel mechanism by which apoE can be regulated and therefore could be useful in designing apoE directed AD therapeutic approaches.

  3. A Study on the Inhibitory Potency of Protease Inhibitors in Porcine Colostrum

    Institute of Scientific and Technical Information of China (English)

    ZHOU Qi; HE Rui-guo; LI Xiang; LIAO Sheng-rong; ZHOU Wu; DU Jin-ping; KONG Ni-jia

    2003-01-01

    Porcine colostrum and milk were separated into the acid-soluble fraction (SF) and casein frac-tion (CF) by centrifuge. Trypsin and chymotrypsin inhibitory capacity in porcine colostrum, milk and theircomponents were determined by incubating bovine trypsin or chymotrypsin in a medium. The inhibition of in-sulin-like growth factor Ⅰ (IGF-Ⅰ) and epidermal growth factor (EGF) degradation in pig small intestinal con-tents by porcine colostrum was measured by incubating iodinated IGF-Ⅰ or EGF. Degradation of labeled IGF-Ⅰor EGF was determined by monitoring the generation of radioactivity soluble in 30% trichloroacetic acid(TCA). The results showed that porcine colostrum had high levels of trypsin and chymotrypsin inhibitory ac-tivity and increased the stability of IGF-Ⅰ and EGF in pig intestinal contents. The SF was higher in inhibitorypotency than CF. The present study revealed that the protease inhibitors in porcine colostrum, milk-derivedand colostrum-specific, existed mainly in SF.

  4. A Study on Heat Resistance and Initial Characterization of Protease Inhibitors in Porcine Colostrum

    Institute of Scientific and Technical Information of China (English)

    ZHOU Qi; HE Rui-guo; YUAN Qian-hua; LI Xiang; LIAO Sheng-rong; ZHOU Wu; KONG Ni-jia

    2003-01-01

    Porcine colostrum was separated into the acid-soluble fraction (SF) and casein fraction (CF) by acidifying followed by centrifuge. SF was further separated by liquid chromatography and anisotropic membrane filtration. Capacities of the SF or CF of porcine colostrum, to inhibit trypsin and chymotrypsin activity and to inhibit the epidermal growth factor (EGF) degradation in pig small intestinal contents, were determined under different heat treatments. The study showed that trypsin inhibitors in porcine colostrum survived heat treatments of 100℃ water bath for up to 10 min, but exposure to boiling water bath for 30 min significantly decreased the inhibitory activity. Compared with the trypsin inhibitors, the chymotrypsin inhibitors were more heat-sensitive. SF was more heat-sensitive than CF. Separation of the SF of porcine colostrum by liquid chromatography and anisotropic membrane filtration revealed that the porcine colostrum protease inhibitors, those had the capacity to inhibit the trypsin-chymotrypsin activity and enhanced the stability of EGF in the gastrointestinal(GI) lumen of weaned pigs, existed mainly in SF, milk-derived, were a group of heat-labile small proteins with molecular weight of 10 000-50 000.

  5. Expression and inhibitory activity analysis of a 25-kD Bowman-Birk protease inhibitor in rice

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Rice Bowman-Birk inhibitors (RBBI), with one (8 kD) or two homologous domains (16 kD), were found to be effective trypsin inhibitors in vitro. In this study, we demonstrate that the 25-kD protein corresponding to the three-domain RBBI indeed exists in rice in planta, and that the RBBIs are regulated by development and wounding. We also found by inhibitory activity assay that the 3:13 disulfide bond, but not the 4:5 disulfide bond, suppresses the trypsin-inhibitory activity, and the D3 domain of RBBI3-1 has no inhibitory activity against trypsin, chymotrypsin, paparin or subtilisin. Mutation analyses showed that conversion from Lys to Leu or Tyr in the N-terminal P1 site in D1 domain did not create chymotrypsin-inhibitory activity, suggesting that the structure of the reactive loop in D1 domain hinder the new inhibitory specificity at P1 site, and the chymotrypsin-inhibitory activity might need the participation of other structures, e.g. 3:13 disulfide bond.

  6. cDNAs for a chymotrypsinogen-like protein from two strains of Plodia interpunctella.

    Science.gov (United States)

    Zhu, Y C; Oppert, B; Kramer, K J; McGaughey, W H; Dowdy, A K

    1997-12-01

    Gut proteinases are involved in the solubilization and activation of insecticidal toxins produced by Bacillus thuringiensis and may also be involved in resistance development. Approximately threefold lower chymotrypsin-like enzyme activity was observed in a Bt(entomocidus)-resistant strain of the Indianmeal moth, Plodia interpunctella, than that in the Bt-susceptible strain. Because chymotrypsin-like proteinases are involved in Bt protoxin activation in P. interpunctella, we compared cDNA sequences, mRNA expression levels, and genomic DNA for chymotrypsin-like enzymes in Bt-susceptible and Bt-resistant strains of P. interpunctella. To isolate cDNA coding for chymotrypsinogen-like proteinases, a probe was developed using polymerase chain reaction (PCR) amplification of a cDNA library from the Bt-susceptible strain using a vector primer and a degenerate primer corresponding to a conserved sequence in the active site of serine proteinases. This probe was used to screen cDNA libraries from resistant and susceptible strains. Predicted amino acid sequences from cDNA clones of each strain share similarity with sequences of chymotrypsin-like proteinases and are most similar to a chymotrypsin-like proteinase from the tobacco hornworm, Manduca sexta. cDNAs for putative chymotrypsinogen-like proteins, from both Bt-susceptible and Bt-resistant strains of P. interpunctella share an identical open reading frame of 846 nucleotides. The encoded proteins contain amino acid sequence motifs of serine proteinase active sites, disulfide-bridge cysteine residues, and both zymogen activation and signal peptides. A difference between these cDNAs was observed only in the untranslated region where a substitution of guanine for adenine occurred in the Bt-resistant strain. Southern and Northern blotting analyses indicated that there are no major differences in chymotrypsinogen-like genomic organization and mRNA expression in the two strains. These data suggest that chymotrypsinogen

  7. Bowman-Birk protease inhibitor from Vigna unguiculata seeds enhances the action of bradykinin-related peptides.

    Science.gov (United States)

    da Cunha Morales Álvares, Alice; Schwartz, Elisabeth Ferroni; Amaral, Nathalia Oda; Trindade, Neidiane Rosa; Pedrino, Gustavo Rodrigues; Silva, Luciano Paulino; de Freitas, Sonia Maria

    2014-01-01

    The hydrolysis of bradykinin (Bk) by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI) and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M-1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus) ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ. PMID:25361421

  8. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. [Ohio State Univ., Columbus, OH (United States)]|[Lawrence Berkeley Lab., CA (United States)

    1995-12-31

    The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonyl amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.

  9. Heterologous Expression of Three Plant Serpins with Distinct Inhibitory Specificities

    DEFF Research Database (Denmark)

    Dahl, Søren Weis; Rasmussen, Søren Kjærsgård; Hejgaard, Jørn

    1996-01-01

    For the first time, inhibitory plant serpins, including WSZ1 from wheat, BSZ4, and the previously unknown protein BSZx from barley, have been expressed in Escherichia coli, and a procedure for fast purification of native plant serpins has been developed, BSZx, BSZ4, and WSZ1 were assayed for inhi...... favorable P-2 Leu. BSZ4 inhibited cathepsin G (k(a) = 2.7 x 10(4) M(-1) s(-1)) at P-1 Met but was hydrolyzed by trypsin and chymotrypsin. The three plant serpins formed stable SDS-resistant complexes with the proteinases in accordance with the kinetic data......., the apparent rate constant for chymotrypsin inhibition at P-2 (k(a) = 9.4 x 10(5) M(-1) s(-1)) was only four times lower than for trypsin at P-1 (k(a) = 3.9 X 10(6) M(-1) s(-1)), and the apparent inhibition stoichiometries were close to 1. Furthermore, our data suggest that cathepsin G was inhibited by BSZx (k......(a) = 3.9 x 10(6) M(-1) s(-1)) at both the P-1 Arg and P-2 Leu. These results indicate a unique adaptability of the reactive center loop of BSZx. WSZ1 inhibited chymotrypsin (k(a) = 1.1 x 10(5) M(-1) s(-1)) and cathepsin G (k(a) = 7.6 x 10(3) M(-1) s(-1)) at P-2 Gln and not, as for BSZx, at the more...

  10. An Investigation into the Gastrointestinal Stability of Exenatide in the Presence of Pure Enzymes, Everted Intestinal Rings and Intestinal Homogenates.

    Science.gov (United States)

    Sun, Yanan; Wang, Mengshu; Sun, Bingxue; Li, Feng; Liu, Shubo; Zhang, Yong; Zhou, Yan; Chen, Yan; Kong, Wei

    2016-01-01

    The purpose of this study was to investigate the gastrointestinal stability of exenatide to determine the key factor(s) contributing to peptide degradation during the oral delivery process. The effects of pH and various digestive enzymes on the degradation kinetics of exenatide were determined. Moreover, the degradation clearances of peptide were also examined using rat everted intestinal rings and intestinal homogenates from various intestinal locations. Exenatide was comparatively stable within a pH range of 1.2-8. However, obvious degradation was observed in the presence of digestive enzymes. The order of enzymes, in terms of ability to degradate exenatide, was chymotrypsin>aminopeptidase N>carboxypeptidase A>trypsin>pepsin. Chymotrypsin showed the greatest ability to degrade exenatide (half-life t1/2, 5.784×10(-2) h), whereas aminopeptidase N and carboxylpeptidase A gave t1/2 values of 3.53 and 10.16 h, respectively. The degradation of exenatide was found to be peptide concentration- and intestinal site-dependent, with a lower clearance in the upper part of the duodenum and the lower part of the ileum. When using intestinal homogenates as enzyme sources, the order, in terms of peptide degradation ability, was ileum>jejunum>duodenum. However, no significant difference was observed in the remaining peptide concentrations throughout 2 h of incubation, which may be due to the involvement of cytosolic enzymes. These results revealed key factors contributing to peptide degradation, and suggest that the inhibition of chymotrypsin and site-specific delivery of exenatide might be advantageous in overcoming metabolic obstacles during its oral delivery.

  11. Bowman-Birk Protease Inhibitor from Vigna unguiculata Seeds Enhances the Action of Bradykinin-Related Peptides

    Directory of Open Access Journals (Sweden)

    Alice da Cunha M. Álvares

    2014-10-01

    Full Text Available The hydrolysis of bradykinin (Bk by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M−1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ.

  12. Designing artificial enzymes from scratch: Experimental study and mesoscale simulation

    Science.gov (United States)

    Komarov, Pavel V.; Zaborina, Olga E.; Klimova, Tamara P.; Lozinsky, Vladimir I.; Khalatur, Pavel G.; Khokhlov, Alexey R.

    2016-09-01

    We present a new concept for designing biomimetic analogs of enzymatic proteins; these analogs are based on the synthetic protein-like copolymers. α-Chymotrypsin is used as a prototype of the artificial catalyst. Our experimental study shows that in the course of free radical copolymerization of hydrophobic and hydrophilic monomers the target globular nanostructures of a "core-shell" morphology appear in a selective solvent. Using a mesoscale computer simulation, we show that the protein-like globules can have a large number of catalytic centers located at the hydrophobic core/hydrophilic shell interface.

  13. Improving the stability of alpha-conotoxin AuIB through N-to-C cyclization

    DEFF Research Database (Denmark)

    Armishaw, Christopher J; Jensen, Anders A; Balle, Lena D;

    2011-01-01

    Modification of alpha-conotoxin frameworks through cyclization via an oligopeptide linker has previously been shown as an effective strategy for improving in vivo stability. We have extended this strategy by investigating cyclic analogs of alpha-conotoxin AuIB, a selective alpha3beta4 nicotinic...... acetylcholine receptor antagonist, to examine a range of oligopeptide linker lengths on the oxidative formation of disulfide bonds, activity at nicotinic acetylcholine receptors, and stability to degradation by chymotrypsin. Upon non-directed random oxidation, the ribbon isomer formed preferentially...

  14. Human lymphokine-activated killer (LAK) cells: III. Effect of L-phenylalanine methyl ester on LAK cell activation from human peripheral blood mononuclear cells: possible protease involvement of monocytes, natural killer cells and LAK cells.

    Science.gov (United States)

    Leung, K H

    1991-01-01

    We have shown that depletion of monocytes from human peripheral blood mononuclear cells (PBMC) by L-phenylalanine methyl ester (PheOMe) enhanced lymphokine-activated killer cell (LAK) generation by recombinant interleukin-2 (rIL-2) at high cell density. In this study, we have investigated the mechanism of action of PheOMe on LAK activation by using trypsin, chymotrypsin, tosylphenylalaninechloromethanol (TPCK, a chymotrypsin inhibitor), tosyl-L-lysinechloromethane (TLCK, a trypsin inhibitor), phenylalaninol (PheOH), and benzamidine. PBMC were treated with 1-5 mM PheOMe for 40 min at room temperature in combination with the various agents, washed and assessed for their effects on natural killer (NK) activity against K562 cells and monocyte depletion. The treated cells were then cultured with or without rIL-2 for 3 days. LAK cytotoxicity was assayed against 51Cr-labeled K562 and Raji tumor target cells. TPCK at 10 micrograms/ml partially inhibited depletion of monocytes by PheOMe. TLCK did not prevent depletion of monocytes nor inhibition of NK activity induced by PheOMe. TPCK and TLCK inhibited NK activity by themselves. TPCK but not TLCK inhibited rIL-2 induction of LAK cells. On the other hand, PheOH and benzamidine (analogs of PheOMe) lacked any effect on monocyte depletion but abrogated the inhibitory effect of PheOMe on NK activity. They had no effect on rIL-2 activation of LAK activity enhanced by PheOMe. Trypsin potentiated the inhibitory effect of PheOMe on NK activity and monocyte depletion. Trypsin partially inhibited IL-2 activation of LAK activity enhanced by PheOMe. Chymotrypsin had little effect on NK activity but prevented the inhibitory effect of PheOMe on NK activity. It had little effect on monocyte depletion induced by PheOMe. PheOMe was hydrolysed by monocytes and chymotrypsin to Phe and methanol as determined by HPLC. TPCK inhibited hydrolysis of PheOMe by monocytes. Our data suggest that the effects of PheOMe on monocytes, NK cells and LAK

  15. Isomeric control of protein recognition with amino acid- and dipeptide-functionalized gold nanoparticles.

    Science.gov (United States)

    You, Chang-Cheng; Agasti, Sarit S; Rotello, Vincent M

    2008-01-01

    Amino acid and dipeptide-functionalized gold nanoparticles (NPs) possessing L/D-leucine and/or L/D-phenylalanine residues have been constructed in order to target the surfaces of alpha-chymotrypsin (ChT) and cytochrome c (CytC). Isothermal titration calorimetry (ITC) was conducted to evaluate the binding thermodynamics and selectivity of these NP-protein interactions. The chirality of the NP end-groups substantially affects the resultant complex stability, with up to 20-fold differences seen between particles of identical hydrophobicity, demonstrating that structural information from the ligands can be used to control protein recognition. PMID:17972262

  16. Synthesis of Cholecystokinin Peptide CCK-4 Exclusively by Enzymatic Methods

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Summary: The synthesis of CCK-4 (H-Trp-Met-Asp-Phe-NH2) by using enzymes exclusively wasdescribed. As protection group for the amino group we used the Phenylacetyl group (Phac) whichhad been cleaved at the end of the synthesis with Penicillin G Amidase (PGA) without affectingthe peptide bonds. Thus, beginning with Phac-Trp-OH we had successfully synthesized the targetpeptide with following 4 enzymes, α-Chymotrypsin, Papain, Thermolysin and PGA in four reac-tion steps. All reactions were carried out in aqueous buffer in reasonable yields (>65 %). FAB-MS or FD-MS verified the correct molecular mass of all peptides.

  17. Conformations of Proteins in Equilibrium

    Energy Technology Data Exchange (ETDEWEB)

    Micheletti, Cristian; Banavar, Jayanth R.; Maritan, Amos

    2001-08-20

    We introduce a simple theoretical approach for an equilibrium study of proteins with known native-state structures. We test our approach with results on well-studied globular proteins, chymotrypsin inhibitor (2ci2), barnase, and the alpha spectrin SH3 domain, and present evidence for a hierarchical onset of order on lowering the temperature with significant organization at the local level even at high temperatures. A further application to the folding process of HIV-1 protease shows that the model can be reliably used to identify key folding sites that are responsible for the development of drug resistance.

  18. Protein Structure Determination Using Chemical Shifts

    DEFF Research Database (Denmark)

    Christensen, Anders Steen

    In this thesis, a protein structure determination using chemical shifts is presented. The method is implemented in the open source PHAISTOS protein simulation framework. The method combines sampling from a generative model with a coarse-grained force field and an energy function that includes...... chemical shifts. The method is benchmarked on folding simulations of five small proteins. In four cases the resulting structures are in excellent agreement with experimental data, the fifth case fail likely due to inaccuracies in the energy function. For the Chymotrypsin Inhibitor protein, a structure...

  19. Glyphosate on digestive enzymes activity in piava (Leporinus obtusidens

    Directory of Open Access Journals (Sweden)

    Joseânia Salbego

    2014-09-01

    Full Text Available The effects of glyphosate, a nonselective herbicide (1.0 or 5.0mg L-1 on digestive enzymes activity (stomach and intestine were evaluated in juveniles of piava (Leporinus obtusidens after 90 days of exposure. The activity of acid protease, trypsin, chymotrypsin and amylase increased with the increase of glyphosate concentration. These results indicate that glyphosate affects digestive enzyme activities in this species, and may be an indicator of poor nutrient availability when fish survive in herbicide-contaminated water.

  20. Antinutritional factors in anasazi and other pinto beans (Phaseolus vulgaris L.).

    Science.gov (United States)

    Weder, J K; Telek, L; Vozári-Hampe, M; Saini, H S

    1997-01-01

    Antinutritional factors of anasazi bean were compared to traditional pinto bean (Phaseolus vulgaris L.). Anasazi beans contained less (p0.05) in stachyose and raffinose content were found between the two bean types; verbascose was not detected at all. Significant (plectin content were observed between anasazi and pinto bean. The lectins of anasazi beans were classified as non toxic and those of the pinto beans as toxic types. No differences (p>0.05) in inhibitor activity against human and bovine trypsin and chymotrypsin were found between the two bean types. PMID:9527344

  1. Pivotal Role for α1-Antichymotrypsin in Skin Repair*

    OpenAIRE

    Hoffmann, Daniel C.; Textoris, Christine; Oehme, Felix; Klaassen, Tobias; Goppelt, Andreas; Römer, Axel; Fugmann, Burkhard; Davidson, Jeffrey M.; Werner, Sabine; Krieg, Thomas; Eming, Sabine A.

    2011-01-01

    α1-Antichymotrypsin (α1-ACT) is a specific inhibitor of leukocyte-derived chymotrypsin-like proteases with largely unknown functions in tissue repair. By examining human and murine skin wounds, we showed that following mechanical injury the physiological repair response is associated with an acute phase response of α1-ACT and the mouse homologue Spi-2, respectively. In both species, attenuated α1-ACT/Spi-2 activity and gene expression at the local wound site was associated with severe wound h...

  2. A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro

    Directory of Open Access Journals (Sweden)

    Suarez Jorge E

    2000-01-01

    Full Text Available The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720 which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD were found to be critical for peptide binding to erythrocytes.

  3. Serpins in plants and green algae

    DEFF Research Database (Denmark)

    Roberts, Thomas Hugh; Hejgaard, Jørn

    2008-01-01

    . Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary relationship between these serpins and those of plants remains unknown. Plant serpins are potent inhibitors...... of mammalian serine proteinases of the chymotrypsin family in vitro but, intriguingly, plants and green algae lack endogenous members of this proteinase family, the most common targets for animal serpins. An Arabidopsis serpin with a conserved reactive centre is now known to be capable of inhibiting...

  4. Molecular Recognition of Cobalt(III)-ligated Peptides by Serine Proteases: The Role of Electrostatic Effects

    DEFF Research Database (Denmark)

    Bagger, Sven; Wagner, Kim

    1998-01-01

    A series of peptides with a positively charged cobalt(III)-complex group attached to the carboxylate terminal was synthesized. The behaviour of these metallopeptides as acceptor nucleophiles in acyl transfer reactions catalyzed by the three serine proteases bovine pancreatic à-chymotrypsin, porcine...... pancreatic trypsin, and proteinase K from Tritirachium album was examined. The efficiency of the substrates was assessed by kinetic measurement of the partition between aminolysis and hydrolysis. The results are discussed with special reference to coulombic interactions between the metal-ligated substrates...... and charged residues on the enzyme surfaces. The idea of using the metallopeptides in practical enzymatic peptide synthesis is put forward....

  5. Metal organic frameworks mimicking natural enzymes: a structural and functional analogy.

    Science.gov (United States)

    Nath, Ipsita; Chakraborty, Jeet; Verpoort, Francis

    2016-07-25

    In this review, we have portrayed the structure, synthesis and applications of a variety of biomimetic MOFs from an unprecedented angle. Synthetic MOF analogues of five distinct enzymes: phosphotriesterase, hydrogenase, cytochrome P450, chymotrypsin and carbonic anhydrase, have been discussed with their skeletal comparison to actual enzymatic active sites as reference, and an explanation of catalytic pathways from the mechanistic cycle of the corresponding enzymes is depicted. We demonstrated critically each of the five discrete situations by assimilating available benchmark researches in an attempt to provide a concise literature source on the ingenious design strategies and versatile biomimetic applications of this domain of materials. PMID:27251115

  6. Preparation and scaling up of a low phenylalanine enzymatic hydrolysate of bovine whey proteins Preparação e escalonamento de um hidrolisado enzimático de proteínas do soro de leite bovino

    OpenAIRE

    Marilisa Guimarães Lara; Clarice Izumi; Lewis Joel Greene; Luciano Vilela; Osvaldo Freitas

    2005-01-01

    We describe the preparation of pancreatic enzymes hydrolysate of milk whey proteins containing low levels of aromatic amino acids. Pancreatin and trypsin/chymotrypsin (6.3% w/w protein) when used to hydrolyze whey proteins for 27 h at 37±2 ºC, released 74% of the Phe, 100% of the Tyr and 100% of the Trp as free amino acids. Most of the free aromatic amino acids present in 2 kg hydrolysate were separated from the remaining peptides and other amino acids by gel filtration on a 15 liter Sephadex...

  7. Light-induced import of the chromoprotein, phytochrome, into mitochondria

    Science.gov (United States)

    Serlin, B. S.; Roux, S. J.

    1986-01-01

    Mitochondria extracted from plants that were irradiated with actinic light in vivo have associated with them the chromoprotein, phytochrome. This phytochrome retains its native subunit size of 124 kDa after proteolytic treatment of the mitochondria with trypsin and chymotrypsin. This result suggests that phytochrome is not exposed on the outer surface of the outer mitochondrial membrane. Phytochrome, so protected, is not found to be associated with mitochondria derived from unirradiated plants. The possibility that the photoactivation of phytochrome induces a conformational change in its structure which facilitates its transport into the mitochondrion is discussed.

  8. Purification and characterization of bacteriocin like substance produced from bacillus lentus with perspective of a new biopreservative for food preservation

    International Nuclear Information System (INIS)

    Molecular weight of bacteriocin like substance (BLIS) of a new strain of Bacillus lentus 121 was found to be approximately 11 kDa. Purification of BLIS was attained by single step gel exclusion chromatography. BLIS was characterized by studying the inhibitory spectrum. It was active at broad pH range, high temperature and high NaCl concentration and showed sensitivity to proteolytic enzymes like trypsin, alpha-chymotrypsin and papain, the characters desirable for food preservation. BLIS extended the shelf stability of milk upto 21 days as a biopreservative. (author)

  9. Synthesis and extended activity of triazole-containing macrocyclic protease inhibitors

    DEFF Research Database (Denmark)

    Pehere, A.D.; Pietsch, M.; Gütschow, M.;

    2013-01-01

    Peptide-derived protease inhibitors are an important class of compounds with the potential to treat a wide range of diseases. Herein, we describe the synthesis of a series of triazole- containing macrocyclic protease inhibitors pre-organized into a b-strand conformation and an evaluation...... of their activity against a panel of proteases. Acyclic azidoalkyne-based aldehydes are also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards calpain II, cathepsin L and S, and the 20S proteasome chymotrypsin-like activity. Some of the first examples of highly potent...

  10. Investigation of larvae digestive β-glucosidase and proteases of the tomato pest Tuta absoluta for inhibiting the insect development.

    Science.gov (United States)

    Sellami, S; Jamoussi, K

    2016-06-01

    The tomato leaf miner Tuta absoluta is one of the most devastating pests for tomato crops. Digestive proteases and β-glucosidase enzymes were investigated using general and specific substrates and inhibitors. Maximal β-glucosidase and proteolytic activities occurred at temperature and pH optima of 30 and 40°C, 5 and 10-11 unit of pH, respectively. Zymogram analysis showed the presence of distinguished β-glucosidase exhibiting a specific activity of about 183 ± 15 µmol min-1 mg-1. In vitro inhibition experiments suggested that serine proteases were the primary gut proteases. Gel based protease inhibition assays demonstrated that the 28 and 73 kDa proteases might be trypsin-like and chymotrypsin-like enzymes, respectively. Overall gut trypsin-like and chymotrypsin-like activities were evaluated to be about 27.2 ± 0.84 and 1.68 ± 0.03 µmol min-1 mg-1, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that T. absoluta gut serine proteases are responsible for Bacillus thuringiensis Cry insecticidal proteins proteolysis. Additionally, bioassays showed that T. absoluta larvae development was more affected by the β-glucosidases inhibitor (D-glucono-δ-lactone) than the serine proteases inhibitor (soybean trypsin inhibitor). These results are of basic interest since they present interesting data of β-glucosidases and gut serine proteases of T. absoluta larvae. PMID:26898349

  11. Effects of dietary pectin and fat on the small intestinal contents and exocrine pancreas of rats.

    Science.gov (United States)

    Forman, L P; Schneeman, B O

    1980-10-01

    The effects of dietary pectin and fat level on digestive enzyme activities in the pancreas and small intestine and on intestinal bile acid levels were investigated. In unfed rats, dietary pectin did not influence the pancreatic enzymes studied, but a higher level of corn oil in the diet lowered the amylase activity in the pancreas, increased pancreatic lipase activity and slightly lowered the chymotrypsin and trypsin activities. Diet did not change the dry weight of the pancreas. In the fed rats, dietary pectin increased the dry weight of the small gut wash plus the mucosal scraping. Dietary pectin increased the small intestinal lipase and chymotrypsin levels and at the low level of fat only, increased amylase and trypsin activities in the small intestine of fed rats. Intestinal lipase levels were higher and amylase levels lower in rats consuming the high level of corn oil. These results indicate that changes in dietary fat level led to changes in the amylase and lipase content of secreted pancreatic juice and that differences in absorption associated with diets containing pectin could be the result of increased material in the small intestine.

  12. Digestive stimulant action of three Indian spice mixes in experimental rats.

    Science.gov (United States)

    Platel, Kalpana; Rao, Alkananda; Saraswathi, G; Srinivasan, K

    2002-12-01

    The present study examined the favourable influence of three spice mixes derived from a few commonly consumed spices of known digestive stimulant action on digestive enzymes of pancreas and small intestine, and on bile secretion and composition in experimental rats. The common ingredients of these mixes were coriander, turmeric, red chilli, black pepper and cumin, while the spice mix II additionally had ginger, and spice mix III contained onion. All the three spice mixes favourably enhanced the activities of pancreatic lipase, chymotrypsin and amylase when consumed during the diet. In addition, these spice mixes brought about a pronounced stimulation of bile flow and of bile acid secretion. Among the three spice mixes examined, spice mix III which is customized so as to include spices that are desirable from the point of view of stimulation of digestion, had the highest stimulatory influence particularly on bile secretion, bile acid output and the activities of pancreatic enzymes. While activities of pancreatic lipase, amylase and chymotrypsin were elevated by 40, 16 and 77%, respectively, the bile volume as well as the bile acid secretion were almost doubled in spice mix III treatment. The higher secretion of bile especially with an elevated level of bile acids and a beneficial stimulation of pancreatic digestive enzymes, particularly of lipase could probably be the two mechanisms by which these combinations of spices aid in digestion.

  13. Proteolytically-induced changes of secondary structural protein conformation of bovine serum albumin monitored by Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy

    Science.gov (United States)

    Güler, Günnur; Vorob'ev, Mikhail M.; Vogel, Vitali; Mäntele, Werner

    2016-05-01

    Enzymatically-induced degradation of bovine serum albumin (BSA) by serine proteases (trypsin and α-chymotrypsin) in various concentrations was monitored by means of Fourier transform infrared (FT-IR) and ultraviolet circular dichroism (UV-CD) spectroscopy. In this study, the applicability of both spectroscopies to monitor the proteolysis process in real time has been proven, by tracking the spectral changes together with secondary structure analysis of BSA as proteolysis proceeds. On the basis of the FTIR spectra and the changes in the amide I band region, we suggest the progression of proteolysis process via conversion of α-helices (1654 cm- 1) into unordered structures and an increase in the concentration of free carboxylates (absorption of 1593 and 1402 cm- 1). For the first time, the correlation between the degree of hydrolysis and the concentration of carboxylic groups measured by FTIR spectroscopy was revealed as well. The far UV-CD spectra together with their secondary structure analysis suggest that the α-helical content decreases concomitant with an increase in the unordered structure. Both spectroscopic techniques also demonstrate that there are similar but less spectral changes of BSA for the trypsin attack than for α-chymotrypsin although the substrate/enzyme ratio is taken the same.

  14. Identification of a new series of amides as non-covalent proteasome inhibitors.

    Science.gov (United States)

    Scarbaci, Kety; Troiano, Valeria; Micale, Nicola; Ettari, Roberta; Tamborini, Lucia; Di Giovanni, Carmen; Cerchia, Carmen; Grasso, Silvana; Novellino, Ettore; Schirmeister, Tanja; Lavecchia, Antonio; Zappalà, Maria

    2014-04-01

    Proteasome inhibition has emerged as an important therapeutic strategy for the treatment of multiple myeloma (MM) and some forms of lymphoma, with potential application in other types of cancers. 20S proteasome consists of three different catalytic activities known as chymotrypsin-like (ChT-L), trypsin-like (T-L), and, post-glutamyl peptide hydrolyzing (PGPH) or caspase-like (C-L), which are located respectively on the β5, β2, and β1 subunits of each heptameric β rings. Currently a wide number of covalent proteasome inhibitors are reported in literature; however, the less widely investigated non-covalent inhibitors might be a promising alternative to employ in therapy, because of the lack of all drawbacks and side-effects related to irreversible inhibition. In the present work we identified a series of amides, two of which (1b and 1f) are good candidates to non-covalent inhibition of the chymotrypsin-like activity of the β5 proteasome subunit. The non-covalent binding mode was corroborated by docking simulations of the most active inhibitors 1b, 1f and 2h into the yeast 20S proteasome crystal structure. PMID:24561716

  15. The cytotoxic effect of Bowman-Birk isoinhibitors, IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases.

    Science.gov (United States)

    Clemente, Alfonso; Moreno, Francisco Javier; Marín-Manzano, Maria del Carmen; Jiménez, Elisabeth; Domoney, Claire

    2010-03-01

    Bowman-Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health-promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non-malignant colonic fibroblast CCD-18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9+/-2.3 and 48.3+/-3.5 microM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose-dependent manner, with cells becoming blocked in the G0-G1 phase. Chemically inactive soybean BBI had a weak but non-significant effect on the proliferation of HT29 cells. The anti-proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI-like proteins. PMID:19885848

  16. Development of digestive enzyme activity in spotted rose snapper, Lutjanus guttatus (Steindachner, 1869) larvae.

    Science.gov (United States)

    Moguel-Hernández, I; Peña, R; Nolasco-Soria, H; Dumas, S; Zavala-Leal, I

    2014-06-01

    We describe digestive enzyme activity during the larval development of spotted rose snapper, Lutjanus guttatus. Trypsin, chymotrypsin, leucine aminopeptidase, pepsin, amylase, lipase, and acid and alkaline phosphatase activities were evaluated using spectrophotometric techniques from hatching through 30 days. The spotted rose snapper larvae present the same pattern of digestive enzyme activity previously reported for other species in which pancreatic (i.e., trypsin, chymotrypsin, amylase, and lipase) and intestinal (i.e., acid and alkaline phosphatases and leucine aminopeptidase) enzymatic activities are present from hatching allowing the larvae to digest and absorb nutrients in the yolk-sac and live prey by the time of first feeding. The digestive and absorption capacity of the spotted rose snapper increases during the larval development. A significant increase in individual activity of all enzymes occurs at 20 DAH, and around 25 DAH, the juvenile-type of digestion is observed with the appearance of pepsin secreted by the stomach, suggesting that maturation of the digestive function occurs around 20-25 DAH. Our results are in agreement with a previous suggestion that early weaning may be possible from 20 DAH. However, the patterns of enzymatic activities reported in our study should be considered during the formulation of an artificial diet for early weaning of the spotted rose snapper.

  17. Bacillus thuringiensis endotoxins active against Chilo partellus and Glossina morsitans morsitans

    International Nuclear Information System (INIS)

    Bacillus thuringiensis crystal endotoxins were isolated by centrifugation on linear sucrose gradients. Analysis of the crystals by gel electrophoresis revealed that the major component of the Chilo partellus active crystal endotoxin was a protein of Mr ∼ 130 kilodalton. The Glossina morsitans morsitans active crystal endotoxin gave a major protein band of Mr ∼ 120 kilodalton. Upon solubilization under alkaline pH and reducing conditions, the C. partellus and G. m. morsitans crystal endotoxin yielded protoxins of Mr ∼ 63 and Mr ∼ 64 kilodalton, respectively. Activation of the C. partellus protoxin with bovine trypsin resulted in no apparent change in the molecular weight. However, treatment with bovine chymotrypsin or C. partellus midgut homogenate resulted in a shift in the molecular weight of the protoxin to a toxin of Mr ∼ 60 kilodalton. Similarly, treatment of G.m. morsitans protoxin with bovine trypsin gave a toxin of Mr ∼ 62 kilodalton, but bovine chymotrypsin gave a toxin of Mr ∼ 60 kilodalton. Staining with periodic acid Schiff reagent revealed that both the crystal endotoxins were glycosylated. The carbohydrate moieties were of the high mannose type, as shown by staining with fluorescein isothiocyanate conjugated-concanavalin A. Rabbit antibodies against C. partellus protoxin cross-reacted with the G. m. morsitans toxin. (author). 19 refs, 5 figs

  18. Caldecrin:A pancreas-derived hypocalcemic factor,regulates osteoclast formation and function

    Institute of Scientific and Technical Information of China (English)

    Mineko; Tomomura; Akito; Tomomura

    2015-01-01

    Caldecrin was originally isolated from the pancreas as afactor that reduced serum calcium levels. This secreted serine protease has chymotrypsin-like activity and is also known as chymotrypsin C; it belongs to the elastase family. Although intravenous administration of caldecrin decreases the serum calcium concentration even when its protease activity is blocked,this effect does require cleavage of caldecrin’s pro-peptide by trypsin,converting it to the mature enzyme. Ectopic intramuscular expression of caldecrin prevented bone resorption in ovariectomized mice. Caldecrin inhibited parathyroid hormone-stimulated calcium release from fetal mouse long bone organ cultures. Furthermore,caldecrin suppressed the formation of osteoclasts from bone marrow cells by inhibiting the receptor activator of nuclear factor-k B ligand(RANKL)-stimulated phospholipase Cγ-calcium oscillation-calcineurinnuclear factor of activated T-cells,cytoplasmic 1 pathway. Caldecrin also suppressed the bone resorption activity of mature osteoclasts by preventing RANKL-stimulated Src activation,calcium entry,and actin ring formation. In vivo and in vitro studies have indicated that caldecrin is a unique multifunctional protease with anti-osteoclastogenic activities that are distinct from its protease activity. Caldecrin might be a potential therapeutic target for the treatment of osteolytic diseases such as osteoporosis and osteoarthritis. This mini-review describes caldecrin’s historical background and its mechanisms of action.

  19. Scalable purification of the lantibiotic nisin and isolation of chemical/enzymatic cleavage fragments suitable for semi-synthesis.

    Science.gov (United States)

    Slootweg, Jack C; Liskamp, Rob M J; Rijkers, Dirk T S

    2013-11-01

    Herein, we describe a scalable purification of the lantibiotic nisin via an extraction/precipitation approach using a biphasic system, which can be carried out up to 40-80 gram scale. This approach results in an at least tenfold enrichment of commercially available preparations of nisin, which usually contain only 2.5% of the desired peptide, to allow further purification by preparative HPLC. As a follow-up study, the enriched nisin sample was digested either by trypsin or chymotrypsin, or treated by CNBr, and these reactions were monitored by LC-MS to identify and characterize the obtained fragments. Two previously unknown cleavage sites have been identified: Asn20-Met21 and Met21-Lys22 for trypsin and chymotrypsin, respectively. Furthermore, a novel and convenient enzymatic approach to isolate the native nisin C-ring [nisin fragment (13-20)] was uncovered. Finally, by means of preparative HPLC, nisin fragments (1-12), (1-20), (22-34), and (22-31) could be isolated and will be used in a semi-synthesis approach to elucidate the role of each fragment in the mode of action of nisin as an antimicrobial peptide.

  20. Prolonged ingestion of prehydrolyzed whey protein induces little or no change in digestive enzymes, but decreases glutaminase activity in exercising rats.

    Science.gov (United States)

    Nery-Diez, Ana Cláudia C; Carvalho, Iara R; Amaya-Farfán, Jaime; Abecia-Soria, Maria Inés; Miyasaka, Célio K; Ferreira, Clécio da S

    2010-08-01

    Because consumption of whey protein hydrolysates is on the increase, the possibility that prolonged ingestion of whey protein hydrolysates affect the digestive system of mammals has prompted us to evaluate the enzymatic activities of pepsin, leucine-aminopeptidase, chymotrypsin, trypsin, and glutaminase in male Wistar rats fed diets containing either a commercial whey isolate or a whey protein hydrolysate with medium degree of hydrolysis and to compare the results with those produced by physical training (sedentary, sedentary-exhausted, trained, and trained-exhausted) in the treadmill for 4 weeks. The enzymatic activities were determined by classical procedures in all groups. No effect due to the form of the whey protein in the diet was seen in the activities of pepsin, trypsin, chymotrypsin, and leucine-aminopeptidase. Training tended to increase the activity of glutaminase, but exhaustion promoted a decrease in the trained animals, and consumption of the hydrolysate decreased it even further. The results are consistent with the conclusion that chronic consumption of a whey protein hydrolysate brings little or no modification of the proteolytic digestive system and that the lowering of glutaminase activity may be associated with an antistress effect, counteracting the effect induced by training in the rat. PMID:20482282

  1. Separation and purification of enzymes by continuous pH-parametric pumping

    Energy Technology Data Exchange (ETDEWEB)

    Huang, S.Y.; Lin, C.K.; Juang, L.Y.

    1985-10-01

    Trypsin and chymotrypsin were separated from porcine pancreas extract by continuous pH-parametric pumping. CHOM (chicken ovomucoid) was convalently bound to laboratory-prepared crab chitin with glutaraldehyde to form an affinity adsorbent of trypsin. The pH levels of top and bottom feeds were 8.0 and 2.5, respectively. Similar inhibitor, DKOM (duck ovomucoid), and pH levels 8.0 and 2.0 for top and bottom feeds, respectively, were used for separation and purification of chymotrypsin. e-Amino caproyl-D-tryptophan methyl ester was coupled to chitosan to form an affinity adsorbent for stem bromelain. The pH levels were 8.7 and 3.0. Separation continued fairly well with high yield, e.g., 95% recovery of trypsin after continuous pumping of 10 cycles. Optimum operational conditions for concentration and purification of these enzymes were investigated. The results showed that the continuous pH-parametric pumping coupled with affinity chromatography is effective for concentration and purification of enzymes. 19 references.

  2. Purification and properties of two protease inhibitors from rat skin inhibiting papain and other SH-proteases.

    Science.gov (United States)

    Järvinen, M

    1976-01-01

    Two papain inhibitors, I1 and I2, from rat skin extract were purified by affinity chromatography on KSCN-modified papain-agarose gel and by gel filtration on Sephadex G-100. I1 had a molecular weight of 74 000, a pI of 4.6, and it contained 4% of carbohydrates. I1 inhibited papain, ficin, bromelain, rat skin benzoylarginine-2-naphthylamide hydrolase, and to a minor extent, rat skin cathepsin C and bovine trypsin. Bovine chymotrypsin or rat skin cathepsin D were not inhibited and benzoylarginine-2-naphthylamide hydrolase was inhibited only at alkaline pH. An inhibitor corresponding to I1 was present in various rat tissues and also in serum. A similar inhibitor was present in the skin of cat, rabbit, guinea pig, and man. I2 had a molecular weight of 13 400, a pI of 4.9 and it contained no carbohydrates. I2 inhibited all thiol proteases tested, but not trypsin, chymotrypsin, or rat skin cathepsin D. I2 formed an equimolar complex with papain and benzoylarginine-2-naphthylamide hydrolase. I2 was present in rat skin, muscle, lung, and small intestine, but not in kidney, liver, or serum. A similar inhibitor was found in skin extracts of cat, rabbit, guinea pig, and man.

  3. Mutagen formation in a model beef boiling system. II. Effects of proteolysis and comparison of soluble fractions from several protein sources

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, R.T.; Shore, V.; Fultz, E.

    1984-01-01

    Fried beef and commercial beef extracts contain Ames/Salmonella frameshift mutagens that form at relatively low temperatures (100-200/sup 0/C). To investigate the types of natural components in beef muscle that give rise to frameshift mutagenic activity, a model boiling system was devised and it was demonstrated that all of the Salmonella TA1538 activity is formed from H/sub 2/O-soluble, < 500 molecular wt compounds that are present in round steak supernatant fractions (S/sub 1/ and S/sub 2/). S/sub 2/ is derived from S/sub 1/, the soluble fraction of homogenized beef, by a brief 30 min boil and centrifugation. The authors report that proteolysis of beef S/sub 1/ with papain, trypsin, or chymotrypsin (+/- carboxypeptidase A) increases the mutagenic activity of boiled S/sub 2/ by 1.8-4.5 fold over the baseline range of 90-100 TA1538 revertants/10/sup 8/ bacteria/g dry beef/14 h at pH 4.0. Sequential treatment of beef S/sub 1/ with chymotrypsin followed by carboxypeptidase A is the most efficient mutagen enhancing digestion (415 revertants/g dry beef or 7.7 revertants/mg of S/sub 2/ soluble protein). 56 references, 7 figures, 5 tables.

  4. Influence of the spacer on the inhibitory effect of different polycarbophil-protease inhibitor conjugates.

    Science.gov (United States)

    Marschütz, M K; Veronese, F M; Bernkop-Schnürch, A

    2001-09-01

    Within the present study various polycarbophil (PCP)-serine protease inhibitor conjugates were synthesized and the influence of different spacers on their inhibitory efficacy was evaluated in vitro. Results demonstrated that 4.2+/-0.15 units (n=3; +/-SD) of alpha-chymotrypsin were inhibited by 50% utilizing 0.86% (w/v) of a PCP-tetramethylenediamine (TMDA)-chymostatin 20:1 conjugate. In contrast, only 0.6+/-0.05 units (n=3; +/-SD) of alpha-chymotrypsin were inhibited by a corresponding PCP-poly(ethylene glycol) (PEG)-chymostatin conjugate. Inhibitory effects of PCP-TMDA-antipain and -elastatinal conjugates towards trypsin and elastase, respectively, were also significantly higher (P<0.05) than those of corresponding PCP-PEG-inhibitor conjugates. Hence, the great impact of the molecular size as well as the structure of the spacer on resulting polymer-inhibitor conjugates could be demonstrated. The small and rigid C4-spacer TMDA (molecular weight (MW) 161.1) was thereby shown to be highly advantageous over a long, hydrophilic and flexible PEG-diamine spacer (MW 3400). Results obtained should provide helpful basic knowledge for the development of mucoadhesive polymer-inhibitor conjugates used as auxiliary agents for the oral administration of peptide drugs.

  5. Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

    Science.gov (United States)

    García-Fernández, Rossana; Ziegelmüller, Patrick; González, Lidice; Mansur, Manuel; Machado, Yoan; Redecke, Lars; Hahn, Ulrich; Betzel, Christian; Chávez, María de Los Ángeles

    2016-07-01

    The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity. PMID:26993255

  6. Early Changes in Costameric and Mitochondrial Protein Expression with Unloading Are Muscle Specific

    Directory of Open Access Journals (Sweden)

    Martin Flück

    2014-01-01

    Full Text Available We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL and soleus (SOL muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS and subjected to fibre typing and measures for costameric (FAK and FRNK, mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1, and MHCI protein and RNA content. Mean cross-sectional area (MCSA of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05≤P<0.10. FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P=0.029. SOL muscle demonstrated a specific reduction in UCP3 content (-23%; P = 0.012. Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

  7. Early changes in costameric and mitochondrial protein expression with unloading are muscle specific.

    Science.gov (United States)

    Flück, Martin; Li, Ruowei; Valdivieso, Paola; Linnehan, Richard M; Castells, Josiane; Tesch, Per; Gustafsson, Thomas

    2014-01-01

    We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS) and subjected to fibre typing and measures for costameric (FAK and FRNK), mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1), and MHCI protein and RNA content. Mean cross-sectional area (MCSA) of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05 ≤ P < 0.10). FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P = 0.029). SOL muscle demonstrated a specific reduction in UCP3 content (-23%; P = 0.012). Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

  8. Proteolytic Activation of Bacillus thuringiensis Cry2Ab through a Belt-and-Braces Approach.

    Science.gov (United States)

    Xu, Lian; Pan, Zhi-Zhen; Zhang, Jing; Liu, Bo; Zhu, Yu-Jing; Chen, Qing-Xi

    2016-09-28

    Proteolytic processing of Bacillus thuringiensis (Bt) crystal toxins by insect midgut proteases plays an essential role in their insecticidal toxicities against target insects. In the present study, proteolysis of Bt crystal toxin Cry2Ab by Plutella xylostella L. midgut proteases (PxMJ) was evaluated. Both trypsin and chymotrypsin were identified involving the proteolytic activation of Cry2Ab and cleaving Cry2Ab at Arg(139) and Leu(144), respectively. Three Cry2Ab mutants (R139A, L144A, and R139A-L144A) were constructed by replacing residues Arg(139), Leu(144), and Arg(139)-Leu(144) with alanine. Proteolysis assays revealed that mutants R139A and L144A but not R139A-L144A could be cleaved into 50 kDa activated toxins by PxMJ. Bioassays showed that mutants R139A and L144A were highly toxic against P. xylostella larvae, while mutant R139A-L144A was almost non-insecticidal. Those results demonstrated that proteolysis by PxMJ was associated with the toxicity of Cry2Ab against P. xylostella. It also revealed that either trypsin or chymotrypsin was enough to activate Cry2Ab protoxin. This characteristic was regarded as a belt-and-braces approach and might contribute to the control of resistance development in target insects. Our studies characterized the proteolytic processing of Cry2Ab and provided new insight into the activation of this Bt toxin.

  9. Serpins in fruit and vegetative tissues of apple (Malus domestica): expression of four serpins with distinct reactive centres and characterisation of a major inhibitory seed form, MdZ1b

    DEFF Research Database (Denmark)

    Hejgaard, Jørn; Laing, W.A.; Marttila, S.;

    2005-01-01

    Most serpins irreversibly inhibit serine proteinases of the chymotrypsin family using a suicide-substrate-based mechanism. Serpins are present in all domains of life, but physiological functions in the plant kingdom are yet to be elucidated. Inhibitory properties of many abundant cereal grain...... and characterised kinetically. MdZ1b was a fast inhibitor of bovine and porcine trypsin (second-order association rate constant k(a) similar to 4 x 10(6) M-1 s(-1) and stoichiometry of inhibition SI = 1). Human plasmin and urokinase-type plasminogen activator (u-PA), but not thrombin, were inhibited at lower rates...... (k(a) similar to 10(4) M-1 s(-1)). Chymotrypsin was inhibited at the same site (k(a) similar to 4 x 10(3) M-1 s(-1)), but a significant part of MdZ1b was cleaved as substrate (SI > 2). Unexpectedly, the MdZ1b-trypsin complex was relatively short-lived with a first-order dissociation rate constant k...

  10. Inducible expression pattern of rice Bowman-Birk inhibitor gene Os WIP1-2 and its protease inhibitory activity

    Institute of Scientific and Technical Information of China (English)

    CHEN Jun; LIU Jing; GUO Lei; QU Lijia; CHEN Zhangliang; GU Hongya

    2004-01-01

    The WIP1-2 gene was cloned from rice. It belongs to the Bowman-Birk inhibitor gene family. Northern blot showed that expression of this gene was induced by wounding and jasmonic acid (JA). It indicates that the OsWIP1 gene plays an important role in the rice defense system. The OsWIP1-2 was cloned into pET28a and expressed in E. Coli. Its expressed product was purified in the form of fusion protein and tested for the inhibitory activities against trypsin and chymotrypsin. It was found that the fusion protein could inhibit chymotrypsin, but not trypsin. It was also found that the His tag at its C-terminal affected its inhibitory activity significantly. The fusion protein with a natural C-terminal had the inhibitory activity, while no inhibitory activity was detected in the fusion protein with a (His)6-tag at its C-terminal. This implies that extra amino acid residues at the C-terminal of OsWIP1-2 may interfere with its correct folding. The inhibitory assay indicated that the members of rice Bowman-Birk inhibitor gene family probably differentiated both in their structure and function.

  11. High-level expression and characterization of two serine protease inhibitors from Trichinella spiralis.

    Science.gov (United States)

    Zhang, Zhaoxia; Mao, Yixian; Li, Da; Zhang, Yvhan; Li, Wei; Jia, Honglin; Zheng, Jun; Li, Li; Lu, Yixin

    2016-03-30

    Serine protease inhibitors (SPIs) play important roles in tissue homeostasis, cell survival, development, and host defense. So far, SPIs have been identified from various organisms, such as animals, plants, bacteria, poxviruses, and parasites. In this study, two SPIs (Tsp03044 and TspAd5) were identified from the genome of Trichinella spiralis and expressed in Escherichia coli. Sequence analysis revealed that these two SPIs contained essential structural motifs, which were well conserved within the tumor-infiltrating lymphocytes (TIL) and serpin superfamily. Based on protease inhibition assays, the recombinant Tsp03044 showed inhibitory effects on trypsin, α-chymotrypsin, and pepsin, while the recombinant TspAd5 could effectively inhibit the activities of α-chymotrypsin and pepsin. Both these inhibitors showed activity between 28 and 48 °C. The expression levels of the two SPIs were also determined at different developmental stages of the parasite with real-time PCR. Our results indicate that Tsp03044 and TspAd5 are functional serine protease inhibitors. PMID:26921036

  12. Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm.

    Directory of Open Access Journals (Sweden)

    Jizhen Wei

    Full Text Available Crystalline (Cry proteins from Bacillus thuringiensis (Bt are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50 for a laboratory-selected resistant strain (LF120 divided by the LC50 for its susceptible parent strain (LF, the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

  13. Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm.

    Science.gov (United States)

    Wei, Jizhen; Liang, Gemei; Wang, Bingjie; Zhong, Feng; Chen, Lin; Khaing, Myint Myint; Zhang, Jie; Guo, Yuyuan; Wu, Kongming; Tabashnik, Bruce E

    2016-01-01

    Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins. PMID:27257885

  14. Proteolysis of bovine beta-lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature-modified protein and yields fragments with low immunoreactivity

    DEFF Research Database (Denmark)

    Iametti, S.; Rasmussen, P.; Frøkiær, Hanne;

    2002-01-01

    Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 C at neutral pH. At these temperatures beta-lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, beta-lactoglobulin......Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 C at neutral pH. At these temperatures beta-lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, beta......-lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. High-temperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large-sized breakdown intermediates...... formed in the early steps of hydrolysis indicated that both enzymes effectively hydrolyzed some regions of beta-lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein. The immunochemical properties of the products of beta...

  15. Pathogenesis of mucosal injury in the blind loop syndrome.

    Science.gov (United States)

    Jonas, A; Krishnan, C; Forstner, G

    1978-11-01

    Bacterial extracts were prepared from cultures originating in chronic self-filling intestinal blind loops in rats. Their ability to remove active maltase molecules from isolated brush border membranes was studied in vitro. Twelve strains in 51 tested, belonging to one of three species, Bacteroides fragilis, Clostridium perfringens, and Streptococcus fecalis, possessed maltase-releasing activity. The ability to remove maltase correlated well with the ability to hydrolyze p-nitrophenyl-tert-butyloxycarbonyl-l-alaninate (NBA), an ester substrate rapidly hydrolyzed by elastase, but not with substrated favored by tryhsin and chymotrypsin. Maltase-releasing activity from C. perfringens was strongly inhibited by soybean trypsin inhibitor and to a lesser extent by lima bean trypsin inhibitor. Of four chloromethylketone active-site directed inhibitors tested with specificities for elastase, trypsin, and chymotrypsin, inhibition was maximal with elastase-specific inhibitors. In two species, activity was shown to be heat sensitive, and to be inhibited by concentration of the extract. In one species maltase-releasing activity was shown to be due to an enzyme of molecular weight at least 66,000 with the capacity to remove lactase, sucrase, and alkaline phosphatase, as well as maltase. The results indicate that anaerobic or facultatively anaerobic species, previously identified with the pathology of of the blind loop syndrome, contain proteases which are capable of removing components of the intestinal surface membrane. These proteases appear to have elastase-like substrate specificity and may be involved in the etiology of disaccharidase deficiency in bacterial overgrowth syndromes.

  16. Mechanisms of evasion of Schistosoma mansoni schistosomula to the lethal activity of complement

    Directory of Open Access Journals (Sweden)

    F. Juarez Ramalho-Pinto

    1992-01-01

    Full Text Available Schistosomula of Schistosoma mansoni became resistant to antibody-dependent complement damage in vitro after pre-incubation with normal human erythrocytes (NHuE whatever the ABO or Rh blood group. Resistant parasites were shown to acquire host decay accelerating factor (DAF , a 70 kDa glycoprotein attached to the membrane of NHue by a GPI anchor. IgG2a mAb anti-human DAF (IA10 immunoprecipitated a 70 kDa molecule from 125I-labeled schistosomula pre-incubated with NHuE and inhibited their resistance to complement-dependent killing in vtro. Incubationof schistosomula with erytrocytes from patients with paroxsimal nocturnal hemoglobinuria (PNHE or SRBC, wich are DAF-deficient, did not protect the parasites from complement lesion. Supernatant of 100,000 x g collected from NHuE incubated for 24 h in defined medium was shown to contain a soluble form of DAF and to protect schistosomula from complement killing. Schistosomula treated with trypsin before incubation with NHuE ghosts did not become resistant to complement damage. On the other hand, pre-treatment with chymotrypsin did not interfere with the acquisition of resistance by the schistosomula. These results indicate that, in vitro, NHuE DAF can be transferred to schistosomula in a soluble form and that the binding of this molecule to the parasite surface is dependent upon trypsin-sensitive chymotrypsin-insensitive polipeptide(s present on the surface of the worm.

  17. PEGylated human plasma fibronectin is proteolytically stable, supports cell adhesion, cell migration, focal adhesion assembly, and fibronectin fibrillogenesis.

    Science.gov (United States)

    Zhang, Chen; Hekmatfar, Sogol; Ramanathan, Anand; Karuri, Nancy W

    2013-01-01

    Delayed wound healing in many chronic wounds has been linked to the degradation of fibronectin (FN) by abnormally high protease levels. We sought to develop a proteolytically stable and functionally active form of FN. For this purpose, we conjugated 3.35 kDa polyethylene glycol diacrylate (PEGDA) to human plasma fibronectin (HPFN). Conjugation of PEGDA to HPFN or HPFN PEGylation was characterized by an increase of approximately 16 kDa in the average molecular weight of PEGylated HPFN compared to native HPFN in SDS-PAGE gels. PEGylated HPFN was more resistant to α chymotrypsin or neutrophil elastase digestion than native HPFN: after 30 min incubation with α chymotrypsin, 56 and 90% of native and PEGylated HPFN respectively remained intact. PEGylated HPFN and native HPFN supported NIH 3T3 mouse fibroblast adhesion and spreading, migration and focal adhesion formation in a similar manner. Fluorescence microscopy showed that both native and PEGylated HPFN in the culture media were assembled into extracellular matrix (ECM) fibrils. Interestingly, when coated on surfaces, native but not PEGylated HPFN was assembled into the ECM of fibroblasts. The proteolytically stable PEGylated HPFN developed herein could be used to replenish FN levels in the chronic wound bed and promote tissue repair.

  18. Antioxidant and antimicrobial activity of lecithin free egg yolk protein preparation hydrolysates obtained with digestive enzymes

    Directory of Open Access Journals (Sweden)

    Aleksandra Zambrowicz

    2012-12-01

    Full Text Available ABSTRACT:Several biological activities have now been associated with egg protein- derived peptides, including antihypertensive, antimicrobial, immunomodulatory, anticancer and antioxidantactivities, highlighting the importance of these biopeptides in human health, and disease prevention and treatment. Special attention has been given to peptides with antioxidant and antimicrobial activities as a new source of natural preservatives in food industry. In this study, the antioxidant properties of the egg-yolk protein by-product (YP hydrolysates were evaluated based on their radical scavenging capacity (DPPH, Fe2+chelating effect and ferric reducing power (FRAP. Furthermore, antimicrobial properties of obtained hydrolysates against Bacillus species were studied. The degrees (DHs of hydrolysis for 4h hydrolysates were: 19.1%, 13.5% and 13.0%, for pepsin, chymotrypsin and trypsin, respectively. Pepsin was the most effective in producing the free amino groups (1410.3 μmolGly/g. The RP-HPLC profiles of the protein hydrolysates showed differences in the hydrophobicity of the generated peptides.Trypsin hydrolysate obtained after 4h reaction demonstrated the strongest DPPH free radical scavenging activity (0.85 µmol Troloxeq/mg. Trypsin and chymotrypsin hydrolysates obtained after 4h reaction exhibited 4 times higher ferric reducing capacity than those treated bypepsin. The hydrolysis products obtained from YP exhibited significant chelating activity. The 4h trypsin hydrolysate exhibited weak antimicrobial activity against B. subtilis B3; B. cereus B512; B. cereus B 3p and B. laterosporum B6.

  19. Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease

    DEFF Research Database (Denmark)

    Studdert, C A; Herrera Seitz, M K; Plasencia, I;

    2001-01-01

    A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.5-12), is rat......A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.......5-12), is rather thermophilic (optimal activity at 60 degrees C in 1-2 M NaCl) and is dependent on high salt concentrations for activity and stability (1-2 M NaCl or KCl). Polyclonal antibodies were raised against the purified protease. In Western blots, they presented no cross-reactivity with culture medium from...... other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests...

  20. Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Surface and enzyme effects.

    Science.gov (United States)

    Ward, Keeran; Xi, Jingshu; Stuckey, David C

    2015-12-01

    The use of non-ionic colloidal liquid aphrons (CLAs) as a support for enzyme immobilisation was investigated. Formulation required the mixing of an aqueous-surfactant solution with a relatively non-polar solvent-surfactant solution, forming a solvent droplet surrounded by a thin stabilised aqueous film (soapy shell). Studies utilising anionic surfactants have showed increased retention, however, very little have been understood about the forces governing immobilisation. This study seeks to determine the effects of enzyme properties on CLA immobilisation by examining a non-ionic/non-polar solvent system comprised of two non-ionic surfactants, Tween 20 and 80, mineral oil and the enzymes lipase, aprotinin and α-chymotrypsin. From these results it was deduced that hydrophobic interactions strongly governed immobilisation. Confocal Scanning Laser Microscopy (CSLM) revealed that immobilisation was predominantly achieved by surface adsorption attributed to hydrophobic interactions between the enzyme and the CLA surface. Enzyme surface affinity was found to increase when added directly to the formulation (pre-manufacture addition), as opposed to the bulk continuous phase (post-manufacture addition), with α-chymotrypsin and aprotinin being the most perturbed, while lipase was relatively unaffected. The effect of zeta potential on immobilisation showed that enzymes adsorbed better closer to their pI, indicating that charge minimisation was necessary for immobilisation. Finally, the effect of increasing enzyme concentration in the aqueous phase resulted in an increase in adsorption for all enzymes due to cooperativity between protein molecules, with saturation occurring faster at higher adsorption rates.

  1. Digestive enzyme activity and mRNA level of trypsin in embryonic redclaw crayfish, Cherax quadricarnatus

    Institute of Scientific and Technical Information of China (English)

    LUO Wen; ZHAO Yunlong; ZHOU Zhongliang; AN Chuanguang; MA Qiang

    2008-01-01

    The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR (FQ-PCR) methods.The results show that the activities of trypsin and chymotrypsin had two different change patterns.Trypsin specific activity increased rapidly in the early stages of development and still remained high in preparation for the hatch stage.However,chymotrypsin activity peaked in stage 4 of embryonic development and decreased significantly in the last stage.The mRNA level of trypsin was elevated in all stages and two peak values were observed in stages 2 and 5 respectively.The results indicate that trypsin is very important for the utilization of the yolk during embryonic development and for the assimilation of dietary protein for larvae.The gene of trypsin is probably regulated at transcriptional level.The mRNA levels of trypsin can reflect not only trypsin activity,but also the regulatory mechanism for expression of trypsin gene to a certain degree.

  2. Proteasomal activities in the claw muscle tissue of European lobster, Homarus gammarus, during larval development.

    Science.gov (United States)

    Götze, Sandra; Saborowski, Reinhard

    2011-10-01

    Decapod crustaceans grow discontinuously and gain size through complex molt processes. The molt comprises the loss of the old cuticle and, moreover, substantial reduction and re-organization of muscles and connective tissues. In adult lobsters, the muscle tissue of the massive claws undergoes significant atrophy of 40-75% before ecdysis. The degradation of this tissue is facilitated by calcium-dependent proteases and by the proteasome, an intra-cellular proteolytic multi-enzyme complex. In contrast to the adults, the involvement of the proteasome during the larval development is yet not validated. Therefore, we developed micro-methods to measure the 20S and the 26S proteasomal activities within mg- and sub-mg-quantities of the larval claw tissue of the European lobster, Homarus gammarus. Within the three larval stages (Z1-3) we distinguished between sub-stages of freshly molted/hatched (post-molt), inter-molt, and ready to molt (pre-molt) larvae. Juveniles were analyzed in the post-molt and in the inter-molt stage. The trypsin-like, the chymotrypsin-like, and the peptidyl-glutamyl peptide hydrolase activity (PGPH) of the 20S proteasome increased distinctly from freshly hatched larvae to pre-molt Z1. During the Z2 stage, the activities were highest in the post-molt animals, decreased in the inter-molt animals and increased again in the pre-molt animals. A similar but less distinct trend was evident in the Z3 stages. In the juveniles, the proteasomal activities decreased toward the lowest values. A similar pattern was present for the chymotrypsin-like activity of the 26S proteasome. The results show that the proteasome plays a significant role during the larval development of lobsters. This is not only reflected by the elevated activities, but also by the continuous change of the trypsin/chymotrypsin-ratio which may indicate a shift in the subunit composition of the proteasome and, thus, a biochemical adjustment to better cope with elevated protein turnover rates

  3. Effect of orally administered betel leaf (Piper betle Linn.) on digestive enzymes of pancreas and intestinal mucosa and on bile production in rats.

    Science.gov (United States)

    Prabhu, M S; Platel, K; Saraswathi, G; Srinivasan, K

    1995-10-01

    The influence of two varieties of betel leaf (Piper betle Linn.) namely, the pungent Mysore and non-pungent Ambadi, was examined on digestive enzymes of pancreas and intestinal mucosa and on bile secretion in experimental rats. The betel leaves were administered orally at two doses which were either comparable to human consumption level or 5 times this. The results indicated that while these betel leaves do not influence bile secretion and composition, they have a significant stimulatory influence on pancreatic lipase activity. Besides, the Ambadi variety of betel leaf has a positive stimulatory influence on intestinal digestive enzymes, especially lipase, amylase and disaccharidases. A slight lowering in the activity of these intestinal enzymes was seen when Mysore variety of betel leaf was administered, and this variety also had a negative effect on pancreatic amylase. Further, both the betel leaf varieties have shown decreasing influence on pancreatic trypsin and chymotrypsin activities.

  4. ¬Enzyme Inhibition Studies on N-Substituted Sulfonamides Derived from m-phenetidine

    Directory of Open Access Journals (Sweden)

    *Aziz-ur-Rehman

    2013-06-01

    Full Text Available Organic synthesis of various compounds followed by biological activities is the going on methodology in the world for pharmacological evaluation. The undertaken research is the synthesis of N-(3-ethoxyphenyl-4-methylbenzenesulfonamide (3 through condensation reaction of m-phenetidine (1 and 4-methylbenzenesulfonyl chloride (2 using basic aqueous media of sodium carbonate. Further, the synthesized compound 3 was reacted with different alkyl/aralkyl halides (4a-j using DMF as aprotic polar solvent and NaH as a base to yield 5a-j compounds. The synthesized molecules were characterized from their spectral data. The synthesized compounds were evaluated against cholinesterase (AChE and BChE, lipoxygenase (LOX, urease, chymotrypsin and tyrosinase enzymes; and found to be the moderate inhibitor against tyrosinase enzyme.

  5. The potential of papain and alcalase enzymes and process optimizations to reduce allergenic gliadins in wheat flour.

    Science.gov (United States)

    Li, Ying; Yu, Jianmei; Goktepe, Ipek; Ahmedna, Mohamed

    2016-04-01

    The objectives of this study were to select effective enzymes that catalyze the hydrolysis of allergenic proteins, gliadins, in wheat flour and to optimize the enzymatic treatment conditions. Six proteases were tested. Hydrolyzed samples were tested for residual gliadin concentrations and in vitro allergenicity. The hydrolysis conditions of wheat protein by the effective enzymes were optimized by central composite design. Results showed that alcalase from Bacillus licheniformis, and papain from latex of papaya fruit had greater ability to reduce gliadin content of wheat flour than flavourzyme, pepsin, trypsin or α-chymotrypsin. The sequential-treatment of wheat flour by alcalase-papain was more effective in reducing gliadin content than single enzyme treatment. Under the optimal conditions of sequential enzymatic treatment, gliadin was almost completely removed, resulting in the flour extract showing lowest IgE-binding. Therefore, this could be a promising biotechnology for preparing low allergenic wheat products. PMID:26593625

  6. A Kazal-Type Serine Protease Inhibitor from the Defense Gland Secretion of the Subterranean Termite Coptotermes formosanus Shiraki.

    Directory of Open Access Journals (Sweden)

    Horia Negulescu

    Full Text Available Coptotermes formosanus is an imported, subterranean termite species with the largest economic impact in the United States. The frontal glands of the soldier caste termites comprising one third of the body mass, contain a secretion expelled through a foramen in defense. The small molecule composition of the frontal gland secretion is well-characterized, but the proteins remain to be identified. Herein is reported the structure and function of one of several proteins found in the termite defense gland secretion. TFP4 is a 6.9 kDa, non-classical group 1 Kazal-type serine protease inhibitor with activity towards chymotrypsin and elastase, but not trypsin. The 3-dimensional solution structure of TFP4 was solved with nuclear magnetic resonance spectroscopy, and represents the first structure from the taxonomic family, Rhinotermitidae. Based on the structure of TFP4, the protease inhibitor active loop (Cys(8 to Cys(16 was identified.

  7. Primary structures of three highly acidic ribosomal proteins S6, S12 and S15 from the archaebacterium Halobacterium marismortui.

    Science.gov (United States)

    Kimura, J; Arndt, E; Kimura, M

    1987-11-16

    The amino acid sequences of three extremely acidic ribosomal proteins, S6, S12, and S15, from Halobacterium marismortui have been determined. The sequences were obtained by the sequence analysis of peptides derived by enzymatic digestion with trypsin. Stapylococcus aureus protease and chymotrypsin, as well as by cleavage with dilute HCl. The proteins, S6, S12 and S15, consist of 116, 147 and 102 amino acid residues, and have molecular masses of 12,251, 16,440 and 11,747 Da, respectively. Comparison of the amino acid sequences of these proteins with ribosomal protein sequences of other organisms revealed that halobacterial protein S12 has homology with the eukaryotic protein S16A from Saccharomyces cerevisiae, while S15 is significantly related to the Xenopus laevis S19 protein. No homology was found between these halobacterial proteins and any eubacterial ribosomal proteins.

  8. Effect of Exogenous MJA Treatment of Tea Plants on the Growth of Geometrid Larvae

    Institute of Scientific and Technical Information of China (English)

    GUI Lian-you; CHEN Zong-mao; LIU Shu-sheng

    2005-01-01

    The effect of tea plant Camellia sinensis induced by exogenous methyl jasmonate (MJA) on lipoxygenase (LOX), polyphenol oxidase (PPO) and proteinase inhibitor (PI) activity in the leaves of tea plants, as well as the growth and midgut proteinase activity of the geometrid Ectropis obliqua larvae were studied. MJA significantly induced LOX, PPO and PI activity in leaves of tea plants. When geometrid larvae have fed on leaves of tea plants treated with MJA, the activities of the high alkaline trypsin-like enzyme and chymotrypsin-like enzyme in their midgut were significantly inhibited, but the activities of the low alkaline trypsin-like enzyme in their midgut were unaffected, leading to imbalance between different types of proteinase activity in the midgut of the larvae and in turn, the growth were inhibited. These chains of response may be an important mechanism of the direct resistance induced by MJA-treatment of tea plant on geometrid larvae.

  9. Molecular cloning, expression, purification and crystallographic analysis of PRRSV 3CL protease

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Xinsheng; Feng, Youjun [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Graduate University, Chinese Academy of Sciences, Beijing 100049 (China); Zhao, Tiezhu [China Animal Disease Control Center, Beijing 100094 (China); Peng, Hao; Yan, Jinghua [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Qi, Jianxun [Graduate University, Chinese Academy of Sciences, Beijing 100049 (China); Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Jiang, Fan [Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Tian, Kegong, E-mail: tiankg@263.net [China Animal Disease Control Center, Beijing 100094 (China); Gao, Feng, E-mail: tiankg@263.net [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); College of Biological Sciences, China Agricultural University, Beijing 100094 (China)

    2007-08-01

    Recombinant PRRSV 3CL protease was crystallized and the crystals diffracted to 2.1 Å resolution. 3CL protease, a viral chymotrypsin-like proteolytic enzyme, plays a pivotal role in the transcription and replication machinery of many RNA viruses, including porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the full-length 3CL protease from PRRSV was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that diffracted to 2.1 Å resolution and belong to space group C2, with unit-cell parameters a = 112.31, b = 48.34, c = 42.88 Å, β = 109.83°. The Matthews coefficient and the solvent content were calculated to be 2.49 Å{sup 3} Da{sup −1} and 50.61%, respectively, for one molecule in the asymmetric unit.

  10. Ochratoxin A labelled with /sup 14/C or /sup 3/H in the phenylalanine moiety

    Energy Technology Data Exchange (ETDEWEB)

    Hult, K.

    1986-08-01

    Ochratoxin A is a mycotoxin produced by Aspergillus and Penicillium moulds. The toxin is an amide formed between phenylalanine and the isocoumarin acid ochratoxin ..cap alpha... Ochratoxin A with high specific radioactivity can be obtained, by substitution of the natural phenylalanine for /sup 14/C of /sup 3/H-labelled phenylalanine. Labelled ochratoxin A was formed by condensation of labelled phenylalanine ethylester with an anhydride derivative of ochratoxin ..cap alpha... The intermediate O-acetyl ochratoxin C was hydrolysed with pig liver esterase and chymotrypsin to afford ochratoxin A. The overall yield of radioactivity was 50%. Specific activities up to 500 Ci/mol and 100 ci/mmol for /sup 14/C and /sup 3/H respectively were obtained.

  11. Phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs.

    Science.gov (United States)

    Luna-González, Antonio; Maeda-Martínez, Alfonso N; Vargas-Albores, Francisco; Ascencio-Valle, Felipe; Robles-Mungaray, Miguel

    2003-10-01

    Phenoloxidase (PO) activity was studied in larval and juvenile homogenates and in the plasma and haemocytes of adult Crassostrea gigas, Argopecten ventricosus, Nodipecten subnodosus, and Atrina maura. Samples were tested for the presence of PO activity by incubation with the substrate L-3, 4-dihydroxyphenylalanine using trypsin, alpha-chymotrypsin, laminarin, lipopolysaccharides (LPS), and sodium dodecyl sulphate (SDS) to elicit activation of prophenoloxidase (proPO) system. PO activity was not detected in larval homogenate. In juvenile homogenate, PO activity was found only in C. gigas and N. subnodosus. PO activity was present in adult samples and was enhanced by elicitors in the plasma of all species tested, but in haemocyte lysate supernatant (HLS) of only N. subnodosus. Activation of proPO by laminarin was suppressed by a protease inhibitor cocktail (P-2714) in plasma and HLS of all species tested.

  12. Exocrine pancreatic secretion is stimulated in piglets fed Fish oil compared with those fed Coconut Oil or Lard

    DEFF Research Database (Denmark)

    Hedemann, Mette Skou; Pedersen, Asger Roer; Engberg, Ricarda M.

    2001-01-01

    An experiment was conducted to study the effect of feeding diets containing fat sources with different fatty acid composition (fish oil, coconut oil or lard, 10 g/100 g diet) on exocrine pancreatic secretion in piglets after weaning. A total of 16 barrows were weaned at 4 wk of age; 3 d later...... the coconut oil or lard diets. The output [U/(h. kg(0.75))] of lipase was higher in piglets fed fish oil than in piglets fed lard or coconut oil. The output of colipase was greater in piglets fed fish oil and coconut oil than in those fed lard. The dietary treatments did not affect the output of carboxylester...... hydrolase. The output of trypsin was significantly lower in piglets fed lard than in piglets fed fish oil or coconut oil diets and the output of carboxypeptidase B was greater in those fed the fish oil diet. Protein, chymotrypsin, carboxypeptidase A, elastase and amylase outputs did not differ among...

  13. On enzymatic activity in organic solvents as a function of enzyme history

    Energy Technology Data Exchange (ETDEWEB)

    Ke, T.; Klibanov, A.M. [Massachusetts Inst. of Tech., Cambridge, MA (United States). Dept. of Chemistry

    1998-03-20

    Catalytic activities of {alpha}-chymotrypsin and subtilisin Carlsberg in various hydrous organic solvents were measured as a function of how the enzyme suspension had been prepared. In one method, lyophilized enzyme was directly suspended in the solvent containing 1% water. In another, the enzyme was precipitated from its aqueous solution by a 100-fold dilution with an anhydrous solvent. In most cases, the reaction rate in a given nonaqueous enzymatic system strongly (up to an order of magnitude) depended on the mode of enzyme preparation. The magnitude of this dependence was markedly affected by the nature of the solvent and enzyme. A mechanistic hypothesis proposed to explain the observed dependencies was verified in additional experiments in which the water contents and enzyme history were further varied.

  14. Comparison of amino acid sequences of the trypsin inhibitors from taro (Colocasia esculenta), giant taro (Alocasia macrorrhiza) and giant swamp taro (Cyrtosperma chamissonis).

    Science.gov (United States)

    Peng, L; Bradbury, J H; Hammer, B C; Shaw, D C

    1993-09-01

    The amino acid sequences of the trypsin inhibitors from taro Colocasia esculenta var. esculenta and giant swamp taro Cyrtosperma chamissonis have been determined and are compared with the protein sequence of the trypsin/chymotrypsin inhibitor from giant taro Alocasia macrorrhiza. Both inhibitors display polymorphism and there is evidence of two components in the giant swamp taro. The positional identity between the proteins is highest at 73-75% for the comparison of the giant taro (GT) with the polymorphic forms of the taro (T) inhibitors and lowest at 56-58% for the pairs of taro and giant swamp taro (GST) proteins. The comparisons show that the inhibitors from T and GT are more related to each other than to GST, which supports their taxonomic classification into different tribes. Location of the P1 site for the trypsin inhibitors of aroids is different from that of other Kunitz-type inhibitors and could be at Leu56.

  15. Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities.

    Science.gov (United States)

    Shrestha, Madan K; Peri, Irena; Smirnoff, Patricia; Birk, Yehudith; Golan-Goldhirsh, Avi

    2002-09-25

    The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.

  16. Effects of in vitro ozone treatment on proteolysis of purified rubisco from two hybrid poplar clones. [Populus maximowizii x trichocarpa

    Energy Technology Data Exchange (ETDEWEB)

    Landry, L.G.; Pell, E.J. (Pennsylvania State Univ., University Park (USA))

    1989-04-01

    Plants exposed to ozone (O{sub 3}) exhibited symptoms of premature senescence, including early decline in quantity of rubisco. O{sub 3}-induced oxidation may cause changes in protein conformation of rubisco, resulting in enhanced proteolysis. To test this hypothesis, rubisco was purified from two hybrid clones of Populus maximowizii x trichocarpa, clones 388 and 245, and treated in vitro with O{sub 3} or air. Rubisco was then challenged with bromelain, papain, chymotrypsin, carboxypeptidase A, or endoproteinase Glu-C and percent degradation measured by SDS-PAGE and densitometric scanning of the gels. Degree of rubisco sensitivity to oxidation may be related to available sulfhydryl (SH) groups on the protein. The number of SH groups in native and denatured rubisco was measured for purified rubisco of both clones by DTNB titration method. The relationship between sensitivity to proteolysis and number and availability of SH groups is discussed.

  17. Nutritional significance of lectins and enzyme inhibitors from legumes.

    Science.gov (United States)

    Lajolo, Franco M; Genovese, Maria Inés

    2002-10-23

    Legumes have natural components, such as lectins, amylase, and trypsin inhibitors, that may adversely affect their nutritional properties. Much information has already been obtained on their antinutritional significance and how to inactivate them by proper processing. Chronic ingestion of residual levels is unlikely to pose risks to human health. On the other hand, the ability of these molecules to inhibit some enzymes such as trypsin, chymotrypsin, disaccharidases, and alpha-amylases, to selectively bind to glycoconjugates, and to enter the circulatory system may be a useful tool in nutrition and pharmacology. Trypsin inhibitors have also been studied as cancer risk reducing factors. These components seem to act as plant defense substances. However, increased contents may represent an impairment of the nutritional quality of legumes because these glycoproteins and the sulfur-rich protease inhibitors have been shown to be poorly digested and to participate in chemical reactions during processing reducing protein digestibility, a still unsolved question. PMID:12381157

  18. The Cobalamin-binding Protein in Zebrafish is an Intermediate Between the Three Cobalamin-binding Proteins in Human

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Nexø, Ebba

    2012-01-01

    In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current...... knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin...... (like human IF, but unlike human HC and TC). The cobalamin analogue, cobinamide, bound weaker to the zebrafish cobalamin binder than to human HC, but stronger than to human TC and IF. Affinity for another analogue, adenosyl-pseudo-cobalamin was low compared with human HC and TC, but high compared...

  19. Valle Agricola lentil, an unknown lentil (Lens culinaris Medik.) seed from Southern Italy as a novel antioxidant and prebiotic source.

    Science.gov (United States)

    Landi, Nicola; Pacifico, Severina; Piccolella, Simona; Di Giuseppe, Antonella M A; Mezzacapo, Maria C; Ragucci, Sara; Iannuzzi, Federica; Zarrelli, Armando; Di Maro, Antimo

    2015-09-01

    In order to promote 'Valle Agricola' lentil, an autochthonous lentil of the Campania Region, a thorough investigation of its biochemical and nutritional properties has been carried out. The macronutrient content (proteins, carbohydrates and lipids), free and total amino acids, and unsaturated fatty acids were determined. The antioxidant capability of raw 'Valle Agricola' lentils, as well as of boiled ones, was estimated in terms of their total phenol content (TPC), ORAC value, and free radical scavenging capacities using DPPH and ABTS assays. The data obtained evidenced that the boiling process slightly decreased Valle Agricola lentil's antioxidant power. Furthermore, when trypsin and chymotrypsin inhibitory activities were measured, a large decrease of the levels of anti-nutritional factors was estimated. In order to have a phytochemical overview of this autochthonous lentil seed, LC-ESI-MS/MS analysis was applied to raw and boiled lentil extracts. Flavonol glycosides and free flavanols, as well as typical seed prebiotic saccharides, were the most representative constituents. PMID:26222801

  20. [The function of the hydrokinetic and ekbolic pancreas after acute pancreatitis].

    Science.gov (United States)

    Tympner, F; Domschke, W; Rösch, W; Koch, H; Demling, L

    1976-11-01

    Thirteen patients were investigated 3-4 weeks (I. examination) and 3-5 months (II. examination) after the ceasing of the signs symptoms of an acute pancreatitis concerning their hydrokinetic and ekbolic pancreas-functions. In addition, the pancreatic ducts were demonstrated by ERCP. Reduction of the secretion were shown in 31% at the I. examination and in 23% at the II. examination. There were significant differences in the outputs of bicarbonate, trypsine and chymotrypsine compared to healthy test persons at the I. examination. At the II. examination, however, no significant differences could be observed compared to healthy test persons. The exocrine function of the pancreas was getting worse during the interval between the I. and II. examination in 2 cases but it became normal in 5 cases. Pathological alterations at the pancreativ-duct-system were not demonstrable shown at all. PMID:1007339

  1. The Concentration and Yield of Hordein and some Lysine-Rich Proteins as Influenced by the lys gene of Hiproly Barley

    DEFF Research Database (Denmark)

    Balasaraswathi, R.; Køie, B; Doll, Hans

    1984-01-01

    -amylase and chymotrypsin inhibitors 1 and 2 was increased 2–6 fold. The yield of these proteins was significantly increased in the lines having the lys gene. However, the absolute increase in the yield of these lysinerich proteins was less than the absolute decrease in hordein yield.......A line having the high-lysine gene lys of ‘Hiproly’ was crossed with a normal variety, and homozygous offspring lines were derived from chromosome-doubled haploids made from the F1. The intluence of the lys gene was tested by comparing the offspring lines having the lys gene with the lines having...... the corresponding normal gene. The lys lines had 16% lower grain yield and 13% lower single seed weight than the corresponding normal lines. The concentration of hordein in the protein and the yield of this storage protein were strongly reduced in the lys lines. On the contrary, the concentration of protein Z, β...

  2. IMP Dehydrogenase: Structural Schizophrenia and an Unusual Base

    Energy Technology Data Exchange (ETDEWEB)

    Hedstrom,L.; Gan, L.

    2006-01-01

    Textbooks describe enzymes as relatively rigid templates for the transition state of a chemical reaction, and indeed an enzyme such as chymotrypsin, which catalyzes a relatively simple hydrolysis reaction, is reasonably well described by this model. Inosine monophosphate dehydrogenase (IMPDH) undergoes a remarkable array of conformational transitions in the course of a complicated catalytic cycle, offering a dramatic counterexample to this view. IMPDH displays several other unusual mechanistic features, including an Arg residue that may act as a general base catalyst and a dynamic monovalent cation site. Further, IMPDH appears to be involved in 'moon-lighting' functions that may require additional conformational states. How the balance between conformational states is maintained and how the various conformational states interconvert is only beginning to be understood.

  3. Characterisation and partial purification of proteolytic enzymes from sardine by-products to obtain concentrated hydrolysates.

    Science.gov (United States)

    Castro-Ceseña, Ana Bertha; del Pilar Sánchez-Saavedra, M; Márquez-Rocha, Facundo J

    2012-11-15

    A procedure to recover proteases and lipases from the by-products of Monterey sardine (Sardinops sagax caerulea) has been developed, comprising 2 steps: a centrifugation at low temperature to eliminate more than 90% of the initial fat content, and an acetone precipitation step. After this treatment, enzymatic activity increased by 33.8% for lipase, 15.5% for trypsin, 14.8% for chymotrypsin, 93.4% for aminopeptidase, and 19.7% for pepsin. The extents of hydrolysis of fish by-product proteins by endogenous enzyme by-product extract, viscera concentrate extract, and commercial Alcalase® were 62%, 85%, and 28%, respectively. The two extract preparations from sardine by-product (viscera and by-product concentrate extracts) produced 3-fold greater hydrolysis than with the commercial enzyme. The recovery of enzyme concentrates from sardine waste has both ecological and economical advantages for the fish industry. PMID:22868132

  4. Nutritional quality of extruded kidney bean (Phaseolus vulgaris L. var. Pinto) and its effects on growth and skeletal muscle nitrogen fractions in rats.

    Science.gov (United States)

    Marzo, F; Alonso, R; Urdaneta, E; Arricibita, F J; Ibáñez, F

    2002-04-01

    The influence of extrusion cooking on the protein content, amino acid profile, and concentration of antinutritive compounds (phytic acid, condensed tannins, polyphenols, trypsin, chymotrypsin, alpha-amylase inhibitors, and hemagglutinating activity) in kidney bean seeds (Phaseolus vulgaris L. var. Pinto) was investigated. Growing male rats were fed diets based on casein containing raw or extruded kidney beans with or without methionine supplementation for 8 or 15 d. Rates of growth, food intake, and protein efficiency ratio were measured and the weight of the gastrocnemius muscle and the composition of its nitrogenous fraction was determined. Extrusion cooking reduced (P cooking improved food intake and utilization by the rats and they gained BW. Supplementation of extruded kidney bean with methionine further enhanced (P food conversion efficiency and growth. However, BW gains and muscle composition still differed (P < 0.01) from those of rats fed a high-quality protein.

  5. Stabilization of porcine pancreatic elastase crystals by glutaraldehyde cross-linking.

    Science.gov (United States)

    Hofbauer, Stefan; Brito, José A; Mulchande, Jalmira; Nogly, Przemyslaw; Pessanha, Miguel; Moreira, Rui; Archer, Margarida

    2015-10-01

    Elastase is a serine protease from the chymotrypsin family of enzymes with the ability to degrade elastin, an important component of connective tissues. Excessive elastin proteolysis leads to a number of pathological diseases. Porcine pancreatic elastase (PPE) is often used for drug development as a model for human leukocyte elastase (HLE), with which it shares high sequence identity. Crystals of PPE were grown overnight using sodium sulfate and sodium acetate at acidic pH. Cross-linking the crystals with glutaraldehyde was needed to resist the soaking procedure with a diethyl N-(methyl)pyridinyl-substituted oxo-β-lactam inhibitor. Crystals of PPE bound to the inhibitor belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 51.0, b = 58.3, c = 74.9 Å, and diffracted to 1.8 Å resolution using an in-house X-ray source.

  6. Assessment of the non-protein amino acid BMAA in Mediterranean mussel Mytilus galloprovincialis after feeding with estuarine cyanobacteria.

    Science.gov (United States)

    Baptista, Mafalda S; Vasconcelos, Rita G W; Ferreira, Paula C; Almeida, C Marisa R; Vasconcelos, Vitor M

    2015-08-01

    To determine whether 2-amino-3-methylaminopropanoic acid (BMAA) could be taken up by marine organisms from seawater or their diet mussels Mytilus galloprovincialis, collected from the North Atlantic Portuguese shore, were exposed to seawater doped with BMAA standard (for up to 48 h) or fed with cyanobacteria (for up to 15 days). Mussels were able to uptake BMAA when exposed to seawater. Mussels fed with cyanobacteria Synechocystis salina showed a rise in BMAA concentration during feeding and a decline in concentration during the subsequent depuration period. Cells from the gills and hepatopancreas of mussels fed with S. salina showed lessened metabolic activity in mussels fed for longer periods of time. A hot acidic digestion (considered to account for total BMAA) was compared with a proteolytic digestion, using pepsin, trypsin and chymotrypsin. The latter was able to extract from mussels approximately 30% of total BMAA. Implications for BMAA trophic transfers in marine ecosystems are discussed.

  7. Identification of a novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine beta-lactoglobulin.

    Science.gov (United States)

    Mullally, M M; Meisel, H; FitzGerald, R J

    1997-02-01

    The angiotensin-I-converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine beta-lactoglobulin (beta-lg) was investigated. Intact beta-lg essentially did not inhibit ACE while the tryptic digest gave an 84.3% inhibition of ACE. Peptide material eluting between 20 and 25% acetonitrile during C18 solid-phase extraction of the beta-lg tryptic digest inhibited ACE by 93.6%. This solid-phase extraction fraction was shown by mass spectroscopy to contain beta-lg f(142-148). This peptide had an ACE IC50 value of 42.6 micromol/l. The peptide was resistant to further digestion with pepsin and was hydrolysed to a very low extent with chymotrypsin. The contribution of specific amino acid residues within the peptide to ACE inhibitory activity and the potential application of this peptide as a nutraceutical is discussed.

  8. Specificity of proteinase K at P2 to P3' sub-sites and its comparison to other serine proteases.

    Science.gov (United States)

    Qasim, Mohammad A

    2014-01-01

    Specificity of the commercially important serine protease, proteinase K, has been investigated by measuring free energies of association of proteinase K with turkey ovomucoid third domain inhibitor variants at contact positions P2, P1, P1', P2', and P3'. Correlations of these values were run with similar values that have been obtained for six other serine proteases. Among the six proteases, subtilisin Carlsberg shows a near perfect correlation (Pearson Product correlation coefficient = 0.93 to 0.99) with proteinase K at all of these positions. Proteinase K has only 35% sequence identity with subtilisin Carlsberg, yet, the two enzymes are nearly identical in their specificity at P2 to P3' positions. With other serine proteases such as bovine chymotrypsin, human leukocyte elastase, porcine pancreatic elastase, Streptomyces griseus protease A and B, proteinase K showed relatively poor or no correlation.

  9. Inhibitory plant serpins with a sequence of three glutamine residues in the reactive center

    DEFF Research Database (Denmark)

    Hejgaard, Jørn

    2005-01-01

    of three Gln residues at P3-P1 or P2-P1' were isolated from barley and wheat grain, respectively. Barley BSZ3 was an irreversible inhibitor of chymotrypsin, with a second-order association rate constant for complex formation k(a)' of the order of 10(4) M-1 s(-1) ; however, only a minor fraction...... for proteinases that specifically degrade storage prolamins containing Gln-rich repetitive sequences, most likely for digestive proteinases of insect pests or fungal pathogens that infect cereals. An assembled full-length amino acid sequence of a serpin expressed in cotton boll fiber (GaZ1) included conserved...... regions essential for serpin-proteinase interaction, suggesting inhibitory capacity at a putative reactive center P2-P2' with a sequence of four Gln residues....

  10. Differential gene expression for suicide-substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley

    DEFF Research Database (Denmark)

    Roberts, T.H.; Marttila, S.; Rasmussen, S.K.;

    2003-01-01

    Proteins of the serpin superfamily (similar to43 kDa) from mature cereal grains are in vitro suicide-substrate inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (kDa), the biological functions...... of plant serpins are unknown. Expression studies of genes encoding members of three subfamilies of serpins (BSZx, BSZ4 and BSZ7) in developing grain and vegetative tissues of barley (Hordeum vulgare L.) showed that transcripts encoding BSZx, which inhibits distinct proteinases at overlapping reactive...... their irreversible inhibitory mechanism in the inhibition of exogenous proteinases capable of breaking down seed storage proteins, and in the defence of specific cell types in vegetative tissues....

  11. /sup 125/I-peptide mapping of protein III isolated from four strains of Neisseria gonorrhoeae

    Energy Technology Data Exchange (ETDEWEB)

    Judd, R.C.

    1982-08-01

    Gonococcal outer-membrane protein I (PI) and PIII were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from reduced and unreduced whole-cell and outer-membrane lysates of four strains of nonpiliated (P-), transparent (O-) Neisseria gonorrhoeae. These proteins were radioiodinated and digested with alpha-chymotrypsin. The resultant /sup 125/I-peptides were then resolved by high-voltage thin-layer electrophoresis, followed by ascending thin-layer chromatography, and visualized by autoradiography. Results corroborated previous observations regarding the structural relationships of PIs having different apparent subunit molecular weights. All PIIIs had very similar apparent primary structures, regardless of the strain from which they were isolated, the source (i.e., whole cells or outer membranes), or the reduction state of the sodium dodecyl sulfate lysates. By the techniques used, it appeared that PIII is structurally similar in all of the gonococcal strains studied, even though each strain had structurally unique PIs.

  12. The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran [correction of brain] of rice (Oryza sativa L.) seeds.

    Science.gov (United States)

    Ohtsubo, K; Richardson, M

    1992-08-31

    A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the alpha-amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Cellulose and Toyopearl CM-650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S-alkylated protein, and by manual DABITC/PITC micro-sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, elastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds. PMID:1511747

  13. Expressed sequence tags from larval gut of the European corn borer (Ostrinia nubilalis: Exploring candidate genes potentially involved in Bacillus thuringiensis toxicity and resistance

    Directory of Open Access Journals (Sweden)

    Crespo Andre LB

    2009-06-01

    Full Text Available Abstract Background Lepidoptera represents more than 160,000 insect species which include some of the most devastating pests of crops, forests, and stored products. However, the genomic information on lepidopteran insects is very limited. Only a few studies have focused on developing expressed sequence tag (EST libraries from the guts of lepidopteran larvae. Knowledge of the genes that are expressed in the insect gut are crucial for understanding basic physiology of food digestion, their interactions with Bacillus thuringiensis (Bt toxins, and for discovering new targets for novel toxins for use in pest management. This study analyzed the ESTs generated from the larval gut of the European corn borer (ECB, Ostrinia nubilalis, one of the most destructive pests of corn in North America and the western world. Our goals were to establish an ECB larval gut-specific EST database as a genomic resource for future research and to explore candidate genes potentially involved in insect-Bt interactions and Bt resistance in ECB. Results We constructed two cDNA libraries from the guts of the fifth-instar larvae of ECB and sequenced a total of 15,000 ESTs from these libraries. A total of 12,519 ESTs (83.4% appeared to be high quality with an average length of 656 bp. These ESTs represented 2,895 unique sequences, including 1,738 singletons and 1,157 contigs. Among the unique sequences, 62.7% encoded putative proteins that shared significant sequence similarities (E-value ≤ 10-3with the sequences available in GenBank. Our EST analysis revealed 52 candidate genes that potentially have roles in Bt toxicity and resistance. These genes encode 18 trypsin-like proteases, 18 chymotrypsin-like proteases, 13 aminopeptidases, 2 alkaline phosphatases and 1 cadherin-like protein. Comparisons of expression profiles of 41 selected candidate genes between Cry1Ab-susceptible and resistant strains of ECB by RT-PCR showed apparently decreased expressions in 2 trypsin-like and 2

  14. Stable and biocompatible cystine knot peptides from the marine sponge Asteropus sp.

    Science.gov (United States)

    Su, Mingzhi; Li, Huayue; Wang, Haibo; Kim, Eun La; Kim, Hyung Sik; Kim, Eun-Hee; Lee, Jaewon; Jung, Jee H

    2016-07-01

    Two new cystine knot peptides, asteropsins F (ASPF) and G (ASPG), were isolated from the marine sponge Asteropus sp. ASPF and ASPG are composed of 33 and 32 amino acids, respectively, and contain six cysteines which are involved in three disulfide bonds. They shared the characteristic features of the asteropsin family, such as, N-terminal pyroglutamate modification, incorporation of cis prolines, and the unique anionic profile, which distinguish them from other knottin families. Tertiary structures of the peptides were determined by high resolution NMR. ASPF and ASPG were found to be remarkably resistant not only to digestive enzymes (chymotrypsin, pepsin, elastase, and trypsin) but also to thermal degradation. In addition, these peptides were pharmacologically inert; non-hemolytic to human and fish red blood cells, non-stimulatory to murine macrophage cells, and nontoxic in vitro or in vivo. These observations support their stability and biocompatibility as suitable carrier scaffolds for the design of oral peptide drug. PMID:27189887

  15. Isoforms of agrin are widely expressed in the developing rat and may function as protease inhibitors.

    Science.gov (United States)

    Biroc, S L; Payan, D G; Fisher, J M

    1993-09-17

    The agrin family of extracellular matrix proteins may be important in the formation of the neuromuscular junction. Using in situ hybridization with a probe recognizing all agrin isoforms, we demonstrate that it is widely expressed during mammalian embryogenesis. In the developing rat, particularly high levels of expression are found in the dorsal root and cranial ganglia, gut, whisker rudiments, penis, snout, teeth, retina, hippocampus, cerebral cortex and the lining of brain ventricles. Functional analysis of the recombinant rat protein shows that it is a potent inhibitor of the proteases trypsin, chymotrypsin and plasmin but not thrombin or the plasminogen activators. We conclude that agrin and its isoforms may play multiple roles in mammalian development including the regulation of proteolysis in the extracellular matrix.

  16. The Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish

    Science.gov (United States)

    Rojas, Ana; Doolittle, Russell F.

    2003-01-01

    Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic "chymotrypsin family" (Group S1A by th e 1998 Barrett nomenclature) has an unusual phylogenetic distribution , being especially common in animals, entirely absent from plants and protists, and rare among fungi. The distribution in Bacteria is larg ely restricted to the genus Streptomyces, although a few isolated occ urrences in other bacteria have been reported. The family may be enti rely absent from Archaea. Although more than a thousand sequences have been reported for enzymes of this type from animals, none of them ha ve been from early diverging phyla like Porifera or Cnidaria, We now report the existence of Group SlA serine proteases in a sponge (phylu m Porifera) and a jellyfish (phylum Cnidaria), making it safe to conc lude that all animal groups possess these enzymes.

  17. Digestive enzymatic patterns as possible biomarkers of endocrine disruption in the red mullet (Mullus barbatus): A preliminary investigation.

    Science.gov (United States)

    Caruso, Gabriella; De Pasquale, Francesca; Mita, Damiano Gustavo; Micale, Valeria

    2016-04-15

    During two seasonal trawl surveys (April and October, 2012), red mullet specimens were caught from two sites of the northern Sicilian coast (Western Mediterranean), characterized by different degrees of pollution, to assess whether their digestive enzymes could be cost-effective diagnostic tools for endocrine disruption. Pepsin, chymotrypsin, carboxypeptidases A and B, amylase and lipase were measured in the digestive tract of each fish. During both samplings, significant differences in the digestive enzymatic patterns of fish collected from the two sites were found. In April, pepsin and lipase contents were significantly lower in fish from the most impacted site than in those from the reference site. In October, the enzymatic patterns showed trends different from spring, with controversial results for carboxypeptidases A and B and amylase. Pepsin and lipase patterns suggest a detrimental effect played by organic pollutants and the use of these enzymes as possible biomarkers of exposure to endocrine disruptors. PMID:26971230

  18. Differential Synthesis in Vitro of Barley Aleurone and Starchy Endosperm Proteins

    DEFF Research Database (Denmark)

    Mundy, John; Hejgaard, Jørn; Hansen, Annette;

    1986-01-01

    RNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA(3). B and C hordein polypeptides and the salt-soluble proteins beta-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2......), the alpha-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, beta-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells......, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus...

  19. Human seminal proteinase and prostate-specific antigen are the same protein

    Indian Academy of Sciences (India)

    Abdul Waheed; Md Imtaiyaz Hassan; Robert L Van Etten; Faizan Ahmad

    2008-06-01

    Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and compared. Both are glycoproteins of 32–34 kDa with protease activities. Based on some physicochemical, enzymatic and immunological properties, it is concluded that these proteins are in fact identical. The protein exhibits properties similar to kallikrein-like serine protease, trypsin, chymotrypsin and thiol acid protease. Tests of the activity of the enzyme against some potential natural and synthetic substrates showed that bovine serum albumin was more readily hydrolysed than casein. The results of this study should be useful in purifying and assaying this protein. Based on published studies and the present results, the broad proteolytic specificity of human seminal proteinase suggests a role for this protein in several physiological functions.

  20. Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera:Cerambycidae)

    Institute of Scientific and Technical Information of China (English)

    Brian David Shaw; John Tane Christeller

    2009-01-01

    The protein digestive capability oftbe larvae of the longhorn beetle (Oemona hirta,Coleoptera:Cerambycidae,Fabricius,1775) was investigated.This species feeds only on wood where there is a high proportion of vascular tissue.The pH of the midgut,the major digestive organ,was alkaline and protein hydrolysis was maximal at alkaline pH.Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases,trypsin and chymotrypsin-like activity,and the exopeptidase,leucine aminopeptidase and the pH curves corresponded to that with protein substrate.Studies using a range ofsefine protease inhibitors as well as specific inhibitors ofmetalloproteases,cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids.Control of these insect pests using protease inhibitors is discussed.

  1. Complete separation of racemic p-boronophenylalanine by high performance liquid chromatography with crown ether-coated reversed-phase packings

    International Nuclear Information System (INIS)

    Since the L-form of p-boronophenylalanine (p-bpa) has been shown to be more efficiently incorporated into melanoma cells than racemic p-bpa separation of racemic p-bpa into its stereoisomers is an important subject. One of the preparative methods used to resolve racemic p-bpa involves the use of α-chymotrypsin. However, there has been a problem in that optical purity of resolved L- or D-p-bpa products was not easily determined. In this paper, the authors describe a method which can be used to confirm the optical purity of b-pba using high performance liquid chromatography (HPLC) with crown ether-coated reversed-phase packings

  2. Structural and functional characterization of proteinase inhibitors from seeds of Cajanus cajan (cv. ICP 7118).

    Science.gov (United States)

    Swathi, Marri; Lokya, Vadthya; Swaroop, Vanka; Mallikarjuna, Nalini; Kannan, Monica; Dutta-Gupta, Aparna; Padmasree, Kollipara

    2014-10-01

    Proteinase inhibitors (C11PI) from mature dry seeds of Cajanus cajan (cv. ICP 7118) were purified by chromatography which resulted in 87-fold purification and 7.9% yield. SDS-PAGE, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrum and two-dimensional (2-D) gel electrophoresis together resolved that C11PI possessed molecular mass of 8385.682 Da and existed as isoinhibitors. However, several of these isoinhibitors exhibited self association tendency to form small oligomers. All the isoinhibitors resolved in Native-PAGE and 2-D gel electrophoresis showed inhibitory activity against bovine pancreatic trypsin and chymotrypsin as well as Achaea janata midgut trypsin-like proteases (AjPs), a devastating pest of castor plant. Partial sequences of isoinhibitor (pI 6.0) obtained from MALDI-TOF/TOF analysis and N-terminal sequencing showed 100% homology to Bowman-Birk Inhibitors (BBIs) of leguminous plants. C11PI showed non-competitive inhibition against trypsin and chymotrypsin. A marginal loss (<15%) in C11PI activity against trypsin at 80 (°)C and basic pH (12.0) was associated with concurrent changes in its far-UV CD spectra. Further, in vitro assays demonstrated that C11PI possessed significant inhibitory potential (IC50 of 78 ng) against AjPs. On the other hand, in vivo leaf coating assays demonstrated that C11PI caused significant mortality rate with concomitant reduction in body weight of both larvae and pupae, prolonged the duration of transition from larva to pupa along with formation of abnormal larval-pupal and pupal-adult intermediates. Being smaller peptides, it is possible to express C11PI in castor to protect them against its devastating pest A. janata. PMID:25093261

  3. Proteolytic Activation of Bacillus thuringiensis Cry2Ab through a Belt-and-Braces Approach.

    Science.gov (United States)

    Xu, Lian; Pan, Zhi-Zhen; Zhang, Jing; Liu, Bo; Zhu, Yu-Jing; Chen, Qing-Xi

    2016-09-28

    Proteolytic processing of Bacillus thuringiensis (Bt) crystal toxins by insect midgut proteases plays an essential role in their insecticidal toxicities against target insects. In the present study, proteolysis of Bt crystal toxin Cry2Ab by Plutella xylostella L. midgut proteases (PxMJ) was evaluated. Both trypsin and chymotrypsin were identified involving the proteolytic activation of Cry2Ab and cleaving Cry2Ab at Arg(139) and Leu(144), respectively. Three Cry2Ab mutants (R139A, L144A, and R139A-L144A) were constructed by replacing residues Arg(139), Leu(144), and Arg(139)-Leu(144) with alanine. Proteolysis assays revealed that mutants R139A and L144A but not R139A-L144A could be cleaved into 50 kDa activated toxins by PxMJ. Bioassays showed that mutants R139A and L144A were highly toxic against P. xylostella larvae, while mutant R139A-L144A was almost non-insecticidal. Those results demonstrated that proteolysis by PxMJ was associated with the toxicity of Cry2Ab against P. xylostella. It also revealed that either trypsin or chymotrypsin was enough to activate Cry2Ab protoxin. This characteristic was regarded as a belt-and-braces approach and might contribute to the control of resistance development in target insects. Our studies characterized the proteolytic processing of Cry2Ab and provided new insight into the activation of this Bt toxin. PMID:27598769

  4. Pyrrolidine dithiocarbamate-zinc(II) and -copper(II) complexes induce apoptosis in tumor cells by inhibiting the proteasomal activity.

    Science.gov (United States)

    Milacic, Vesna; Chen, Di; Giovagnini, Lorena; Diez, Alejandro; Fregona, Dolores; Dou, Q Ping

    2008-08-15

    Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for subsequent apoptosis induction. We first showed that zinc(II) chloride was able to inhibit the chymotrypsin-like activity of a purified 20S proteasome with an IC(50) value of 13.8 microM, which was less potent than copper(II) chloride (IC(50) 5.3 microM). We then compared the potencies of a pyrrolidine dithiocarbamate (PyDT)-zinc(II) complex and a PyDT-copper(II) complex to inhibit cellular proteasomal activity, suppress proliferation and induce apoptosis in various human breast and prostate cancer cell lines. Consistently, zinc complex was less potent than copper complex in inhibiting the proteasome and inducing apoptosis. Additionally, zinc and copper complexes appear to use somewhat different mechanisms to kill tumor cells. Zinc complexes were able to activate calpain-, but not caspase-3-dependent pathway, while copper complexes were able to induce activation of both proteases. Furthermore, the potencies of these PyDT-metal complexes depend on the nature of metals and also on the ratio of PyDT to the metal ion within the complex, which probably affects their stability and availability for interacting with and inhibiting the proteasome in tumor cells. PMID:18501397

  5. Physiology of digestion and the molecular characterization of the major digestive enzymes from Periplaneta americana.

    Science.gov (United States)

    Tamaki, Fábio K; Pimentel, André C; Dias, Alcides B; Cardoso, Christiane; Ribeiro, Alberto F; Ferreira, Clélia; Terra, Walter R

    2014-11-01

    Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the digestive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplanetaamericana was searched for sequences of digestive enzymes. The selected sequences were manually curated. The complete or nearly complete sequences showing all characteristic motifs and highly expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two α- and three β-glucosidases, one β-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 5 trypsins, and none β-glucanase. Electrophoretic and enzymological data agreed with transcriptome data in showing that there is a single β-galactosidase, two α-glucosidases, one preferring as substrate maltase and the other aryl α-glucoside, and two β-glucosidases. Chromatographic and enzymological data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack of β-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A counter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase and trypsin are released by both merocrine and apocrine secretion

  6. Proteasome functioning in breast cancer: connection with clinical-pathological factors.

    Directory of Open Access Journals (Sweden)

    Elena E Shashova

    Full Text Available Breast cancer is one of four oncology diseases that are most widespread in the world. Moreover, breast cancer is one of leading causes of cancer-related deaths in female population within economically developed regions of the world. So far, detection of new mechanisms of breast cancer development is very important for discovery of novel areas in which therapy approaches may be elaborated. The objective of the present study is to investigate involvement of proteasomes, which cleave up to 90% of cellular proteins and regulate numerous cellular processes, in mechanisms of breast cancer development. Proteasome characteristics in 106 patient breast carcinomas and adjacent tissues, as well as relationships of detected proteasome parameters with clinical-pathological factors, were investigated. Proteasome chymotrypsin-like activity was evaluated by hydrolysis of fluorogenic peptide Suc-LLVY-AMC. The expression of proteasome subunits was studied by Western-blotting and immunohistochemistry. The wide range of chymotrypsin-like activity in tumors was detected. Activity in tumors was higher if compared to adjacent tissues in 76 from 106 patients. Multiple analysis of generalized linear models discovered that in estrogen α-receptor absence, tumor growth was connected with the enhanced expression of proteasome immune subunit LMP2 and proteasome activator PA700 in tumor (at 95% confidence interval. Besides, by this analysis we detected some phenomena in adjacent tissue, which are important for tumor growth and progression of lymph node metastasis in estrogen α-receptor absence. These phenomena are related to the enhanced expression of activator PA700 and immune subunit LMP7. Thus, breast cancer development is connected with functioning of immune proteasome forms and activator PA700 in patients without estrogen α-receptors in tumor cells. These results could indicate a field for search of new therapy approaches for this category of patients, which has the

  7. Impact of Q139R substitution of MEB4-Cry2Aa toxin on its stability, accessibility and toxicity against Ephestia kuehniella.

    Science.gov (United States)

    Nouha, Abdelmalek; Sameh, Sellami; Fakher, Frikha; Slim, Tounsi; Souad, Rouis

    2015-11-01

    The Bacillus thuringiensis subsp. kurstaki strain MEB4 was previously found to be highly toxic to Ephestia kuehniella. SDS-PAGE analysis of the recombinant strain DH5α (pBS-cry2Aa-MEB4) showed that Cry2Aa-MEB4 delta-endotoxins were forming inclusion bodies, and were 2.75 fold more toxic towards E. kuehniella than those of Cry2Aa-BNS3. Besides to the 65kDa active toxin, proteolysis activation of Cry2Aa-BNS3 protein with E. kuehniella midgut juice generated an extra proteolysis form of 49kDa, which was the result of another chymotrypsin cleavage located in Leu144. The amino acid sequences alignment of Cry2Aa-MEB4 and Cry2Aa-BNS3 showed that among the different 15 amino acids, the Q139R substitution was found to be interesting. In fact, due to its presence within the loop α3-α4, the chymotrypsin-like protease was unable to access to its site in Cry2Aa-MEB4, resulting to the production of only the 65kDa form. The accessible surface and the stability studies of the structure model of the Cry2Aa-BNS3-49 form showed a lower hydrophobicity surface due to the omission of 144 amino acids from the N-terminal comparing with the active Cry2Aa-MEB4 protein. All these features caused the diminishing of Cry2Aa-BNS3 toxicity towards E. kuehniella. PMID:26321422

  8. Biopotency of serine protease inhibitors from cowpea (Vigna unguiculata) seeds on digestive proteases and the development of Spodoptera littoralis (Boisduval).

    Science.gov (United States)

    Abd El-latif, Ashraf Oukasha

    2015-05-01

    Serine protease inhibitors (PIs) have been described in many plant species and are universal throughout the plant kingdom, where trypsin inhibitors is the most common type. In the present study, trypsin and chymotrypsin inhibitory activity was detected in the seed flour extracts of 13 selected cultivars/accessions of cowpea. Two cowpea cultivars, Cream7 and Buff, were found to have higher trypsin and chymotrypsin inhibitory potential compared to other tested cultivars for which they have been selected for further purification studies using ammonium sulfate fractionation and DEAE-Sephadex A-25 column. Cream7-purified proteins showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) corresponding to molecular mass of 17.10 and 14.90 kDa, while the purified protein from Buff cultivar showed a single band corresponding mass of 16.50 kDa. The purified inhibitors were stable at temperature below 60°C and were active at wide range of pH from 2 to 12. The kinetic analysis revealed noncompetitive type of inhibition for both inhibitors against both enzymes. The inhibitor constant (Ki ) values suggested high affinity between inhibitors and enzymes. Purified inhibitors were found to have deep and negative effects on the mean larval weight, larval mortality, pupation, and mean pupal weight of Spodoptera littoralis, where Buff PI was more effective than Cream7 PI. It may be concluded that cowpea PI gene(s) could be potential insect control protein for future studies in developing insect-resistant transgenic plants. PMID:25524889

  9. Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish.

    Science.gov (United States)

    Yildirim, Z; Johnson, M G

    1998-04-01

    Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, alpha-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 degrees C for 15, 30 and 60 min, or 121 degrees C for 15 min. Lactococcin R remained active after storage at -20 and -70 degrees C for 3 months and after exposure to a pH of 2-9. The molecular weight of lactococcin R was about 2.5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml-1) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0.5% glucose, at 6.5-7.0 initial pH values, 30 degrees C temperature and 18-24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6-7 (95%). Crude lactococcin R at 1280 AU ml-1 was bactericidal, reducing colony counts of Listeria monocytogenes by 99.98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2.5 kDa vs 3.4 kDa, and in being sensitive to pepsin and alpha-chymotrypsin to which nisin is resistant. PMID:9633097

  10. Effects of Fungal Pancreatic Enzymes on the Function of Islet Cells in Syrian Golden Hamsters

    Directory of Open Access Journals (Sweden)

    Fumiaki Nozawa

    2013-05-01

    Full Text Available Context Our previous studies showed that porcine pancreatic enzymes in Syrian golden hamsters with peripheral insulin resistance normalizes the plasma insulin level, reduces the size of enlarged islets and inhibits the increased DNA synthesis in the beta-cell of islets. Objective In order to exclude the possibility that these effects was attributed to some contaminants of this crude material, we tested the effect of purified fungal pancreatic enzyme (FPE that contains primarily amylase and lipase without (FPE and with addition of chymotrypsin (FPE+chy. Material and methods In a pilot study we tested the effect of different doses of FPE given in drinking water on insulin level, islet size and DNA synthesis of islet cells in hamsters with induced peripheral insulin resistance by a high fat diet. The most effective dose of FPE on these parameters was used in a long-term experiment with FPE and FPE+chy in hamsters fed a high-fat diet for 36 or 40 weeks. Results In the pilot study a dose of 2 g/kg body weight was found to be optimal for controlling the body weight, normalizing plasma insulin level, the size of islets, the DNA synthesis and the number of insulin cells in the islets. These data were produced in the long-term study, where steatorrhea was also inhibited. Addition of chymotrypsin had no effects on these parameters. Conclusion Pancreatic lipase and amylase appear to be responsible for the observed effects and offer a safe and effective natural product for the treatment of pancreatic diseases, including acute pancreatitis, chronic pancreatic, cystic fibrosis and any conditions associated with peripheral insulin resistance, including obesity and type 2 diabetes. The possible mechanism of the action is discussed.

  11. Crosstalk between the proteasome system and autophagy in the clearance of α-synuclein

    Institute of Scientific and Technical Information of China (English)

    Fang YANG; Ya-ping YANG; Cheng-jie MAO; Ling LIU; Hui-fen ZHENG; Li-fang HU; Chun-feng LIU

    2013-01-01

    A growing body of evidence suggests that α-synuclein accumulation may play an important role in the pathogenesis of Parkinson's disease.The aim of this study was to investigate the roles of the proteasome and autophagy pathways in the clearance of wild-type and mutant α-synuclein in PC12 cells.Methods:PC12 cells overexpressing either wild-type or A30P mutant α-synuclein were treated with the proteasome inhibitor epoxomicin,the macroautophagy inhibitor 3-MA and the macroautophagy activator rapamycin alone or in combination.The cell viability was assessed using MTT assay.Immunofluorescence and Western blot analysis were used to detect the level of α-synuclein,LAMP-2A,E1 activase,and E2 ligase in the cells.Chymotrypsin-like proteasomal activity was measured using a commercial kit.Results:When the proteasome and macroautophagy in the wild-type and mutant cells were inhibited with epoxomicin and 3-MA,respectively,the cell viability was significantly decreased,and the α-synuclein level was increased.Both epoxomicin and 3-MA activated the chaperone-mediated autophagy (CMA) by increasing the level of the CMA-limiting enzyme LAMP-2A.Furthermore,3-MA or epoxomicin significantly decreased chymotrypsin-like proteasomal activity.3-MA or epoxomicin did not change E1 activase expression in either mutant or wild-type cells,but increased E2 ligase expression,especially when used together.Macroautophagy inducer rapamycin increased the cell viability and reduced epoxomicin-induced α-synuclein accumulation.Interestingly,CMA was also activated by rapamycin.Conclusion:Our results demonstrate the existence of complex crosstalk between different forms of autophagy and between autophagy and the proteasome pathway in the clearance of α-synuclein in PC12 cells.

  12. Molecular study on copper-mediated tumor proteasome inhibition and cell death.

    Science.gov (United States)

    Xiao, Yan; Chen, Di; Zhang, Xia; Cui, Qiuzhi; Fan, Yuhua; Bi, Caifeng; Dou, Q Ping

    2010-07-01

    The metal ion copper is a cofactor essential for maintaining normal biological and physical functions in human beings. High copper levels have been found in variety of tumor tissues and are involved in tumor angiogenesis processes. The ubiquitin-proteasome system plays an important role in cell growth and apoptosis and has been shown as a novel target for cancer therapy. We previously reported that some organic copper complexes can inhibit the proteasomal chymotrypsin-like activity and induce apoptosis in human cancer cells and xenograft models. In the current study, we investigated the effect of oxidation status of copper, Cu(I) or Cu(II), on inhibition of proteasome activity, induction of apoptosis, and induction of reactive oxygen species (ROS) in human cancer cells. We report four major findings here: i) both Cu(I) and Cu(II) could inhibit the chymotrypsin-like activity of purified 20S proteasome, but Cu(I) was more potent than Cu(II), ii) purified 20S proteasome protein was able to reduce Cu(II) to Cu(I), suggesting that Cu(I) is the oxidation status of copper that directly reacts with the proteasome, iii) when complexed with the copper ligand neocuproine, Cu(I) showed higher ability to induce ROS production in cancer cells, compared with Cu(II), iv) addition of a ROS scavenger in the cancer cell culture-blocked copper-induced ROS generation, but did not overcome copper-mediated proteasome-inhibitory and cell death-inducing events, demonstrating the ROS-independent proteasome-inhibitory property of copper complexes.

  13. Changes in digestive enzyme activities during larval development of Chinese loach Paramisgurnus dabryanus (Dabry de Thiersant, 1872).

    Science.gov (United States)

    Zhang, Yun-Long; Wu, Qiao-Wan; Hu, Wei-Hua; Wang, Fan; Zhao, Zhong-Bo; He, Hui; Shao, Wei-Han; Fan, Qi-Xue

    2015-12-01

    The digestive physiology of Chinese loach (Paramisgurnus dabryanus) was studied by assessing the specific and total activities of different pancreatic (trypsin, chymotrypsin, amylase and lipase), gastric (pepsin) and intestinal (alkaline phosphatase and leucine-aminopeptidase) enzymes from hatching to 40 days after hatching (DAH). Larvae were reared at 24.4 ± 0.4 °C and fed with rotifers from mouth opening (4 DAH) to 15 DAH, from 10 to 35 DAH with Cladocera and from 30 to 40 DAH with compound diet. Enzyme activities for trypsin, chymotrypsin, amylase and lipase were detected before the onset of exogenous feeding, indicating that these enzymes were genetically pre-programmed. Most of the pancreatic enzyme specific activities increased until 20 DAH and decreased thereafter. The pepsin activity of Chinese loach was firstly detected at 30 DAH, indicating the appearance of functional gastric gland. Alkaline phosphatase specific activity was detected from hatching onward, showed marked increase and reached the second peak at 20 DAH, while a gradual increase in specific leucine-aminopeptidase activity was observed until the end of the experiment. Accordingly, the larvae of Chinese loach possess a functional digestive system before the onset of exogenous feeding and the digestive capacity gradually increases as development progresses. The abrupt increase in intestinal enzyme activities between 10 and 20 DAH demonstrates onset of juvenile-like digestive mode in Chinese loach larvae. The increase in pepsin activity after 30 DAH indicates the shift from alkaline to acidic digestion in Chinese loach larvae, which may be considered as the onset of weaning. PMID:26232086

  14. Strategy for sensor based on fluorescence emission red shift of conjugated polymers: applications in pH response and enzyme activity detection.

    Science.gov (United States)

    Tang, Yanli; Liu, Yue; Cao, Ali

    2013-01-15

    A new strategy was developed and applied in monitoring pH response and enzyme activity based on fluorescence emission red shift (FERS) of the conjugated polymer PPP-OR10 induced by the inner filter effect (IFE) of nitrobenzene derivatives. Neutral poly(p-phenylenes) functionalized with oligo(oxyethylene) side chains (PPP-OR10) was designed and synthesized by the Suzuki cross-coupling reaction. Nitrobenzene derivatives display different light absorption activities in the acidic or basic form due to adopting different electron-transition types. When environmental pH is higher than their pK(a) values, nitrobenzene derivatives exhibit strong absorbance around 400 nm, which is close to the maximal emission of polymer PPP-OR10. As a result, the maximal emission wavelength of PPP-OR10/nitrobenzene derivatives red shifts with the pH value increasing. Apparently, the IFE plays a very important role in this case. A new method has been designed that takes advantage of this pH-sensitive platform to sensor α-chymotrypsin (ChT) based on the IFE of p-nitroaniline, since the absorption spectrum of p-nitroaniline, the ChT-hydrolyzed product of N-benzoyl-L-tyrosine-p-nitroaniline (BTNA), overlaps with the emission spectrum of PPP-OR10. In addition, the present approach can detect α-chymotrypsin with a detection limit of 0.1 μM, which is lower than that of the corresponding absorption spectroscopy method. Furthermore, the pH response and enzyme detections can be carried out in 10% serum, which makes this new FERS-based strategy promising in applications in more complex conditions and a broader field.

  15. Characterization of immunoglobulins through analysis of N-glycopeptides by MALDI-TOF MS.

    Science.gov (United States)

    Komatsu, Emy; Buist, Marjorie; Roy, Rini; Gomes de Oliveira, Andrey Giovanni; Bodnar, Edward; Salama, Apolline; Soulillou, Jean-Paul; Perreault, Hélène

    2016-07-15

    The aim of this report is to emphasize the role, usefulness and power of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the analysis of glycoforms of antibodies (Abs) through their proteolytic glycopeptides. Abs are complex biomolecules in which glycans hold determinant properties and thus need to be thoroughly characterized following Ab production by recombinant methods or Ab collection from human/animal serum or tissue. In spite of the great robustness of MALDI-TOF MS in terms of tolerance to impurities, the analysis of Abs and Ab components using this technique requires extensive sample preparation involving all or some of chromatography, solid phase extraction, enzymatic modification, and chemical derivatization. This report focuses on a monoclonal Ab produced in cell culture, as well as on a polyclonal human immunoglobulin (Ig) G obtained commercially and a polyclonal porcine IgG obtained from serum. A method is first provided to separate Ab protein chain components (light chains, heavy chains) by gel electrophoresis, which is useful for instance for protein-A eluates of Igs either from cell culture or biological samples. This allows for in-gel proteolytic digestion of the protein gel band(s) of choice for further MS characterization. Also discussed is the more conventional in-solution overnight digestion method used here with each of two proteolytic enzymes, i.e. trypsin and chymotrypsin. The overnight method is in turn compared with a much faster approach, that of digesting Abs with trypsin or chymotrypsin through the action of microwave heating. For method comparison, glycopeptides are fractionated from digestion mixtures using mostly C-18 cartridges for simplicity, although this enrichment procedure is also compared with other published procedures. The advantages of MALDI tandem mass spectrometry are highlighted for glycopeptide analysis, and lastly an esterification method applied to glycopeptides is

  16. 酶催化合成CCK-4三肽片段%Enzymatic synthesis of a CCK-4 tripeptide fragment

    Institute of Scientific and Technical Information of China (English)

    郭丽; 吕子敏; Heiner Eckstein

    2003-01-01

    Objective To synthesize a tripeptide derivative Phac-Met-Asp(OMe)-Phe -NH2, which is a fragment of the gastrin C-terminal tetrapeptide CCK-4, by enzymatic reaction. Methods Three free enzymes, α-chymotrypsin, papain and thermolysin from acyl donor Phac-Met-OCam was involved in three steps. The choice of appropriate enzymes and solvents was selected.Results All enzymatic reactions were obtained in reasonable yields (63%-92%). FAB-MS and FD-MS verified the correct molecular mass of the peptides. Conclusion Studies on the α-chymotrypsin catalyzed coupling reaction between Phac-Met-OCam and H-Asp(OMe)2 have focused on the low water content media. By papain catalyzed saponification of Phac-Met-Asp(OMe)2, α-methyl ester of aspartic acid is selectively hydrolyzed to retain β-methyl ester, and Phac-Met-Asp (OMe)-OH and H-Phc-NH2 can be coupled efficiently by thermolysin.%目的探讨酶法合成胃泌激素CCK-4 C-末端片段三肽衍生物Phac-Met-Asp(OMe)-Phe-NH2.方法以Phac-Met-OCam为起始原料,采用三种蛋白酶,即α-糜蛋白酶、木瓜蛋白酶、嗜热菌蛋白酶(α-chymotrypsin,papain,thermolysin),经过三步酶促反应得到目标三肽化合物;研究了酶催化条件下Met-Asp肽键的形成以及天门冬氨酸双酯中α-酯的酶催化条件下的选择性水解;对酶以及反应介质的选择等酶促合成条件进行了研究.结果三步酶促反应均可得到较合适的收率(63%~92%),产物经FAB-MS、FD-MS质谱确认.结论以α-糜蛋白酶为酶催化剂,在含有1.5%0.05 mol/LT^s-HCl缓冲液的乙酸乙酯介质中,Phac-Met-OCam和H-Asp(OMe)2缩合可得到收率大于63%的缩合产物;木瓜蛋白酶催化下可选择性水解Phac-Met-Asp(OMe)2中的α-酯而保留β-酯;嗜热菌蛋白酶能有效地催化Phac-Met-Asp(OMe)-OH与H-Phe-NH2生成肽键.

  17. Zinc oxide-montmorillonite hybrid influences diarrhea, intestinal mucosal integrity, and digestive enzyme activity in weaned pigs.

    Science.gov (United States)

    Hu, Caihong; Song, Juan; You, Zhaotong; Luan, Zhaoshuang; Li, Weifen

    2012-11-01

    One hundred-eighty piglets (Duroc × Landrace × Yorkshire), with an average initial weight of 7.4 kg weaned at 27 ± 1 days of age, were used to evaluate the effects of dietary zinc oxide-montmorillonite hybrid (ZnO-MMT) on growth performance, diarrhea, intestinal mucosal integrity, and digestive enzyme activity. All pigs were allotted to five treatments and fed with the basal diets supplemented with 0, 250, 500, and 750 mg/kg of Zn as ZnO-MMT or 2,000 mg/kg of Zn as ZnO. The results showed that supplementation with 500 or 750 mg/kg of Zn from ZnO-MMT and 2,000 mg/kg of Zn from ZnO improved average daily gain, enhanced average daily feed intake, decreased fecal scores at 4, 8, and 14 days postweaning, reduced intestinal permeability which was evident from the reduced lactulose recovery and urinary lactulose/mannitol ratio, and improved the activities of protease, amylase, lipase, trypsin, and chymotrypsin both in pancreas and small intestinal contents of pigs as compared with the control. Supplemental 250 mg/kg of Zn from ZnO-MMT also decreased fecal scores at 8 and 14 days postweaning, decreased urinary lactulose/mannitol ratio, and improved chymotrypsin activity in pancreas and small intestinal contents as well as protease activity in small intestinal contents compared with control. Moreover, the above indexes of weanling pigs fed with 500 or 750 mg/kg of Zn as ZnO-MMT did not differ from those fed with 2,000 mg/kg of Zn as ZnO. The results demonstrated that supplementation with 500 or 750 mg/kg of Zn from ZnO-MMT was as efficacious as 2,000 mg/kg of Zn from ZnO in improving growth performance, alleviating postweaning diarrhea, and enhancing intestinal mucosal integrity and the digestive enzyme activities in pancreas and small intestinal contents of pigs. The results that feeding lower concentrations of ZnO-MMT to weanling pigs maintained performance will be beneficial for the environment and for sustaining swine production. PMID:22539019

  18. Protein sources and digestive enzyme activities in jundiá (Rhamdia quelen Fontes protéicas e atividade de enzimas digestivas em jundiás (Rhamdia quelen

    Directory of Open Access Journals (Sweden)

    Rafael Lazzari

    2010-01-01

    Full Text Available Digestive enzymes activity influence feed utilization by fish, and its understanding is important to optimize diet formulation. This study reports the digestive enzyme activities of jundiá juveniles fed diets with protein sources. Fish were fed six experimental diets for 90 days: MBY (meat and bone meal + sugar cane yeast, SY (soybean meal + sugar cane yeast, S (soybean meal, MBS (meat and bone meal + soybean meal, FY (fish meal + sugar cane yeast and FS (fish meal + soybean meal, and then sampled every 30 days and assayed in two intestine sections for digestive enzymes - trypsin, chymotrypsin and amylase - activities; gastric protease was assayed in the stomach. Digestive and hepatosomatic index, intestinal quotient, digestive tract length and weight gain were also measured. Trypsin and chymotrypsin activities were higher (p As enzimas digestivas influenciam a utilização dos alimentos em peixes, e seu conhecimento é importante para otimizar a formulação de dietas. Este trabalho descreve a atividade de enzimas digestivas em juvenis de jundiá alimentados com fontes protéicas. Os peixes foram alimentados com seis dietas (90 dias: MBY (farinha de carne e ossos + levedura de cana, SY (farelo de soja + levedura de cana, S (farelo de soja, MBS (farinha de carne e ossos + farelo de soja, FY (farinha de peixe + levedura de cana e FS (farinha de peixe + farelo de soja. A cada 30 dias, foram analisadas as enzimas digestivas (tripsina, quimiotripsina e amilase no intestino. No estômago, foi mensurada a protease ácida. Foram estimados os índices digestivo e hepato-somático, quociente intestinal, comprimento do trato digestório e ganho em peso. As atividades de tripsina e quimiotripsina foram maiores (p < 0,0001 nos peixes alimentados com as dietas com fontes de origem animal (MBY, MBS, FY e FS. As proteases alcalinas foram afetadas negativamente pelo farelo de soja nas dietas. A atividade de amilase apresentou grandes variações. Os peixes

  19. SjAPI, the first functionally characterized Ascaris-type protease inhibitor from animal venoms.

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    Zongyun Chen

    Full Text Available BACKGROUND: Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear. PRINCIPAL FINDINGS: Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI, Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2, Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI, and Buthus martensii Ascaris-type protease inhibitor (BmAPI. The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues "AAV" and might be a useful template to produce new serine protease inhibitors. CONCLUSIONS/SIGNIFICANCE: To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the

  20. Hydrogen-ion titrations of amino acids and proteins in solutions containing concentrated electrolyte

    Energy Technology Data Exchange (ETDEWEB)

    Fergg, F. [Technische Universitaet Muenchen (Germany); Kuehner, D.E.; Blanch, H.W.; Prausnitz, J.M. [Lawrence Berkeley Lab., CA (United States)

    1994-12-01

    This report describes a first attempt to quantify the net charge as a function of solution pH for lysozyme and {alpha}-chymotrypsin at 0.1 M, 1.0 M and 3.0 M ionic strength, (IS). The calculations are based on the residue (titratable group) pK{sub a}`s in the amino-acid sequence of the protein. To determine these pK{sub a}`s, a simple theory was used which assumes that the pK{sub a}`s are independent from each other in the protein and are equal to their pK{sub a} values in free amino-acid solution (Independent-Site Theory, IST). Residue pK{sub a}`s were obtained from amino-acid hydrogen-ion titrations at three different KCl concentrations corresponding to 0.1M, 1.0M and 3.0M ionic strength. After construction of a suitable apparatus, the experimental procedure and data reduction were computerized to perform a large number of titrations. Most measured pK{sub a}`s showed high reproducibility (the difference of pK{sub a} values observed between two experiments was less than 0.05). For IS = 0.1M, observed pK{sub a}`s agreed with literature values to within a few hundredths of a pH unit. Furthermore, the ionic-strength dependence of the pK{sub a}`s followed the trends reported in the literature, viz. pK{sub a} values decrease with increasing ionic strength until they reach a minimum at about IS = 0.5M. At still higher IS, pK{sub a}`s increase as the ionic strength rises to 3M. The known pK{sub a}`s of all titratable groups in a protein were used with the IST to give a first approximation of how the protein net charge varies with pH at high ionic strength. A comparison of the titration curves based on the IST with experimental lysozyme and {alpha}-chymotrypsin titration data indicates acceptable agreement at IS = 0.1M. However, comparison of measured and calculated titration curves at IS = 1M and IS = 3M indicates only quantitative agreement.

  1. Kinetics and mechanism of proteinase-binding of pregnancy zone protein (PZP). Appearance of sulfhydryl groups in reactions with proteinases.

    Science.gov (United States)

    Christensen, U; Sottrup-Jensen, L; Simonsen, M

    1992-01-01

    Proteinase binding by pregnancy zone protein (PZP), an alpha-macroglobulin involves bait region cleavages, association of dimeric-PZP into tetrameric and reaction of internal gamma-glutamyl-beta-cysteinyl thiol esters of PZP with proteinase side chains. The product is an equimolar enzyme-PZP(tetramer) covalently linked complex with four free sulfhydryl groups. The kinetics of the appearances of sulfhydryl groups during the reaction of PZP with chymotrypsin has been investigated using stopped-flow and conventional mixing techniques over a broad concentration range. Thiol ester cleavages followed double exponential decays corresponding with two steps. The faster one resulted in the appearance of three sulfhydryl groups with an observed rate constant, k(obs) = k1.1 + k1.2 delta E, dependent on the excess concentration of chymotrypsin, delta E, and k1.1 = 0.03 s-1 and k1.2 = 4 x 10(4) M-1 s-1. The last sulfhydryl group appeared in a slower step, with similar concentration dependence and k2.1 approximately 0.003 s-1 and k2.2 approximately 5 x 10(3) M-1s-1. Covalent binding of the enzyme apparently was simultaneous with the faster thiol ester cleavage step. Based on these and previous results a model of the reaction mechanism of the proteinase binding reaction of PZP is proposed. It consists of four major steps: (i) Bait region cleavage of PZP-dimers by the enzyme, (ii) fast association of enzyme-PZP(dimer) species with native PZP or with another enzyme-PZP(dimer) compound resulting in release of one of the associated enzyme molecules (iii) reaction of an average of three thiol esters of the enzyme-PZP(tetramer) intermediate with the associated internal enzyme molecule or with an external one. In this step one enzyme molecule becomes covalently linked to the PZP-(tetramer), three sulfhydryl groups appear and the enzymic activity of the bound enzyme molecule decreases to the level of that of the final complex. (iv) Hydrolysis of the last thiol ester and in the presence of

  2. In vivo and in vitro effect of Acacia nilotica seed proteinase inhibitors on Helicoverpa armigera (Hübner) larvae

    Indian Academy of Sciences (India)

    S Ramesh Babu; B Subrahmanyam; Srinivasan; I M Santha

    2012-06-01

    Acacia nilotica proteinase inhibitor (AnPI) was isolated by ammonium sulphate precipitation followed by chromatography on DEAE-Sephadex A-25 and resulted in a purification of 10.68-fold with a 19.5% yield. Electrophoretic analysis of purified AnPI protein resolved into a single band with molecular weight of approximately 18.6+1.00 kDa. AnPI had high stability at different pH values (2.0 to 10.0) except at pH 5.0 and are thermolabile beyond 80°C for 10 min. AnPI exhibited effective against total proteolytic activity and trypsin-like activity, but did not show any inhibitory effect on chymotrypsin activity of midgut of Helicoverpa armigera. The inhibition kinetics studies against H. armigera gut trypsin are of non-competitive type. AnPI had low affinity for H. armigera gut trypsin when compared to SBTI. The partially purified and purified PI proteins-incorporated test diets showed significant reduction in mean larval and pupal weight of H. armigera. The results provide important clues in designing strategies by using the proteinase inhibitors (PIs) from the A. nilotica that can be expressed in genetically engineered plants to confer resistance to H. armigera.

  3. Pancreatic metaplasia in the gastro-achlorhydria in WTC-dfk rat, a potassium channel Kcnq1 mutant.

    Science.gov (United States)

    Kuwamura, M; Okajima, R; Yamate, J; Kotani, T; Kuramoto, T; Serikawa, T

    2008-07-01

    The WTC-deafness Kyoto (dfk) rat is a new mutant characterized by deafness and abnormal, imbalanced behavior. WTC-dfk rats carry an intragenic deletion at the Kcnq1 gene; KCNQ1 plays an important role in K(+) homeostasis, and the mutation of Kcnq1 causes a cardiac long QT syndrome in humans. Here, we studied stomach lesions in these WTC-dfk rats. The most characteristic pathologic feature in the stomach was the appearance of hypertrophic gastric glands in the stomach body. The hypertrophic cells had many eosinophilic granules in their cytoplasm, and these granules were stained red with Azan stain; stained positively for trypsinogen, amylase, and chymotrypsin; and did not stain positively for pepsinogen when using immunohistochemical analysis. These staining results suggested a metaplasia toward a pancreatic acinar cells. Extensive fibrosis was found in the bottom part of the mucosa of 34-week-old WTC-dfk rats, suggesting a progression of stomach lesions with aging. Although cells that were positive for proliferating cell nuclear antigen were restricted in the area of the glandular neck in WTC control rats, positive cells in WTC-dfk rats were scattered throughout the mucosa. The parietal cells in WTC-dfk rats were negative for KCNQ1 immunohistochemical analysis. These findings indicate that a deficiency in rat Kcnq1 provokes an abnormal proliferation and differentiation of gastric glandular cells.

  4. The amino acid sequence of Escherichia coli cyanase.

    Science.gov (United States)

    Chin, C C; Anderson, P M; Wold, F

    1983-01-10

    The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides. The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines. Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides. The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350). Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit. The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.

  5. A serine proteinase inhibitor isolated from Tamarindus indica seeds and its effects on the release of human neutrophil elastase.

    Science.gov (United States)

    Fook, J M S L L; Macedo, L L P; Moura, G E D D; Teixeira, F M; Oliveira, A S; Queiroz, A F S; Sales, M P

    2005-05-01

    Proteinaceous inhibitors with high inhibitory activities against human neutrophil elastase (HNE) were found in seeds of the Tamarind tree (Tamarindus indica). A serine proteinase inhibitor denoted PG50 was purified using ammonium sulphate and acetone precipitation followed by Sephacryl S-300 and Sephadex G-50 gel filtration chromatographies. Inhibitor PG50 showed a Mr of 14.9 K on Sephadex G-50 calibrated column and a Mr of 11.6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PG50 had selective activity while cysteine proteinases (papain and bromelain) and serine proteinases (porcine pancreatic elastase and bovine chymotrypsin) were not inhibited, it was strongly effective against serine proteinases such as bovine trypsin and isolated human neutrophil elastase. The IC50 value was determined to be 55.96 microg.mL-1. PG50 showed neither cytotoxic nor haemolytic activity on human blood cells. After pre-incubation of PG50 with cytochalasin B, the exocytosis of elastase was initiated using PAF and fMLP. PG50 exhibited different inhibition on elastase release by PAF, at 44.6% and on release by fMLP, at 28.4%. These results showed that PG50 preferentially affected elastase release by PAF stimuli and this may indicate selective inhibition on PAF receptors.

  6. New Prenylated Aeruginosin, Microphycin, Anabaenopeptin and Micropeptin Analogues from a Microcystis Bloom Material Collected in Kibbutz Kfar Blum, Israel

    Directory of Open Access Journals (Sweden)

    Shira Elkobi-Peer

    2015-04-01

    Full Text Available Thirteen new and eighteen known natural products were isolated from a bloom material of an assembly of various Microcystis spp. collected in November, 2008, from a commercial fishpond near Kibbutz Kfar Blum, the Jordan Valley, Israel. The new natural products included the prenylated aeruginosin KB676 (1, microphycin KB921 (2, anabaenopeptins KB906 (3 and KB899 (4 and micropeptins KB928 (5, KB956 (6, KB970A (7, KB970B (8, KB984 (9, KB970C (10, KB1048 (11, KB992 (12 and KB1046 (13. Their structures were elucidated primarily by interpretation of their 1D and 2D nuclear magnetic resonance spectra and high-resolution mass spectrometry. Marfey’s and chiral-phase high performance liquid chromatography methods were used to determine the absolute configurations of their chiral centers. Aeruginosin KB676 (1 contains the rare (2S,3aS,6S,7aS-Choi and is the first prenylated aeruginosin derivative described in the literature. Compounds 1 and 5–11 inhibited trypsin with sub-μM IC50s, while Compounds 11–13 inhibited chymotrypsin with sub-μM IC50s. The structures and biological activities of the new natural products and our procedures of dereplication are described.

  7. Synthesis and Resolution of Ring-substituted phenylalanines%苯环取代苯丙氨酸的合成及拆分

    Institute of Scientific and Technical Information of China (English)

    梁远军; 刘克良; 于文胜; 赵玉芬

    2001-01-01

    Seven racemic aromatic ring modified phenylalanines wer e sy nthesized using aniline derivatives as raw materials. The amino acids were resol ved with α--chymotrypsin or subtilisin. D-and L-amino acids were separ ated from each other. The chemical structures of the amino acids were characteri zed by IR, 1 HNMR, EA and optical rotation. The procedure mentioned here coul d be scaled up for producing the amino acids.%以苯胺类化合物为起始原料,经重氮化、丙烯酸加成、氨解 等反应合成了7种消旋苯环取代苯丙氨酸类氨基酸,并采用α-糜蛋白酶或枯草杆菌蛋白酶 对所有氨基酸进行了拆分,其结构分别通过红外光谱、核磁共振、元素分析、熔点、旋光等 分析得到确证。该法可以较大规模合成此类氨基酸。

  8. Development and in vitro characterisation of an oral self-emulsifying delivery system for daptomycin.

    Science.gov (United States)

    Zupančič, Ožbej; Partenhauser, Alexandra; Lam, Hung Thanh; Rohrer, Julia; Bernkop-Schnürch, Andreas

    2016-01-01

    It was the aim of this study to develop an oral self-emulsifying drug delivery system (SEDDS) for the peptide drug daptomycin exhibiting an anionic net charge. Drug lipophilicity was increased by hydrophobic ion pairing with cationic surfactant dodecylamine hydrochloride in molar ratio of surfactant to peptide 5:1. Log P (octanol/water) of -5.0 was even raised to +4.8 due to complexation with dodecylamine hydrochloride. Various SEDDS formulations were developed and characterised regarding emulsification properties, droplet size, polydispersity index and zeta potential. When the daptomycin dodecylamine complex (DAP/DOA) was dissolved in a formulation containing 35% Dermofeel MCT, 30% Capmul MCM and 35% Cremophor RH40, a maximum payload of even 8.0% (w/w) corresponding to 5.5% pure daptomycin was achieved. The formulation was degraded by lipase within 90min. Release studies of daptomycin from this formulation emulsified in 50mM phosphate buffer pH6.8 demonstrated a sustained drug release for at least six hours. Moreover, SEDDS exhibited also mucus permeating properties as well as a protective effect towards drug degradation by α-chymotrypsin. According to these results, SEDDS containing 8% DAP/DOA complex may be considered as a new potential oral delivery system for daptomycin. PMID:26485536

  9. Purification and properties of elastolytic enzyme from Flavobacterium immotum.

    Science.gov (United States)

    Ozaki, H; Shiio, I

    1975-01-01

    Elastolytic enzyme was purified and crystallized from culture fluid of Flavobacterium immotum No. 9-35. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was determined by Sephadex G-100 gel filtration to be 13,000. The isoelectric point was between pH 8.3 and 8.9. The optimum pH of the enzyme was 7.2 for elastolytic activity. The purified enzyme showed not only elastolytic activity, but also non-specific proteolytic activity against various other proteins. Milk-clotting activity was also observed. The enzyme did not act on keratin, collagen, or fourteen amino acid esters, including N-benzoyl-L-alanine methyl ester, N-benzoyl-L-arginine ethyl ester, and N-acetyl-L-tyrosine ethyl ester, which were typical substrates of pancreatic elastase [EC 3.4.21.11], trypsin [EC 3.4.21.4], and chymotrypsin [EC 3.4.21.1], respectively. However, the enzyme selectively hydrolyzed elastin when both elastin and albumin were present in the reaction mixture. The enzyme was inhibited by o-phenanthroline and various heavy metals such as cadmium, lead, zinc, and mercury. Various inhibitors, such as diisopropyl phosphofluoridate, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, trypsin inhibitor, iodoacetamide, etc., had no effect on the elastolytic activity.

  10. Characterization of certain proteinase isoenzymes produced by benign and virulent strains of Bacteroides nodosus.

    Science.gov (United States)

    Green, R S

    1985-11-01

    Three proteinase isoenzymes from one benign strain of Bacteroides nodosus and five proteinase isoenzymes from each of two virulent strains of B. nodosus were purified by horizontal slab polyacrylamide gel electrophoresis. The purified isoenzymes hydrolysed casein, collagen I, collagen III, elastin, alpha-elastin, fibrinogen, gelatin, haemoglobin and alpha-keratin. The pH optima of all the isoenzymes lay between 7.25 and 9.5, the range of 8.75-9.25 being common to all. The isoenzymes were inhibited by phenylmethylsulphonyl fluoride, diphenylcarbamyl chloride, L-(1-tosylamide-2-phenyl)ethyl chloromethyl ketone, EGTA and EDTA, indicating that they were chymotrypsin-like serine proteinases that require a metal ion for stability or activity. EDTA inhibition was not reversed by addition of Ca2+ or Mg2+. Some isoenzymes were activated by Mg2+, Ca2+, Cr3+ and Se4+ and all were inhibited by Fe2+, Co2+, Cu2+, Zn2+, Cd2+ and Hg2+. Isoenzymes from benign strains had a lower temperature stability, losing all activity at 55 degrees C, whereas those from virulent strains lost all activity at 60 degrees C.

  11. Evaluation of copper-dependent proteasome-inhibitory and apoptosis-inducing activities of novel pyrrolidine dithiocarbamate analogues.

    Science.gov (United States)

    Yu, Zhiyong; Wang, Fei; Milacic, Vesna; Li, Xiaofeng; Cui, Qiuzhi Cindy; Zhang, Bin; Yan, Bing; Dou, Q Ping

    2007-12-01

    Apoptosis has a central role in the pathogenesis of many human diseases, one of which is cancer. One of the most important strategies to regulate apoptosis is via the ubiquitin-proteasome pathway. It has been shown that inhibition of proteasomal chymotrypsin-like activity is a strong apoptosis-inducing stimulus and that actively proliferating cancer cells are more sensitive to proteasome inhibitors than normal or untransformed cells. Dithioscarbamates are a class of metal-chelating compounds with various applications in medicine. We reported previously that certain members of dithiocarbamates, such as pyrrolidine dithiocarbamate (PDTC), diethyldithiocarbamate and disulfiram, are able to bind with tumor cellular copper, forming an active complex with proteasome-inhibitory, apoptosis-inducing and anti-cancer activities. In the current study, we synthesized eight PDTC analogues with substitutions made to the pyrrolidine ring and studied their structure-activity relationships. We found that substitution of the pyrrolidine ring with piperidine had almost no effect on their proteasome-inhibitory and anti-proliferative potencies in human breast cancer cells. However, after the pyrrolidine ring was substituted with morpholine, the activity of the mixtures slightly decreased but was completely lost when piperazine with the attached ethyl group was used for the substitution. This structure-activity relationship was confirmed by the results generated with the corresponding copper complexes. Our data further support the novel concept of using accumulated copper in human cancer cells as a selective approach for chemotherapy. PMID:17982703

  12. Generation, Fractionation, and Characterization of Iron-Chelating Protein Hydrolysate from Palm Kernel Cake Proteins.

    Science.gov (United States)

    Zarei, Mohammad; Ghanbari, Rahele; Tajabadi, Naser; Abdul-Hamid, Azizah; Bakar, Fatimah Abu; Saari, Nazamid

    2016-02-01

    Palm kernel cake protein was hydrolyzed with different proteases namely papain, bromelain, subtilisin, flavourzyme, trypsin, chymotrypsin, and pepsin to generate different protein hydrolysates. Peptide content and iron-chelating activity of each hydrolysate were evaluated using O-phthaldialdehyde-based spectrophotometric method and ferrozine-based colorimetric assay, respectively. The results revealed a positive correlation between peptide contents and iron-chelating activities of the protein hydrolysates. Protein hydrolysate generated by papain exhibited the highest peptide content of 10.5 mM and highest iron-chelating activity of 64.8% compared with the other hydrolysates. Profiling of the papain-generated hydrolysate by reverse phase high performance liquid chromatography fractionation indicated a direct association between peptide content and iron-chelating activity in most of the fractions. Further fractionation using isoelectric focusing also revealed that protein hydrolysate with basic and neutral isoelectric point (pI) had the highest iron-chelating activity, although a few fractions in the acidic range also exhibited good metal chelating potential. After identification and synthesis of papain-generated peptides, GGIF and YLLLK showed among the highest iron-chelating activities of 56% and 53%, whereas their IC50 were 1.4 and 0.2 μM, respectively. PMID:26720491

  13. Agglutination of human erythrocytes by the interaction of Zn(2+)ion with histidine-651 on the extracellular domain of band 3.

    Science.gov (United States)

    Kiyotake, Kento; Ochiai, Hideharu; Yamaguchi, Takeo

    2016-05-01

    Clustering of band 3, chloride/bicarbonate exchanger, has been reported in Zn(2+)-treated human erythrocytes. However, the agglutination of human erythrocytes is also induced by the interaction of Zn(2+)ion with histidine on band 3. Identification of histidine that interacts with Zn(2+)ion remains to be determined. The Zn(2+)-induced agglutination of human erythrocytes was unaffected by chymotrypsin cleavage of the small loop region containing His-547 in the extracellular domain of band 3. On the other hand, papain digestion of the large loop region containing His-651 in band 3 inhibited such Zn(2+)-induced agglutination. Moreover, Zn(2+)-induced erythrocyte agglutination was inhibited by the peptide (ARGWVIHPLG) containing His-651, but not by the peptide such as ARGWVIRPLG, which His-651 was substituted by arginine. Among 10 kinds of animal erythrocytes tested, interestingly, no agglutination by Zn(2+)ions was observed in cow cells only that the forth amino acid in the upstream from His-669 on the large loop of cow band 3 is aspartate (Asp-665) instead of glycine. As expected, the agglutination of human erythrocytes by Zn(2+) ions was inhibited in the presence of aspartate. These data indicate that the interaction of Zn(2+) ion with His-651 residue of band 3 plays an important role in the Zn(2+)-induced agglutination of human erythrocytes. PMID:26859120

  14. Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

    Directory of Open Access Journals (Sweden)

    Thomas A Prohaska

    Full Text Available The serine protease inhibitor protein C inhibitor (PCI is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4 M(-1 s(-1. Low molecular weight (LMWH and unfractionated heparin (UFH slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml. By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

  15. Nucleotide-Induced Conformational Changes in Escherichia coli DnaA Protein Are Required for Bacterial ORC to Pre-RC Conversion at the Chromosomal Origin.

    Science.gov (United States)

    Saxena, Rahul; Vasudevan, Sona; Patil, Digvijay; Ashoura, Norah; Grimwade, Julia E; Crooke, Elliott

    2015-01-01

    DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket. Upon limited proteolysis with trypsin or chymotrypsin ADP-DnaA, but not ATP-DnaA generated relatively stable proteolytic fragments of various sizes. Examined sites of limited protease susceptibility that differ between ATP-DnaA and ADP-DnaA largely reside in the amino terminal half of DnaA. The concentration of adenine nucleotide needed to induce conformational changes, as detected by these protease susceptibilities of DnaA, coincides with the conversion of an inactive bacterial origin recognition complex (bORC) to a replication efficient pre-replication complex (pre-RC) at the E. coli chromosomal origin of replication (oriC). PMID:26610483

  16. Nucleotide-Induced Conformational Changes in Escherichia coli DnaA Protein Are Required for Bacterial ORC to Pre-RC Conversion at the Chromosomal Origin

    Directory of Open Access Journals (Sweden)

    Rahul Saxena

    2015-11-01

    Full Text Available DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket. Upon limited proteolysis with trypsin or chymotrypsin ADP-DnaA, but not ATP-DnaA generated relatively stable proteolytic fragments of various sizes. Examined sites of limited protease susceptibility that differ between ATP-DnaA and ADP-DnaA largely reside in the amino terminal half of DnaA. The concentration of adenine nucleotide needed to induce conformational changes, as detected by these protease susceptibilities of DnaA, coincides with the conversion of an inactive bacterial origin recognition complex (bORC to a replication efficient pre-replication complex (pre-RC at the E. coli chromosomal origin of replication (oriC.

  17. Synthesis and characterization of functional elastomeric poly(ester amide) co-polymers.

    Science.gov (United States)

    Jokhadze, G; Machaidze, M; Panosyan, H; Chu, C C; Katsarava, R

    2007-01-01

    A new family of random co-poly(ester amides)s (co-PEAs) having reactive pendant functional carboxylic acid groups were synthesized by co-polycondensation of di-p-toluenesulfonic acid salts of bis-(L-alpha-amino acid (L-leucine and/or L-phenylalanine)) alpha,omega-alkylene diesters with active diesters of dicarboxylic acids using di-p-toluenesulfonic acid salt of L-lysine benzyl ester as a co-monomer. The lateral benzyl ester groups in the L-lysine segment of co-PEAs were subsequently transformed into free COOH groups by catalytic hydrogenolysis using Pd black as a catalyst. The co-PEA-based polyacids obtained, as well as the original co-PEA having lateral benzyl ester groups were characterized by standard methods. In vitro biodegradation studies in the presence of hydrolases like alpha-chymotrypsin and lipase showed significant enzymatic-catalyzed biodegradation of these co-PEAs. These co-PEA-based polyacids were used for covalent attachment of iminoxyl radicals (4-amino-TEMPO) and in vitro biodegradation of 4-aminoTEMPO attached polymer was studied along with releasing kinetic of iminoxyl radical. PMID:17540117

  18. Pancreatic function testing:Here to stay for the 21st century

    Institute of Scientific and Technical Information of China (English)

    John G Lieb II; Peter V Draganov

    2008-01-01

    The diagnosis of Chronic Pancreatitis (CP) is based on the detection of abnormal structure or function of the diseased pancreas.The pancreatic function tests more accurately determine the presence of CP than tests of structure,especially for early stage disease.The function tests can be divided into two categories:noninvasive and invasive.The invasive "tube" tests can reliably detect mild,early CP,but are only available at a few referral centers and tend to be poorly tolerated by patients.The non-invasive tests are easy to obtain,but tend to perform poorly in patients with early,mild disease.Therefore,no one test is useful in all clinical situations,and a detailed understanding of the rational,pathophysiologic basis,strengths,and limitations of various tests is needed.This review highlights the role of various pancreatic function tests in the diagnosis of CP including fecal fat analysis,fecal elastase,fecal chymotrypsin,serum trypsin,the secretin stimulation test,the cholecystokinin (CCK) stimulation test,the combined secretin-CCK stimulation test,the intraductal and endoscopic secretin stimulation tests,and the functional magnetic resonance imaging of the pancreas after secretin stimulation.

  19. Efficacy of Wobe-Mugos {sup registered} E for reduction of oral mucositis after radiotherapy. Results of a prospective, randomized, placebo-controlled, triple-blind phase III multicenter study

    Energy Technology Data Exchange (ETDEWEB)

    Doerr, W.; Herrmann, T. [Univ. of Technology, Dresden (Germany). Dept. of Radiotherapy and Radiooncology, Medical Faculty Carl Gustav Carus

    2007-03-15

    Purpose: To investigate the efficacy and safety of Wobe-Mugos {sup registered} E (proteolytic enzymes) for amelioration of early side effects of radiotherapy for head-and-neck tumors, particularly oral mucositis. Patients and Methods: The study was a prospective, randomized, multicenter, placebo-controlled, triple-blind phase III study with parallel groups. 69 patients with carcinomas of the oropharynx or the oral cavity were enrolled between 1996 and 2000 in five centers; 54 of these were recruited in Dresden. Of the 69 patients, 61 (Dresden: 46) were available for analysis. The proteolytic enzymes tested (Wobe-Mugos {sup registered} E) comprised papain 100 mg, trypsin 40 mg, and chymotrypsin 40 mg. Results: Wobe-Mugos {sup registered} E was well tolerated. For the maximum mucositis scores, no statistically significant differences were found between the placebo and the verum group. The average mucositis score over weeks 1-6 revealed a significant difference in favor of the placebo arm, based on an earlier onset of mucositis in the Wobe-Mugos {sup registered} E group. Conclusion: The present study failed to demonstrate any effect of treatment with Wobe-Mugos {sup registered} E on radiotherapy side effects in patients treated for head-and-neck tumors. In particular, there was no beneficial effect on radiation-induced early oral mucositis. (orig.)

  20. Orchestration of an uncommon maturation cascade of the house dust mite protease allergen quartet

    Directory of Open Access Journals (Sweden)

    Marie-Eve eDumez

    2014-03-01

    Full Text Available In more than 20% of the world population, sensitization to house dust mite (HDM allergens triggers typical allergic diseases such as allergic rhinitis and asthma. Amongst the 23 mite allergen groups hitherto identified, groups 1 are cysteine proteases belonging to the papain-like family whereas groups 3, 6 and 9 are serine proteases displaying trypsin, chymotrypsin and collagenolytic activities, respectively. While these proteases are more likely to be involved in the mite digestive system, they also play critical roles in the initiation and in the chronicity of the allergic response notably through the activation of innate immune pathways. All these allergenic proteases are expressed in mite as inactive precursor form. Until recently, the exact mechanisms of their maturation into active proteases remained to be fully elucidated. Recent breakthroughs in the understanding of the activation mechanisms of mite allergenic protease precursors have highlighted an uncommon and unique maturation pathway orchestrated by group 1 proteases that tightly regulates the proteolytic activities of groups 1, 3, 6 and 9 through complex intra- or intermolecular mechanisms. This review presents and discusses the currently available knowledge of the activation mechanisms of group 1, 3, 6 and 9 allergens of Dermatophagoides pteronyssinus laying special emphasis on their localization, regulation and interconnection.

  1. PRELIMINARY DETECTION OF BACTERIOCIN-LIKE INHIBITORY SUBSTANCES PRODUCED BY ENTEROCOCCUS SPECIESISOLATED FROM DIFFERENT SOURCES

    Directory of Open Access Journals (Sweden)

    Snehal P Nemade and M Musaddiq

    2012-06-01

    Full Text Available Some lactic acid bacteria and particularly species belonging to the genus Enetrococcus are known to produce bacteriocin like inhibitory substance (BLIS. Usually they are small cationic peptide with bactericidal activity. The antimicrobial peptide produced by bacteria that deserve considerable interest for their use as natural and non-toxic food preservatives. The use of bacteriocin is among the new approaches as it has major potential in preservatives. Broad spectrum activities against prominent pathogens make it an issue of medical interest. The ability to produce such a biocompound may play role in providing an ecological advantage on non-bacteriocin producer species. 34 strains of Enterococci were isolated from different sources. These strains were identified to species: E. faecalis and E. faecium. Direct antimicrobial activity against indicator strain S. aureus was detected in 34 of the tested isolates. From these, only 7 displayed strong inhibitory activity against this indicator strain. The antimicrobial activity was altered after treatment with trypsin, α-chymotrypsin, papain which confirms the proteinaceous nature of the inhibition. This fact suggests that bacteriocin-like substance produced by Enterococcus strains may find application as biopreservatives in food products. Hence, the focus here is put on bacteriocin like substance screened by Enterococcus species isolated from different sources

  2. Measuring kinetic isotope effects in enzyme reactions using time-resolved electrospray mass spectrometry.

    Science.gov (United States)

    Liuni, Peter; Olkhov-Mitsel, Ekaterina; Orellana, Arturo; Wilson, Derek J

    2013-04-01

    Kinetic isotope effect (KIE) measurements are a powerful tool for studying enzyme mechanisms; they can provide insights into microscopic catalytic processes and even structural constraints for transition states. However, KIEs have not come into widespread use in enzymology, due in large part to the requirement for prohibitively cumbersome experimental procedures and daunting analytical frameworks. In this work, we introduce time-resolved electrospray ionization mass spectrometry (TRESI-MS) as a straightforward, precise, and inexpensive method for measuring KIEs. Neither radioisotopes nor large amounts of material are needed and kinetic measurements for isotopically "labeled" and "unlabeled" species are acquired simultaneously in a single "competitive" assay. The approach is demonstrated first using a relatively large isotope effect associated with yeast alcohol dehydrogenase (YADH) catalyzed oxidation of ethanol. The measured macroscopic KIE of 2.19 ± 0.05 is consistent with comparable measurements in the literature but cannot be interpreted in a way that provides insights into isotope effects in individual microscopic steps. To demonstrate the ability of TRESI-MS to directly measure intrinsic KIEs and to characterize the precision of the technique, we measure a much smaller (12)C/(13)C KIE associated specifically with presteady state acylation of chymotrypsin during hydrolysis of an ester substrate.

  3. Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

    Science.gov (United States)

    Platis, Dimitris; Labrou, Nikolaos E

    2008-03-01

    Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants. PMID:18307162

  4. Adaptive evolution of threonine deaminase in plant defense against insect herbivores

    Energy Technology Data Exchange (ETDEWEB)

    Gonzales-Vigil, Eliana; Bianchetti, Christopher M.; Phillips, Jr., George N.; Howe, Gregg A. (MSU); (UW)

    2011-11-07

    Gene duplication is a major source of plant chemical diversity that mediates plant-herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to {alpha}-ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and related Solanum species contain a duplicated TD paralog (TD2) that is coexpressed with a suite of genes involved in herbivore resistance. Analysis of TD2-deficient tomato lines showed that TD2 has a defensive function related to Thr catabolism in the gut of lepidopteran herbivores. During herbivory, the regulatory domain of TD2 is removed by proteolysis to generate a truncated protein (pTD2) that efficiently degrades Thr without being inhibited by Ile. We show that this proteolytic activation step occurs in the gut of lepidopteran but not coleopteran herbivores, and is catalyzed by a chymotrypsin-like protease of insect origin. Analysis of purified recombinant enzymes showed that TD2 is remarkably more resistant to proteolysis and high temperature than the ancestral TD1 isoform. The crystal structure of pTD2 provided evidence that electrostatic interactions constitute a stabilizing feature associated with adaptation of TD2 to the extreme environment of the lepidopteran gut. These findings demonstrate a role for gene duplication in the evolution of a plant defense that targets and co-opts herbivore digestive physiology.

  5. Emodin potentiates the antiproliferative effect of interferon α/β by activation of JAK/STAT pathway signaling through inhibition of the 26S proteasome.

    Science.gov (United States)

    He, Yujiao; Huang, Junmei; Wang, Ping; Shen, Xiaofei; Li, Sheng; Yang, Lijuan; Liu, Wanli; Suksamrarn, Apichart; Zhang, Guolin; Wang, Fei

    2016-01-26

    The 26S proteasome is a negative regulator of type I interferon (IFN-α/β) signaling. Inhibition of the 26S proteasome by small molecules may be a new strategy to enhance the efficacy of type I IFNs and reduce their side effects. Using cell-based screening assay for new 26S proteasome inhibitors, we found that emodin, a natural anthraquinone, was a potent inhibitor of the human 26S proteasome. Emodin preferably inhibited the caspase-like and chymotrypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Computational modeling showed that emodin exhibited an orientation/conformation favorable to nucleophilic attack in the active pocket of the β1, β2, and β5 subunits of the 26S proteasome. Emodin increased phosphorylation of STAT1, decreased phosphorylation of STAT3 and increased endogenous gene expression stimulated by IFN-α. Emodin inhibited IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Emodin also sensitized the antiproliferative effect of IFN-α in HeLa cervical carcinoma cells and reduced tumor growth in Huh7 hepatocellular carcinoma-bearing mice. These results suggest that emodin potentiates the antiproliferative effect of IFN-α by activation of JAK/STAT pathway signaling through inhibition of 26S proteasome-stimulated IFNAR1 degradation. Therefore, emodin warrants further investigation as a new means to enhance the efficacy of IFN-α/β. PMID:26683360

  6. Inhibitory effect of metal ions on alkaline mesentericopeptidase.

    Science.gov (United States)

    Raykova, D; Dorovska-Taran, V; Blagoev, B

    1981-01-01

    The effect of AG+, Cu2+, Cd2+, Co2+ and Ni2+ on the activity of alkaline mesentericopeptidase (EC 3.4.21.-) has been studied. Ag+, Cu2+ and Cd2+ were found to be reversible non-competitive inhibitors of the enzyme. The pH-dependence of Ki for Ag+-inhibition is sigmoidal with a pKa near 6. The Kilim values, calculated for the pH-independent region of the metal-enzyme inhibition, are close to the corresponding dissociation constants of metal-imidazole complexes, thus implying that the inhibitory effect of metal ions on enzyme activity is due to complex formation with the imidazole group of the active site histidine. The method of the two-component inhibition showed that Cu2+ and Ag+ bind to the same ligand of the enzyme molecule. The addition of Cu2+ decreases the rate of deacylation of the hydrolysis of p-nitrophenyl valerate, catalyzed by alkaline mesentericopeptidase in contrast to alpha-chymotrypsin where the acylation step is affected. PMID:7016196

  7. Detection and preliminary characterization of a narrow spectrum bacteriocin produced by Lactobacillus pentosus K2N7 from Thai traditional fermented shrimp (Kung-Som

    Directory of Open Access Journals (Sweden)

    Nisit Watthanasakphuban

    2016-02-01

    Full Text Available A total of 48 lactic acid bacteria (LAB exhibited antagonistic activity against Lactobacillus sakei subsp. sakei JCM 1157 or Staphylococcus aureus DMST 8840. Only strain K2N7 was selected for characterization of bacteriocin activity. It was identified as Lactobacillus pentosus based on 16S rDNA analysis. The maximum bacteriocin production was detected in early stationary phase of growth. It was found to be sensitive to proteolytic enzymes (trypsin, proteinase K, pronase E and -chymotrypsin. The bacteriocin K2N7 was heat stable (2 h at 100ºC and retained activity over a wide pH range (2.0-12.0. Bacteriocin K2N7 has a narrow inhibitory spectrum restricted to genus Lactobacillus including Lactobacillus plantarum D6SM3, a bacterial strain known to cause overfermentation in Kung-Som. The peptide was purified by 60% ammonium sulphate precipitation followed by sequential cation exchange chromatography and hydrophobic interaction characteristic. The molecular mass of bacteriocin K2N7 (2.017 kDa was determined by matrix-assisted laser desorption/ionization time-offlight mass spectrometry analysis (MALDI-TOF MS.

  8. Tissue factor pathway inhibitor-2 may interact with nuclear protein RASSF1C

    Institute of Scientific and Technical Information of China (English)

    Xudong Chen; Zhenwu Li; Jin Zhang; Zuohua Mao; Duan Ma; Huijun Wang

    2012-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region,three tandem Kunitz-type domains,and a positively charged carboxyterminal tail.Human TFPI-2 (hTFPI-2) inhibits a broad spectrum of serine proteinases (including trypsin,plasmin,plasma kallikrein,XIa,and chymotrypsin) almost exclusively via its first Kunitz-type domain,and potentially plays an important role in the regulation of extracellular matrix digestion and remodeling [1].Reduced TFPI-2 synthesis has been related to numerous pathophysiological processes such as inflammation,angiogenesis,atherosclerosis [2,3],retinal degeneration,and tumor growth/metastasis [4-6].It has been suggested that TFPI-2 is a tumor suppressor gene in some cancers [7,8].However,the specific physiological functions of hTFPI-2 in humans are unclear,particularly its interactions with other proteins.To better understand the physiological function of hTFPI-2,we used yeast two-hybrid system screening and bioinformatics analysis to identify its interacting proteins and confirm its interactions with nuclear protein RASSF1C using confocal microscopy and co-immunoprecipitation.

  9. Chronic Pancreatitis Associated with the p.G208A Variant of PRSS1 Gene in a European Patient

    Directory of Open Access Journals (Sweden)

    Eszter Hegyi

    2014-01-01

    Full Text Available Context The major etiologic factor of chronic pancreatitis in adults is excessive alcohol consumption, whereas among Children structural anomalies, systemic and metabolic disorders, and genetic factors are prevalent. Mutations in the cationic trypsinogen gene (PRSS1 cause hereditary pancreatitis, while mutations in serine protease inhibitor Kazal type 1 (SPINK1, cystic fibrosis transmembrane conductance regulator (CFTR and chymotrypsin C (CTRC genes have been shown to associate with chronic pancreatitis as independent risk factors. Case report We present a case of 13-year-old boy with idiopathic chronic pancreatitis. Given the unexplained attacks of pancreatitis since early childhood and despite the negative family history, molecular-genetic analysis of four pancreatitis susceptibility genes (PRSS1, SPINK1, CTRC and CFTR was performed. The boy was found to carry the c.623G>C (p.G208A mutation of the PRSS1 gene and the c.180C>T (p.G60G mutation of the CTRC gene, both in heterozygous state. These mutations are considered as contributing risk factors for chronic pancreatitis. Conclusions In children with idiopathic chronic pancreatitis genetic causes should be considered, even in absence of positive family history. To the best of our knowledge, this is the first description of a European patient with chronic pancreatitis associated with the p.G208A mutation of PRSS1 gene. This mutation was previously reported only in Asian subjects and is thought to be a unique genetic cause of pancreatitis in Asia.

  10. Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein

    International Nuclear Information System (INIS)

    The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein

  11. Discovery of salt taste enhancing arginyl dipeptides in protein digests and fermented fish sauces by means of a sensomics approach.

    Science.gov (United States)

    Schindler, Alexander; Dunkel, Andreas; Stähler, Frauke; Backes, Michael; Ley, Jakob; Meyerhof, Wolfgang; Hofmann, Thomas

    2011-12-14

    As enzymatic digests of fish proteins were recently reported to enhance salt taste, the fish protein protamine was digested by chymotrypsin and trypsin and subsequently screened for candidate salt taste modulating (STM) peptides. To achieve this, first, a two-step sensory assay was developed and demonstrated to be a rather suitable tool for the detection of salt taste enhancers and the "quantitation" of their salt taste enhancing activity on the basis of isointensities with reference solutions. By means of activity-guided fractionation using ultrafiltration, gel permeation chromatography, and hydrophilic liquid interaction chromatography in combination with the sensory assay for STM activity assessment, a series of arginyl dipeptides, with RP, RA, AR, RG, RS, RV, VR, and RM being the most active, as well as l-arginine were found as salt taste enhancing molecules in fish protamine digests. For the first time, HPLC-MS/MS analysis on a PFP and a HILIC stationary phase, respectively, enabled the quantitative analysis of the arginyl peptides in a series of commercial and laboratory-made protein hydrolysates as well as fermented fish sauces.

  12. Selective Uptake and Refolding of Globular Proteins in Coacervate Microdroplets.

    Science.gov (United States)

    Martin, Nicolas; Li, Mei; Mann, Stephen

    2016-06-14

    Intrinsic differences in the molecular sequestration of folded and unfolded proteins within poly(diallyldimethylammonium) (PDDA)/poly(acrylate) (PAA) coacervate microdroplets are exploited to establish membrane-free microcompartments that support protein refolding, facilitate the recovery of secondary structure and enzyme activity, and enable the selective uptake and exclusion of folded and unfolded biomolecules, respectively. Native bovine serum albumin, carbonic anhydrase, and α-chymotrypsin are preferentially sequestered within positively charged coacervate microdroplets, and the unfolding of these proteins in the presence of increasing amounts of urea results in an exponential decrease in the equilibrium partition constants as well as the kinetic release of unfolded molecules from the droplets into the surrounding continuous phase. Slow refolding in the presence of positively charged microdroplets leads to the resequestration of functional proteins and the restoration of enzymatic activity; however, fast refolding results in protein aggregation at the droplet surface. In contrast, slow and fast refolding in the presence of negatively charged PDDA/PAA droplets gives rise to reduced protein aggregation and misfolding by interactions at the droplet surface to give increased levels of protein renaturation. Together, our observations provide new insights into the bottom-up design and construction of self-assembling microcompartments capable of supporting the selective uptake and refolding of globular proteins.

  13. Secapin, a bee venom peptide, exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities.

    Science.gov (United States)

    Lee, Kwang Sik; Kim, Bo Yeon; Yoon, Hyung Joo; Choi, Yong Soo; Jin, Byung Rae

    2016-10-01

    Bee venom contains a variety of peptide constituents that have various biological, toxicological, and pharmacological actions. However, the biological actions of secapin, a venom peptide in bee venom, remain largely unknown. Here, we provide the evidence that Asiatic honeybee (Apis cerana) secapin (AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. The recombinant mature AcSecapin-1 peptide was expressed in baculovirus-infected insect cells. AcSecapin-1 functions as a serine protease inhibitor-like peptide that has inhibitory effects against plasmin, elastases, microbial serine proteases, trypsin, and chymotrypsin. Consistent with these functions, AcSecapin-1 inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products, thus indicating the role of AcSecapin-1 as an anti-fibrinolytic agent. AcSecapin-1 also inhibited both human neutrophil and porcine pancreatic elastases. Furthermore, AcSecapin-1 bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi and gram-positive and gram-negative bacteria. Taken together, our data demonstrated that the bee venom peptide secapin has multifunctional roles as an anti-fibrinolytic agent during fibrinolysis and an anti-microbial agent in the innate immune response. PMID:27208884

  14. Topography of the simian rotavirus nonstructural glycoprotein (NS28) in the endoplasmic reticulum membrane.

    Science.gov (United States)

    Chan, W K; Au, K S; Estes, M K

    1988-06-01

    The simian rotavirus SA11 genome segment 10 codes for a nonstructural glycoprotein, NS28, that has been hypothesized to be involved in budding of viral particles into the endoplasmic reticulum (ER) membrane. Previous studies had suggested that NS28 is an integral membrane protein of the ER, possibly a transmembrane protein. We have examined the topography of NS28 inserted in microsomal membranes following cell-free translation of genome segment 10 transcripts. These transcripts were obtained either by hybrid selection of mRNA synthesized by the endogenous viral RNA polymerase or by in vitro transcription of genome segment 10 cDNA using SP6 polymerase. Full-length and truncated gene 10 transcripts were translated in a cell-free system supplemented with dog pancreatic microsomes. The existence of a cytoplasmic domain of the translation product was demonstrated by protease protection experiments. An 18,000 (18K) mol wt glycosylated polypeptide was protected from digestion with proteinase K and trypsin, whereas chymotrypsin digestion yielded a 23K mol wt glycosylated polypeptide. Correlation of these biochemical data with the known sequence of NS28 suggests that a 10K mol wt hydrophilic, carboxy-terminal fragment (from amino acid number 86 to amino acid number 175) of this glycoprotein is exposed on the cytoplasmic side of the ER membrane. A model of how NS28 folds in the ER membrane is proposed. PMID:2835861

  15. Studies on terrein as a new class of proteasome inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Demasi, M.; Felicio, A.L.; Lima, C. [Instituto Butantan, Sao Paulo, SP (Brazil); Pacheco, A.O.; Leite, H.G.; Andrade, L.H. [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Quimica], e-mail: marimasi@butantan.gov.br

    2010-07-01

    The proteasome is an intracellular multicatalytic protease involved in the cell cycle regulation, signaling response, antigen presentation and apoptosis. Since proteasome inhibitors promote cell death by apoptosis, they have been proposed as new anti-tumoral drugs. Terrein, a secondary metabolite secreted by the fungus Aspergillus terreus, was firstly described in 1935. In the present work we report that terrein isolated through the screening for inhibitors of the 20S proteasome showed inhibitory effect upon both chymotrypsin- and trypsin-like activities of the multicatalytic core particle, the 20S proteasome. Despite of the high inhibitory concentration determined in vitro, that verified by incubating cells (fibroblasts and a pulmonary tumor cell line) in the presence of terrein was 4-fold lower indicating the proteasome as a selective intracellular target. Moreover, terrein promoted apoptotic cell death on both fibroblasts and pulmonary tumor cell line tested. Although terrein concentrations (mM range) necessary to elicit apoptosis in the cellular models herein tried were high when compared to those (muM and nM range) of other inhibitors recently described, its chemical structure is not correlated to any other inhibitor reported thus far. Therefore, the present results point out for the possibility of exploring terrein as a new molecular fragment for the development of synthetic proteasome inhibitors. (author)

  16. Computational model for protein unfolding simulation

    Science.gov (United States)

    Tian, Xu-Hong; Zheng, Ye-Han; Jiao, Xiong; Liu, Cai-Xing; Chang, Shan

    2011-06-01

    The protein folding problem is one of the fundamental and important questions in molecular biology. However, the all-atom molecular dynamics studies of protein folding and unfolding are still computationally expensive and severely limited by the time scale of simulation. In this paper, a simple and fast protein unfolding method is proposed based on the conformational stability analyses and structure modeling. In this method, two structure-based conditions are considered to identify the unstable regions of proteins during the unfolding processes. The protein unfolding trajectories are mimicked through iterative structure modeling according to conformational stability analyses. Two proteins, chymotrypsin inhibitor 2 (CI2) and α -spectrin SH3 domain (SH3) were simulated by this method. Their unfolding pathways are consistent with the previous molecular dynamics simulations. Furthermore, the transition states of the two proteins were identified in unfolding processes and the theoretical Φ values of these transition states showed significant correlations with the experimental data (the correlation coefficients are >0.8). The results indicate that this method is effective in studying protein unfolding. Moreover, we analyzed and discussed the influence of parameters on the unfolding simulation. This simple coarse-grained model may provide a general and fast approach for the mechanism studies of protein folding.

  17. Generation, Fractionation, and Characterization of Iron-Chelating Protein Hydrolysate from Palm Kernel Cake Proteins.

    Science.gov (United States)

    Zarei, Mohammad; Ghanbari, Rahele; Tajabadi, Naser; Abdul-Hamid, Azizah; Bakar, Fatimah Abu; Saari, Nazamid

    2016-02-01

    Palm kernel cake protein was hydrolyzed with different proteases namely papain, bromelain, subtilisin, flavourzyme, trypsin, chymotrypsin, and pepsin to generate different protein hydrolysates. Peptide content and iron-chelating activity of each hydrolysate were evaluated using O-phthaldialdehyde-based spectrophotometric method and ferrozine-based colorimetric assay, respectively. The results revealed a positive correlation between peptide contents and iron-chelating activities of the protein hydrolysates. Protein hydrolysate generated by papain exhibited the highest peptide content of 10.5 mM and highest iron-chelating activity of 64.8% compared with the other hydrolysates. Profiling of the papain-generated hydrolysate by reverse phase high performance liquid chromatography fractionation indicated a direct association between peptide content and iron-chelating activity in most of the fractions. Further fractionation using isoelectric focusing also revealed that protein hydrolysate with basic and neutral isoelectric point (pI) had the highest iron-chelating activity, although a few fractions in the acidic range also exhibited good metal chelating potential. After identification and synthesis of papain-generated peptides, GGIF and YLLLK showed among the highest iron-chelating activities of 56% and 53%, whereas their IC50 were 1.4 and 0.2 μM, respectively.

  18. Molecular dynamics investigation of the ionic liquid/enzyme interface: application to engineering enzyme surface charge.

    Science.gov (United States)

    Burney, Patrick R; Nordwald, Erik M; Hickman, Katie; Kaar, Joel L; Pfaendtner, Jim

    2015-04-01

    Molecular simulations of the enzymes Candida rugosa lipase and Bos taurus α-chymotrypsin in aqueous ionic liquids 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium ethyl sulfate were used to study the change in enzyme-solvent interactions induced by modification of the enzyme surface charge. The enzymes were altered by randomly mutating lysine surface residues to glutamate, effectively decreasing the net surface charge by two for each mutation. These mutations resemble succinylation of the enzyme by chemical modification, which has been shown to enhance the stability of both enzymes in ILs. After establishing that the enzymes were stable on the simulated time scales, we focused the analysis on the organization of the ionic liquid substituents about the enzyme surface. Calculated solvent charge densities show that for both enzymes and in both solvents that changing positively charged residues to negative charge does indeed increase the charge density of the solvent near the enzyme surface. The radial distribution of IL constituents with respect to the enzyme reveals decreased interactions with the anion are prevalent in the modified systems when compared to the wild type, which is largely accompanied by an increase in cation contact. Additionally, the radial dependence of the charge density and ion distribution indicates that the effect of altering enzyme charge is confined to short range (≤1 nm) ordering of the IL. Ultimately, these results, which are consistent with that from prior experiments, provide molecular insight into the effect of enzyme surface charge on enzyme stability in ILs. PMID:25641162

  19. Influence of reactive oxygen species on the enzyme stability and activity in the presence of ionic liquids.

    Directory of Open Access Journals (Sweden)

    Pankaj Attri

    Full Text Available In this paper, we have examined the effect of ammonium and imidazolium based ionic liquids (ILs on the stability and activity of proteolytic enzyme α-chymotrypsin (CT in the presence of cold atmospheric pressure plasma jet (APPJ. The present work aims to illustrate the state of art implementing the combined action of ILs and APPJ on the enzyme stability and activity. Our circular dichroism (CD, fluorescence and enzyme activity results of CT have revealed that buffer and all studied ILs {triethylammonium hydrogen sulphate (TEAS from ammonium family and 1-butyl-3-methyl imidazolium chloride ([Bmim][Cl], 1-methylimidazolium chloride ([Mim][Cl] from imidazolium family} are notable to act as protective agents against the deleterious action of the APPJ, except triethylammonium dihydrogen phosphate (TEAP ammonium IL. However, TEAP attenuates strongly the deleterious action of reactive oxygen species (ROS created by APPJ on native structure of CT. Further, TEAP is able to retain the enzymatic activity after APPJ exposure which is absent in all the other systems.This study provides the first combined effect of APPJ and ILs on biomolecules that may generate many theoretical and experimental opportunities. Through this methodology, we can utilise both enzyme and plasma simultaneously without affecting the enzyme structure and activity on the material surface; which can prove to be applicable in various fields.

  20. Expression of Aedes trypsin-modulating oostatic factor on the virion of TMV: A potential larvicide.

    Science.gov (United States)

    Borovsky, Dov; Rabindran, Shailaja; Dawson, William O; Powell, Charles A; Iannotti, Donna A; Morris, Timothy J; Shabanowitz, Jeffry; Hunt, Donald F; DeBondt, Hendrik L; DeLoof, Arnold

    2006-12-12

    We report the engineering of the surface of the tobacco mosaic virus (TMV) virion with a mosquito decapeptide hormone, trypsin-modulating oostatic factor (TMOF). The TMV coat protein (CP) was fused to TMOF at the C terminus by using a read-through, leaky stop codon that facilitated expression of CP and chimeric CP-TMOF (20:1 ratio) that were coassembled into virus particles in infected Nicotiana tabacum. Plants that were infected with the hybrid TMV RNA accumulated TMOF to levels of 1.3% of total soluble protein. Infected tobacco leaf discs that were fed to Heliothis virescens fourth-instar larvae stunted their growth and inhibited trypsin and chymotrypsin activity in their midgut. Purified CP-TMOF virions fed to mosquito larvae stopped larval growth and caused death. Because TMV has a wide host range, expressing TMV-TMOF in plants can be used as a general method to protect them against agricultural insect pests and to control vector mosquitoes.

  1. Cyclic pentapeptide analogs based on endomorphin-2 structure: cyclization studies using liquid chromatography combined with on-line mass spectrometry and tandem mass spectrometry.

    Science.gov (United States)

    Piekielna, Justyna; Kluczyk, Alicja; Perlikowska, Renata; Janecka, Anna

    2014-05-01

    The cyclization of linear analogs based on endomorphin-2 structure, Tyr/Dmt-d-Lys-Phe-Phe-Asp-NH2 and Tyr/Dmt-d-Cys-Phe-Phe-Cys-NH2 (where Dmt=2',6'-dimethyltyrosine), resulting in obtaining lactam or disulfide derivatives, was studied using liquid chromatography combined with on-line mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). In case of cyclization via an amide bond, the formation of the cyclic monomers, cyclic but not linear dimers and even traces of cyclic trimers was observed. Disulfide bridge containing peptides was obtained by the solid-phase synthesis of the linear sequences, followed by either in-solution or on-resin cyclization. In case of the in-solution cyclization, the expected cyclic monomers were the only products. When oxidation of the cysteine residues was performed when the peptides were still on the resin, cyclic monomer and two cyclodimers, parallel and antiparallel, were found. Digestion of the isolated cyclodimers with α-chymotrypsin allowed for their unambiguous identification. The comparison of the cyclic monomer/dimer ratios for analogs with Tyr versus Dmt in position 1 revealed that the presence of the exocyclic Dmt favored formation of the cyclic monomer, most likely due to the increased steric bulk of this amino acid side-chain as compared with Tyr. PMID:24525024

  2. [Evaluation of reference genes for quantitative real-time PCR normalization in cotton bollworm, Helicoverna armigera].

    Science.gov (United States)

    Chandra, G Sharath; Asokan, R; Manamohan, M; Kumar, N K K; Sita, T

    2014-01-01

    Reverse-transcription quantitative real-time PCR (RT-qPCR), a sensitive technique is being extensively employed in quantification of gene expression. However this requires normalization with suitable reference gene (RG) which is crucial in minimizing inter sample variations. Information regarding suitable RG is scarce in general and more so in insects, including the cotton bollworm, Helicoverpa armigera, an economically important pest. In management of this pest RNA interference (RNAi), is perceived as a potential tool, which is achieved by double-stranded RNA (dsRNA) delivery. These studies demand accurate quantification of gene silencing. In this study we assessed the suitability of five RGs viz. β-actin (ACTB), 18S rRNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB) and elongation fator-1-alfa (EF1-α) for gene expression studies in dsRNA treatment and across different developmental stages of H. armigera and ranked using geNorm, NormFinder and BestKeeper software programs. Data analysis revealed that best ranked RGs were varied in dsRNA treatment and in developmental stages. Under dsRNA treatment, 18S and GAPDH were more stable whereas, TUB and GAPDH were more stable across developmental stages. We also demonstrate that inappropriate selection of RG led to erroneous estimation of the target gene, chymotrypsin, expression. These results facilitate accurate quantification of gene expression in H. armigera.

  3. Probing structural heterogeneities and conformational fluctuations of biopolymers

    Energy Technology Data Exchange (ETDEWEB)

    Laurence, T; Kong, X; Jaeger, M; Weiss, S

    2004-12-15

    We study protein and nucleic acid structure and dynamics using single-molecule fluorescence resonance energy transfer measurements with alternating-laser excitation. Freely diffusing molecules are sorted into subpopulations based on stoichiometry, detecting donor and acceptor coincidence for periods over 100 {micro}s-1 ms. Faster (< 100 {micro}s) fluctuating distance distributions are studied within these subpopulations using time-resolved single photon counting measurements. We find that short double-stranded DNA (dsDNA) is more flexible than expected from persistence lengths measured on long dsDNA. We find that the electrostatic portion of the persistence length of single-stranded poly-dT varies as the ionic strength (I) to the -1/2 power (I{sup -1/2}). Lastly, we find that the unfolded protein Chymotrypsin Inhibitor 2 (CI2) is unstructured at high denaturant. However, in the presence of folded CI2 (at lower denaturant), unfolded CI2 is more compact and displays larger distance fluctuations, possibly due to unsuccessful attempts to cross the folding barrier.

  4. Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Werb, Z.; Aggeler, J.

    1978-04-01

    We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

  5. New poly(ester urea) derived from L-leucine: electrospun scaffolds loaded with antibacterial drugs and enzymes.

    Science.gov (United States)

    Díaz, Angélica; del Valle, Luis J; Tugushi, David; Katsarava, Ramaz; Puiggalí, Jordi

    2015-01-01

    Electrospun scaffolds from an amino acid containing poly(ester urea) (PEU) were developed as promising materials in the biomedical field and specifically in tissue engineering applications. The selected poly(ester urea) was obtained with a high yield and molecular weight by reaction of phosgene with a bis(α-aminoacyl)-α,ω-diol-diester monomer. The polymer having L-leucine, 1,6-hexanediol and carbonic acid units had a semicrystalline character and relatively high glass transition and melting temperatures. Furthermore it was highly soluble in most organic solvents, an interesting feature that facilitated the electrospinning process and the effective incorporation of drugs with bactericidal activity (e.g. biguanide derivatives such as clorhexidine and polyhexamethylenebiguanide) and enzymes (e.g. α-chymotrypsin) that accelerated the degradation process. Continuous micro/nanofibers were obtained under a wide range of processing conditions, being diameters of electrospun fibers dependent on the drug and solvent used. Poly(ester urea) samples were degradable in media containing lipases and proteinases but the degradation rate was highly dependent on the surface area, being specifically greater for scaffolds with respect to films. The high hydrophobicity of new scaffolds had repercussions on enzymatic degradability since different weight loss rates were found depending on how samples were exposed to the medium (e.g. forced or non-forced immersion). New scaffolds were biocompatible, as demonstrated by adhesion and proliferation assays performed with fibroblast and epithelial cells.

  6. Digestive enzyme activity of two stonefly species (Insecta, Plecoptera) and their feeding habits.

    Science.gov (United States)

    de Figueroa, J M Tierno; Trenzado, C E; López-Rodríguez, M J; Sanz, A

    2011-11-01

    The digestive enzymes of two stoneflies species, Hemimelaena flaviventris and Isoperla morenica, were studied for the first time. These species are temporary water inhabitants and exhibit great feeding plasticity. Although they are traditionally referred to as predators, a previous study revealed that H. flaviventris incorporates some diatoms into its diet in addition to feeding usually on several prey, and I. morenica (in that study under the name of I. curtata) only feeds on animals occasionally. The enzymatic activities of digestive amylase, lipase, protease, trypsin and chymotrypsin were determined for each species at the same developmental stage. The results show that H. flaviventris has a greater digestive enzymatic pool and higher relative and absolute protease, lipase and trypsin activities than I. morenica. The latter has a relative higher amylase activity. As higher amylase activity is typical of phytophagous species and higher protease activity typical of carnivorous species; these results reveal that H. flaviventris is a more efficient zoophagous species than I. morenica. The ecological implications of these findings, including the higher secondary production of H. flaviventris in its habitat, are discussed.

  7. A new Kunitz-type plasmin inhibitor from scorpion venom.

    Science.gov (United States)

    Ding, Li; Wang, Xiaobo; Liu, Hongyan; San, Mingkui; Xu, Yue; Li, Jian; Li, Shan; Cao, Zhijian; Li, Wenxin; Wu, Yingliang; Chen, Zongyun

    2015-11-01

    Kunitz-type peptides from venomous animals are an important source of lead drug candidates towards human plasmin, a target of protease-associated diseases. However, no Kunitz-type plasmin inhibitor from venomous scorpion has been characterized. Here, we first investigated plasmin inhibiting activities of eight known Kunitz-type scorpion toxins Hg1, BmKTT-1, BmKTT-2, BmKTT-3, LmKTT-1a, LmKTT-1b, LmKTT-1c and BmKPI, and found a new plasmin inhibitor BmKTT-2, a Kunitz-type toxin peptide from the scorpion Buthus martensi karch. Protease inhibitory activity assay showed that BmKTT-2 potently inhibited plasmin with a Ki value of 8.75 ± 2.05 nM. Structure-function relationship studies between BmKTT-2 and plasmin showed that BmKTT-2 is a classical Kunitz-type plasmin inhibitor: Lys13 in BmKTT-2 is the P1 site, and Ala14 in BmKTT-2 is the P1' site. Interestingly, BmKTT-2 has potent inhibiting activities towards three important digestive serine proteases trypsin, chymotrypsin and elastase, suggesting a good stability for administering oral medications. To the best of our knowledge, BmKTT-2 is the first Kunitz-type human plasmin inhibitor from scorpion venom, providing novel insights into drug developments targeting human plasmin protease. PMID:26363290

  8. Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human μ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines.

    Science.gov (United States)

    Rosa, Mònica; Bech-Serra, Joan Josep; Canals, Francesc; Zajac, Jean Marie; Talmont, Franck; Arsequell, Gemma; Valencia, Gregorio

    2015-08-01

    Human μ-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR. PMID:26090583

  9. Purification and Characterization of a Novel Kazal-Type Trypsin Inhibitor from the Leech of Hirudinaria manillensis

    Directory of Open Access Journals (Sweden)

    Yanmei Lai

    2016-07-01

    Full Text Available Kazal-type serine proteinase inhibitors are found in a large number of living organisms and play crucial roles in various biological and physiological processes. Although some Kazal-type serine protease inhibitors have been identified in leeches, none has been reported from Hirudinaria manillensis, which is a medically important leech. In this study, a novel Kazal-type trypsin inhibitor was isolated from leech H. manillensis, purified and named as bdellin-HM based on the sequence similarity with bdellin-KL and bdellin B-3. Structural analysis revealed that bdellin-HM was a 17,432.8 Da protein and comprised of 149 amino acid residues with six cysteines forming three intra-molecular disulfide bonds. Bdellin-HM showed similarity with the Kazal-type domain and may belong to the group of “non-classical” Kazal inhibitors according to its CysI-CysII disulfide bridge position. Bdellin-HM had no inhibitory effect on elastase, chymotrypsin, kallikrein, Factor (F XIIa, FXIa, FXa, thrombin and plasmin, but it showed a potent ability to inhibit trypsin with an inhibition constant (Ki of (8.12 ± 0.18 × 10−9 M. These results suggest that bdellin-HM from the leech of H. manillensis plays a potent and specific inhibitory role towards trypsin.

  10. Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

    Science.gov (United States)

    Platis, Dimitris; Labrou, Nikolaos E

    2008-03-01

    Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.

  11. Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals

    Directory of Open Access Journals (Sweden)

    Caroline B. F. Mourão

    2013-06-01

    Full Text Available The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K+ channel blockers are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K+ channel blocking activity was also compared.

  12. Liposomes containing glycocholate as potential oral insulin delivery systems: preparation, in vitro characterization, and improved protection against enzymatic degradation

    Science.gov (United States)

    Niu, Mengmeng; Lu, Yi; Hovgaard, Lars; Wu, Wei

    2011-01-01

    Background: Oral delivery of insulin is challenging and must overcome the barriers of gastric and enzymatic degradation as well as low permeation across the intestinal epithelium. The present study aimed to develop a liposomal delivery system containing glycocholate as an enzyme inhibitor and permeation enhancer for oral insulin delivery. Methods: Liposomes containing sodium glycocholate were prepared by a reversed-phase evaporation method followed by homogenization. The particle size and entrapment efficiency of recombinant human insulin (rhINS)-loaded sodium glycocholate liposomes can be easily adjusted by tuning the homogenization parameters, phospholipid:sodium glycocholate ratio, insulin:phospholipid ratio, water:ether volume ratio, interior water phase pH, and the hydration buffer pH. Results: The optimal formulation showed an insulin entrapment efficiency of 30% ± 2% and a particle size of 154 ± 18 nm. A conformational study by circular dichroism spectroscopy and a bioactivity study confirmed the preserved integrity of rhINS against preparative stress. Transmission electron micrographs revealed a nearly spherical and deformed structure with discernable lamella for sodium glycocholate liposomes. Sodium glycocholate liposomes showed better protection of insulin against enzymatic degradation by pepsin, trypsin, and α-chymotrypsin than liposomes containing the bile salt counterparts of sodium taurocholate and sodium deoxycholate. Conclusion: Sodium glycocholate liposomes showed promising in vitro characteristics and have the potential to be able to deliver insulin orally. PMID:21822379

  13. Semi-synthesis of biologically active nisin hybrids composed of the native lanthionine ABC-fragment and a cross-stapled synthetic DE-fragment.

    Science.gov (United States)

    Slootweg, Jack C; Peters, Nienke; Quarles van Ufford, H Linda C; Breukink, Eefjan; Liskamp, Rob M J; Rijkers, Dirk T S

    2014-10-01

    The antimicrobial peptide nisin is a promising template for designing novel peptide-based antibiotics to improve its drug-like properties. First steps in that direction represent the synthesis of hybrid nisin derivatives that contain a native nisin ABC-part and synthesized cross-stapled DE-ring fragments and are described here. The biological activity of the newly synthesized nisin derivatives was evaluated in order to compare the bioactivity of the synthetic DE-ring containing mimic and native lanthionine-bridged DE-ring containing nisin. The native nisin ABC-ring system was obtained via chymotrypsin digestion of full-length nisin, and was subsequently functionalized at the C-terminal carboxylate with two different amino alkyne moieties. Next, nisin hybrids were successfully prepared using Cu(I)-catalyzed azide alkyne cycloaddition 'click' chemistry by chemo-selective ligation of the ABC-alkyne with the N-terminal azido functionalized dicarba-DE ring mimic. The newly synthesized compounds were active as potent lipid II binders and retained antimicrobial activity in a growth inhibition assay. However, pore formation was not observed, possibly either due to the different character of the 'staples' as compared to the parent sulfides, or due to the triazole moiety as a sub-optimal amide bond isostere.

  14. Effects of diets containing vegetable protein concentrates on performance and activity of digestive enzymes in silver catfish (Rhamdia quelen

    Directory of Open Access Journals (Sweden)

    Naglezi de Menezes Lovatto

    2014-02-01

    Full Text Available The purpose of study was to evaluate the effect of using protein concentrates crambe and sunflower meal in the diet of silver catfish juveniles, as substitute for animal protein source. A total of 300 silver catfish had been separate in 15 experimental units of 280 L, totaling five treatments with three replications. We evaluated two levels (25% and 50% replacement of the meat and bone meal by protein concentrates of crambe and sunflower meals. Evaluated growth parameters, biological index and digestive enzymes in fish. There was no statistical difference for mass (g and standard length (cm, but the fish diet CPFCr-25% had greater total length (cm. No difference in dry matter, crude protein and total protein deposited (calculated. However, there was a higher concentration of ash in the carcass of the animals fed the control diet and CPFCr-50% in relation to diet CPFG- 50%, in addition, higher levels of lipids in fish fed diet CPFG-50%. No significant differences for hepatosomatic index, digestive somatic index and intestinal quotient of animals subjected to different treatments. The activity of digestive enzymes trypsin and chymotrypsin did not change. There was increased activity of acid protease. The quantitative and qualitative increase in protein concentration from this fraction allows the use of bran protein concentrates crambe and sunflower as substitutes for animal protein source.

  15. EFFECTS OF FIVE NEW COMPOUNDS ON THE LARVAL GROWTH AND DIGESTIVE PHYSIOLOGY OF THE ASIATIC CORN BORER, OSTRINIA FURNA CALIS LARVAE

    Institute of Scientific and Technical Information of China (English)

    JiaHe; Zeng-guangYan; Zhi-shengJiang

    2003-01-01

    Five new compounds were tested on the growth and antifeeding activity compared with toosendanin against fifth instar larvae Ostrinia furtuwalis. The activities of two proteases, a weak alkaline trypsine-like enzyme and a chymotrypsin-like enzyme, in the midgut of Ostriniafurnacalis larvae were also measured. Experimental restilts suggest that when incorporated into an artificial diet at the concentration of 500mg/kg, the antifeeding activities of toosendanin, C19 , C23 , C24 , C26 , C28 were 51.16%, 57.61%, 4.28%, 51.08%, 36.73% and 51.67%,respectively, C19, C24, C28 had no significant difference with toosendanin. At 20mg/kg, the larval growth were remarkably suppressed by CI9, C26, C28, the inhibition of C28 was close to toosendanin in 48 h. The two proteases were activated by toosendanin and C28 while they were inhibited in 48 h but activated in 24 h by C19, C24 and C26,In this paper, the related functions and mechanisms were discussed.

  16. Partial Purification and Characterization of the Mode of Action of Enterocin S37: A Bacteriocin Produced by Enterococcus faecalis S37 Isolated from Poultry Feces

    Science.gov (United States)

    Belguesmia, Y.; Choiset, Y.; Prévost, H.; Dalgalarrondo, M.; Chobert, J.-M.; Drider, D.

    2010-01-01

    The aim of this research was to purify and characterize the mode of action of enterocin S37, a bacteriocin produced by Enterococcus faecalis S37, a strain recently isolated from the chicken feces. Enterocin S37 has a molecular weight comprised between 4 and 5 kDa. It remained active after 1 h at 80oC and at pH values ranging from 4.0 to 9.0. Furthermore, cell-free supernatant of Enterococcus faecalis S37 and purified enterocin S37 were active against Gram-positive bacteria including Listeria monocytogenes EGDe, L. innocua F, Enterococcus faecalis JH2-2, and Lactobacillus brevis F145. The purification of enterocin S37 was performed by ammonium sulfate precipitation followed up by hydrophobic-interaction chromatography procedures. Treatment of enterocin S37 with proteinase K, α-chymotrypsin, and papain confirmed its proteinaceous nature, while its treatment with lysozyme and lipase resulted in no alteration of activity. Enterocin S37 is hydrophobic, anti-Listeria and likely acting by depletion of intracellular K+ ions upon action on KATP channels. This study contributed to gain more insights into the mode of action of enterocins. PMID:20811593

  17. Partial Purification and Characterization of the Mode of Action of Enterocin S37: A Bacteriocin Produced by Enterococcus faecalis S37 Isolated from Poultry Feces

    Directory of Open Access Journals (Sweden)

    Y. Belguesmia

    2010-01-01

    Full Text Available The aim of this research was to purify and characterize the mode of action of enterocin S37, a bacteriocin produced by Enterococcus faecalis S37, a strain recently isolated from the chicken feces. Enterocin S37 has a molecular weight comprised between 4 and 5 kDa. It remained active after 1 h at 80oC and at pH values ranging from 4.0 to 9.0. Furthermore, cell-free supernatant of Enterococcus faecalis S37 and purified enterocin S37 were active against Gram-positive bacteria including Listeria monocytogenes EGDe, L. innocua F, Enterococcus faecalis JH2-2, and Lactobacillus brevis F145. The purification of enterocin S37 was performed by ammonium sulfate precipitation followed up by hydrophobic-interaction chromatography procedures. Treatment of enterocin S37 with proteinase K, -chymotrypsin, and papain confirmed its proteinaceous nature, while its treatment with lysozyme and lipase resulted in no alteration of activity. Enterocin S37 is hydrophobic, anti-Listeria and likely acting by depletion of intracellular K+ ions upon action on KATP channels. This study contributed to gain more insights into the mode of action of enterocins.

  18. Effects of 2-alkynyladenosine derivatives on intraocular pressure in rabbits.

    Science.gov (United States)

    Konno, Takashi; Ohnuma, Shin-ya; Uemoto, Kazuhiro; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Endo, Kazuki; Hosokawa, Tomokazu; Nakahata, Norimichi

    2004-02-23

    We evaluated the activities of 2-alkynyladenosine derivatives, relatively selective adenosine A2 receptor agonists, in the intraocular pressure regulation in rabbits. An adenosine A2 receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680) decreased intraocular pressure, while another A2 receptor agonist 2-(phenylamino)adenosine transiently increased it. The first group of 2-alkynyladenosine derivatives (1-hexyn-1-yl derivatives) caused a transient increase followed by decrease in intraocular pressure, while the second group (1-octyn-1-yl and 6-cyano-1-hexyn-1-yl derivatives) only decreased it. The second group is also effective in the ocular hypertensive models induced by water-loading and alpha-chymotrypsin. The outflow facility was increased by a 1-octyn-1-yl derivative. Both increase and decrease in intraocular pressure induced by 2-alkynyladenosine derivatives were inhibited by an adenosine A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine, but not by an adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropyl xanthine. These findings suggest that 2-alkynyladenosine derivatives may affect intraocular pressure via adenosine A2 receptor, and 2-alkynyladenosine derivative-induced ocular hypotension is due to the increase of outflow facility.

  19. Effect of chymase on intraocular pressure in rabbits.

    Science.gov (United States)

    Konno, Takashi; Maruichi, Midori; Takai, Shinji; Oku, Hidehiro; Sugiyama, Tetsuya; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Ikeda, Tsunehiko; Miyazaki, Mizuo

    2005-11-01

    Chymase is a chymotrypsin-like serine protease that is stored exclusively in the secretory granules of mast cells and converts big endothelins to endothelin-1 (1-31). The aim of this study was to evaluate the effect of chymase on intraocular pressure in rabbits. Chymase injection (3 and 10 mU) resulted in a trend toward increased intraocular pressure and a significant increase in intraocular pressure at a dose of 10 mU compared with the control. A specific chymase inhibitor, Suc-Val-Pro-Phe(P)(OPh)(2), attenuated the ocular hypertension induced by chymase. Endothelin-1 (1-31) also caused ocular hypertension, which was inhibited by a selective endothelin ET(A) receptor antagonist, cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123). Moreover, chymase-induced ocular hypertension was inhibited by BQ-123. These results suggest that chymase influences the regulation of intraocular pressure, and it is likely that the formation of endothelin-1 (1-31) and subsequent activation of endothelin ET(A) receptors are involved in the development of ocular hypertension induced by chymase.

  20. Effect of processing methods on compositional evaluation of underutilized legume, Parkia roxburghii G. Don (yongchak) seeds.

    Science.gov (United States)

    Sathya, Arumugam; Siddhuraju, Perumal

    2015-10-01

    The present study has been undertaken to analyze the effect of various processing methods like (i) soaking followed by autoclaving with (a) ash, (b) sodium bicarbonate, (c) sugar and (d) water; (ii) dry heating and (iii) fermentation on nutritional and antinutritional components of under-utilized tree legume Parkia roxburghii. The applied methods were found to enhance the protein (15-36 %) and lipid content (11-69 %) and to decrease the other proximal components. All the methods significantly reduced the antinutrients viz. condensed tannins, phytate, saponins, trypsin inhibitors, chymotrypsin inhibitors and lectins. Exceptionally, increased content was documented on total phenolics (117-207 %) and tannins (171-257 %). These reduced antinutritional loads have led to an increase in protein (9-20 %) and starch digestibility (75-254 %). Fermented kernels, the best processed form showed characteristic leguminous pattern for content and composition of amino acids, fatty acids and minerals. So knowledge gathering and exploration of nutritionally balanced under-utilized legumes would enhance food and nutritional security. PMID:26396363

  1. Antimicrobial Activity of a Halocidin-Derived Peptide Resistant to Attacks by Proteases ▿

    Science.gov (United States)

    Shin, Yong Pyo; Park, Ho Jin; Shin, Seo Hwa; Lee, Young Shin; Park, Seungmi; Jo, Sungho; Lee, Yong Ho; Lee, In Hee

    2010-01-01

    Cationic antimicrobial peptides (AMPs) have attracted a great deal of interest as a promising candidate for a novel class of antibiotics that might effectively treat recalcitrant infections caused by a variety of microbes that are resistant to currently available drugs. However, the AMPs are inherently limited in that they are inevitably susceptible to attacks by proteases generated by human and pathogenic microbes; this vulnerability severely hinders their pharmaceutical use in human therapeutic protocols. In this study, we report that a halocidin-derived AMP, designated HG1, was found to be resistant to proteolytic degradation. As a result of its unique structural features, HG1 proved capable of preserving its antimicrobial activity after incubation with trypsin, chymotrypsin, and human matrix metalloprotease 7 (MMP-7). Additionally, HG1 was observed to exhibit profound antimicrobial activity in the presence of fluid from human skin wounds or proteins extracted from the culture supernatants of Staphylococcus aureus and Pseudomonas aeruginosa. Greater understanding of the structural motifs of HG1 required for its protease resistance might provide feasible ways to solve the problems intrinsic to the development of an AMP-based antibiotic. PMID:20385874

  2. Quantitative Correlation of Conformational Binding Enthalpy with Substrate Specificity of Serine Proteases.

    Science.gov (United States)

    Waldner, Birgit J; Fuchs, Julian E; Huber, Roland G; von Grafenstein, Susanne; Schauperl, Michael; Kramer, Christian; Liedl, Klaus R

    2016-01-21

    Members of the same protease family show different substrate specificity, even if they share identical folds, depending on the physiological processes they are part of. Here, we investigate the key factors for subpocket and global specificity of factor Xa, elastase, and granzyme B which despite all being serine proteases and sharing the chymotrypsin-fold show distinct substrate specificity profiles. We determined subpocket interaction potentials with GRID for static X-ray structures and an in silico generated ensemble of conformations. Subpocket interaction potentials determined for static X-ray structures turned out to be insufficient to explain serine protease specificity for all subpockets. Therefore, we generated conformational ensembles using molecular dynamics simulations. We identified representative binding site conformations using distance-based hierarchical agglomerative clustering and determined subpocket interaction potentials for each representative conformation of the binding site. Considering the differences in subpocket interaction potentials for these representative conformations as well as their abundance allowed us to quantitatively explain subpocket specificity for the nonprime side for all three example proteases on a molecular level. The methods to identify key regions determining subpocket specificity introduced in this study are directly applicable to other serine proteases, and the results provide starting points for new strategies in rational drug design.

  3. Effect of alternative crosslinking techniques on the enzymatic degradation of bovine pericardia and their calcification.

    Science.gov (United States)

    Vasudev, S C; Chandy, T

    1997-06-01

    The in vitro calcification and enzymatic degradation of bovine pericardia (BP) after a series of surface treatments were studied as a function of exposure time. The degradation of these treated surfaces was monitored by scanning electron micrography and tensile strength measurements. Polyethylene glycol-(PEG) grafted BP and glutaraldehyde-(GA) treated BPs retained maximum stability in collagenase digestion compared with SDS-treated BP. The ability of alpha chymotrypsin, bromelain, esterase, trypsin, and collagenase to modulate the degradation of SDS-, GA-, PEG-, Carbodiimide-, and glycidylether-treated BPs also was investigated. Incubation of various enzymes to these crosslinked pericardia variably reduced the tensile strength of these tissues. It is conceivable that chemical treatments of pericardial tissues might have altered their physical and chemical configuration and the subsequent degradation properties. In vitro calcification studies showed a substantial reduction in the calcification profile of PEG-grafted bovine pericardia compared to other treated tissues. Furthermore, the biocompatibility aspects of pericardial tissues were established by platelet adhesion and octane contact angle. In conclusion, it seems that the surface modification of bovine pericardia via GA-PEG grafting may provide new ways of controlling biodegradation and calcification.

  4. Inhibitory effects of a Kunitz-type inhibitor from Pithecellobium dumosum (Benth) seeds against insect-pests' digestive proteinases.

    Science.gov (United States)

    Rufino, Fabiola P S; Pedroso, Vanessa M A; Araujo, Jonalson N; França, Anderson F J; Rabêlo, Luciana M A; Migliolo, Ludovico; Kiyota, Sumika; Santos, Elizeu A; Franco, Octavio L; Oliveira, Adeliana S

    2013-02-01

    Pithecellobium dumosum is a tree belonging to the Mimosoideae subfamily that presents various previously characterized Kunitz-type inhibitors. The present study provides a novel Kunitz-trypsin inhibitor isoform purified from P. dumosum seeds. Purification procedure was performed by TCA precipitation followed by a trypsin-Sepharose chromatography and a further reversed-phase HPLC. Purified inhibitor (PdKI-4) showed enhanced inhibitory activity against bovine trypsin and chymotrypsin. Furthermore, PdKI-4 showed remarkable inhibitory activity against serine proteases from the coleopterans Callosobruchus maculatus and Zabrotes subfasciatus, and the lepidopterans Alabama argillacea and Telchin licus. However, PdKI-4 was unable to inhibit porcine pancreatic elastase, pineapple bromelain and Carica papaya papain. SDS-PAGE showed that PdKI-4 consisted of a single polypeptide chain with molecular mass of 21 kDa. Kinetic studies demonstrated that PdKI-4 is probably a competitive inhibitor with a Ki value of 5.7 × 10(-10) M for bovine trypsin. PdKI-4 also showed higher stability over a wide range of temperature (37-100 °C) and pH (2-12). N-termini sequence was obtained by Edman degradation showing higher identity with other Mimosoideae subfamily Kunitz-type inhibitor members. In summary, data here reported indicate the biotechnological potential of PdKI-4 for development of products against insect-pests.

  5. Production of Defatted Palm Kernel Cake Protein Hydrolysate as a Valuable Source of Natural Antioxidants

    Directory of Open Access Journals (Sweden)

    Mohammad Zarei

    2012-06-01

    Full Text Available The aim of this study was to produce a valuable protein hydrolysate from palm kernel cake (PKC for the development of natural antioxidants. Extracted PKC protein was hydrolyzed using different proteases (alcalase, chymotrypsin, papain, pepsin, trypsin, flavourzyme, and bromelain. Subsequently, antioxidant activity and degree of hydrolysis (DH of each hydrolysate were evaluated using DPPH• radical scavenging activity and O-phthaldialdehyde spectrophotometric assay, respectively. The results revealed a strong correlation between DH and radical scavenging activity of the hydrolysates, where among these, protein hydrolysates produced by papain after 38 h hydrolysis exhibited the highest DH (91 ± 0.1% and DPPH• radical scavenging activity (73.5 ± 0.25% compared to the other hydrolysates. In addition, fractionation of the most effective (potent hydrolysate by reverse phase high performance liquid chromatography indicated a direct association between hydrophobicity and radical scavenging activity of the hydrolysates. Isoelectric focusing tests also revealed that protein hydrolysates with basic and neutral isoelectric point (pI have the highest radical scavenging activity, although few fractions in the acidic range also exhibited good antioxidant potential.

  6. Purification and characterization of kininogens from sheep plasma.

    Science.gov (United States)

    Baba, Shahid P; Zehra, Sadaf; Bano, Bilqees

    2005-02-01

    High molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) have been purified from sheep (Avis Arias) plasma in three steps involving ammonium sulphate precipitation, column chromatography on Sephacryl-300HR and ion exchange chromatography on DEAE cellulose. HMWK gave a single band on native and SDS-PAGE with a molecular weight corresponding to 280 kDa. Under reducing conditions purified HMWK was again resolved to a single band with molecular weight corresponding to 140 kDa indicative of its dimeric nature. LMWK was resolved into two isoforms named as LMWK1 and LMWK2, with an apparent molecular weight of 68 kDa. The yield of HMWK, LMWK1 and 2 was about 8.1, 5.63 and 10.65 respectively. HMWK, LMWK1 and 2 strongly inhibited activities of ficin and papain but not of trypsin, chymotrypsin and bromelain. Ki values estimated for HMWK with papain and ficin was 0.8 and 0.6 nM respectively. Ki values estimated for LMWK1 and 2 with papain were 2.40 and 2.00 nM respectively. Binding of HMWK, LMWK1 and 2 to activated papain were accompanied by pronounced changes in secondary and tertiary structure that are compatible with perturbations of environment of aromatic residues.

  7. Proteolytic activity in the midgut of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae, Pomegranate carob moth

    Directory of Open Access Journals (Sweden)

    M Ranjbar

    2011-07-01

    Full Text Available In this study, the proteolytic activity in the midgut of Ectomyelois ceratoniae as the major pest of pomegranate was investigated to find nature of specific proteases and their properties for adopting possible pest management procedure. It was found that fourth and fifth instar larvae had the highest proteolytic activity as well as specific proteinases including, elastase, trypsin-like, chymotrypsin-like and two exopeptidases. The optimal pH of general protease was 10 and 9 for azocasein, casein and hemoglobin as substrate. The optimal temperature of the total proteolytic activity in the midgut of E. ceratoniae was found 30 and 35 °C by using azocasein and casein as substrates, respectively. In case of hemoglobin, the enzyme showed the highest enzymatic activity at temperatures from 15 to 35 °C. There was no enhancement in the proteolytic activity by using different cations but SDS, citric acid and mercaptoetahnol significantly decreased the proteolytic activity in the midgut of E. ceratoniae. Using specific proteolytic inhibitors including PMSF, TLCK, TPCK, E-64, DTT and phenanthroline revealed presence of serine proteases as the major proteases in the midgut of E. ceratoniae.

  8. Concentric Förster resonance energy transfer imaging.

    Science.gov (United States)

    Wu, Miao; Algar, W Russ

    2015-08-18

    Concentric Förster resonance energy transfer (cFRET) configurations based on semiconductor quantum dots (QDs) are promising probes for biological sensing because they offer multiplexing capability in a single vector with robust ratiometric detection by exploiting a network of FRET pathways. To expand the scope and utility of cFRET probes, it is necessary to develop and validate cFRET imaging methodology. In this technical note, we present such a methodology using a protease-sensitive cFRET configuration that comprises a green-emitting QD, Alexa Fluor 555 (A555), and Alexa Fluor 647 (A647). Photoluminescence (PL) images were acquired with three filter-based emission channels to permit measurement of A555/QD and A647/QD PL ratios. With reference to calibration samples, these PL ratios were used to calculate quantitative progress curves for proteolytic activity in regions of interest in the acquired images. Importantly, the imaging methodology reproduces quantitative results obtained with a monochromator-based fluorescence plate reader. Spatiotemporal resolution is demonstrated by tracking the activity of two prototypical proteases, trypsin and chymotrypsin, as they diffuse down the length of a capillary. This methodology is expected to enable the future use of cFRET probes for cellular sensing and other imaging assays. PMID:26214686

  9. Antiproteolytic and leishmanicidal activity of Coccinia grandis (L.) Voigt leaf extract against Leishmania donovani promastigotes.

    Science.gov (United States)

    Das, Partha; Paik, Dibyendu; Pramanik, Asmita; De, Tripti; Chakraborti, Tapati

    2015-11-01

    In visceral leishmaniasis (VL), development of alternative safe therapeutic strategy is gaining paramount wherein natural components of plant origin have prominence. We explored Coccinia grandis (L.) Voigt, a medicinal plant known in traditional folk medicine, for its antileishmanial efficacy. SDS-PAGE analysis of the C. grandis leaf extract (Cg-Ex) showed few protein bands about 14-66 kDa among which three (64.8, 55.8 and 15.3 kDa) were identified as serine protease inhibitors by reverse zymography. Since the virulence of Leishmania is also attributed by serine proteases, objective of the present study was to evaluate in vitro antileishmanial activity of Cg-Ex, targeting Leishmania donovani serine protease(s). Inhibition study of Cg-Ex in gelatin-zymogram and spectrophotometric assay revealed its strong inhibitory activity against bovine trypsin rather than chymotrypsin, and also showed significant inhibition of L. donovani serine protease(s). Further, studies with Cg-Ex were extended to estimate its antileishmanial efficacy with half maximal inhibitory concentration (IC50) at 308.0 ± 2.42 μg/ml along with significant morphological alterations. The results have demonstrated the potential of the serine protease inhibitor rich fraction of the C. grandis leaf extract against visceral leishmaniasis. PMID:26669017

  10. Transformation of human cathelicidin LL-37 into selective, stable, and potent antimicrobial compounds.

    Science.gov (United States)

    Wang, Guangshun; Hanke, Mark L; Mishra, Biswajit; Lushnikova, Tamara; Heim, Cortney E; Chittezham Thomas, Vinai; Bayles, Kenneth W; Kielian, Tammy

    2014-09-19

    This Letter reports a family of novel antimicrobial compounds obtained by combining peptide library screening with structure-based design. Library screening led to the identification of a human LL-37 peptide resistant to chymotrypsin. This d-amino-acid-containing peptide template was active against Escherichia coli but not methicillin-resistant Staphylococcus aureus (MRSA). It possesses a unique nonclassic amphipathic structure with hydrophobic defects. By repairing the hydrophobic defects, the peptide (17BIPHE2) gained activity against the ESKAPE pathogens, including Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species. In vitro, 17BIPHE2 could disrupt bacterial membranes and bind to DNA. In vivo, the peptide prevented staphylococcal biofilm formation in a mouse model of catheter-associated infection. Meanwhile, it boosted the innate immune response to further combat the infection. Because these peptides are potent, cell-selective, and stable to several proteases, they may be utilized to combat one or more ESKAPE pathogens. PMID:25061850

  11. Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: purification and enzyme kinetic properties.

    Science.gov (United States)

    Yadav, Vikash Kumar; Chhikara, Nirmal; Gill, Kamaldeep; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

    2013-08-01

    The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research. PMID:23619703

  12. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    International Nuclear Information System (INIS)

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, 3H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a 3H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of 3H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A4, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each

  13. Preparation and characterization of singly-substituted sulfhydryl derivatives of cytochrome c

    International Nuclear Information System (INIS)

    Sulfydryl derivatives of horse heart cytochrome c have been prepared by reaction with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3'-dithiopropionate, which modifies lysine epsilon - amino groups. The mixture of products containing cytochromes c derivatized at different lysines was resolved by HPLC using a cation exchange column. The resulting purified mono-derivatives were identified as follows: the sulfhydryl group of the modified lysine was alkylated using [3H]-iodoacetic acid, this alkylated derivative was digested with chymotrypsin, and the labeled peptide was isolated by HPLC and its amino acid composition determined. Kinetic analysis of each derivative's reactivity with cytochrome c oxidase revealed a pattern of inhibition similar to that observed for the carboxydinitrophenyl-derivatives of horse heart cytochrome c. By reacting the sulfhydryl group with N-(iodoethyl)trifluoroacetamide, the original charge of the cytochrome c is restored. The effects on the reactivity of cytochrome c with cytochrome c oxidase caused by the change in dipole and by steric hindrance from the derivatization can then be distinguished. These derivatives have the advantage that various labels (photoaffinity, fluorescent, etc.) can readily be attached specifically to each of the modified lysines, and are being used in variety of studies

  14. Digestive enzymes from workers and soldiers of termite Nasutitermes corniger.

    Science.gov (United States)

    Lima, Thâmarah de Albuquerque; Pontual, Emmanuel Viana; Dornelles, Leonardo Prezzi; Amorim, Poliana Karla; Sá, Roberto Araújo; Coelho, Luana Cassandra Breitenbach Barroso; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes

    2014-10-01

    The digestive apparatus of termites may have several biotechnological applications, as well as being a target for pest control. This report discusses the detection of cellulases (endoglucanase, exoglucanase, and β-glucosidase), hemicellulases (β-xylosidase, α-l-arabinofuranosidase, and β-d-xylanase), α-amylase, and proteases (trypsin-like, chymotrypsin-like, and keratinase-type) in gut extracts from Nasutitermes corniger workers and soldiers. Additionally, the effects of pH (3.0-11.0) and temperature (30-100°C) on enzyme activities were evaluated. All enzymes investigated were detected in the gut extracts of worker and soldier termites. Endoglucanase and β-xylanase were the main cellulase and hemicellulase, respectively. Zymography for proteases of worker extracts revealed polypeptides of 22, 30, and 43kDa that hydrolyzed casein, and assays using protease inhibitors showed that serine proteases were the main proteases in worker and soldier guts. The determined enzyme activities and their response to different pH and temperature values revealed that workers and soldiers contained a distinct digestive apparatus. The ability of these termites to efficiently digest the main components of lignocellulosic materials stimulates the purification of gut enzymes. Further investigation into their biotechnological potential as well as whether the enzymes detected are produced by the termites or by their symbionts is needed. PMID:25026598

  15. Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-gingerol in cervical cancer cells.

    Science.gov (United States)

    Rastogi, Namrata; Duggal, Shivali; Singh, Shailendra Kumar; Porwal, Konica; Srivastava, Vikas Kumar; Maurya, Rakesh; Bhatt, M L B; Mishra, Durga Prasad

    2015-12-22

    Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer. PMID:26621832

  16. DNA Microarray Analysis on the Genes Differentially Expressed in the Liver of the Pufferfish, Takifugu rubripes, Following an Intramuscular Administration of Tetrodotoxin

    Directory of Open Access Journals (Sweden)

    Takuya Matsumoto

    2014-10-01

    Full Text Available Pufferfish accumulate tetrodotoxin (TTX mainly in the liver and ovary. This study aims at investigating the effect of TTX accumulation in the liver of cultured specimens of torafugu Takifugu rubripes on the hepatic gene expression by microarray analysis on Day 5 after the intramuscular administration of 0.25 mg TTX/kg body weight into the caudal muscle. TTX was detected in the liver, skin and ovary in the TTX-administered individuals. The total amount of TTX accumulated in the body was 67 ± 8% of the administered dose on Day 5. Compared with the buffer-administered control group, a total of 59 genes were significantly upregulated more than two-fold in the TTX-administered group, including those encoding chymotrypsin-like elastase family member 2A, transmembrane protein 168 and Rho GTP-activating protein 29. In contrast, a total of 427 genes were downregulated by TTX administration, including those encoding elongation factor G2, R-spondin-3, nuclear receptor activator 2 and fatty acyl-CoA hydrolase precursor. In conclusion, our results demonstrate that the intramuscular administration of TTX changes the expression of hepatic genes involved in various signaling pathways.

  17. Bacteriocin production by Carnobacterium piscicola LV 61.

    Science.gov (United States)

    Schillinger, U; Stiles, M E; Holzapfel, W H

    1993-11-26

    Carnobacterium piscicola LV 61 produces a bacteriocin designated piscicolin 61, that is heat resistant, active over a wide pH range and inactivated by alpha-chymotrypsin, pepsin, trypsin and papain. It is effective against strains of the genera Carnobacterium, Enterococcus and Listeria. Other lactic acid bacteria tested were less sensitive or resistant to piscicolin. It is produced at temperatures from 1 to 30 degrees C. Maximum bacteriocin activity was detected after the culture had entered the stationary phase of growth and when the culture was grown in a medium with an initial pH between 8.0 and 9.0. The same high amounts of bacteriocin could be obtained in a culture at a constant pH of 6.5. No bacteriocin was produced at pH 5.0. Peptone in the growth medium promotes bacteriocin production, whereas meat and yeast extract did not influence the amounts of bacteriocin produced. Bacteriocin production and immunity are probably encoded by a 22 kb plasmid. PMID:8312140

  18. Polycarbophil-cysteine conjugates as platforms for oral polypeptide delivery systems.

    Science.gov (United States)

    Bernkop-Schnürch, A; Thaler, S C

    2000-07-01

    The purpose of the present study was to evaluate the potential of polycarbophil-cysteine conjugates as carrier systems for orally administered peptide and protein drugs. Mediated by a carbodiimide, cysteine was covalently attached to polycarbophil. The properties of resulting conjugates, displaying 35-50 microM thiol groups per gram of polymer, to bind polypeptides and to inhibit pancreatic proteases was evaluated in vitro. Results demonstrated that only some polypeptides are immobilized to the polycarbophil-cysteine conjugate. Due to the covalent attachment of cysteine to polycarbophil, the inhibitory effect of the polymer toward carboxypeptidase A (EC 3.4. 17.1) and carboxypeptidase B (EC 3.4.17.2) could be significantly (p polycarbophil could be improved by the covalent attachment of cysteine, the raised inhibitory effect seems to be based on the complexation of this divalent cation from the enzyme structure. Whereas the covalent attachment of cysteine on polycarbophil had no influence on the enzymatic activity of trypsin (EC 3.4.21.4) and elastase (EC 3.4.21. 36), the inhibitory effect of the polymer-cysteine conjugate toward chymotrypsin (EC 3.4.21.1) was significantly (p polycarbophil-cysteine conjugates seem to be a promising tool in protecting orally administered therapeutic polypeptides, which are not bound to the polymer, from presystemic metabolism in the intestine.

  19. The proteasomes of two marine decapod crustaceans, European lobster (Homarus gammarus) and Edible crab (Cancer pagurus), are differently impaired by heavy metals.

    Science.gov (United States)

    Götze, Sandra; Bose, Aneesh; Sokolova, Inna M; Abele, Doris; Saborowski, Reinhard

    2014-05-01

    The intracellular ubiquitin-proteasome system is a key regulator of cellular processes involved in the controlled degradation of short-living or malfunctioning proteins. Certain diseases and cellular dysfunctions are known to arise from the disruption of proteasome pathways. Trace metals are recognized stressors of the proteasome system in vertebrates and plants, but their effects on the proteasome of invertebrates are not well understood. Since marine invertebrates, and particularly benthic crustaceans, can be exposed to high metal levels, we studied the effects of in vitro exposure to Hg(2+), Zn(2+), Cu(2+), and Cd(2+) on the activities of the proteasome from the claw muscles of lobsters (Homarus gammarus) and crabs (Cancer pagurus). The chymotrypsin like activity of the proteasome of these two species showed different sensitivity to metals. In lobsters the activity was significantly inhibited by all metals to a similar extent. In crabs the activities were severely suppressed only by Hg(2+) and Cu(2+) while Zn(2+) had only a moderate effect and Cd(2+) caused almost no inhibition of the crab proteasome. This indicates that the proteasomes of both species possess structural characteristics that determine different susceptibility to metals. Consequently, the proteasome-mediated protein degradation in crab C. pagurus may be less affected by metal pollution than that of the lobster H. gammarus. PMID:24721378

  20. ENZYME ACTIVITIES IN TRICHOPHYTON RUBRUM, TRICHOPHYTON MENTAGROPHYTES AND TRICHOPHYTON VERRUCOSUM

    Directory of Open Access Journals (Sweden)

    F.Zaini

    1991-12-01

    Full Text Available Since most of the studies on chemical compounds of dermatophytes have shown the existence of a relationship between their pathogenisity and proteolytec enzymes. Activities of 19 different enzymes in viable mycelia and cytoplasmic extracts of T.rubrum (CETr, T.mentagrophytes (CETm and T.verrucosum (CETv were investigated by the API-Zym System. The results showed that Viable mycelia of T.rubrm and T.mentagrophytes had valine arylamidase and cystine arylamidase activity where as no such activity was observed in CETr and CETm or in the viable mycelia and cytoplasmic extracts of T.verrucosum. Also the viable mycelia of T.rubrum showed α-mannosidase activity; gowever this enzyme was not detected in CETr, but was active in both the viable mycelia and CETv and CETm. Finally, only in viable mycelia and CETv and CETm was lipase (C14 activity observed. Furthermore none of the viable mycelia of these three species showed any α-galactosidase, β-galactosidase, α-fucosidase,β-glucoronidase, trypsin or chymotrypsin activity.

  1. Solution structure of PMP-C: a new fold in the group of small serine proteinase inhibitors.

    Science.gov (United States)

    Mer, G; Hietter, H; Kellenberger, C; Renatus, M; Luu, B; Lefèvre, J F

    1996-04-26

    The solution structure and the disulfide pairings of a 36-residue proteinase inhibitor isolated from the insect Locusta migratoria have been determined using NMR spectroscopy and simulated annealing calculations. The peptide, termed PMP-C, was previously shown to inhibit bovine alpha-chymotrypsin as well as human leukocyte elastase, and was also found to block high-voltage-activated Ca2+ currents in rat sensory neurones. PMP-C has a prolate ellipsoid shape and adopts a tertiary fold hitherto unobserved in the large group of small "canonical" proteinase inhibitors. The over-all fold consists mainly of three strands arranged in a right-handed twisted, antiparallel, beta-sheet that demarcates a cavity, together with a linear amino-terminal segment oriented almost perpendicular to the three strands of the beta-sheet. Inside the cavity a phenyl ring constitutes the centre of a hydrophobic core. The proteinase binding loop is located in the carboxy-terminal part of the molecule, between two cysteine residues involved in disulfide bridges. Its conformation resembles that found in other small canonical proteinase inhibitors. A comparison of PMP-C structure with the recently published solution structure of the related peptide PMP-D2 shows that the most significant differences are complementary changes involved in the stabilization of similar folds. This comparison led us to review the structure of PMP-D2 and to identify two salt bridges in PMP-D2.

  2. Molecular cloning, bioinformatics analysis and functional characterization of HWTX-XI toxin superfamily from the spider Ornithoctonus huwena.

    Science.gov (United States)

    Jiang, Liping; Deng, Meichun; Duan, Zhigui; Tang, Xing; Liang, Songping

    2014-04-01

    Spider venom contains a very valuable repertoire of natural resources to discover novel components for molecular diversity analyses and therapeutic applications. In this study, HWTX-XI toxins from the spider venom glands of Ornithoctonus huwena which are Kunitz-type toxins (KTTs) and were directly cloned, analyzed and functionally characterized. To date, the HWTX-XI superfamily consists of 38 members deduced from 121 high-quality expressed sequence tags, which is the largest spider KTT superfamily with significant molecular diversity mainly resulted from cDNA tandem repeats as well as focal hypermutation. Among them, HW11c40 and HW11c50 may be intermediate variants between native Kunitz toxins and sub-Kunitz toxins based on evolutionary analyses. In order to elucidate their biological activities, recombinant HW11c4, HW11c24, HW11c27 and HW11c39 were successfully expressed, further purified and functionally characterized. Both HW11c4 and HW11c27 display inhibitory activities against trypsin, chymotrypsin and kallikrein. Moreover, HW11c4 is also an inhibitor relatively specific for Kv1.1 channels. HW11c24 and HW11c39 are found to be inactive on chymotrysin, trypsin, kallikrein, thrombin and ion channels. These findings provide molecular evidence for toxin diversification of the HWTX-XI superfamily and useful molecular templates of serine protease inhibitors and ion channel blockers for the development of potentially clinical applications.

  3. Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein

    Energy Technology Data Exchange (ETDEWEB)

    Pothoulakis, C.; LaMont, J.T.; Eglow, R.; Gao, N.; Rubins, J.B.; Theoharides, T.C.; Dickey, B.F. (Boston Univ. School of Medicine, MA (USA))

    1991-07-01

    The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.

  4. Purification of a Kunitz-type inhibitor from Acacia polyphyllaDC seeds: characterization and insecticidal properties against Anagasta kuehniella Zeller (Lepidoptera: Pyralidae).

    Science.gov (United States)

    Machado, Suzy Wider; de Oliveira, Caio Fernando Ramalho; Bezerra, Cezar da Silva; Freire, Maria das Graças Machado; Regina Kill, Marta; Machado, Olga Lima Tavares; Marangoni, Sergio; Macedo, Maria Ligia Rodrigues

    2013-03-13

    Anagasta kuehniella is a polyphagous pest that causes economic losses worldwide. This species produces serine proteases as its major enzymes for protein digestion. In this study, a new serine-protease inhibitor was isolated from Acacia polyphylla seeds (AcKI).Further analysis revealed that AcKI is formed by two polypeptide chains with a relative molecular mass of ∼20 kDa. The effects of AcKI on the development, survival, and enzymatic activity of Anagasta kuehniella larvae were evaluated, by incorporating AcKI in an artificial diet. Bioassays revealed a reduction in larval weight of ∼50% with the lower concentration of AcKI used in the study (0.5%). Although additionalassays showed an increase in endogenous trypsin and chymotrypsin activities, with a degree of AcKI-insensivity, AcKI produces an anti nutritional effect on A. kuehniella, indicating AcKI as a promising bioinsecticide protein for engineering plants that are resistant to insect pests.

  5. Prevalence of S and Z alpha 1-antitrypsin mutations in patients with pancreatic diseases in Serbian population

    Directory of Open Access Journals (Sweden)

    Nikolić Aleksandra

    2010-01-01

    Full Text Available One of the key points in research of pancreatic disease pathology is further elucidation of the role of proteases and antiproteases, since their imbalance can lead to pancreatic injury. Alpha 1-antitrypsin (AAT is one of the most important serum inhibitors of proteolytic enzymes, including pancreatic enzymes trypsin, chymotrypsin and elastase. It is speculated that mutations in the AAT gene may influence the onset and the development of pancreatic disease. The presence of the most common AAT mutations Z and S was analyzed in 160 patients with pancreatic diseases (50 patients with pancreatic cancer, 50 patients with chronic pancreatitis and 60 patients with type 2 diabetes mellitus and 129 healthy individuals by PCR-mediated site-directed mutagenesis (PSM method. One patient with pancreatic cancer was found to be a carrier of Z mutation, as well as one patient with type 2 diabetes mellitus. One patient with chronic pancreatitis was found to be a carrier of S mutation. The common AAT mutations were statistically significantly over-represented in patients with pancreatic diseases (3 of 160 patients, allelic frequency 0.9% than in the control group (1 of 129 individuals, allelic frequency 0.4%. The results of this study, requiring confirmation, suggest that common AAT mutations Z and S may be associated with a modest increase in susceptibility to the development of pancreatic disease.

  6. 利用cDNA-AFLP技术研究副溶血弧菌感染下拟穴青蟹的基因差异表达%The differential gene expression of Scylla paramamosain upon Vibrio parahaemolyticus infection by cDNA-AFLP

    Institute of Scientific and Technical Information of China (English)

    李升康; 朱烁; 张昭; 温小波; 李远友

    2012-01-01

    Mud crab Scylla paramamosains is widely cultured in brackish and seawater ponds along the coast of southeast China including Shantou. In recent years, mud crab cultured in Niutianyang reclaiming coastal area of Shantou suffered from serious diseases, especially the bacterial diseases (such as Vibrio parahaemolyticus), which results in high mortality and great economic loss. In present study, V.parahaemolyticus isolated from Shantou Niutianyang crab culture area was injected to 5.paramamosains to study the differential gene expression of the mud crab by cDNA-AFLP technology. Furthermore, 20 fragments that exhibited differential expression were discovered and sequenced. The results show that 6 fragments are homologous to the known functional genes, 5 of which are up-regulated. They are involved in energy metabolism including arginine kinase, phosphoenolpymvate-carboxykinase and glyceraldehydes 3 phosphate dehydrogenase, arginyl-tRNA synthetase involved in transport progress, as well as the chymotrypsin-like proteinase involved in defense response. The other 8 fragments were identified to be unknown function genes. The result here shows that the response of S.paramamosain upon V. parahaemolyticus infection is involved in multiple pathways and needs further investigation.

  7. Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis.

    Science.gov (United States)

    Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M

    2016-07-01

    Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. PMID:27226576

  8. Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy.

    Science.gov (United States)

    Pozzi, Nicola; Bystranowska, Dominika; Zuo, Xiaobing; Di Cera, Enrico

    2016-08-26

    The coagulation factor prothrombin has a complex spatial organization of its modular assembly that comprises the N-terminal Gla domain, kringle-1, kringle-2, and the C-terminal protease domain connected by three intervening linkers. Here we use single molecule Förster resonance energy transfer to access the conformational landscape of prothrombin in solution and uncover structural features of functional significance that extend recent x-ray crystallographic analysis. Prothrombin exists in equilibrium between two alternative conformations, open and closed. The closed conformation predominates (70%) and features an unanticipated intramolecular collapse of Tyr(93) in kringle-1 onto Trp(547) in the protease domain that obliterates access to the active site and protects the zymogen from autoproteolytic conversion to thrombin. The open conformation (30%) is more susceptible to chymotrypsin digestion and autoactivation, and features a shape consistent with recent x-ray crystal structures. Small angle x-ray scattering measurements of prothrombin wild type stabilized 70% in the closed conformation and of the mutant Y93A stabilized 80% in the open conformation directly document two envelopes that differ 50 Å in length. These findings reveal important new details on the conformational plasticity of prothrombin in solution and the drastic structural difference between its alternative conformations. Prothrombin uses the intramolecular collapse of kringle-1 onto the active site in the closed form to prevent autoactivation. The open-closed equilibrium also defines a new structural framework for the mechanism of activation of prothrombin by prothrombinase. PMID:27435675

  9. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  10. Proteasome Assay in Cell Lysates

    Science.gov (United States)

    Maher, Pamela

    2016-01-01

    The ubiquitin-proteasome system (UPS) mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes including tumorigenesis, inflammation and cell death (Ciechanover, 2005; Kisselev and Goldberg, 2001). A number of recent studies have shown that proteasome activity is decreased in a variety of neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke as well as during normal aging (Chung et al., 2001; Ciechanover and Brundin, 2003; Betarbet et al., 2005). This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Protein clearance by the UPS involves two sequential reactions. The first is the tagging of protein lysine residues with ubiquitin (Ub) and the second is the subsequent degradation of the tagged proteins by the proteasome. We herein describe an assay for the second of these two reactions (Valera et al., 2013). This assay uses fluorogenic substrates for each of the three activities of the proteasome: chymotrypsin-like activity, trypsin-like activity and caspase-like activity. Cleavage of the fluorophore from the substrate by the proteasome results in fluorescence that can be detected with a fluorescent plate reader.

  11. PG-2, a Potent AMP against Pathogenic Microbial Strains, from Potato (Solanum tuberosum L cv. Gogu Valley Tubers Not Cytotoxic against Human Cells

    Directory of Open Access Journals (Sweden)

    Yoonkyung Park

    2013-02-01

    Full Text Available In an earlier study, we isolated potamin-1 (PT-1, a 5.6-kDa trypsin-chymotrypsin protease inhibitor, from the tubers of a potato strain (Solanum tuberosum L cv. Gogu Valley. We established that PT-1 strongly inhibits pathogenic microbial strains, but not human bacterial strains, and that its sequence shows 62% homology with a serine protease inhibitor. In the present study, we isolated an antifungal and antibacterial peptide with no cytotoxicity from tubers of the same potato strain. The peptide (peptide-G2, PG-2 was isolated using salt-extraction, ultrafiltration and reverse-phase high performance liquid chromatography (RP-HPLC. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS showed the protein to have a molecular mass of 3228.5 Da, while automated Edman degradation showed the N-terminal sequence of PG-2 to be LVKDNPLDISPKQVQALCTDLVIRCMCCC-. PG-2 exhibited antimicrobial activity against Candida albicans, a human pathogenic yeast strain, and Clavibacter michiganensis subsp. michiganensis, a plant late blight strain. PG-2 also showed antibacterial activity against Staphylococcus aureus, but did not lyse human red blood cells and was thermostable. Overall, these results suggest PG-2 may be a good candidate to serve as a natural antimicrobial agent, agricultural pesticide and/or food additive.

  12. Purification and some properties of reovirus-like particles from leafhoppers and their possible involvement in wallaby ear disease of maize.

    Science.gov (United States)

    Boccardo, G; Hatta, T; Francki, R I; Grivell, C J

    1980-01-30

    Reovirus-like particles, occurring in association with viroplasms, crystalline arrays and tubules, in the cytoplasm of Cicadulina bimaculata capable of inducing wallaby ear disease in maize, were purified from the insects by differential centrifugation, treatment with the nonionic detergent, Nonidet P-40, and sucrose density gradient centrifugation. The purified particles have a double-shelled icosahedral structure about 70 nm in diameter with external projections (A spikes) about 10 nm long located at the 12 vertices. These intact particles (IPs) are morphologically similar to those of Fiji disease virus (FDV), but are more stable. Cores were produced by enzymatic digestion of IPs with alpha-chymotrypsin. The cores are icosahedra about 57 nm in diameter with projections (B spikes) located at the 12 vertices, resembling those of FDV and cytoplasmic polyhedrosis virus. Immunization of a rabbit with purified IPs resulted in the production of antibodies specific to IPs, cores, and dsRNA. Immunoelectron microscopic investigations revealed that there is no relationship between this virus and FDV, maize rough dwarf, oat sterile dwarf, pangola stunt, and rice ragged stunt viruses, all members of the genus Fijivirus in the family Reoviridae. The nucleic acid extracted from partially purified virus was resolved into 10 segments by polyacrylamide gel electrophoresis. Reovirus-like particles or viroplasms could not be detected in thin sections of maize seedlings colonized by C. bimaculata showing wallaby ear symptoms. In the light of these data the possible etiology of wallaby ear disease is discussed. PMID:18631635

  13. Characterization of protein-protein interactions by isothermal titration calorimetry.

    Science.gov (United States)

    Velazquez-Campoy, Adrian; Leavitt, Stephanie A; Freire, Ernesto

    2015-01-01

    The analysis of protein-protein interactions has attracted the attention of many researchers from both a fundamental point of view and a practical point of view. From a fundamental point of view, the development of an understanding of the signaling events triggered by the interaction of two or more proteins provides key information to elucidate the functioning of many cell processes. From a practical point of view, understanding protein-protein interactions at a quantitative level provides the foundation for the development of antagonists or agonists of those interactions. Isothermal Titration Calorimetry (ITC) is the only technique with the capability of measuring not only binding affinity but the enthalpic and entropic components that define affinity. Over the years, isothermal titration calorimeters have evolved in sensitivity and accuracy. Today, TA Instruments and MicroCal market instruments with the performance required to evaluate protein-protein interactions. In this methods paper, we describe general procedures to analyze heterodimeric (porcine pancreatic trypsin binding to soybean trypsin inhibitor) and homodimeric (bovine pancreatic α-chymotrypsin) protein associations by ITC.

  14. Transduction of proteins into leishmania tarentolae by formation of non-covalent complexes with cell-penetrating peptides.

    Science.gov (United States)

    Keller, Andrea-Anneliese; Breitling, Reinhard; Hemmerich, Peter; Kappe, Katarina; Braun, Maria; Wittig, Berith; Schaefer, Buerk; Lorkowski, Stefan; Reissmann, Siegmund

    2014-02-01

    Cell-penetrating peptides (CPPs) are used to transport peptides, proteins, different types of ribonucleic acids (or mimics of these molecules), and DNA into live cells, both plant and mammalian. Leishmania belongs to the class of protozoa having, in comparison to mammalian cells, a different lipid composition of the membrane, proteoglycans on the surface, and signal pathways. We investigated the uptake of two different and easily detectable proteins into the non-pathogenic strain Leishmania tarentolae. From the large number of CPPs available, six and a histone were chosen specifically for their ability to form non-covalent complexes. For Leishmania we used the enzyme β-galactosidase and fluorescent labeled bovine serum albumin as cargoes. The results are compared to similar internalization studies using mammalian cells [Mussbach et al., ]. Leishmania cells can degrade CPPs by a secreted and membrane-bound chymotrypsin-like protease. Both cargo proteins were internalized with sufficient efficiency and achieved intramolecular concentrations similar to mammalian cells. The transport efficiencies of the CPPs differed from each other, and showed a different rank order for both cargoes. The intracellular distribution of fluorescent-labeled bovine serum albumin showed highest concentrations in the nucleus and kinetoplast. Leishmania are susceptible to high concentrations of some CPPs, although comparably dissimilar to mammalian cells. MPG-peptides are more cytotoxic in Leishmania than in mammalian cells, acting as antimicrobial peptides. Our results contribute to a better understanding of molecular interactions in Leishmania cells and possibly to new treatments of leishmaniasis.

  15. Interface of covalently bonded phospholipids with a phosphorylcholine head: characterization, protein nonadsorption, and further functionalization.

    Science.gov (United States)

    Ferez, Lynda; Thami, Thierry; Akpalo, Edefia; Flaud, Valérie; Tauk, Lara; Janot, Jean-Marc; Déjardin, Philippe

    2011-09-20

    Surface anchored poly(methylhydrosiloxane) (PMHS) thin films on oxidized silicon wafers or glass substrates were functionalized via the SiH hydrosilylation reaction with the internal double bonds of 1,2-dilinoleoyl-sn-glycero-3-phosphorylcholine (18:2 Cis). The surface was characterized by X-ray photoelectron spectroscopy, contact angle measurements, atomic force microscopy, and scanning electron microscopy. These studies showed that the PMHS top layer could be efficiently modified resulting in an interfacial high density of phospholipids. Grafted phospholipids made the initially hydrophobic surface (θ = 106°) very hydrophilic and repellent toward avidin, bovine serum albumin, bovine fibrinogen, lysozyme, and α-chymotrypsin adsorption in phosphate saline buffer pH 7.4. The surface may constitute a new background-stable support with increased biocompatibility. Further possibilities of functionalization on the surface remain available owing to the formation of interfacial SiOH groups by Karstedt-catalyzed side reactions of SiH groups with water. The presence of interfacial SiOH groups was shown by zeta potential measurements. The reactivity and surface density of SiOH groups were checked by fluorescence after reaction of a monoethoxy silane coupling agent bearing Alexa as fluorescent probe.

  16. High-sensitivity detection of PSA by time-resolved fluorometry with Europium chelate

    Science.gov (United States)

    Nahm, Kie B.; Jeong, Jin H.; Kim, Byoung C.; Kim, Jae H.; Kim, Young M.; Jeong, Dong S.; Oh, Sang W.; Choi, Eui Y.; Ko, Dong S.

    2006-01-01

    Prostate-specific antigen (PSA) is an androgen-dependent glycoprotein protease (M.W. 33 kDa) and a member of kallikrein super-family of serine protease, and has chymotrypsin-like enzymatic activity. It is synthesized by the prostate epithelial cells and found in the prostate gland and seminal plasma as a major protein. It is widely used as a clinical marker for diagnosis, screening, monitoring and prognosis of prostate cancer. In normal male adults, the concentration of PSA in the blood is below 4 ng/ml and this value increases in patients with the prostate cancer or the benign prostatic hyperplasia (BPH) due to its leakage into the circulatory system. As such, systematic monitoring of the PSA level in the blood can provide critical information about the progress of the prostatic disease. We have fabricated a bread-board time resolved fluorescence system that could detect a concentration of Prostate Specific Antigen t-PSA) at clinically meaningful level in plasma as well as in whole blood sample. We chose Europium chelates as the fluorescence markers to attach to the PSA for its long decay lifetime and relative photostability. We have simplified the electronic circuits considerably by employing a MCS. With this setup, we have successfully proved that PSA concentration of 4pg/mL can be detected with acceptable reliability.

  17. Fluorescence of prostate-specific antigen as measured with a portable 1D scanner

    Science.gov (United States)

    Kim, Byeong C.; Jeong, Jin H.; Jeong, Dong S.; Kim, Young M.; Oh, Sang W.; Choi, Eui Y.; Kim, Jae H.; Nahm, Kie B.

    2005-01-01

    Prostate-specific antigen (PSA) is an androgen-dependent glycoprotein protease (M.W. 33 kDa) and a member of kallikrein super-family of serine protease, and has chymotrypsin-like enzymatic activity. It is synthesized by the prostate epithelial cells and found in the prostate gland and seminal plasma as a major protein. It is widely used as a clinical marker for diagnosis, screening, monitoring and prognosis of prostate cancer. In normal male adults, the concentration of PSA in the blood is below 4 ng/ml and this value increases in patients with the prostate cancer or the benign prostatic hyperplasia (BPH) due to its leakage into the circulatory system. As such, systematic monitoring of the PSA level in the blood can provide critical information about the progress of the prostatic disease. We have developed a compact integral system that can quantitatively measure the concentration of total PSA in human blood. This system utilizes the fluorescence emitted from the dye molecules attached to PSA molecules after appropriate immunoassay-based processing. Developed for the purpose of providing an affordable means of fast point-of-care testing of the prostate cancer, this system proved to be able to detect the presence of the PSA at the level of 0.18 ng/ml in less than 12 minutes, with the actual measurement taking less than 2 minutes. The design concept for this system is presented together with the result for a few representative samples.

  18. Mitochondrial Malfunctioning, Proteasome Arrest and Apoptosis in Cancer Cells by Focused Intracellular Generation of Oxygen Radicals

    Directory of Open Access Journals (Sweden)

    Ilaria Postiglione

    2015-08-01

    Full Text Available Photofrin/photodynamic therapy (PDT at sub-lethal doses induced a transient stall in proteasome activity in surviving A549 (p53+/+ and H1299 (p53−/− cells as indicated by the time-dependent decline/recovery of chymotrypsin-like activity. Indeed, within 3 h of incubation, Photofrin invaded the cytoplasm and localized preferentially within the mitochondria. Its light activation determined a decrease in mitochondrial membrane potential and a reversible arrest in proteasomal activity. A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease. Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete. We evaluated whether combining PDT (which is a treatment for killing tumor cells, per se, and inducing proteasome arrest in the surviving ones with Bortezomib doses capable of sustaining the stall would protract the arrest with sufficient time to induce apoptosis in remaining cells. The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis.

  19. The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs.

    Science.gov (United States)

    Zimic, Mirko; Pajuelo, Mónica; Gilman, Robert H; Gutiérrez, Andrés H; Rueda, Luis D; Flores, Myra; Chile, Nancy; Verástegui, Manuela; Gonzalez, Armando; García, Héctor H; Sheen, Patricia

    2012-01-15

    Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection.

  20. Purification and characterization of albumin from frog skin of Duttaphrynus melanostictus.

    Science.gov (United States)

    Zhang, Ying-Xia; Chen, Cong-Wei; Wang, Manchuriga; Wei, Shuang-Shuang; Guan, Huai; Chi, Ting-Ting; Qi, Xing-Zhu; Hu, Wen-Ting

    2011-10-01

    Following determination of trypsin inhibitory activity, a serine protease inhibitor was purified and characterized from frog Duttaphrynus melanostictus serum. It was identified as serum albumin, with molecular weight of 67 kDa (DmA-serum). Different from bovine serum albumin, DmA-serum potently inhibited trypsin with similar K(i) values around 1.6 × 10⁻⁷ M. No inhibitory effect on thrombin, chymotrypsin, elastase and subtilisin was observed under the assay conditions. The N-terminal amino acid is EAEPHSRI. Subsequently, a protein with same N-terminal amino acid was purified from skin, termed as DmA-skin. However, DmA-skin is distinct from DmA-serum by binding of a haem b (0.5 mol/mol protein), and with low trypsin inhibitory activity. Frog albumin is distributed in frog skin and exhibited trypsin inhibitory activity, suggesting that it plays important roles in skin physiological functions, like water economy, metabolite exchange and osmoregulation, etc. PMID:21858423

  1. Purification and antioxidant properties of bigeye tuna (Thunnus obesus) dark muscle peptide on free radical-mediated oxidative systems.

    Science.gov (United States)

    Je, Jae-Young; Qian, Zhong-Ji; Lee, Sang-Hoon; Byun, Hee-Guk; Kim, Se-Kwon

    2008-12-01

    To produce bioactive peptides from by-products of fish processing, bigeye tuna dark muscle was hydrolyzed using various enzymes (alcalase, alpha-chymotrypsin, neutrase, papain, pepsin, and trypsin), and the hydrolysates were evaluated for antioxidant activity. Considering the results of degree of hydrolysis and antioxidant activities, peptic hydrolysate was used for further studies to identify a potent antioxidant peptide. Antioxidant peptide was purified using consecutive chromatographic methods and was identified as being H-Leu-Asn-Leu-Pro-Thr-Ala-Val-Tyr-Met-Val-Thr-OH (MW 1,222 Da) by quantitative time-of-flight electrospray ionization mass spectrometry. Purified antioxidant peptide from bigeye tuna dark muscle (APTDM) was investigated for its antioxidant activities using both free radical scavenging effects and polyunsaturated fatty acid (PUFA) peroxidation inhibitory activity. The results showed that APTDM effectively quenched with low 50% inhibitory concentration values compared to vitamin C as a positive control against four different free radicals: 1,1-diphenyl-2-picrylhydrazyl, hydroxyl, superoxide, and alkyl radical. APTDM also inhibited PUFA peroxidation in a linoleic acid emulsion system, and the activity was similar to that of alpha-tocopherol. We further investigated its antioxidant activities on cellular systems, and the results showed that APTDM significantly scavenged cellular radicals and enhanced the viability of tert-butyl hydroperoxide-induced cytotoxicity. These results indicate that APTDM or a peptide fraction containing APTDM would be a beneficial ingredient for functional food and/or pharmaceuticals. PMID:19053853

  2. Insulin-like substance and insulin-degrading complex of hemolysate of human erythrocytes

    International Nuclear Information System (INIS)

    A lysate of human erythrocytes was fractionated on gel-filtration resins of different types and immunoreactive insulin, the insulinase activity and the effect of individual fractions on the insulinase activity was determined in the fractions obtained. It was established that the hemolysate contains a complex of insulin-metabolizing compounds, including an insulin-like substance, insulinase, and an inhibitor and activator of the insulinase activity. The insulin-like substance coincided with native insulin in site of elution from a column of Sephadex G-50 and its concentration in the lysate exceeded that of insulin in the blood plasma. Insulinase, which has a molecular weight of about 100,000, cleaved [125I] insulin to fragments soluble in trichloroacetic acid, but had no effect on hypophyseal proteins and glycoprotein hormones. The insulinase activity was inhibited by low temperatures, atropine, and a newly discovered intraerythrocytic proteinase inhibitor, which also inhibits the serine proteinases trypsin and chymotrypsin. A substance eluted from a column of Sephadex G-100 in the region of low-molecular-weight substances increased the insulinase activity. The elution curve of substances with proteinase-inhibiting and insulinase-activating activities indicates that there is more than one inhibitory and activating factor. The results of the studies suggest that the insulin-degrading complex in human erythrocytes acts as a regulator of the insulin level in the blood plasma. It is also possible that the insulin-like substance is produced in the cytosol of the erythrocytes

  3. Stepwise Versus Concerted Mechanisms in General-Base Catalysis by Serine Proteases.

    Science.gov (United States)

    Uritsky, Neta; Shokhen, Michael; Albeck, Amnon

    2016-01-26

    General-base catalysis in serine proteases still poses mechanistic challenges despite decades of research. Whether proton transfer from the catalytic Ser to His and nucleophilic attack on the substrate are concerted or stepwise is still under debate, even for the classical Asp-His-Ser catalytic triad. To address these key catalytic steps, the transformation of the Michaelis complex to tetrahedral complex in the covalent inhibition of two prototype serine proteases was studied: chymotrypsin (with the catalytic triad) inhibition by a peptidyl trifluoromethane and GlpG rhomboid (with Ser-His dyad) inhibition by an isocoumarin derivative. The sampled MD trajectories of averaged pKa  values of catalytic residues were QM calculated by the MD-QM/SCRF(VS) method on molecular clusters simulating the active site. Differences between concerted and stepwise mechanisms are controlled by the dynamically changing pKa  values of the catalytic residues as a function of their progressively reduced water exposure, caused by the incoming ligand.

  4. L-Ornithine Schiff base-copper and -cadmium complexes as new proteasome inhibitors and apoptosis inducers in human cancer cells.

    Science.gov (United States)

    Zhang, Zhongyu; Bi, Caifeng; Fan, Yuhua; Zhang, Nan; Deshmukh, Rahul; Yan, Xingchen; Lv, Xiuwen; Zhang, Pengfei; Zhang, Xia; Dou, Q Ping

    2015-01-01

    Ubiquitin-proteasome system (UPS) plays a crucial role in many cellular processes such as cell cycle, proliferation and apoptosis. Aberrant activation of UPS may result in cellular transformation or other altered pathological conditions. Previous studies have shown that metal-based complexes could inhibit proteasome activity and induce apoptosis in certain human cancer cells. In the current study, we report that the cadmium and copper complexes with heterocycle-ornithine Schiff base are potent inhibitors of proteasomal chymotrypsin-like (CT-like) activity, leading to induction of apoptosis in cancer cells. Two novel copper-containing complexes and two novel cadmium-containing complexes with different heterocycle-ornithine Schiff base structures as ligands were synthesized and characterized. We found that complexes Cu1, Cd1 and Cd2 show proteasome-inhibitory activities in human breast cancer MDA-MB-231 and human prostate cancer LNCaP cells, resulting in the accumulation of p27, a natural proteasome substrate and other ubiquitinated proteins, followed by the induction of apoptosis. Our results suggest that metal complexes with heterocycle-ornithine Schiff base have proteasome-inhibitory capabilities and have the potential to be developed into novel anticancer drugs.

  5. First isolation and antinociceptive activity of a lipid transfer protein from noni (Morinda citrifolia) seeds.

    Science.gov (United States)

    Campos, Dyély C O; Costa, Andrea S; Lima, Amanda D R; Silva, Fredy D A; Lobo, Marina D P; Monteiro-Moreira, Ana Cristina O; Moreira, Renato A; Leal, Luzia K A M; Miron, Diogo; Vasconcelos, Ilka M; Oliveira, Hermógenes D

    2016-05-01

    In this study a novel heat-stable lipid transfer protein, designated McLTP1, was purified from noni (Morinda citrifolia L.) seeds, using four purification steps which resulted in a high-purified protein yield (72 mg McLTP1 from 100g of noni seeds). McLTP1 exhibited molecular masses of 9.450 and 9.466 kDa, determined by electrospray ionisation mass spectrometry. The N-terminal sequence of McLTP1 (AVPCGQVSSALSPCMSYLTGGGDDPEARCCAGV), as analysed by NCBI-BLAST database, revealed a high degree of identity with other reported plant lipid transfer proteins. In addition, this protein proved to be resistant to pepsin, trypsin and chymotrypsin digestion. McLTP1 given intraperitoneally (1, 2, 4 and 8 mg/kg) and orally (8 mg/kg) caused an inhibition of the writhing response induced by acetic acid in mice. This protein displayed thermostability, retaining 100% of its antinociceptive activity after 30 min incubation at 80 °C. Pretreatment of mice with McLTP1 (8 mg/kg, i.p. and p.o.) also decreased neurogenic and inflammatory phases of nociception in the formalin test. Naloxone (2 mg/kg, i.p.) antagonised the antinociceptive effect of McLTP1 suggesting that the opioid mechanisms mediate the analgesic properties of this protein.

  6. Induction of inflammatory cytokines by cartilage extracts.

    Science.gov (United States)

    Merly, Liza; Simjee, Shabana; Smith, Sylvia L

    2007-03-01

    Shark cartilage extracts were examined for induction of cytokines and chemokines in human peripheral blood leukocytes. Primary leukocyte cultures were exposed to a variety of aqueous and organic extracts prepared from several commercial brands of shark cartilage. From all commercial sources of shark cartilage tested the acid extracts induced higher levels of TNFalpha than other extracts. Different commercial brands of shark cartilage varied significantly in cytokine-inducing activity. TNFalpha induction was seen as early as 4 h and IFNgamma at detectable levels for up to four days. Shark cartilage extracts did not induce physiologically significant levels of IL-4. Results suggest that shark cartilage, preferentially, induces Th1 type inflammatory cytokines. When compared to bovine cartilage extract, collagen, and chondroitin sulfate, shark cartilage induced significantly higher levels of TNFalpha. Treatment with digestive proteases (trypsin and chymotrypsin) reduced the cytokine induction response by 80%, suggesting that the active component(s) in cartilage extracts is proteinaceous. The induction of Th1 type cytokine response in leukocytes is a significant finding since shark cartilage, taken as a dietary supplement for a variety of chronic degenerative diseases, would be contraindicated in cases where the underlying pathology of the chronic condition is caused by inflammation. PMID:17276897

  7. The effect of stirring and seeding on the AcPheLeuNH(2) synthesis and crystallization in a reversed micellar system.

    Science.gov (United States)

    Feliciano; Dias; Prazeres

    2000-08-01

    The present work describes the enzymatic synthesis and simultaneous crystallization of the dipeptide AcPheLeuNH(2) by alpha-chymotrypsin in a reversed micellar system of tetradecyltrimethylammonium bromide (TTAB)/heptane/octanol/carbonate buffer. The low solubility of the dipeptide in the micellar solution led to the formation and growth of needle-like crystals during the synthesis as soon as supersaturation was achieved. The crystallization process then followed a typical pattern, proceeding in three phases: nucleation, de-supersaturation, and re-equilibrium of saturation. Crystallization was followed by visual observation with an optical microscope, and the increase of crystal number and size was confirmed. Experiments showed that the supersaturation concentration decreases with the addition of AcPheLeuNH(2) seeds before the reaction, and also with a decrease of the stirring speed. It was also observed that the increase of both seed concentration and stirring advances the start of crystallization, so that the dipeptide is more quickly removed from solution. The consequent decrease in its loss through hydrolysis causes an increase in its yield. Both stirring and seeding could constitute important generic strategies for promoting crystallization of more soluble dipeptides during their synthesis in similar reversed micellar systems.

  8. Enterococci isolated from farm ostriches and their relation to enterocins.

    Science.gov (United States)

    Lauková, Andrea; Kandričáková, Anna; Ščerbová, Jana; Strompfová, Viola

    2016-07-01

    The present study focuses on the detection of enterococci in ostrich faeces. Forty-six bacterial colonies from 140 ostriches were identified at the species level using the MALDI-TOF MS identification system. According to the score value evaluation, they were allotted to the species Enterococcus hirae, Enterococcus faecium and Enterococcus mundtii confirmed also by phenotypic testing. Dominated species E. hirae (34 strains) were submitted to more detailed testing. Those strains E. hirae produced either no or only slight amount of the enzymes related to disorders (N-acetyl-β-glucosaminidase, β-glucuronidase, α-chymotrypsin, trypsin). Most of the strains were not hemolytic. They did not harbour the hiracin-producing gene. Five E. hirae strains harboured virulence factor gene gelE; however, they were phenotypically gelatinase negative. They also harboured other virulence factor genes such as esp, efaAfm and ccf. E. hirae strains were mostly sensitive to antibiotics and those resistant at least to one antibiotic were sensitive to enterocins (200-25,600 AU/mL). This study represents original and novel results concerning the enterococcal microflora in ostriches; enterococci in ostriches have not been described in detail up to now; sensitivity to enterocins of E. hirae strains harbouring virulence factor genes to enterocins is also new. PMID:26603748

  9. Using cornstarch in microparticulate diets for larvicultured tropical gar (Atractosteus tropicus).

    Science.gov (United States)

    Frías-Quintana, C A; Domínguez-Lorenzo, J; Álvarez-González, C A; Tovar-Ramírez, D; Martínez-García, R

    2016-04-01

    Aquaculture in Mexico has been developed by the cultivation of commercial species. In Tabasco, the cultivation of native species is mainly limited by the lack of nutrition studies to support its crop profitability. Among these species is the tropical gar (Atractosteus tropicus), which has great potential for cultivation. However, the nutritional value of carbohydrates in diets for this species which contribute to improved growth and survival, have not been evalulated,. Thus, in the present investigation, isoprotein and isolipid diets have been designed based on the substitution of cellulose by corn starch (D1: 0% starch-15% cellulose, D2: 7.5% starch-7.5% cellulose and D3: 15% starch-0% cellulose) and compared with a commercial trout diet (45% protein and 16% lipids). A total of 1800 larvae (0.008 ± 0.002 g and 10.5 ± LT 0.126 mm) were used, distributed in a recirculation system in order to evaluate growth and survival for 30 days. The results show higher growth and survival of 97% of larvae fed the D3 diet, while cannibalism in the species was mitigated. Major digestive enzyme activities occurred (acid protease, alkaline protease, trypsin, chymotrypsin, leucine aminopeptidase, carboxypeptidase A, lipase, α-glucosidase and amylase) for larvae fed D3. It is concluded that the contribution of corn starch (15%) replacing cellulose in the diet improves growth and survival of this species. PMID:26573856

  10. A vitellogenin polyserine cleavage site: highly disordered conformation protected from proteolysis by phosphorylation.

    Science.gov (United States)

    Havukainen, Heli; Underhaug, Jarl; Wolschin, Florian; Amdam, Gro; Halskau, Øyvind

    2012-06-01

    Vitellogenin (Vg) is an egg-yolk precursor protein in most oviparous species. In honeybee (Apis mellifera), the protein (AmVg) also affects social behavior and life-span plasticity. Despite its manifold functions, the AmVg molecule remains poorly understood. The subject of our structure-oriented AmVg study is its polyserine tract - a little-investigated repetitive protein segment mostly found in insects. We previously reported that AmVg is tissue specifically cleaved in the vicinity of this tract. Here, we show that, despite its potential for an open, disordered structure, AmVg is unexpectedly resistant to trypsin/chymotrypsin digestion at the tract. Our findings suggest that multiple phosphorylation plays a role in this resilience. Sequence variation is highly pronounced at the polyserine region in insect Vgs. We demonstrate that sequence differences in this region can lead to structural variation, as NMR and circular dichroism (CD) evidence assign different conformational propensities to polyserine peptides from the honeybee and the jewel wasp Nasonia vitripennis; the former is extended and disordered and the latter more compact and helical. CD analysis of the polyserine region of bumblebee Bombus ignitus and wasp Pimpla nipponica supports a random coil structure in these species. The spectroscopic results strengthen our model of the AmVg polyserine tract as a flexible domain linker shielded by phosphorylation. PMID:22573762

  11. Effect of chymase on intraocular pressure in rabbits.

    Science.gov (United States)

    Konno, Takashi; Maruichi, Midori; Takai, Shinji; Oku, Hidehiro; Sugiyama, Tetsuya; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Ikeda, Tsunehiko; Miyazaki, Mizuo

    2005-11-01

    Chymase is a chymotrypsin-like serine protease that is stored exclusively in the secretory granules of mast cells and converts big endothelins to endothelin-1 (1-31). The aim of this study was to evaluate the effect of chymase on intraocular pressure in rabbits. Chymase injection (3 and 10 mU) resulted in a trend toward increased intraocular pressure and a significant increase in intraocular pressure at a dose of 10 mU compared with the control. A specific chymase inhibitor, Suc-Val-Pro-Phe(P)(OPh)(2), attenuated the ocular hypertension induced by chymase. Endothelin-1 (1-31) also caused ocular hypertension, which was inhibited by a selective endothelin ET(A) receptor antagonist, cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123). Moreover, chymase-induced ocular hypertension was inhibited by BQ-123. These results suggest that chymase influences the regulation of intraocular pressure, and it is likely that the formation of endothelin-1 (1-31) and subsequent activation of endothelin ET(A) receptors are involved in the development of ocular hypertension induced by chymase. PMID:16253233

  12. Effects of 2-alkynyladenosine derivatives on intraocular pressure in rabbits.

    Science.gov (United States)

    Konno, Takashi; Ohnuma, Shin-ya; Uemoto, Kazuhiro; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Endo, Kazuki; Hosokawa, Tomokazu; Nakahata, Norimichi

    2004-02-23

    We evaluated the activities of 2-alkynyladenosine derivatives, relatively selective adenosine A2 receptor agonists, in the intraocular pressure regulation in rabbits. An adenosine A2 receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680) decreased intraocular pressure, while another A2 receptor agonist 2-(phenylamino)adenosine transiently increased it. The first group of 2-alkynyladenosine derivatives (1-hexyn-1-yl derivatives) caused a transient increase followed by decrease in intraocular pressure, while the second group (1-octyn-1-yl and 6-cyano-1-hexyn-1-yl derivatives) only decreased it. The second group is also effective in the ocular hypertensive models induced by water-loading and alpha-chymotrypsin. The outflow facility was increased by a 1-octyn-1-yl derivative. Both increase and decrease in intraocular pressure induced by 2-alkynyladenosine derivatives were inhibited by an adenosine A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine, but not by an adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropyl xanthine. These findings suggest that 2-alkynyladenosine derivatives may affect intraocular pressure via adenosine A2 receptor, and 2-alkynyladenosine derivative-induced ocular hypotension is due to the increase of outflow facility. PMID:14985053

  13. Uranium quantification in semen by inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Todorov, Todor I; Ejnik, John W; Guandalini, Gustavo; Xu, Hanna; Hoover, Dennis; Anderson, Larry; Squibb, Katherine; McDiarmid, Melissa A; Centeno, Jose A

    2013-01-01

    In this study we report uranium analysis for human semen samples. Uranium quantification was performed by inductively coupled plasma mass spectrometry. No additives, such as chymotrypsin or bovine serum albumin, were used for semen liquefaction, as they showed significant uranium content. For method validation we spiked 2g aliquots of pooled control semen at three different levels of uranium: low at 5 pg/g, medium at 50 pg/g, and high at 1000 pg/g. The detection limit was determined to be 0.8 pg/g uranium in human semen. The data reproduced within 1.4-7% RSD and spike recoveries were 97-100%. The uranium level of the unspiked, pooled control semen was 2.9 pg/g of semen (n=10). In addition six semen samples from a cohort of Veterans exposed to depleted uranium (DU) in the 1991 Gulf War were analyzed with no knowledge of their exposure history. Uranium levels in the Veterans' semen samples ranged from undetectable (<0.8 pg/g) to 3350 pg/g. This wide concentration range for uranium in semen is consistent with known differences in current DU body burdens in these individuals, some of whom have retained embedded DU fragments.

  14. Solution Structure of an Amyloid-Forming Protein During Photoinitiated Hexamer-Dodecamer Transitions Revealed Through Small-Angle Neutron Scattering

    Energy Technology Data Exchange (ETDEWEB)

    Hamill,A.; Wang, S.; Lee, Jr., C.

    2007-01-01

    Shape-reconstruction analysis applied to small angle neutron scattering (SANS) data is used to determine the in vitro conformations of {alpha}-chymotrypsin oligomers that form as a result of partial unfolding with a photoresponsive surfactant. In the presence of the photoactive surfactant under visible light, the native oligomers (dimers or compact hexamers) rearrange into expanded corkscrew-like hexamers. Converting the surfactant to the photopassive form with UV light illumination causes the hexamers to laterally aggregate and intertwine into dodecamers with elongated, twisted conformations containing cross-sectional dimensions similar to amyloid protofilaments. Secondary-structure measurements with FT-IR indicate that this photoinduced hexamer-to-dodecamer association occurs through intermolecular {beta} sheets stabilized with hydrogen bonds, similar to amyloid formation. Traditional structural characterization techniques such as X-ray crystallography and NMR are not easily amenable to the study of these non-native protein conformations; however, SANS is ideally suited to the study of these associated intermediates, providing direct observation of the mechanism of oligomeric formation in an amyloid-forming protein. Combined with photoinitiated hexamer-to-dodecamer associations in the presence of the photoresponsive surfactant, this study could provide unique insight into the amyloidosis disease pathway, as well as novel disease treatment strategies.

  15. Generation of a 90 000 molecular weight fragment from human plasma angiotensin-I-converting enzyme by enzymatic or alkaline hydrolysis.

    Science.gov (United States)

    Yotsumoto, H; Lanzillo, J J; Fanburg, B L

    1983-12-12

    A catalytically active Mr 90 000 fragment was generated from native Mr 140 000 human plasma angiotensin-I-converting enzyme after treatment with reagents that induced a perturbation of the native tertiary conformation. Treatment of converting enzyme with 6 M urea produced an aggregation of molecules that was susceptible to proteolysis by either trypsin, chymotrypsin or Staphylococcus aureus V8 proteinase to generate the Mr 90 000 converting enzyme. Also, 1 M ammonium hydroxide, pH 11.3, or 0.01 M sodium hydroxide, pH 11.3, cleaved converting enzyme to the Mr 90 000 fragment. Degradation was not an autocatalytic phenomenon, since it was not prevented by inhibition of converting enzyme with EDTA. The enzymatically mediated, but not the alkaline mediated, cleavage was inhibited by specific converting enzyme inhibitors captopril and Merck L-154,826. This suggests that captopril and Merck L-154,826 can prevent converting-enzyme degradation by preserving a conformation that does not have sites exposed to proteolytic enzymes. This conformation may mimic the native conformation which is quite resistant to serine proteinases.

  16. A Serpin Released by an Entomopathogen Impairs Clot Formation in Insect Defense System

    Science.gov (United States)

    Hao, YouJin; Balasubramanian, Natesan; Jing, Yingjun; Montiel, Rafael; Faria, Tiago Q.; Brito, Rui M.; Simões, Nelson

    2013-01-01

    Steinernema carpocapsae is an entomopathogenic nematode widely used for the control of insect pests due to its virulence, which is mainly attributed to the ability the parasitic stage has to overcome insect defences. To identify the mechanisms underlying such a characteristic, we studied a novel serpin-like inhibitor (sc-srp-6) that was detected in a transcriptome analysis. Recombinant Sc-SRP-6 produced in Escherichia coli had a native fold of serpins belonging to the α-1-peptidase family and exhibited inhibitory activity against trypsin and α-chymotrypsin with Ki of 0.42×10−7 M and 1.22×10−7 M, respectively. Functional analysis revealed that Sc-SRP-6 inhibits insect digestive enzymes, thus preventing the hydrolysis of ingested particles. Moreover, Sc-SRP-6 impaired the formation of hard clots at the injury site, a major insect defence mechanism against invasive pathogens. Sc-SRP-6 does not prevent the formation of clot fibres and the activation of prophenoloxidases but impairs the incorporation of the melanin into the clot. Binding assays showed a complex formation between Sc-SRP-6 and three proteins in the hemolymph of lepidopteran required for clotting, apolipophorin, hexamerin and trypsin-like, although the catalytic inhibition occurred exclusively in trypsin-like. This data allowed the conclusion that Sc-SRP-6 promotes nematode virulence by inhibiting insect gut juices and by impairing immune clot reaction. PMID:23874900

  17. Antioxidative effect of yak milk caseinates hydrolyzed with three different proteases

    Directory of Open Access Journals (Sweden)

    Santosh Kumar

    2013-09-01

    Full Text Available Aim: Yak milk is a type of milk that people are less familiar with due to its remote geographical location which may havesignificant effects on composition, microbiota and hydrolytic outcome. Present work was designed with the aim to evaluatethe antioxidative effect of peptides derived from yak milk caseinate on hydrolysis with three different proteases.Materials and Methods: In this investigation Yak milk casein was hydrolyzed by three commercially available proteases(Trypsin, Pepsin and chymotrypsin. These hydrolysates collected at different hydrolysis times (30 min, 60 min, 90 min, 120min, 150 min, 180 min, 210 min, 240 min, 270 min, 300 min, 330 min and 360 min were assayed for their antioxidant activitywith respect to the effect of incubation period.Results: Among all the enzyme hydrolysates, the tryptic hydrolysates showed highest antioxidant activity followed bychymotryptic hydrolysates. Further, the peptide samples showing highest activity were subjected to RP-HPLC for their partialcharacterization. Tryptic and peptic hydrolysates produced peaks mainly in the region of hydrophillic solvent indicating thepresence of hydrophillic peptides/peptides.Conclusion: The results indicated that yak milk casein could be a resource to generate antioxidative peptides and be used asmultifunctional active ingredients for many value-added functional foods as well as a traditional food protein.

  18. Protease inhibitors from marine actinobacteria as a potential source for antimalarial compound.

    Directory of Open Access Journals (Sweden)

    L Karthik

    Full Text Available The study was planned to screen the marine actinobacterial extract for the protease inhibitor activity and its anti- Pf activity under in vitro and in vivo conditions. Out of 100 isolates, only 3 isolates exhibited moderate to high protease inhibitor activities on trypsin, chymotrypsin and proteinase K. Based on protease inhibitor activity 3 isolates were chosen for further studies. The potential isolate was characterized by polyphasic approach and identified as Streptomyces sp LK3 (JF710608. The lead compound was identified as peptide from Streptomyces sp LK3. The double-reciprocal plot displayed inhibition mode is non-competitive and it confirms the irreversible nature of protease inhibitor. The peptide from Streptomyces sp LK3 extract showed significant anti plasmodial activity (IC50: 25.78 µg/ml. In in vivo model, the highest level of parasitemia suppression (≈ 45% was observed in 600 mg/kg of the peptide. These analyses revealed no significant changes were observed in the spleen and liver tissue during 8 dpi. The results confirmed up-regulation of TGF-β and down regulation of TNF-α in tissue and serum level in PbA infected peptide treated mice compared to PbA infection. The results obtained infer that the peptide possesses anti- Pf activity activity. It suggests that the extracts have novel metabolites and could be considered as a potential source for drug development.

  19. Inhibition of coagulation factors by recombinant barley serpin BSZx

    DEFF Research Database (Denmark)

    Dahl, Søren Weis; Rasmussen, S.K.; Petersen, L..C.;

    1996-01-01

    Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol, Chem., in press), We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas...... as substrate, Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P-1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k(ass) = 1-2 x 10(2) M(-1) s(-1)) whereas factor XIIa...... and leukocytes, a fungal trypsin and three subtilisins, Thrombin, plasma kallikrein, factor VIIa/tissue factor and factor Xa were inhibited by BSZx at heparin independent association rates (k(ass)) of 4.5 x 10(3)-1.3 x 10(5) M(-1) s(-1) at 22 degrees C. Only factor Xa turned a significant fraction of BSZx over...

  20. Surface peptide mapping of protein I and protein III of four strains of Neisseria gonorrhoeae

    Energy Technology Data Exchange (ETDEWEB)

    Judd, R.C.

    1982-08-01

    Whole cells and isolated outer membranes (OMs) of four strains of gonococci were surface radioiodinated with either lactoperoxidase or Iodogen (Pierce Chemical Co., Rockford, Ill.). These preparations were solubilized in sodium dodecyl sulfate and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface-radioiodinated protein I (PI) and PIII bands were excised from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and digested with alpha-chymotrypsin, and the resultant /sup 125/I-peptide fragments were resolved by high-voltage electrophoresis and thin-layer chromatography (i.e., surface peptide mapping). Radioemitting peptidic fragments were visualized by autoradiography. Results demonstrated that the PI molecule of each gonococcal strain studied had unique iodinatable peptides exposed on the surface of whole cells and OMs, whereas PIIIs appeared to have the same portion of the molecule exposed on the surface of bacteria or OMs, regardless of the gonococcal strain from which they were isolated. Many more radiolabeled peptides were seen in surface peptide maps of PIs from radiolabeled OMs than in those from radioiodinated whole cells, whereas different peptidic fragments were seen in the surface peptide maps of PIIIs from radiolabeled OMs than were seen in those from radiolabeled whole cells. These data suggest that PI may contribute strain-specific antigenic determinants and PIII may contribute cross-reactive determinants and that the surface exposure of PI and PIII is different in isolated OMs than in the OM of intact gonococci.

  1. First report of a glutamine-rich antifungal peptide with immunomodulatory and antiproliferative activities from family Amaryllidaceae

    International Nuclear Information System (INIS)

    This represents the first report of purification of a glutamine-rich antifungal peptide from family Amarylliaceace. The peptide, designated as nartazin, was purified from the bulbs of the Chinese daffodil Narcissus tazetta var. chinensis by means of ion-exchange chromatography and affinity chromatography. Its molecular mass was 7.1 kDa, as determined by SDS-PAGE and gel filtration. Nartazin stimulated proliferation of mouse splenocytes and bone marrow cells but inhibited proliferation of leukemia L1210 cells. It also inhibited translation in a cell-free rabbit reticulocyte lysate system. The sequence of its first 20 N-terminal residues was characterized by an abundance of glutamine. The peptide possessed antifungal activity on four phytopathogenic fungi. Its activity was retained after incubation with bovine trypsin and chymotrypsin (enzyme: substrate ratio 1:10 w/w) at 37 deg C for 1 h but was attenuated after treatment with proteinase K. The data revealed its pronounced resistance to proteolytic digestion

  2. Biofilms of vaginal Lactobacillus reuteri CRL 1324 and Lactobacillus rhamnosus CRL 1332: kinetics of formation and matrix characterization.

    Science.gov (United States)

    Leccese Terraf, María Cecilia; Juárez Tomás, María Silvina; Rault, Lucie; Le Loir, Yves; Even, Sergine; Nader-Macías, María Elena Fátima

    2016-09-01

    Adhesion and biofilm formation are strain properties that reportedly contribute to the permanence of lactobacilli in the human vagina. The kinetics of biofilm formation and the chemical nature of the biofilm matrix formed by Lactobacillus reuteri CRL (Centro de Referencia para Lactobacilos Culture Collection) 1324 and Lactobacillus rhamnosus CRL 1332, vaginal beneficial strains, were evaluated in this work. Crystal violet-stained microplate assay and techniques of epifluorescence, electron and confocal microscopy were applied. The highest density and complexity of biofilms of both vaginal lactobacilli were observed at 72 h of incubation. Protease, proteinase K, α-chymotrypsin and trypsin treatments efficiently detached L. reuteri CRL 1324 biofilm that was also partially affected by α-amylase. However, L. rhamnosus CRL 1332 biofilm was slightly affected by protease, proteinase K and α-amylase. Confocal microscopy revealed greater amount of polysaccharides in L. rhamnosus CRL 1332 biofilm matrix than in L. reuteri CRL 1324 biofilm matrix. The results indicate that proteins are one of the main components of the L. reuteri CRL 1324 biofilm, while the biofilm matrix of L. rhamnosus CRL 1332 is composed of carbohydrates and proteins. The results obtained support the knowledge, understanding and characterization of two biofilm-forming vaginal Lactobacillus strains. PMID:27146055

  3. Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus

    International Nuclear Information System (INIS)

    The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with [alpha-32P]GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the 32P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the 32P-labeled large subunit and the [35S]methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5 and 3 termini were precisely located by S1 nuclease analysis

  4. HDAC inhibitor L-carnitine and proteasome inhibitor bortezomib synergistically exert anti-tumor activity in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Hongbiao Huang

    Full Text Available Combinations of proteasome inhibitors and histone deacetylases (HDAC inhibitors appear to be the most potent to produce synergistic cytotoxicity in preclinical trials. We have recently confirmed that L-carnitine (LC is an endogenous HDAC inhibitor. In the current study, the anti-tumor effect of LC plus proteasome inhibitor bortezomib (velcade, Vel was investigated both in cultured hepatoma cancer cells and in Balb/c mice bearing HepG2 tumor. Cell death and cell viability were assayed by flow cytometry and MTS, respectively. Gene, mRNA expression and protein levels were detected by gene microarray, quantitative real-time PCR and Western blot, respectively. The effect of Vel on the acetylation of histone H3 associated with the p21(cip1 gene promoter was examined by using ChIP assay and proteasome peptidase activity was detected by cell-based chymotrypsin-like (CT-like activity assay. Here we report that (i the combination of LC and Vel synergistically induces cytotoxicity in vitro; (ii the combination also synergistically inhibits tumor growth in vivo; (iii two major pathways are involved in the synergistical effects of the combinational treatment: increased p21(cip1 expression and histone acetylation in vitro and in vivo and enhanced Vel-induced proteasome inhibition by LC. The synergistic effect of LC and Vel in cancer therapy should have great potential in the future clinical trials.

  5. Binding Quantum Dots to Silk Biomaterials for Optical Sensing

    Directory of Open Access Journals (Sweden)

    Disi Lu

    2015-01-01

    Full Text Available Quantum dots (QDs, have great potential for fabricating optical sensing devices and imaging biomaterial degradation in vivo. In the present study, 2-mercaptoethylamine- (MEA- and mercaptopropionic acid- (MPA- capped CdTe-QDs were physically incorporated in silk films that contained a high content (>30% of crystalline beta-sheet structure. The beta-sheets were induced by the addition of glycerol, water annealing, glycerol/annealing, or treatment with methanol. Incorporation of QDs did not influence the formation of beta-sheets. When the films were extracted with water, most QDs remained associated with the silk, based on the retention of photoluminescence in the silk films and negligible photoluminescence in the extracts. Compared to the solution state, photoluminescence intensity significantly decreased for MEA-QDs but not for MPA-QDs in the silk films, while the emission maximum blue shifted (≈4 nm slightly for both. Further film digestion using protease XIV, alpha-chymotrypsin, and the combination of the two proteases suggested that QDs may be bound to the silk beta-sheet regions but not the amorphous regions. QDs photoluminescence in silk films was quenched when the concentration of hydrogen peroxide (H2O2 was above 0.2-0.3 mM, indicating the QDs-incorporated silk films can be used to report oxidation potential in solution.

  6. Crystallization and preliminary X-ray analysis of a protease inhibitor from the latex of Carica papaya

    Energy Technology Data Exchange (ETDEWEB)

    Azarkan, Mohamed [Université Libre de Bruxelles, Faculty of Medicine, Protein Chemistry Unit, Campus Erasme (CP 609), 808 Route de Lennik, B-1070 Brussels (Belgium); Garcia-Pino, Abel [Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie and Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel (Belgium); Dibiani, Rachid [Université Libre de Bruxelles, Faculty of Medicine, Protein Chemistry Unit, Campus Erasme (CP 609), 808 Route de Lennik, B-1070 Brussels (Belgium); Wyns, Lode; Loris, Remy, E-mail: reloris@vub.ac.be [Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie and Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel (Belgium); Baeyens-Volant, Danielle [Université Libre de Bruxelles, Faculty of Medicine, Protein Chemistry Unit, Campus Erasme (CP 609), 808 Route de Lennik, B-1070 Brussels (Belgium)

    2006-12-01

    The Kunitz-type trypsin/chymotrypsin inhibitor isolated from C. papaya latex has been crystallized using the hanging-drop vapour-diffusion method. Two different crystal forms are observed, diffracting to 2.6 and 1.7 Å. A Kunitz-type protease inhibitor purified from the latex of green papaya (Carica papaya) fruits was crystallized in the presence and absence of divalent metal ions. Crystal form I, which is devoid of divalent cations, diffracts to a resolution of 2.6 Å and belongs to space group P3{sub 1} or P3{sub 2}. This crystal form is a merohedral twin with two molecules in the asymmetric unit and unit-cell parameters a = b = 74.70, c = 78.97 Å. Crystal form II, which was grown in the presence of Co{sup 2+}, diffracts to a resolution of 1.7 Å and belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 44.26, b = 81.99, c = 140.89 Å.

  7. Protease activation in glycerol-based deep eutectic solvents

    Science.gov (United States)

    Zhao, Hua; Baker, Gary A.; Holmes, Shaletha

    2011-01-01

    Deep eutectic solvents (DESs) consisting of mixtures of a choline salt (chloride or acetate form) and glycerol are prepared as easily accessible, biodegradable, and inexpensive alternatives to conventional aprotic cation-anion paired ionic liquids. These DES systems display excellent fluidity coupled with thermal stability to nearly 200 °C. In this work, the transesterification activities of cross-linked proteases (subtilisin and α-chymotrypsin), immobilized on chitosan, were individually examined in these novel DESs. In the 1:2 molar ratio mixture of choline chloride/glycerol containing 3% (v/v) water, cross-linked subtilisin exhibited an excellent activity (2.9 μmo l min−1 g−1) in conjunction with a selectivity of 98% in the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol. These highly encouraging results advocate more extensive exploration of DESs in protease-mediated biotransformations of additional polar substrates and use of DESs in biocatalysis more generally. PMID:21909232

  8. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

    Directory of Open Access Journals (Sweden)

    Leah Theresa Sigle

    2013-09-01

    Full Text Available Sandflies (Diptera: Psychodidae are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2. Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

  9. Primary structure of the mating pheromone Er-1 of the ciliate Euplotes raikovi.

    Science.gov (United States)

    Raffioni, S; Luporini, P; Chait, B T; Disper, S S; Bradshaw, R A

    1988-12-01

    The complete amino acid sequence of the mating pheromone Er-1 purified from Euplotes raikovi homozygous for mat-1 was determined by automated Edman degradation of the whole protein and peptides generated by cyanogen bromide, trypsin, Staphylococcus aureus V8 protease, and chymotrypsin. The proposed sequence is: Asp-Ala-Cys-Glu-Gln-Ala-Ala-Ile-Gln-Cys-Val-Glu-Ser-Ala-Cys-Glu-Ser-Leu- Cys-Thr-Glu-Gly-Glu-Asp-Arg-Thr-Gly-Cys-Tyr-Met-Tyr-Ile-Tyr-Ser-Asn-Cys- Pro-Pro-Tyr-Val The calculated molecular weight is 4411.0, which is in agreement with the averaged mass of 4410.2 obtained by fission fragment ionization mass spectrometry. Previously reported values of the native molecular weight, determined by gel filtration, have ranged from 9,000 to 12,000. Thus, the native structure is likely a dimer (or larger aggregate) of identical subunits with the three disulfide bonds present occurring as intrachain links. Secondary structure predictions suggest a helical structure at the amino terminus. A comparison of the Er-1 amino acid sequence with known protein sequences did not reveal any significant similarities. PMID:3142868

  10. Impaired small-bowel barrier integrity in the presence of lumenal pancreatic digestive enzymes leads to circulatory shock.

    Science.gov (United States)

    Kistler, Erik B; Alsaigh, Tom; Chang, Marisol; Schmid-Schönbein, Geert W

    2012-08-01

    In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small-bowel lumen. It is unresolved, however, whether ischemically mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of nonischemic rats was perfused for 2 h with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase, and lipase. Control (n = 6) and experimental animals perfused with pancreatic enzymes only (n = 6) or single enzymes (n = 3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n = 6) developed mild hypotension (P shock and increase systemic protease activation in the absence of ischemia, implicating bowel mucin disruption as a key event in early ischemia. Digestive enzymes and their products, if allowed to penetrate the gut wall, may trigger multiorgan failure and death.

  11. Cloning of a serine proteinase inhibitor from bovine brain: expression in the brain and characterization of its target proteinases.

    Science.gov (United States)

    Nakaya, N; Nishibori, M; Kawabata, M; Saeki, K

    1996-12-01

    A cDNA encoding of the serine proteinase inhibitor (serpin), B-43, was cloned from the cDNA library of the bovine brain. It encoded 378 amino acids, and the MW of the protein was estimated to be 42.6 kDa, which is consistent with that of the native B-43 purified from the bovine brain. The homology search revealed that B-43 belongs to the ovalbumin branch of the serpin superfamily. Among them, B-43 was most homologous to human placental thrombin inhibitor (PI-6) and its murine counterpart, with the amino acid identity of 76% and 71%, respectively. Northern blot analysis showed that the size of the transcript was 1.4 kb, and that the expression of B-43 in the bovine brain varied depending on the brain regions, i.e. a lower level of expression was observed in the cerebral cortex and the hippocampus compared to the level of expression that was observed in the medulla oblongata. [35S]-labeled B-43 protein was synthesized in vitro by using a rabbit reticulocyte lysate system, which formed complexes with proteinases such as thrombin, trypsin, alpha-chymotrypsin, and 7S nerve growth factor (NGF), but not with urokinase or plasmin. These results, together with the immunohistochemical localization of B-43 in astrocytes and in some neurons which was observed in the previous study suggest that B-43 may be involved in the regulation of serine proteinases present in the brain or extravasated from the blood.

  12. Cyclic pentapeptide analogs based on endomorphin-2 structure: cyclization studies using liquid chromatography combined with on-line mass spectrometry and tandem mass spectrometry.

    Science.gov (United States)

    Piekielna, Justyna; Kluczyk, Alicja; Perlikowska, Renata; Janecka, Anna

    2014-05-01

    The cyclization of linear analogs based on endomorphin-2 structure, Tyr/Dmt-d-Lys-Phe-Phe-Asp-NH2 and Tyr/Dmt-d-Cys-Phe-Phe-Cys-NH2 (where Dmt=2',6'-dimethyltyrosine), resulting in obtaining lactam or disulfide derivatives, was studied using liquid chromatography combined with on-line mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). In case of cyclization via an amide bond, the formation of the cyclic monomers, cyclic but not linear dimers and even traces of cyclic trimers was observed. Disulfide bridge containing peptides was obtained by the solid-phase synthesis of the linear sequences, followed by either in-solution or on-resin cyclization. In case of the in-solution cyclization, the expected cyclic monomers were the only products. When oxidation of the cysteine residues was performed when the peptides were still on the resin, cyclic monomer and two cyclodimers, parallel and antiparallel, were found. Digestion of the isolated cyclodimers with α-chymotrypsin allowed for their unambiguous identification. The comparison of the cyclic monomer/dimer ratios for analogs with Tyr versus Dmt in position 1 revealed that the presence of the exocyclic Dmt favored formation of the cyclic monomer, most likely due to the increased steric bulk of this amino acid side-chain as compared with Tyr.

  13. Syntheses ofγ-fluoro-α-amino acids.

    Science.gov (United States)

    Haufe, G; Kröger, S

    1996-09-01

    Methods for the synthesis of racemic and optically active title compounds are presented. Key step of these four-step procedures is the alkylation with 1-bromo-2-fluoroalkanes of glycine-ester-derived imines in anhydrous medium using lithium diisopropylamide as a base at low temperature or phase transfer catalyzed alkylation with 50% NaOH and triethylbenzylammoniumchloride as the phase transfer catalyst, respectively. Subsequent three-step deprotection gave the free acids in 13-33% overall yield. Deracemization ofγ-fluoro-α-aminobutyric acid methyl and ethyl esters withα-chymotrypsin was shown to give the (-)-enantiomers of the esters and (+)-γ-fluoro-α-aminobutyric acid in >98% ee, while from thetert-butylester the opposite stereochemical result was observed giving the (-)-acid with 88% ee. Optically activeγ-fluoro-α-amino acids were synthesized alternatively by phase transfer catalysis with N-benzyl-cinchonium chloride or using an auxiliary-directed asymmetric alkylation of the imine derived from (R)-(+)-camphor or (R)-(+)-2-hydroxypinan-3-one. These processes gave different enantiomers ofγ-fluoro-α-aminobutyric acid via a monomeric lithium enolate in the first or a dimeric lithium enolate in the second case, respectively. The enantiomeric excess can be improved by lithium/magnesium exchange. PMID:24178725

  14. Anticancer activity and computational modeling of ternary copper (II) complexes with 3-indolecarboxylic acid and 1,10-phenanthroline.

    Science.gov (United States)

    Zhang, Zhen; Wang, Huiyun; Wang, Qibao; Yan, Maocai; Wang, Huannan; Bi, Caifeng; Sun, Shanshan; Fan, Yuhua

    2016-08-01

    Metal-containing compounds have been extensively studied for many years as potent proteasome inhibitors. The 20S proteasome, the main component of the ubiquitin proteasome pathway, is one of the excellent targets in anticancer drug development. We recently reported that several copper complexes were able to inhibit cancer-special proteasome and induce cell death in human cancer cells. However, the involved molecular mechanism is not known yet. We therefore synthesized three copper complexes and investigated their abilities on inhibiting proteasome activity and inducting apoptosis in human breast cancer cells. Furthermore, we employed molecular dockings to analyze the possible interaction between the synthetic copper complexes and the β5 subunit of proteasome which only reflects the chymotrypsin-like activity. Our results demonstrate that three Cu(II) complexes possess potent proteasome inhibition capability in a dose-dependent and time-dependent manner in MDA-MB-231 human breast cancer cells. They could bind to the β5 subunit of the 20S proteasome, which consequently cause deactivation of the proteasome and tumor cell death. The present study is significant for providing important theoretical basis for design and synthesis of anticancer drugs with low toxicity, high efficiency and high selectivity. PMID:27278680

  15. Amino acid sequence and glycosylation of functional unit RtH2-e from Rapana thomasiana (gastropod) hemocyanin.

    Science.gov (United States)

    Stoeva, Stanka; Idakieva, Krasimira; Betzel, Christian; Genov, Nicolay; Voelter, Wolfgang

    2002-03-15

    The complete amino acid sequence of Rapana thomasiana hemocyanin functional unit RtH2-e was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin, and trypsin. The single-polypeptide chain of RtH2-e consists of 413 amino acid residues and contains two consensus sequences NXS/T (positions 11-19 and 127-129), potential sites for N-glycosylation. Monosaccharide analysis of RtH2-e revealed a carbohydrate content of about 1.1% and the presence of xylose, fucose, mannose, and N-acetylglucosamine, demonstrating that only N-linked carbohydrate chains of high-mannose type seem to be present. On basis of the monosaccharide composition and MALDI-MS analysis of native and PNGase-F-treated chymotryptic glycopeptide fragment of RtH2-e the oligosaccharide Man(5)GlcNAc(2), attached to Asn(127), is suggested. Multiple sequence alignments with other molluscan hemocyanin e functional units revealed an identity of 63% to the cephalopod Octopus dofleini and of 69% to the gastropod Haliotis tuberculata. The present results are discussed in view of the recently determined X-ray structure of the functional unit g of the O. dofleini hemocyanin. PMID:11888200

  16. Physico-Chemical and Antifungal Properties of Trypsin Inhibitor from the Seeds of Mucuna Pruriens

    Directory of Open Access Journals (Sweden)

    K. S. Chandrashekharaiah

    2013-12-01

    Full Text Available The trypsin inhibitor has been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CMcellulose and gel-permeation chromatography on Sephadex G-100. The purified Mucuna pruriens trypsin inhibitor (MPTI showed a specific inhibitor activity of 474.66, fold purity of 99.51 and the yield obtained was 22.08%. The homogeneity of the purified preparation was confirmed by PAGE, IEF and SDS–PAGE. The molecular weight determined by gel-permeation chromatography and SDS–PAGE were 12 and 11.6 kDa respectively. The pI of MPTI was found to be 5.6 ± 0.05. MPTI was found to be stable at all temperatures lying between 0 and 90 ºC and pH 2.0 – 10 and hence exhibited high stability. The MPTI was inhibited both trypsin and chymotrypsin showing the double headed nature. Antifungal studies showed inhibitory activity of MPTI against Aspergillus niger and Trichoderma viridae.

  17. Purification and modes of antifungal action by Vicia faba cv. Egypt trypsin inhibitor.

    Science.gov (United States)

    Fang, Evandro Fei; Hassanien, Abdallah Abd Elazeem; Wong, Jack Ho; Bah, Clara Shui Fern; Soliman, Saeed Saad; Ng, Tzi Bun

    2010-10-13

    A new 15 kDa Bowman-Birk type trypsin inhibitor (termed VFTI-E1) from fava beans (Vicia faba cv. Egypt 1) was isolated using liquid chromatography. Though it exhibited substantial homology in N-terminal amino acid sequence to other protease inhibitors, VFTI-E1 showed antiproteolytic activity against trypsin (K(i) 11.9 × 10(-9) M) but hardly any activity against chymotrypsin. It demonstrated antifungal activity toward the filamentous fungus Valsa mali with an IC(50) of 20 μM. The mechanism of its antifungal action toward V. mali included (1) induction of alteration of hyphal morphology, (2) growth inhibition by chitin deposition at hyphal tips, and (3) permeabilization of fungal membrane. The antifungal activity of VFTI-E1 was dependent on the ambient ionic strength as increasing concentrations of NaCl, CaCl(2), and MgCl(2) diminished the activity. The membranolytic action of VFTI-E1 was confined to fungus, but not exerted on human and rabbit erythrocytes. This study sheds light on the mode of hyphal growth inhibitory activity of protease inhibitors with antifungal activity. The antifungal activity of VFTI-E1 amplifies the scope of its potential applications. PMID:20836498

  18. Production, purification and characterization of bacteriocin from Lactobacillus murinus AU06 and its broad antibacterial spectrum

    Institute of Scientific and Technical Information of China (English)

    Sivaramasamy Elayaraja; Neelamegam Annamalai; Packiyam Mayavu; Thangavel Balasubramanian

    2014-01-01

    Objective: To study the production, purification and characterization of bacteriocin fromLactobacillus murinus against fish pathogens.Methods:AU06 isolated from marine sediments and its broad spectrum of inhibition bacteriocin. In addition, purified bacteriocin was tested for its antimicrobial activity against fish pathogens.Results:In the present study, the bacteriocin production was found to be higher at 35 °C, pH The selected strain was used in production, purification and characterized of 6.0 and was purified to 4.74 fold with 55. 38 U/mg of specific activity with the yield of 28.92%. The molecular weight of the purified bacteriocin was estimated as 21 kDa. The purified bacteriocin exhibited complete inactivation of antimicrobial activity when treated with proteinase K, pronase, chymotrypsin, trypsin, pepsin and papain. The purified bacteriocin exhibited broad inhibitory spectrum against both Gram positive and negative bacteria.Conclusions:It is concluded that the ability of bacteriocin in inhibiting a wide-range of pathogenic bacteria is of potential interest for food safety and may have future applications in food preservative.

  19. In silico peptide prediction for antibody generation to recognize 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in genetically modified organisms.

    Science.gov (United States)

    Marani, Mariela M; Costa, Joana; Mafra, Isabel; Oliveira, Maria Beatriz P P; Camperi, Silvia A; Leite, José Roberto de Souza Almeida

    2015-03-01

    For the prospective immunorecognition of 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) as a biomarker protein expressed by transgenic soybean, an extensive in silico evaluation of the referred protein was performed. The main objective of this study was the selection of a set of peptides that could function as potential immunogens for the production of novel antibodies against CP4-EPSPS protein. For this purpose, the protein was in silico cleaved with trypsin/chymotrypsin and the resultant peptides were extensively analyzed for further selection of the best candidates for antibody production. The analysis enabled the successful proposal of four peptides with potential immunogenicity for their future use as screening biomarkers of genetically modified organisms. To our knowledge, this is the first attempt to select and define potential linear epitopes for the immunization of animals and, subsequently, to generate adequate antibodies for CP4-EPSPS recognition. The present work will be followed by the synthesis of the candidate peptides to be incubated in animals for antibody generation and potential applicability for the development of an immunosensor for CP4-EPSPS detection.

  20. Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium

    Directory of Open Access Journals (Sweden)

    Anderson F. Santos

    2013-12-01

    Full Text Available Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9, a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i molecular masses ranging from 30 to 80 kDa, (ii better hydrolytic activities under neutral-alkaline pH range, (iii expression modulated according to the culture age, (iv susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v specific cleavage over the chymotrypsin substrate, and (vi enzymatic stability in the presence of salt (up to 20% NaCl and organic solvents (e.g., ether, isooctane and cyclohexane. The proteases described herein are promising for industrial practices due to its haloalkaline properties.

  1. Momordica charantia trypsin inhibitor Ⅱ inhibits growth and development of Helicoverpa armigera

    Institute of Scientific and Technical Information of China (English)

    Manasi Alok Telang; Prashant Pyati; Mohini Sainani; Vidya Shrikant Gupta; Ashok Prabhakar Giri

    2009-01-01

    Bitter gourd (Momordica charantia L.) seeds contain several squash-type serine proteinase inhibitors (PIs),which inhibit the digestive proteinases of the polyphagous insect pest Helicoverpa armigera.In the present work isolation of a DNA sequence encoding the mature peptide of a trypsin inhibitor McTI-Ⅱ,its cloning and expression as a recombinant protein using Pichia pastoris have been reported.Recombinant McTI-Ⅱinhibited bovine trypsin at 1:1 molar ratio,as expected,but did not inhibit chymotrypsin or elastase.McTI-Ⅱalso strongly inhibited trypsin-like proteinases (81% inhibition) as well as the total proteolytic activity of digestive proteinases (70% inhibition) from the midgut of H.armigera larvae.The insect larvae fed with McTI-Ⅱ-incorporated artificial diet suffered over 70% reduction in the average larval weight after 12 days of feeding.Moreover,ingestion of McTI-Ⅱresulted in 23% mortality in the larval population.The strong antimetabolic activity of McTI-Ⅱtoward H.armigera indicates its probable use in developing insect tolerance in susceptible plants.

  2. Three different conformational states of pregnancy zone protein identified by monoclonal antibodies.

    Science.gov (United States)

    Carlsson-Bosted, L; Moestrup, S K; Gliemann, J; Sottrup-Jensen, L; Stigbrand, T

    1988-05-15

    Human pregnancy zone protein (PZP), related to human alpha 2-macroglobulin, forms dimeric/tetrameric (360/720 kDa) species. PZP binds proteinases which cause the cleavage of internal thiol esters in the molecule. Both the binding of proteinases, i.e. chymotrypsin, (CT) to PZP, forming PZP.CT complexes, or reaction with methylamine (MA) forming PZP.MA complexes, cause transition to a new similar conformational state. Reactivity of selected monoclonal antibodies against PZP towards the three PZP derivatives demonstrated differences in the reactivity pattern. PZP and PZP.MA share one determinant, which is missing on the PZP.CT complex. PZP after transition to PZP.CT, but not to PZP.MA, presents a neodeterminant detected by one of six monoclonal antibodies. The findings demonstrate that at least three different conformational states exist for PZP and its derivatives. Access to discriminating immunochemical tools makes possible an evaluation of the relative abundance of the different complexes in vivo.

  3. Comparative effects of ohmic, induction cooker, and electric stove heating on soymilk trypsin inhibitor inactivation.

    Science.gov (United States)

    Lu, Lu; Zhao, Luping; Zhang, Caimeng; Kong, Xiangzhen; Hua, Yufei; Chen, Yeming

    2015-03-01

    During thermal treatment of soymilk, a rapid incorporation of Kunitz trypsin inhibitor (KTI) into protein aggregates by covalent (disulfide bond, SS) and/or noncovalent interactions with other proteins is responsible for its fast inactivation of trypsin inhibitor activity (TIA). In contrast, the slow cleavage of a single Bowman-Birk inhibitor (BBI) peptide bond is responsible for its slow inactivation of TIA and chymotrypsin inhibitor activity (CIA). In this study, the effects of Ohmic heating (220 V, 50 Hz) on soymilk TIA and CIA inactivation were examined and compared to induction cooker and electric stove heating with similar thermal histories. It was found that: (1) TIA and CIA inactivation was slower from 0 to 3 min, and faster after 3 min as compared to induction cooker and electric stove. (2) The thiol (SH) loss rate was slower from 0 to 3 min, and similar to induction cooker and electric stove after 3 min. (3) Ohmic heating slightly increased protein aggregate formation. (4) In addition to the cleavage of one BBI peptide bond, an additional reaction might occur to enhance BBI inactivation. (5) Ohmic heating was more energy-efficient for TIA and CIA inactivation. (6) TIA and CIA inactivation was accelerated with increasing electric voltage (110, 165, and 220 V) of Ohmic heating. It is likely that the enhanced inactivation of TIA by Ohmic heating is due to its combined electrochemical and thermal effects.

  4. Transcriptional Analysis of The Adaptive Digestive System of The Migratory Locust in Response to Plant Defensive Protease Inhibitors

    Science.gov (United States)

    Spit, Jornt; Holtof, Michiel; Badisco, Liesbet; Vergauwen, Lucia; Vogel, Elise; Knapen, Dries; vanden Broeck, Jozef

    2016-09-01

    Herbivorous insects evolved adaptive mechanisms to compensate for the presence of plant defensive protease inhibitors (PI) in their food. The underlying regulatory mechanisms of these compensatory responses remain largely elusive. In the current study, we investigated the initiation of this adaptive response in the migratory locust, Locusta migratoria, via microarray analysis of gut tissues. Four hours after dietary uptake of PIs, 114 and 150 transcripts were respectively found up- or downregulated. The results suggest a quick trade-off between compensating for potential loss of digestive activity on the one hand, and stress tolerance, defense, and structural integrity of the gut on the other hand. We additionally addressed the role of a group of related upregulated hexamerin-like proteins in the PI-induced response. Simultaneous knockdown of corresponding transcripts by means of RNA interference resulted in a reduced capacity of the locust nymphs to cope with the effects of PI. Moreover, since insect hexamerins have been shown to bind Juvenile Hormone (JH), we also investigated the effect of JH on the proteolytic digestion in L. migratoria. Our results indicate that JH has a stimulatory effect on the expression of three homologous chymotrypsin genes, while knocking down the JH receptor (methoprene tolerant) led to opposite effects.

  5. Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis).

    Science.gov (United States)

    Samac, D; Storey, R

    1981-12-01

    Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination.

  6. Role of peptidases of the intestinal microflora and prey in temperature adaptations of the digestive system in planktivorous and benthivorous fish.

    Science.gov (United States)

    Kuz'mina, V V; Skvortsova, E G; Shalygin, M V; Kovalenko, K E

    2015-12-01

    Many fish enzymatic systems possess limited adaptations to low temperature; however, little data are available to judge whether enzymes of fish prey and intestinal microbiota can mitigate this deficiency. In this study, the activity of serine peptidases (casein-lytic, mainly trypsin and hemoglobin-lytic, mainly chymotrypsin) of intestinal mucosa, chyme and intestinal microflora in four species of planktivorous (blue bream) and benthivorous (roach, crucian carp, perch) was investigated across a wide temperature range (0-70 °C) to identify adaptations to low temperature. At 0 °C, the relative activity of peptidases of intestinal mucosa (microflora (5-12.6%) is considerably less than that of chyme peptidases (up to 40% of maximal activity). The level of peptidase relative activity in crucian carp intestinal microflora was 45% of maximal activity. The shape of t°-function curves of chyme peptidase also differs in fish from different biotopes. Fish from the littoral group are characterized by a higher degree of adaptation of chyme casein-lytic peptidases to functioning at low temperatures as compared to fish from the pelagic group. The role of intestinal microbiota and prey peptidases in digestive system adaptations of planktivorous and benthivorous fish to low temperatures is discussed.

  7. Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

    International Nuclear Information System (INIS)

    Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. WF decreased the viability of C2C12 myotubes, especially at concentrations of 20–25 μg.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model

  8. Biomolecular adsorption behavior on spherical carbon aerogels with various mesopore sizes.

    Science.gov (United States)

    Long, Donghui; Zhang, Rui; Qiao, Wenming; Zhang, Liang; Liang, Xiaoyi; Ling, Licheng

    2009-03-01

    Spherical carbon aerogels (SCAs) with controlled particle size and mesopore size were synthesized by an emulsified sol-gel polymerization of phenol, melamine and formaldehyde. The adsorption rate and capacity of biomolecules with different molecular dimensions, including L-phenylalanine (Phe), vitamin B(12) (VB), alpha-chymotrypsin (Chy) and bovine serum albumin (BSA) onto SCAs were investigated. The mesopore size can be easily tuned in the range from 5 to 10 nm by simply adjusting catalyst concentration in the initial solution and the spherical particle size can be controlled in 50-500 microm by changing stirring speed. The as-prepared SCAs have high specific surface area (>600 m(2)/g) and large pore volume (>1 cm(3)/g). The hardness of SCAs is ca. 10 times as large as that of commercial spherical activated carbon particles. The adsorption rate of VB is strongly depended on the mesopore size and particle size, and show an increasing tread with the increase of mesopore size and the decrease of particle size. For small molecule Phe, the specific surface area is key factor to determine the adsorption capacity, but the adsorption capacity of large molecules (VB, Chy and BSA) is dependent on the pore size of SCAs, which should be suitably larger than the molecule size of biomolecules.

  9. Adaptive changes of pancreatic protease secretion to a short-term vegan diet: influence of reduced intake and modification of protein.

    Science.gov (United States)

    Walkowiak, Jaroslaw; Mądry, Edyta; Lisowska, Aleksandra; Szaflarska-Popławska, Anna; Grzymisławski, Marian; Stankowiak-Kulpa, Hanna; Przysławski, Juliusz

    2012-01-01

    In our previous study, we demonstrated that abstaining from meat, for 1 month, by healthy omnivores (lacto-ovovegetarian model) resulted in a statistical decrease in pancreatic secretion as measured by faecal elastase-1 output. However, no correlation between relative and non-relative changes of energy and nutrient consumption and pancreatic secretion was documented. Therefore, in the present study, we aimed to assess the changes of exocrine pancreatic secretion with a more restrictive dietetic modification, by applying a vegan diet. A total of twenty-one healthy omnivores (sixteen females and five males) participated in the prospective study lasting for 6 weeks. The nutrient intake and faecal output of pancreatic enzymes (elastase-1, chymotrypsin and lipase) were assessed twice during the study. Each assessment period lasted for 7 d: the first before the transition to the vegan diet (omnivore diet) and the second during the last week of the study (vegan diet). The dietary modification resulted in a significant decrease in faecal elastase-1 (P vegan diet resulted in an adaptation of pancreatic protease secretion in healthy volunteers.

  10. MALDI-TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome.

    Science.gov (United States)

    Ruprecht, Benjamin; Roesli, Christoph; Lemeer, Simone; Kuster, Bernhard

    2016-05-01

    Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe-IMAC column chromatography and subjected to LC-MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI-TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC-MS/MS systems was comparable to that of using alternative proteases such as Asp-N, Arg-C, chymotrypsin, Glu-C and Lys-C on just one LC-MS/MS instrument. Notably, MALDI-TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites (∼20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC-MALDI MS/MS can be a useful complement to LC-nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides. PMID:26990019

  11. Synthesis, enzymatic stability and in vitro cytostatic effect of Daunorubicin-GnRH-III derivative dimers.

    Science.gov (United States)

    Schreier, Verena Natalie; Mező, Gábor; Orbán, Erika; Dürr, Claudia; Marquardt, Andreas; Manea, Marilena

    2013-04-01

    Bioconjugates containing chemotherapeutic agents attached to peptide hormones, such as gonadotropin-releasing hormone (GnRH), are developed as drug delivery systems for targeted cancer chemotherapy. We report here the synthesis and biochemical characterization of disulfide bond-linked dimeric bioconjugates in which daunorubicin was coupled via an oxime linkage to aminooxyacetylated GnRH-III ([Glp-His-Trp-Ser-His-Asp-Trp-Lys(DauAoa-Cys)-Pro-Gly-NH2]2; where Glp is pyroglutamic acid and Aoa is aminooxyacetyl) and its derivatives modified in position four by N-Me-Ser and Lys(Ac). The in vitro stability/degradation of the bioconjugates was determined in human serum, as well as in the presence of rat liver lysosomal homogenate and digestive enzymes. All compounds were stable at least for 24h in human serum and in the presence of pepsin and trypsin, while they were degraded by lysosomal enzymes. The daunorubicin-GnRH-III derivative dimers were partly digested by α-chymotrypsin; however, they had increased stability compared to the corresponding monomers, making them potential candidates for oral administration. The in vitro cytostatic effect of the compounds was determined on MCF-7 human breast cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. All daunorubicin-GnRH-III derivative dimers exerted slightly increased in vitro cytostatic effect (IC50 values in low μM range) than the corresponding monomeric bioconjugates.

  12. Valence parity to distinguish c' and z• ions from electron capture dissociation/electron transfer dissociation of peptides: effects of isomers, isobars, and proteolysis specificity.

    Science.gov (United States)

    Mao, Yuan; Tipton, Jeremiah D; Blakney, Greg T; Hendrickson, Christopher L; Marshall, Alan G

    2011-10-15

    Valence parity provides a way to distinguish between N-terminal and C-terminal electron capture dissociation/electron transfer dissociation (ECD/ETD) product ions based on their number of hydrogen plus nitrogen atoms determined by accurate mass measurement and forms a basis for de novo peptide sequencing. The effect of mass accuracy (0.1-1 ppm error) on c'/z(•) overlap and unique elemental composition overlap is evaluated for a database of c'/z(•) product ions each based on all possible amino acid combinations and four subset databases containing the same c' ions but with z(•) ions determined by in silico digestion with trypsin, Glu-C, Lys-C, or chymotrypsin. High mass accuracy reduces both c'/z(•) overlap and unique elemental composition overlap. Of the four proteases, trypsin offers slightly better discrimination between N- and C-terminal ECD/ETD peptides. Interestingly, unique elemental composition overlap curves for c'/c' and z(•)/z(•) peptide ions exhibit discontinuities at certain nominal masses for 0.1-1.0 ppm mass error. Also, as noted in the companion article (Polfer et al. Anal. Chem.2011, DOI: 10.1021/ac201624t), the number of ECD/ETD product ion amino acid compositions as a function of nominal mass increases exponentially with mass but with a superimposed modulation due to higher prevalence of certain elemental compositions.

  13. The R Protein of SARS-CoV: Analyses of Structure and Function Based on Four Complete Genome Sequences of Isolates BJ01-BJ04

    Institute of Scientific and Technical Information of China (English)

    Zuyuan Xu; Zizhang Zhang; Jing Xu; Wei Wei; Jingui Zhu; Haiyan Sun; Xiaowei Zhang; Jun Zhou; Songgang Li; Jun Wang; Jian Wang; Haiqing Zhang; Shengli Bi; Huanming Yang; Xiangjun Tian; Jia Ji; Wei Li; Yan Li; Wei Tian; Yujun Han; Lili Wang

    2003-01-01

    The R (replicase) protein is the uniquely defined non-structural protein (NSP)responsible for RNA replication, mutation rate or fidelity, regulation of transcrip-tion in coronaviruses and many other ssRNA viruses. Based on our completegenome sequences of four isolates (BJ01-BJ04) of SARS-CoV from Beijing, China,we analyzed the structure and predicted functions of the R protein in comparisonwith 13 other isolates of SARS-CoV and 6 other coronaviruses. The entire ORF(open-reading frame) encodes for two major enzyme activities, RNA-dependentRNA polymerase (RdRp) and proteinase activities. The R polyprotein under-goes a complex proteolytic process to produce 15 function-related peptides. Ahydrophobic domain (HOD) and a hydrophilic domain (HID) are newly identifiedwithin NSP1. The substitution rate of the R protein is close to the average ofthe SARS-CoV genome. The functional domains in all NSPs of the R proteingive different phylogenetic results that suggest their different mutation rate underselective pressure. Eleven highly conserved regions in RdRp and twelve cleavagesites by 3CLP (chymotrypsin-like protein) have been identified as potential drugtargets. Findings suggest that it is possible to obtain information about the phy-logeny of SARS-CoV, as well as potential tools for drug design, genotyping anddiagnostics of SARS.

  14. Efficacy of Wobe-Mugos registered E for reduction of oral mucositis after radiotherapy. Results of a prospective, randomized, placebo-controlled, triple-blind phase III multicenter study

    International Nuclear Information System (INIS)

    Purpose: To investigate the efficacy and safety of Wobe-Mugos registered E (proteolytic enzymes) for amelioration of early side effects of radiotherapy for head-and-neck tumors, particularly oral mucositis. Patients and Methods: The study was a prospective, randomized, multicenter, placebo-controlled, triple-blind phase III study with parallel groups. 69 patients with carcinomas of the oropharynx or the oral cavity were enrolled between 1996 and 2000 in five centers; 54 of these were recruited in Dresden. Of the 69 patients, 61 (Dresden: 46) were available for analysis. The proteolytic enzymes tested (Wobe-Mugos registered E) comprised papain 100 mg, trypsin 40 mg, and chymotrypsin 40 mg. Results: Wobe-Mugos registered E was well tolerated. For the maximum mucositis scores, no statistically significant differences were found between the placebo and the verum group. The average mucositis score over weeks 1-6 revealed a significant difference in favor of the placebo arm, based on an earlier onset of mucositis in the Wobe-Mugos registered E group. Conclusion: The present study failed to demonstrate any effect of treatment with Wobe-Mugos registered E on radiotherapy side effects in patients treated for head-and-neck tumors. In particular, there was no beneficial effect on radiation-induced early oral mucositis. (orig.)

  15. Bioactive protein-based nanofibers interact with intestinal biological components resulting in transepithelial permeation of a therapeutic protein.

    Science.gov (United States)

    Stephansen, Karen; García-Díaz, María; Jessen, Flemming; Chronakis, Ioannis S; Nielsen, Hanne Mørck

    2015-11-10

    Proteins originating from natural sources may constitute a novel type of material for use in drug delivery. However, thorough understanding of the behavior and effects of such a material when processed into a matrix together with a drug is crucial prior to further development into a drug product. In the present study the potential of using bioactive electrospun fish sarcoplasmic proteins (FSP) as a carrier matrix for small therapeutic proteins was demonstrated in relation to the interactions with biological components of the intestinal tract. The inherent structural and chemical properties of FSP as a biomaterial facilitated interactions with cells and enzymes found in the gastrointestinal tract and displayed excellent biocompatibility. More specifically, insulin was efficiently encapsulated into FSP fibers maintaining its conformation, and subsequent controlled release was obtained in simulated intestinal fluid. The encapsulation of insulin into FSP fibers provided protection against chymotrypsin degradation, and resulted in an increase in insulin transport to around 12% without compromising the cellular viability. This increased transport was driven by interactions upon contact between the nanofibers and the Caco-2 cell monolayer leading to the opening of the tight junction proteins. Overall, electrospun FSP may constitute a novel material for oral delivery of biopharmaceuticals. PMID:26320547

  16. Versatile Biodegradable Poly(ester amides Derived from α-Amino Acids for Vascular Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Pooneh Karimi

    2010-03-01

    Full Text Available Biodegradable poly(ester amide (PEA biomaterials derived from α-amino acids, diols, and diacids are promising materials for biomedical applications such as tissue engineering and drug delivery because of their optimized properties and susceptibility for either hydrolytic or enzymatic degradation. The objective of this work was to synthesize and characterize biodegradable PEAs based on the α-amino acids L-phenylalanine and L-methionine. Four different PEAs were prepared using 1,4-butanediol, 1,6-hexanediol, and sebacic acid by interfacial polymerization. High molecular weight PEAs with narrow polydispersity indices and excellent film-forming properties were obtained. The incubation of these PEAs in PBS and chymotrypsin indicated that the polymers are biodegradable. Human coronary artery smooth muscle cells were cultured on PEA films for 48 h and the results showed a well-spread morphology. Porous 3D scaffolds fabricated from these PEAs were found to have excellent porosities indicating the utility of these polymers for vascular tissue engineering.

  17. NaC1对三疣梭子蟹消化酶活力的影响%The effects of NaCl on digestive enzyme activities of Portunus trituberculatus

    Institute of Scientific and Technical Information of China (English)

    胡毅; 潘鲁青

    2009-01-01

    The effects of NaCl on the activities ofprotease, trypsin, chymotrypsin, carboxype ptidase A, carboxy-peptidase B and amylase ofPortunus trituberculatus were studied by adjusting the assay mixture (0,0.005, 0.001,0.05, 0.1, 0.5, 1.0 and 2.0 mol/L) through using enzyme analytical method. The experimental results indicated that all enzymes were activated by low NaCI concentration except for carboxypeptidase B. Within the scope of these experimental concentrations, protease has higher activities at NaC1 concentration around 0.1-0.5 mol/L, trypsin has the higher activity at NaC1 concentration around 0.5 mol/L, chymotrypsin has a more high activity at NaC1 concentration around 1 mol/L, carboxypeptidase A has a more high activity at NaC1 concentration around 0.05-0.1 mol/L, carboxypeptidase B was inhibited by NaC1, and has 60% maximum activities at 2 mol/L NaC1. NaC1 activated amylase within the scope of this experimental concentrations, amylase has the highest activity at NaC1 concentration around 0.5 mol/L.%作者采用酶学分析方法研究了反应介质中添加NaC1对三疣梭子蟹(Porturrus tritubrculatus)中肠腺蛋白酶、胰蛋白酶、胰凝乳蛋白酶、羧肽酶A、羧肽酶B、淀粉酶活力的影响,反应介质中NaC1浓度设置为0、0.005、0.01、0.05、0.1、0.5、1.0和2.0 mol/L 8个梯度.实验结果表明,反应介质中添加NaC1对消化酶活力影响显著,除羧肽酶B外,添加NaC1对三疣梭子蟹消化酶均有激活作用,且所有消化酶都有很高的耐盐性.当反应介质中NaC1浓度为0.1~0.5 mol/L时蛋白酶活力最大;0.5 mol/L时胰蛋白酶活力最大;胰凝乳蛋白酶在反应介质中NaC1浓度为1 mol/L时酶活力最大;羧肽酶A在反应介质中NaC1浓度为0.05~0.1 mol/L时酶活力最大;NaC1对羧肽酶B有抑制作用,在反应介质中NaC1浓度为2 mol/L的条件下,只有未添加NaC1时酶活力的60%;NaC1在0~2 mol/L时对淀粉酶均有激活作用,其最大激活能力在0.5mol/L左右.

  18. A biomimetic colorimetric logic gate system based on multi-functional peptide-mediated gold nanoparticle assembly

    Science.gov (United States)

    Li, Yong; Li, Wang; He, Kai-Yu; Li, Pei; Huang, Yan; Nie, Zhou; Yao, Shou-Zhuo

    2016-04-01

    In natural biological systems, proteins exploit various functional peptide motifs to exert target response and activity switch, providing a functional and logic basis for complex cellular activities. Building biomimetic peptide-based bio-logic systems is highly intriguing but remains relatively unexplored due to limited logic recognition elements and complex signal outputs. In this proof-of-principle work, we attempted to address these problems by utilizing multi-functional peptide probes and the peptide-mediated nanoparticle assembly system. Here, the rationally designed peptide probes function as the dual-target responsive element specifically responsive to metal ions and enzymes as well as the mediator regulating the assembly of gold nanoparticles (AuNPs). Taking advantage of Zn2+ ions and chymotrypsin as the model inputs of metal ions and enzymes, respectively, we constructed the peptide logic system computed by the multi-functional peptide probes and outputted by the readable colour change of AuNPs. In this way, the representative binary basic logic gates (AND, OR, INHIBIT, NAND, IMPLICATION) have been achieved by delicately coding the peptide sequence, demonstrating the versatility of our logic system. Additionally, we demonstrated that the three-input combinational logic gate (INHIBIT-OR) could also be successfully integrated and applied as a multi-tasking biosensor for colorimetric detection of dual targets. This nanoparticle-based peptide logic system presents a valid strategy to illustrate peptide information processing and provides a practical platform for executing peptide computing or peptide-related multiplexing sensing, implying that the controllable nanomaterial assembly is a promising and potent methodology for the advancement of biomimetic bio-logic computation.In natural biological systems, proteins exploit various functional peptide motifs to exert target response and activity switch, providing a functional and logic basis for complex cellular

  19. Effect of seaweed supplementation on growth performance, immune and oxidative stress responses in gilthead seabream (Sparus aurata

    Directory of Open Access Journals (Sweden)

    Augusto Cesar dos Santos Queiroz

    2014-06-01

    , total glutathione and catalase responses and digestive capacity (amylase, chymotrypsin, trypsin and lipase. Growth performance parameters showed no significant difference, but all supplemented treatments tended to have better results than control. Plasma peroxidase was improved by dietary seaweed supplementation. Plasm lysozyme increased in R7.5% group when compared to the other dietary groups (Table 1. Amylase, chymotrypsin and trypsin had low activity, while lipase had higher activity. Catalase, glutathione peroxidase, glutathione reductase and glutathione s-transferase were not statistically different among the dietary treatments. Nevertheless, these enzymes tended to have higher activity on supplemented treatments than control. Total glutathione and lipid peroxidation were significantly higher in R2,5% and, R7,5% and M, respectively (Table 1. Results indicate seaweed supplementation may improve immune and antioxidant responses in gilthead seabream. Further studies are needed in order to access the protective effects of dietary seaweed supplementation in fish subjected to stress conditions.

  20. Interactive effects of CO₂ and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria

    Energy Technology Data Exchange (ETDEWEB)

    Götze, Sandra [Alfred Wegener Institute, Helmholtz Centre for Polar, Marine Research, Functional Ecology, 27570 Bremerhaven (Germany); Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223 (United States); Matoo, Omera B. [Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223 (United States); Beniash, Elia [Department of Oral Biology, University of Pittsburgh, Pittsburgh, PA (United States); Saborowski, Reinhard [Alfred Wegener Institute, Helmholtz Centre for Polar, Marine Research, Functional Ecology, 27570 Bremerhaven (Germany); Sokolova, Inna M., E-mail: isokolov@uncc.edu [Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223 (United States)

    2014-04-01

    Highlights: • Elevated PCO₂ enhanced accumulation of Cu and Cd in the gills of mollusks. • The proteasome activities were affected by metals but robust to elevated PCO₂. • Exposure to Cd and Cu had opposite effects on the proteasome activity. • Combined exposure to Cu and elevated PCO₂ negatively affected energy status. - Abstract: Increased anthropogenic emission of CO₂ changes the carbonate chemistry and decreases the pH of the ocean. This can affect the speciation and the bioavailability of metals in polluted habitats such as estuaries. However, the effects of acidification on metal accumulation and stress response in estuarine organisms including bivalves are poorly understood. We studied the interactive effects of CO₂ and two common metal pollutants, copper (Cu) and cadmium (Cd), on metal accumulation, intracellular ATP/ubiquitin-dependent protein degradation, stress response and energy metabolism in two common estuarine bivalves—Crassostrea virginica (eastern oyster) and Mercenaria mercenaria (hard shell clam). Bivalves were exposed for 4–5 weeks to clean seawater (control) and to either 50 μg L⁻¹ Cu or 50 μg L⁻¹ Cd at one of three partial pressures of CO₂ PCO₂ ~395, ~800 and ~1500 μatm) representative of the present-day conditions and projections of the Intergovernmental Panel for Climate Change (IPCC) for the years 2100 and 2250, respectively. Clams accumulated lower metal burdens than oysters, and elevated PCO₂ enhanced the Cd and Cu accumulation in mantle tissues in both species. Higher Cd and Cu burdens were associated with elevated mRNA expression of metal binding proteins metallothionein and ferritin. In the absence of added metals, proteasome activities of clams and oysters were robust to elevated PCO₂, but PCO₂ modulated the proteasome response to metals. Cd exposure stimulated the chymotrypsin-like activity of the oyster proteasome

  1. Innovation of the preparative process of bovine trypsin and its enzymatic degradation effect on the precursor of genetic engineering human insulin%牛胰蛋白酶制备工艺的改进及其对基因工程人胰岛素前体的酶切作用

    Institute of Scientific and Technical Information of China (English)

    冷春生; 李庆伟

    2013-01-01

    目的 对牛胰蛋白酶制备工艺进行改进,制备高纯度牛胰蛋白酶,并对其酶切基因工程人胰岛素前体的作用进行考察.方法 采用粗制后先色谱分离再活化的方法对传统工艺进行改进,对传统工艺制备样品和改进工艺制备样品中的胰蛋白酶和胰凝乳蛋白酶的比活力进行测定和比较;进一步采用这2种样品对基因工程人胰岛素前体进行酶切,对酶切作用进行比较.结果 传统工艺制备样品中胰蛋白酶比活力为212.5 U·mg-1,胰凝乳蛋白酶比活力为20.4 U·mg-1;而改进工艺样品中胰蛋白酶比活力为240.4 U·mg-1,胰凝乳蛋白酶比活力为0.14 U·mg-1.传统工艺制备样品酶切基因工程人胰岛素前体目标产物纯度质量分数为16.3%,改进工艺制备样品酶切基因工程人胰岛素前体目标产物纯度质量分数为32.2%.结论 粗制后先进行SP色谱可实现胰蛋白酶原和胰凝乳蛋白酶原较好的分离,该方法适合于高纯度胰蛋白酶的制备,制备的胰蛋白酶适合作为工具酶进行基因工程人胰岛素前体的酶切.%Objective To isolate and purify bovine trypsin with high purity and determine the activation of the genetic engineering human insulin prosoma with it. Methods The traditional preparative process of bovine trypsin was improved. The crude product was firstly separated by column chromatography and then was activated. The activities of trypsin and chymotrypsin of the samples,from the traditional method and the improved way, were assayed and compared. Then the genetic engineering human insulin precursor was enzy-matically degradated by these 2 samples and the target product was detected by HPLC. The effects of these two samples were compared. Results The specific activity was 212.5 and 20.4 U·mg-1 for trypsin and chymotrypsin in traditionally prepared sample, while that was 240.4 and 0. 14 U · mg-1 in the improved method. Conclusions Trypsinogen and chymotrypsinogen

  2. Interactive effects of CO₂ and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria.

    Science.gov (United States)

    Götze, Sandra; Matoo, Omera B; Beniash, Elia; Saborowski, Reinhard; Sokolova, Inna M

    2014-04-01

    Increased anthropogenic emission of CO2 changes the carbonate chemistry and decreases the pH of the ocean. This can affect the speciation and the bioavailability of metals in polluted habitats such as estuaries. However, the effects of acidification on metal accumulation and stress response in estuarine organisms including bivalves are poorly understood. We studied the interactive effects of CO2 and two common metal pollutants, copper (Cu) and cadmium (Cd), on metal accumulation, intracellular ATP/ubiquitin-dependent protein degradation, stress response and energy metabolism in two common estuarine bivalves-Crassostrea virginica (eastern oyster) and Mercenaria mercenaria (hard shell clam). Bivalves were exposed for 4-5 weeks to clean seawater (control) and to either 50 μg L(-1) Cu or 50 μg L(-1) Cd at one of three partial pressures of CO2 ( [Formula: see text] ∼ 395, ∼ 800 and ∼ 1500 μatm) representative of the present-day conditions and projections of the Intergovernmental Panel for Climate Change (IPCC) for the years 2100 and 2250, respectively. Clams accumulated lower metal burdens than oysters, and elevated [Formula: see text] enhanced the Cd and Cu accumulation in mantle tissues in both species. Higher Cd and Cu burdens were associated with elevated mRNA expression of metal binding proteins metallothionein and ferritin. In the absence of added metals, proteasome activities of clams and oysters were robust to elevated [Formula: see text] , but [Formula: see text] modulated the proteasome response to metals. Cd exposure stimulated the chymotrypsin-like activity of the oyster proteasome at all CO2 levels. In contrast, trypsin- and caspase-like activities of the oyster proteasome were slightly inhibited by Cd exposure in normocapnia but this inhibition was reversed at elevated [Formula: see text] . Cu exposure inhibited the chymotrypsin-like activity of the oyster proteasome regardless of the exposure [Formula: see text] . The effects of metal exposure on

  3. Ontogenetic changes in digestive enzyme activities and the amino acid profile of starry flounder Platichthys stellatus

    Science.gov (United States)

    Song, Zhidong; Wang, Jiying; Qiao, Hongjin; Li, Peiyu; Zhang, Limin; Xia, Bin

    2016-01-01

    Ontogenetic changes in digestive enzyme activities and the amino acid (AA) profile of starry flounder, Platichthys stellatus, were investigated and limiting amino acids were estimated compared with the essential AA profile between larvae and live food to clarify starry flounder larval nutritional requirements. Larvae were collected at the egg stage and 0, 2, 4, 7, 12, 17, 24 days after hatching (DAH) for analysis. Larvae grew from 1.91 mm at hatching to 12.13 mm at 24 DAH. Trypsin and chymotrypsin activities changed slightly by 4 DAH and then increased significantly 4 DAH. Pepsin activity increased sharply beginning 17 DAH. Lipase activity increased significantly 4 DAH and increased progressively with larval growth. Amylase activity was also detected in newly hatched larvae and increased 7 DAH followed by a gradual decrease. High free amino acid (FAA) content was detected in starry flounder eggs (110.72 mg/g dry weight). Total FAA content dropped to 43.29 mg/g in 4-DAH larvae and then decreased gradually to 13.74 mg/g in 24-DAH larvae. Most FAAs (except lysine and methionine) decreased >50% in 4-DAH larvae compared with those in eggs and then decreased to the lowest values in 24-DAH larvae. Changes in the protein amino acid (PAA) profile were much milder than those observed for FAAs. Most PAAs increased gradually during larval development, except lysine and phenylalanine. The percentages of free threonine, valine, isoleucine, and leucine decreased until the end of the trial, whereas the protein forms of these four AAs followed the opposite trend. A comparison of the essential AA composition of live food (rotifers, Artemia nauplii, and Artemia metanauplii) and larvae suggested that methionine was potentially the first limiting AA. These results may help develop starry flounder larviculture methods by solving the AA imbalance in live food. Moreover, the increased digestive enzyme activities indicate the possibility of introducing artificial compound feed.

  4. Involvement of a Serpin serine protease inhibitor (OoSerpin) from mollusc Octopus ocellatus in antibacterial response.

    Science.gov (United States)

    Wei, Xiumei; Xu, Jie; Yang, Jianmin; Liu, Xiangquan; Zhang, Ranran; Wang, Weijun; Yang, Jialong

    2015-01-01

    Serpin is an important member of serine protease inhibitors (SPIs), which is capable of regulating proteolytic events and involving in a variety of physiological processes. In present study, a Serpin homolog was identified from Octopus ocellatus (designated as OoSerpin). Full-length cDNA of OoSerpin was of 1735 bp, containing a 5' untranslated region of 214 bp, a 3' UTR of 282 bp, and an open reading frame of 1239 bp. The open reading frame encoded a polypeptide of 412 amino acids which has a predicted molecular weight of 46.5 kDa and an isoelectric point of 8.52. The OoSerpin protein shares 37% sequence identity with other Serpins from Mus musculus (NP_941373) and Ixodes scapularis (XP_002407493). The existence of a conserved SERPIN domain strongly suggested that OoSerpin was a member of the Serpin subfamily. Expression patterns of OoSerpin, both in tissues and towards bacterial stimulation, were then characterized. The mRNA of OoSerpin was constitutively expressed at different levels in all tested tissues of untreated O. ocellatus, including mantle (lowest), muscle, renal sac, gill, hemocyte, gonad, systemic heart, and hepatopancreas (highest). The transcriptional level of OoSerpin was significantly up-regulated (P<0.01) in O. ocellatus upon bacterial challenges with Vibrio anguillarum and Micrococcus luteus, indicating its involvement in the antibacterial immune response. Furthermore, rOoSerpin, the recombinant protein of OoSerpin, exhibited strong abilities to inhibit proteinase activities of trypsin and chymotrypsin as well as the growth of Escherichia coli. Our results demonstrate that OoSerpin is a potential antibacterial factor involved in the immune response of O. ocellatus against bacterial infection.

  5. Cardiac-specific catalase overexpression rescues anthrax lethal toxin-induced cardiac contractile dysfunction: role of oxidative stress and autophagy

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    Kandadi Machender R

    2012-11-01

    Full Text Available Abstract Background Lethal and edema toxins secreted by Bacillus anthracis during anthrax infection were found to incite serious cardiovascular complications. However, the underlying mechanisms in anthrax lethal toxin-induced cardiac anomalies remain unknown. This study was designed to evaluate the impact of antioxidant enzyme catalase in anthrax lethal toxin-induced cardiomyocyte contractile dysfunction. Methods Wild type (WT and cardiac-specific catalase overexpression mice were challenged with lethal toxin (2 μg/g, intraperotineally (i.p.. Cardiomyocyte contractile and intracellular Ca2+ properties were assessed 18 h later using an IonOptix edge-detection system. Proteasome function was assessed using chymotrypsin-like and caspase-like activities. GFP-LC3 puncta and Western blot analysis were used to evaluate autophagy and protein ubiquitination. Results Lethal toxin exposure suppressed cardiomyocyte contractile function (suppressed peak shortening, maximal velocity of shortening/re-lengthening, prolonged duration of shortening/re-lengthening, and impaired intracellular Ca2+ handling, the effects of which were alleviated by catalase. In addition, lethal toxin triggered autophagy, mitochondrial and ubiquitin-proteasome defects, the effects of which were mitigated by catalase. Pretreatment of cardiomyocytes from catalase mice with the autophagy inducer rapamycin significantly attenuated or ablated catalase-offered protection against lethal toxin-induced cardiomyocyte dysfunction. On the other hand, the autophagy inhibitor 3-MA ablated or significantly attenuated lethal toxin-induced cardiomyocyte contractile anomalies. Conclusions Our results suggest that catalase is protective against anthrax lethal toxin-induced cardiomyocyte contractile and intracellular Ca2+ anomalies, possibly through regulation of autophagy and mitochondrial function.

  6. Digestive enzyme activity in the intestine of Nile tilapia (Oreochromis niloticus L.) under pond and cage farming systems.

    Science.gov (United States)

    Santos, Juliana Ferreira; Soares, Karollina Lopes Siqueira; Assis, Caio Rodrigo Dias; Guerra, Carlos Augusto Martins; Lemos, Daniel; Carvalho, Luiz Bezerra; Bezerra, Ranilson Souza

    2016-10-01

    The effect of different farming systems (cage, pond) upon digestive enzyme activities of Nile tilapia was evaluated. Juvenile Nile tilapia (87.61 ± 1.52 g) were simultaneously cultured in pond and cage systems during 90 days. Cages used nutritional biphasic plan (35 and 32 % crude protein-CP feeds) and ponds used nutritional triphasic plan (35, 32 and 28 % CP feeds). Biometric measurements were monthly performed for adjustments in feeding regimes and removal of intestine tissues to evaluate the performance of enzyme activities. Total proteolytic, amylase and lipase activities were not statistically different between the treatments throughout the periods analyzed (31, 63 and 94 days of culture). However, trypsin and chymotrypsin activities were higher with 31 and 63 days of culture in fish from pond system, suggesting that natural food may have influenced these activities. A positive correlation was observed between the recommended concentration of essential amino acids for Nile tilapia and specific aminopeptidases activity in fish cage system. Substrate-SDS-PAGE revealed 12 active proteolytic bands in both systems. However, integrated density (ID) values were higher in the bands of ponds. Specimens of either cage or pond exhibited five bands of amylolytic activity. Fish from cage and pond systems showed the highest values of ID within 31 days of cultivation. In this study, the complexity of digestive functions could be verified for animals maintained under commercial conditions. Some of the assessed enzymes may show adaptations of their activities and/or expression that allow the fish to achieve a more efficient nutrient assimilation. PMID:27021899

  7. Oncocytic carcinoma of parotid gland: a case report with clinical, immunohistochemical and ultrastructural features

    Directory of Open Access Journals (Sweden)

    Giordano Giovanna

    2006-08-01

    Full Text Available Abstract Background Oncocytic carcinoma is an extremely rare neoplasm of the salivary glands. We report a case of oncocytic carcinoma arising in a parotid gland in a 66-year-old female. Method An excisional biopsy of the parotid tumor was performed. The specimen was submitted for histology and after fixation in formalin solution and inclusion in paraffin, 3–5 μm sections were stained with hematoxylin and eosin for conventional evaluation and Periodic acid Schiff stain. Immunohistochemical studies were performed using antibodies against mitochondrial antigen, keratin, S-100, alpha-actin, vimentin, alpha-1-antichymotrypsin as well as an ultrastructural analysis was performed. Results Frozen sections revealed an infiltrative growth pattern and the diagnosis of a malignant epithelial lesion was made. Permanent sections stained with haematoxylin and eosin revealed a neoplasm that had replaced a wide area of the parotid gland and had invaded subcutaneous adipose tissue. Perineural invasion was evident, but vascular invasion was not found. Neoplastic elements were large, round or polyhedral cells and were arranged in solid sheets, islands and cords. The cytoplasm was abundant, eosinophilic and finely granular. The nuclei were large and located centrally or peripherally. The nucleoli were distinct and large. Periodic acid Schiff stain demonstrated a granular cytoplasm. Immunohistochemistry demonstrated mithochondrial antigen, keratin, and chymotrypsin immunoreactivity in the neoplastic cells. Ultrastructural analysis revealed numerous mitochondria packed into the cytoplasm of the neoplastic cells. Thus, the final diagnosis was that of oncocytic carcinoma of parotid gland. Conclusion This neoplasm shows clinical, microscopical, histological and ultrastructural features of oncocytic carcinoma and this must be considered in the differential diagnosis of other proliferations in the parotid gland with abundant granular cytoplasm and metastatic oncocytic

  8. Ontogenetic changes in digestive enzyme activities and the amino acid profile of starry flounder Platichthys stellatus

    Science.gov (United States)

    Song, Zhidong; Wang, Jiying; Qiao, Hongjin; Li, Peiyu; Zhang, Limin; Xia, Bin

    2016-09-01

    Ontogenetic changes in digestive enzyme activities and the amino acid (AA) profile of starry flounder, Platichthys stellatus, were investigated and limiting amino acids were estimated compared with the essential AA profile between larvae and live food to clarify starry flounder larval nutritional requirements. Larvae were collected at the egg stage and 0, 2, 4, 7, 12, 17, 24 days after hatching (DAH) for analysis. Larvae grew from 1.91 mm at hatching to 12.13 mm at 24 DAH. Trypsin and chymotrypsin activities changed slightly by 4 DAH and then increased significantly 4 DAH. Pepsin activity increased sharply beginning 17 DAH. Lipase activity increased significantly 4 DAH and increased progressively with larval growth. Amylase activity was also detected in newly hatched larvae and increased 7 DAH followed by a gradual decrease. High free amino acid (FAA) content was detected in starry flounder eggs (110.72 mg/g dry weight). Total FAA content dropped to 43.29 mg/g in 4-DAH larvae and then decreased gradually to 13.74 mg/g in 24-DAH larvae. Most FAAs (except lysine and methionine) decreased >50% in 4-DAH larvae compared with those in eggs and then decreased to the lowest values in 24-DAH larvae. Changes in the protein amino acid (PAA) profile were much milder than those observed for FAAs. Most PAAs increased gradually during larval development, except lysine and phenylalanine. The percentages of free threonine, valine, isoleucine, and leucine decreased until the end of the trial, whereas the protein forms of these four AAs followed the opposite trend. A comparison of the essential AA composition of live food (rotifers, Artemia nauplii, and Artemia metanauplii) and larvae suggested that methionine was potentially the first limiting AA. These results may help develop starry flounder larviculture methods by solving the AA imbalance in live food. Moreover, the increased digestive enzyme activities indicate the possibility of introducing artificial compound feed.

  9. The role of autolysis loop in determining the specificity of coagulation proteases

    Directory of Open Access Journals (Sweden)

    L. Yang

    2007-08-01

    Full Text Available We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system of activated protein C (APC with the corresponding loop of factor Xa (fXa renders the APC mutant (APC/fX143-154 susceptible to inhibition by antithrombin (AT in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154. Unlike an ~500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.

  10. Preparation of Glycol-Fosfomycin Modified Zirconia Chromatography Stationary Phases and Its Application to the Separation of Proteins%乙二醇-磷霉素改性氧化锆色谱固定相的制备及其在蛋白质分离中的应用

    Institute of Scientific and Technical Information of China (English)

    张淑琼; 邹凤平; 李烃

    2009-01-01

    A novel glycol-fosfomycin modified zirconia (G-F-ZrO_2) stationary phase was prepared and characterized by elemental analysis, FT-IR and N_2 adsorption. Its chromatographic performance was evaluated using four standard proteins including lysozyme (Lys), ribonuclease-A (Rnase-A), α-chymotrypsin (α-Chy) and Cytochrome C (Cyt-C) as probes, and the effects of salt concentration, pH, salt type in eluents and temperature on retention behavior of proteins were investigated. The results indicate that the stationary phase behaves mainly as a hydrophobic interaction chromatographic packing with salt concentration in the mobile phase above 1.0 moh·L~(-1). At the same time, the retention mechanism between protein and G-F-ZrO_2 stationary phase was also discussed.%合成了分离蛋白质的乙二醇-磷霉素钠改性氧化锆高效液相色谱固定相,通过漫反射红外光谱、元素分析等分析方法对该固定相进行了表征.以溶菌酶、核糖核酸酶A、细胞色素C和糜蛋白酶四种标准碱性蛋白质为探针,系统地考察了固定相的疏水相互作用色谱性能.结果表明,乙二醇-磷霉素改性氧化锆固定相对蛋白质有一定的保留,表现出较高的分离选择性.

  11. Conformational states of a bacterial α2-macroglobulin resemble those of human complement C3.

    Directory of Open Access Journals (Sweden)

    David Neves

    Full Text Available α(2 macroglobulins (α(2Ms are broad-spectrum protease inhibitors that play essential roles in the innate immune system of eukaryotic species. These large, multi-domain proteins are characterized by a broad-spectrum bait region and an internal thioester, which, upon cleavage, becomes covalently associated to the target protease, allowing its entrapment by a large conformational modification. Notably, α(2Ms are part of a larger protein superfamily that includes proteins of the complement system, such as C3, a multi-domain macromolecule which is also characterized by an internal thioester-carrying domain and whose activation represents the pivotal step in the complement cascade. Recently, α(2M/C3-like genes were identified in a large number of bacterial genomes, and the Escherichia coli α(2M homolog (ECAM was shown to be activated by proteases. In this work, we have structurally characterized ECAM by electron microscopy and small angle scattering (SAXS techniques. ECAM is an elongated, flexible molecule with overall similarities to C3 in its inactive form; activation by methylamine, chymotrypsin, or elastase induces a conformational modification reminiscent of the one undergone by the transformation of C3 into its active form, C3b. In addition, the proposed C-terminus of ECAM displays high flexibility and different conformations, and could be the recognition site for partner macromolecules. This work sheds light on a potential bacterial defense mechanism that mimics structural rearrangements essential for activation of the complement cascade in eukaryotes, and represents a possible novel target for the development of antibacterials.

  12. Structural phylogeny by profile extraction and multiple superimposition using electrostatic congruence as a discriminator

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Sandeep [Tata Inst. of Fundamental Research, Bombay (India); Rao, Basuthkar J. [Tata Inst. of Fundamental Research, Bombay (India); Baker, Nathan A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Asgeirsson, Bjarni [Univ. of Iceland, Reykjavik (Iceland)

    2013-04-01

    Phylogenetic analysis of proteins using multiple sequence alignment (MSA) assumes an underlying evolutionary relationship in these proteins which occasionally remains undetected due to considerable sequence divergence. Structural alignment programs have been developed to unravel such fuzzy relationships. However, none of these structure based methods have used electrostatic properties to discriminate between spatially equivalent residues. We present a methodology for MSA of a set of related proteins with known structures using electrostatic properties as an additional discriminator (STEEP). STEEP first extracts a profile, then generates a multiple structural superimposition providing a consolidated spatial framework for comparing residues and finally emits the MSA. Residues that are aligned differently by including or excluding electrostatic properties can be targeted by directed evolution experiments to transform the enzymatic properties of one protein into another. We have compared STEEP results to those obtained from a MSA program (ClustalW) and a structural alignment method (MUSTANG) for chymotrypsin serine proteases. Subsequently, we used PhyML to generate phylogenetic trees for the serine and metallo-β-lactamase superfamilies from the STEEP generated MSA, and corroborated the accepted relationships in these superfamilies. We have observed that STEEP acts as a functional classifier when electrostatic congruence is used as a discriminator, and thus identifies potential targets for directed evolution experiments. In summary, STEEP is unique among phylogenetic methods for its ability to use electrostatic congruence to specify mutations that might be the source of the functional divergence in a protein family. Based on our results, we also hypothesize that the active site and its close vicinity contains enough information to infer the correct phylogeny for related proteins.

  13. [Biochemical diagnostics in acute pancreatitis recognition and outcome predicition].

    Science.gov (United States)

    Olczyk, Paweł; Kozma, Ewa M; Olczyk, Krystyna; Komosińska-Vassev, Katarzyna

    2004-01-01

    Acute pancreatitis (AP) is a common disease associated with an improper activation of pancreatic zymogens leading to autodigestion of the gland and if excessive--to multiple organ dysfunction. Acute necrotizing pancreatitis manifested by 20% of patients with acute pancreatitis is a life threatening disorder requiring subsequent management in intensive care unit. Unfortunately, none of biochemical tests presently used for laboratory assessment of acute pancreatitis at the early stage of the disease is able to estimate accurately: diagnosis, etiology and severity. At present, diagnosis of acute pancreatitis is based on evaluation of serum amylase and lipase activity due to easy availability and simplicity of these enzymatic tests. Low specificity of the mentioned enzymes resulted in studies concerning pancreatic isoamylase, elastase-1, chymotrypsine, procarboxy-peptidase B, trypsinogen-2 and immunoreactive trypsinogen usefulness in the laboratory diagnosis of AP. The prediction of severity in acute pancreatitis using multifactorial scoring systems is cumbersome especially due to their complexity. On the other hand the biochemical method of choice, estimation of serum C reactive protein, is useless in the early phase of disease. Unfortunately, the computed tomography--the most accurate method in severity assessing--is not always available. Recent studies have brought some progress in severity predicting, such as phospholipase A2, cellular immunity markers, cytokines, activation peptides of trypsinogen and carboxypeptidase B, procalcitonine, pancreatitis associated protein and serum amyloid A. All these newly introduced biochemical methods allow to look optimistically into the future of laboratory diagnostics of the acute pancreatitis believing that the problem of diagnosing and predicting the AP severity will be solved. PMID:15850341

  14. Comparison the effect of three commercial enzymes for enzymatic hydrolysis of two substrates (rice bran protein concentrate and soy-been protein) with SDS-PAGE.

    Science.gov (United States)

    Ahmadifard, Nasrollah; Murueta, Julio Humberto Cordova; Abedian-Kenari, Abdolmohammad; Motamedzadegan, Ali; Jamali, Hadi

    2016-02-01

    In this research enzymatic hydrolysis of rice bran protein concentrate (RBPC) and soybean Protein (SBP) as control were studied with 3 commercial enzymes (Alcalase®, Papain and acommercial 3-enzyme cocktail containing of 1.6 mg ml(-1) Trypsin, 3.1 mg ml(-1) Chymotrypsin, 1.3 mg ml(-1)Aminopeptidase (SIGMA P7500) and 7.95 mg ml(-1)pronase type XIV (SIGMA P5147) by the pH stat method. The hydrolysis was carried out at temperature of 28 C, 60 min and pH 8.00. Results were showed that RBPC, and SBP had higher Degree hydrolysis (DH %) with Alcalase® enzyme. Alcalase®had stronger capability for hydrolysis compared to the other tested enzymes. After 60 minute of hydrolysis time, the DH% of Alcalase® for RBPC and SBP was 12.69 and 12.50 %, respectively. In contrast, papain enzyme was showed lowest DH% in three substrates that 1.56 and 1.24 % were for SBP and RBPC, respectively.The hydrolysis of the protein fraction performed the three enzymes on the two substrates was followed in SDS-PAGE. RBPC and SBP showed almost complete digestion with Alcalase® enzyme after 60 minutes. 3-enzyme cocktail enzyme hydrolyzed better the RBPC than the SBP. Papain enzyme had less effect on the two substrates than other 2 enzymes. It was found that Alcalase® has highest capability for hydrolysis compared to other enzyme preparations. The high value protein hydrolysates prepared by Alcalase® can be used as value added ingredients in many food formulations. They are also suitable for a broad range of industrial food applications and also for cosmetic and personal care products. PMID:27162408

  15. Purification and Characterization of Plantaricin JLA-9: A Novel Bacteriocin against Bacillus spp. Produced by Lactobacillus plantarum JLA-9 from Suan-Tsai, a Traditional Chinese Fermented Cabbage.

    Science.gov (United States)

    Zhao, Shengming; Han, Jinzhi; Bie, Xiaomei; Lu, Zhaoxin; Zhang, Chong; Lv, Fengxia

    2016-04-01

    Bacteriocins are ribosomally synthesized peptides with antimicrobial activity produced by numerous bacteria. A novel bacteriocin-producing strain, Lactobacillus plantarum JLA-9, isolated from Suan-Tsai, a traditional Chinese fermented cabbage, was screened and identified by its physiobiochemical characteristics and 16S rDNA sequence analysis. A new bacteriocin, designated plantaricin JLA-9, was purified using butanol extraction, gel filtration, and reverse-phase high-performance liquid chromatography. The molecular mass of plantaricin JLA-9 was shown to be 1044 Da by MALDI-TOF-MS analyses. The amino acid sequence of plantaricin JLA-9 was predicted to be FWQKMSFA by MALDI-TOF-MS/MS, which was confirmed by Edman degradation. This bacteriocin exhibited broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, especially Bacillus spp., high thermal stability (20 min, 121 °C), and narrow pH stability (pH 2.0-7.0). It was sensitive to α-chymotrypsin, pepsin, alkaline protease, and papain. The mode of action of this bacteriocin responsible for outgrowth inhibition of Bacillus cereus spores was studied. Plantaricin JLA-9 had no detectable effects on germination initiation over 1 h on monitoring the hydration, heat resistance, and 2,6-pyridinedicarboxylic acid (DPA) release of spores. Rather, germination initiation is a prerequisite for the action of plantaricin JLA-9. Plantaricin JLA-9 inhibited growth by preventing the establishment of oxidative metabolism and disrupting membrane integrity in germinating spores within 2 h. The results suggest that plantaricin JLA-9 has potential applications in the control of Bacillus spp. in the food industry. PMID:26985692

  16. Soybean β-conglycinin induces inflammation and oxidation and causes dysfunction of intestinal digestion and absorption in fish.

    Directory of Open Access Journals (Sweden)

    Jin-Xiu Zhang

    Full Text Available β-Conglycinin has been identified as one of the major feed allergens. However, studies of β-conglycinin on fish are scarce. This study investigated the effects of β-conglycinin on the growth, digestive and absorptive ability, inflammatory response, oxidative status and gene expression of juvenile Jian carp (Cyprinus carpio var. Jian in vivo and their enterocytes in vitro. The results indicated that the specific growth rate (SGR, feed intake, and feed efficiency were reduced by β-conglycinin. In addition, activities of trypsin, chymotrypsin, lipase, creatine kinase, Na(+,K(+-ATPase and alkaline phosphatase in the intestine showed similar tendencies. The protein content of the hepatopancreas and intestines, and the weight and length of the intestines were all reduced by β-conglycinin. β-Conglycinin increased lipid and protein oxidation in the detected tissues and cells. However, β-conglycinin decreased superoxide dismutase (SOD, catalase (CAT, glutathione-S-transferase (GST, glutathione peroxidase (GPx and glutathione reductase (GR activities and glutathione (GSH content in the intestine and enterocytes. Similar antioxidant activity in the hepatopancreas was observed, except for GST. The expression of target of rapamycin (TOR gene was reduced by β-conglycinin. Furthermore, mRNA levels of interleukin-8 (IL-8, tumor necrosis factor-α (TNF-α, and transforming growth factor-β (TGF-β genes were increased by β-conglycinin. However, β-conglycinin increased CuZnSOD, MnSOD, CAT, and GPx1b gene expression. In conclusion, this study indicates that β-conglycinin induces inflammation and oxidation, and causes dysfunction of intestinal digestion and absorption in fish, and finally reduces fish growth. The results of this study provide some information to the mechanism of β-conglycinin-induced negative effects.

  17. Shikonin Promotes Skin Cell Proliferation and Inhibits Nuclear Factor-κB Translocation via Proteasome Inhibition In Vitro

    Institute of Scientific and Technical Information of China (English)

    Yan Yan; Minao Furumura; Takako Gouya; Atsufumi Iwanaga; Kwesi Teye; Sanae Numata; Tadashi Karashima

    2015-01-01

    Background:Shikonin is a major active chemical component extracted from Lithospermi Radix,an effective traditional herb in various types of wound healing.Shikonin can accelerate granulomatous tissue formation by the rat cotton pellet method and induce neovascularization in granulomatous tissue.The purpose of the study was to investigate its mechanism of action in human skin cells.Methods:MTS assay was used to measure cell growth.The collagen type Ⅰ (COL1) mRNA expression and procollagen type Ⅰ C-peptide (PIP) production were detected by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay,respectively.Immunofluorescence and western blot analyses were carried out to investigate nuclear factor-κB (NF-κB) signaling pathway.Cell-based proteasome activity assay was used to determine proteasome activity.Results:In this study,we found that 10 μmol/L shikonin stimulated the growth of normal human keratinocytes and 1 μmol/L shikonin promoted growth of human dermal fibroblasts.However,shikonin did not directly induce COLI mRNA expression and PIP production in dermal fibroblasts in vitro.In addition,1 μmol/L shikonin inhibited translocation of NF-κB p65 from cytoplasm to nucleus induced by tumor necrosis factor-α stimulation in dermal fibroblasts.Furthermore,shikonin inhibited chymotrypsin-like activity of proteasome and was associated with accumulation ofphosphorylated inhibitor κB-α in dermal fibroblasts.Conclusions:These results suggested that shikonin may promote wound healing via its cell growth promoting activity and suppress skin inflammation via inhibitory activity on proteasome.Thus,shikonin may be a potential therapeutic reagent both in wound healing and inflammatory skin diseases.

  18. Dietary β-glucan (MacroGard®) enhances survival of first feeding turbot (Scophthalmus maximus) larvae by altering immunity, metabolism and microbiota.

    Science.gov (United States)

    Miest, Joanna J; Arndt, Carmen; Adamek, Mikolaj; Steinhagen, Dieter; Reusch, Thorsten B H

    2016-01-01

    Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, β-glucan (MacroGard(®)) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast β-1,3/1,6-glucan in form of MacroGard(®) at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, microbiota analysis, trypsin activity and size measurements. Along with the feeding of β-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard(®) fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by β-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-α and il-1β was observed. We conclude that the administration of MacroGard(®) induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot. PMID:26564474

  19. Magnetic Sephadex as a carrier for enzyme immobilization and drug targeting

    Energy Technology Data Exchange (ETDEWEB)

    Torchilin, V.P.; Papisov, M.I.; Smirnov, V.N.

    1985-04-01

    Magnetic materials were suggested as carriers for protein immobilization about 10 years ago. The main advantage of these carriers is their ability to be concentrated near magnetic terminals upon the application of the external magnetic field. This property is used in technological processes for selective catalyst removal from the reaction mixture (3), in immunological studies for the separation of cells to which magnetic particles are specifically bound modified with antibodies against cell surface components, in experiments for the drug targeting in vivo into appropriate tissues under the action of external magnetic field. There exist a number of methods to obtain porous magnetic carriers, containing immobilized matter not only on the surface, but also in the volume of a particle. Normally, these preparations are obtained by the granule formation from the suspension of ferromagnetic particles in the solution or melt of appropriate high-molecular-weight compound. The drawback of the above-mentioned methods is the pronounced aggregation of ferromagnetic particles. The aggregation does not permit to use concentrated enough suspensions of magnetic particles and causes the formation of the product with a variety of sizes and magnetic properties. We made an attempt to synthesize the magnetic carrier for protein immobilization on the basis of commercial Sephadex porous spheres. Sephadex granules were made magnetic by adsorptional fixation of ferromagnetic particles in its pores. The properties of the ''native'' and ''magnetic'' Sephadexes as carriers for protein immobilization were compared by parallel immobilization on both carriers of alpha-chymotrypsin and T I-albumin. In in vivo experiments we studied the ability of magnetic Sephadex to be concentrated in a desired region of the circulation under the action of external magnetic field.

  20. A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

    Science.gov (United States)

    Poston, Chloe N.; Higgs, Richard E.; You, Jinsam; Gelfanova, Valentina; Hale, John E.; Knierman, Michael D.; Siegel, Robert; Gutierrez, Jesus A.

    2014-07-01

    De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

  1. Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus.

    Directory of Open Access Journals (Sweden)

    Shiwanthi L Ranasinghe

    2015-12-01

    Full Text Available The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE, a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic.

  2. Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus.

    Science.gov (United States)

    Ranasinghe, Shiwanthi L; Fischer, Katja; Zhang, Wenbao; Gobert, Geoffrey N; McManus, Donald P

    2015-12-01

    The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic. PMID:26645974

  3. Recent Advances in Protein Extraction and Chiral Separation of Biomolecules

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Reverse micelles create unique environment in organic media. They are capable of solubilizing hydrophilic biomolecules (e.g., proteins, peptides, amino acids, and DNAs) in their aqueous interior. This feature brings about the practical use of biomaterials in organic media because reverse micelles solubilize them with the intrinsic activity. In this paper, we focus on recent two topics concerning protein extraction and chiral separation of biomolecules using liquid membranes. In the first topic, we present recent attempts to extract proteins from an aqueous solution into isooctane using reverse micelles, and some important operational parameters to achieve an efficient protein transfer are discussed. Furthermore, novel function of reverse micelles as a protein activation medium is introduced. In the reverse micellar phase, denatured proreins were completely reactivated in the reverse micellar solution. The reverse micellar technique is found to be a useful tool not only for protein separation but also for protein refolding. Furthermore, we found that a cyclic ligand carixarene has an extraction ability to set up optimum conditions for protein transfer. In the second topic, we have found that a supported liquid membrane (SLM) encapsulating enzymes shows high enantioselectivity (enantioselective excess value is over 96%) in the transport of racemic pharmaceutical compound ibuprofen. A different experiment also suggests that the α-chymotrypsin-catalyzed reactions droved the enantioselective transport of L-phenylalanine based on the enantioselectivity of the enzyme. The SLM encapsulating the surfactant-enzyme complex enabled the highly enantioselective separation of racemic mixtures. It can be envisioned that arrangement of appropriate enzymes in the SLM system will allow enantioselective separation of various useful organic compounds.

  4. Enzyme-assisted extraction of flavorings and colorants from plant materials.

    Science.gov (United States)

    Sowbhagya, H B; Chitra, V N

    2010-02-01

    From times immemorial, colorants, and flavorings have been used in foods. Color and flavor are the major attributes to the quality of a food product, affecting the appearance and acceptance of the product. As a consequence of the increased demand of natural flavoring and colorant from industries, there is a renewed interest in the research on the composition and recovery of natural food flavors and colors. Over the years, numerous procedures have been proposed for the isolation of aromatic compounds and colors from plant materials. Generally, the methods of extraction followed for aroma and pigment from plant materials are solvent extraction, hydro-distillation, steam distillation, and super critical carbon dioxide extraction. The application of enzymes in the extraction of oil from oil seeds like sunflower, corn, coconut, olives, avocado etc. are reported in literature. There is a great potential for this enzyme-based extraction technology with the selection of appropriate enzymes with optimized operating conditions. Various enzyme combinations are used to loosen the structural integrity of botanical material thereby enhancing the extraction of the desired flavor and color components. Recently enzymes have been used for the extraction of flavor and color from plant materials, as a pre-treatment of the raw material before subjecting the plant material to hydro distillation/solvent extraction. A deep knowledge of enzymes, their mode of action, conditions for optimum activity, and selection of the right type of enzymes are essential to use them effectively for extraction. Although the enzyme hydrolases such as lipases, proteases (chymotrypsin, subtilisin, thermolysin, and papain), esterases use water as a substrate for the reaction, they are also able to accept other nucleophiles such as alcohols, amines, thio-esters, and oximes. Advantages of enzyme-assisted extraction of flavor and color in some of the plant materials in comparison with conventional methods are

  5. Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata

    Directory of Open Access Journals (Sweden)

    Tomoe Furuta

    2016-02-01

    Full Text Available We examined the inhibitory activity of angiotensin I converting enzyme (ACE in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE followed by phycocyanin (PC and allophycocyanin (APC. The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC, and it was demonstrated that the synthetic peptide LRY (IC50: 0.044 μmol has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477–17,638 and β-subunit (Mw: 17,455–18,407. The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and β-subunits. In addition, the LRY sequence was found in the β-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and β-subunits of PE (rPEα and rPEβ, respectively, and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEβ: 87.0%. Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins.

  6. Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles.

    Directory of Open Access Journals (Sweden)

    Karin E Darpel

    Full Text Available Bluetongue virus (BTV and epizootic haemorrhagic disease virus (EHDV are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.. The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin, forming infectious subviral particles (ISVP which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis. We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector, cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species. Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species, can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to

  7. Proteins at fluid interfaces: adsorption layers and thin liquid films.

    Science.gov (United States)

    Yampolskaya, Galina; Platikanov, Dimo

    2006-12-21

    A review in which many original published results of the authors as well as many other papers are discussed. The structure and some properties of the globular proteins are shortly presented, special accent being put on the alpha-chymotrypsin (alpha-ChT), lysozyme (LZ), human serum albumin (HSA), and bovine serum albumin (BSA) which have been used in the experiments with thin liquid films. The behaviour of protein adsorption layers (PAL) is extensively discussed. The dynamics of PAL formation, including the kinetics of adsorption as well as the time evolution of the surface tension of protein aqueous solutions, are considered. A considerable place is devoted to the surface tension and adsorption isotherms of the globular protein solutions, the simulation of PAL by interacting hard spheres, the experimental surface tension isotherms of the above mentioned proteins, and the interfacial tension isotherms for the protein aqueous solution/oil interface. The rheological properties of PAL at fluid interfaces are shortly reviewed. After a brief information about the experimental methods for investigation of protein thin liquid (foam or emulsion) films, the properties of the protein black foam films are extensively discussed: the conditions for their formation, the influence of the electrolytes and pH on the film type and stability, the thermodynamic properties of the black foam films, the contact angles film/bulk and their dynamic hysteresis. The next center of attention concerns some properties of the protein emulsion films: the conditions for formation of emulsion black films, the formation and development of a dimpling in microscopic, circular films. The protein-phospholipid mixed foam films are also briefly considered.

  8. Dietary β-glucan (MacroGard®) enhances survival of first feeding turbot (Scophthalmus maximus) larvae by altering immunity, metabolism and microbiota.

    Science.gov (United States)

    Miest, Joanna J; Arndt, Carmen; Adamek, Mikolaj; Steinhagen, Dieter; Reusch, Thorsten B H

    2016-01-01

    Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, β-glucan (MacroGard(®)) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast β-1,3/1,6-glucan in form of MacroGard(®) at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, microbiota analysis, trypsin activity and size measurements. Along with the feeding of β-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard(®) fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by β-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-α and il-1β was observed. We conclude that the administration of MacroGard(®) induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.

  9. The anti-proliferative effect of TI1B, a major Bowman-Birk isoinhibitor from pea (Pisum sativum L.), on HT29 colon cancer cells is mediated through protease inhibition.

    Science.gov (United States)

    Clemente, Alfonso; Carmen Marín-Manzano, M; Jiménez, Elisabeth; Carmen Arques, M; Domoney, Claire

    2012-08-01

    Bowman-Birk inhibitors (BBI) from legumes, such as soyabean, pea, lentil and chickpea, are naturally occurring plant protease inhibitors which have potential health-promoting properties within the mammalian gastrointestinal tract. BBI can survive both acidic conditions and the action of proteolytic enzymes within the stomach and small intestine, permitting significant amounts to reach the large intestine in active form to exert their reported anti-carcinogenic and anti-inflammatory properties. In a previous study, we reported the ability of a recombinant form of TI1B (rTI1B), representing a major BBI isoinhibitor from pea, to influence negatively the growth of human colorectal adenocarcinoma HT29 cells in vitro. In the present study, we investigate if this effect is related directly to the intrinsic ability of BBI to inhibit serine proteases. rTI1B and a novel engineered mutant, having amino acid substitutions at the P1 positions in the two inhibitory domains, were expressed in the yeast Pichia pastoris. The rTI1B proved to be active against trypsin and chymotrypsin, showing K i values at nanomolar concentrations, whereas the related mutant protein was inactive against both serine proteases. The proliferation of HT29 colon cancer cells was significantly affected by rTI1B in a dose-dependent manner (IC50 = 31 (sd 7) μm), whereas the inactive mutant did not show any significant effect on colon cancer cell growth. In addition, neither recombinant protein affected the growth of non-malignant colonic fibroblast CCD-18Co cells. These findings suggest that serine proteases should be considered as important targets in investigating the potential chemopreventive role of BBI during the early stages of colorectal carcinogenesis. PMID:22916809

  10. [Interconnection of physico-chemical characteristics of organic solvents and their denaturing ability in relation to proteins].

    Science.gov (United States)

    Belova, A B; Mozhaev, V V; Levashov, A V; Sergeeva, M V; Martinek, K; Khmel'nitskiĭ, Iu L

    1991-11-01

    Reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was studied. The regularities of this process were analyzed, employing both experimental and literary data based on the results of kinetic and spectroscopic measurements. In all the systems under study denaturation proceeded in a threshold manner, i. e., an abrupt change in the catalytic and/or spectroscopic properties of the dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of reversible protein denaturation by organic solvents was proposed. This model is based on the widely accepted viewpoint that the undisturbed water shell around the protein globule is necessary for maintaining the dissolved protein in the native state. Quantitative analysis of the model led to an equation establishing a relationship between the threshold concentration of an organic solvent and its physico-chemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation fits well in the experimental data for all the proteins tested. Based on the above thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents (termed as the denaturation capacity or DC) was proposed. Different organic solvents arranged according to their DC values form the DC scale of organic solvents which permits to predict theoretically the threshold concentration of any organic solvent for a given protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1666845

  11. Enzymatic hydrolysis of ovomucoid and the functional properties of its hydrolysates.

    Science.gov (United States)

    Abeyrathne, E D N S; Lee, H Y; Jo, C; Suh, J W; Ahn, D U

    2015-09-01

    Ovomucoid is well known as a "trypsin inhibitor" and is considered to be the main food allergen in egg. However, the negative functions of ovomucoid can be eliminated if the protein is cut into small peptides. The objectives of this study were to hydrolyze ovomucoid using various enzyme combinations, and compare the functional properties of the hydrolysates. Purified ovomucoid was dissolved in distilled water (20 mg/mL) and treated with 1% of pepsin, α-chymotrypsin, papain, and alcalase, singly or in combinations. Sodium sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) results of the hydrolysates indicated that pepsin (OMP), alcalase (OMAl), alcalase+trypsin (OMAlTr), and alcalase+papain (OMAlPa) treatments best hydrolyzed the ovomucoid, and the 4 treatments were selected to determine their functional characteristics. Among the 4 enzyme treatments, hydrolysate from OMAlTr showed the highest iron-chelating and antioxidant activities, while OMP showed higher ACE-inhibitory activity, but lower Fe-chelating activity than the other treatments. However, no difference in the copper-chelating activity among the treatments was found. MS/MS analysis identified numerous peptides from the hydrolysates of OMAlPa and OMAlTr, and majority of the peptides produced were <2 kDa. Pepsin treatment (OMP), however, hydrolyzed ovomucoid almost completely and produced only amino acid monomers, di- and tri-peptides. The ACE-inhibitory, antioxidant and iron-chelating activities of the enzyme hydrolysates were not consistent with the number and size of peptides in the hydrolysates, but we do not have information about the quantity of each peptide present in the hydrolysates at this point. PMID:26195809

  12. Expression and site-directed mutagenesis of human dihydrofolate reductase

    International Nuclear Information System (INIS)

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 → Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by α-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme

  13. Subclinical zinc deficiency impairs pancreatic digestive enzyme activity and digestive capacity of weaned piglets.

    Science.gov (United States)

    Brugger, Daniel; Windisch, Wilhelm M

    2016-08-01

    This study investigated the effects of short-term subclinical Zn deficiency on exocrine pancreatic activity and changes in digestive capacity. A total of forty-eight weaned piglets were fed ad libitum a basal diet (maize and soyabean meal) with adequate Zn supply (88 mg Zn/kg diet) during a 2-week acclimatisation phase. Animals were then assigned to eight dietary treatment groups (n 6) according to a complete randomised block design considering litter, live weight and sex. All pigs were fed restrictively (450 g diet/d) the basal diet but with varying ZnSO4.7H2O additions, resulting in 28·1, 33·6, 38·8, 42·7, 47·5, 58·2, 67·8 and 88·0 mg Zn/kg diet for a total experimental period of 8 d. Pancreatic Zn concentrations and pancreatic activities of trypsin, chymotrypsin, carboxypeptidase A and B, elastase and α-amylase exhibited a broken-line response to stepwise reduction in dietary Zn by declining beneath thresholds of 39·0, 58·0, 58·0, 41·2, 47·5, 57·7 and 58·0 mg Zn/kg diet, respectively. Furthermore, carboxypeptidase B and α-amylase activities were significantly lower in samples with reduced pancreatic Zn contents. Coefficients of faecal digestibility of DM, crude protein, total lipids and crude ash responded similarly to pancreatic enzyme activities by declining below dietary thresholds of 54·7, 45·0, 46·9 and 58·2 mg Zn/kg diet, respectively. In conclusion, (1) subclinical Zn deficiency impaired pancreatic exocrine enzymes, (2) this response was connected to pancreatic Zn metabolism and (3) the decline in catalytic activity impaired faecal digestibility already after 1 week of insufficient alimentary Zn supply and very early before clinical deficiency symptoms arise. PMID:27230230

  14. Preparation of biocatalytic nanofibers with high activity and stability via enzyme aggregate coating on polymer nanofibers

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Byoung Chan; Nair, Sujith; Kim, Jungbae; Kwak, Ja Hun; Grate, Jay W.; Kim, Seong H.; Gu, Man Bock

    2005-04-01

    We have developed a unique approach for the fabrication of enzyme coating on the surface of electrospun polymer nanofibers. This approach employs covalent attachment of seed enzymes onto nanofibers, followed by the glutaraldehyde treatment that crosslinks additional enzymes onto the seed enzyme molecules. These crosslinked enzyme aggregates, covalently attached to the nanofibers via seed enzyme linker, would improve not only the enzyme activity due to increased enzyme loading, but also the enzyme stability. To demonstrate the principle of concept, we fabricated the coating of alpha-chymotrypsin (CT) on the nanofibers electrospun from a mixture of polystyrene and poly(styrene-co-maleic anhydride). The addition of poly(styrene-co-maleic anhydride) makes it much easier to attach the seed enzyme molecules onto electrospun nanofibers without any rigorous functionalization of nanofibers for the attachment of enzymes. The initial activity of final CT coating was 17 and 9 times higher than those of simply-adsorbed CT and covalently-attached CT, respectively. While adsorbed and covalently-attached CT resulted in a serious enzyme leaching during initial incubation in a shaking condition, the CT coating did not show any leaching from the beginning of incubation in the same condition. As a result, the enzyme stability of CT coating was impressively improved with a half-life of 686 days under rigorous shaking while the half-life of covalently-attached CT was only 21 hours. This new approach of enzyme coating with high stability and activity will make a great impact in various applications of enzymes such as bioconversion, bioremediation, and biosensors.

  15. Quinoxaline derivatives: novel and selective butyrylcholinesterase inhibitors.

    Science.gov (United States)

    Zeb, Aurang; Hameed, Abdul; Khan, Latifullah; Khan, Imran; Dalvandi, Kourosh; Choudhary, M Iqbal; Basha, Fatima Z

    2014-01-01

    Alzheimer's disease (AD) is a progressive brain disorder which occurs due to lower levels of acetylcholine (ACh) neurotransmitters, and results in a gradual decline in memory and other cognitive processes. Acetycholinesterase (AChE) and butyrylcholinesterase (BChE) are considered to be primary regulators of the ACh levels in the brain. Evidence shows that AChE activity decreases in AD, while activity of BChE does not change or even elevate in advanced AD, which suggests a key involvement of BChE in ACh hydrolysis during AD symptoms. Therefore, inhibiting the activity of BChE may be an effective way to control AD associated disorders. In this regard, a series of quinoxaline derivatives 1-17 was synthesized and biologically evaluated against cholinesterases (AChE and BChE) and as well as against α- chymotrypsin and urease. The compounds 1-17 were found to be selective inhibitors for BChE, as no activity was found against other enzymes. Among the series, compounds 6 (IC50 = 7.7 ± 1.0 µM) and 7 (IC50 = 9.7 ± 0.9 µM) were found to be the most active inhibitors against BChE. Their IC50 values are comparable to the standard, galantamine (IC50 = 6.6 ± 0.38 µM). Their considerable BChE inhibitory activity makes them selective candidates for the development of BChE inhibitors. Structure-activity relationship (SAR) of this new class of selective BChE inhibitors has been discussed.

  16. Acclimatory responses of the Daphnia pulex proteome to environmental changes. II. Chronic exposure to different temperatures (10 and 20°C mainly affects protein metabolism

    Directory of Open Access Journals (Sweden)

    Madlung Johannes

    2009-04-01

    Full Text Available Abstract Background Temperature affects essentially every aspect of the biology of poikilothermic animals including the energy and mass budgets, activity, growth, and reproduction. While thermal effects in ecologically important groups such as daphnids have been intensively studied at the ecosystem level and at least partly at the organismic level, much less is known about the molecular mechanisms underlying the acclimation to different temperatures. By using 2D gel electrophoresis and mass spectrometry, the present study identified the major elements of the temperature-induced subset of the proteome from differently acclimated Daphnia pulex. Results Specific sets of proteins were found to be differentially expressed in 10°C or 20°C acclimated D. pulex. Most cold-repressed proteins comprised secretory enzymes which are involved in protein digestion (trypsins, chymotrypsins, astacin, carboxypeptidases. The cold-induced sets of proteins included several vitellogenin and actin isoforms (cytoplasmic and muscle-specific, and an AAA+ ATPase. Carbohydrate-modifying enzymes were constitutively expressed or down-regulated in the cold. Conclusion Specific sets of cold-repressed and cold-induced proteins in D. pulex can be related to changes in the cellular demand for amino acids or to the compensatory control of physiological processes. The increase of proteolytic enzyme concentration and the decrease of vitellogenin, actin and total protein concentration between 10°C and 20°C acclimated animals reflect the increased amino-acids demand and the reduced protein reserves in the animal's body. Conversely, the increase of actin concentration in cold-acclimated animals may contribute to a compensatory mechanism which ensures the relative constancy of muscular performance. The sheer number of peptidase genes (serine-peptidase-like: > 200, astacin-like: 36, carboxypeptidase-like: 30 in the D. pulex genome suggests large-scaled gene family expansions that

  17. Influence of the APOE genotype on hepatic stress response: Studies in APOE targeted replacement mice and human liver cells.

    Science.gov (United States)

    Dose, Janina; Nebel, Almut; Piegholdt, Stefanie; Rimbach, Gerald; Huebbe, Patricia

    2016-07-01

    Apolipoprotein E (APOE) is a multifunctional plasma protein mainly acting in lipid metabolism. Human APOE is polymorphic with three major isoforms (APOE2, APOE3 and APOE4). Up to 75% of the body's APOE is produced by the liver. There is increasing evidence from studies in brain-derived cells that APOE4 affects mitochondrial function and biogenesis as well as stress and inflammatory responses - processes, whose disturbances are considered hallmarks of the ageing process. However, although the liver is the main production site of APOE, knowledge about the impact of the APOE genotype on hepatic stress response-related processes is rather limited. Therefore, we studied biomarkers of oxidative status (glutathione levels, 3-nitrotyrosine adducts, protein carbonyl concentration), ER stress (XBP1(S), BiP, DDIT3), proteasome activity, mitochondrial function (respiratory complexes, ATP levels and mitochondrial membrane potential as well as biomarkers of mitochondrial biogenesis, fission and fusion), autophagy (LC3, LAMP2A), apoptosis (BCL2, BAX, CYCS) and DNA damage in the liver of APOE targeted replacement (TR) mice and in Huh7 hepatocytes overexpressing the APOE3 and the APOE4 isoform, respectively. APOE4 mice exhibited a lower chymotrypsin-like and a higher trypsin-like proteasome activity. Levels of protein carbonyls were moderately higher in liver tissue of APOE4 vs. APOE3 mice. Other biomarkers of oxidative stress were similar between the two genotypes. Under basal conditions, the stress-response pathways investigated appeared largely unaffected by the APOE genotype. However, upon stress induction, APOE4 expressing cells showed lower levels of adenosine triphosphate (ATP) and lower mRNA levels of the ATP-generating complex V of the mitochondrial respiratory chain. Overall, our findings provide evidence for a rather low influence of the APOE genotype on the hepatic stress response processes investigated in this study. PMID:27130033

  18. Screening for lactic acid bacteria capable of inhibiting Campylobacter jejuni in in vitro simulations of the broiler chicken caecal environment.

    Science.gov (United States)

    Robyn, J; Rasschaert, G; Messens, W; Pasmans, F; Heyndrickx, M

    2012-12-01

    Thermotolerant Campylobacter spp., specifically Campylobacter jejuni and Campylobacter coli, are the most common bacterial causes of human gastroenteritis in developed countries. Consumption of improperly prepared poultry products and cross contamination are among the main causes of human campylobacteriosis. The aim of this study was to identify lactic acid bacterial (LAB) strains capable of inhibiting C. jejuni growth in initial in vitro trials ('spot-on-lawn' method), as well as in batch fermentation studies mimicking the broiler caecal environment. These experiments served as an indication for using these strains to decrease the capability of Campylobacter to colonise and grow in the chicken caeca during primary production, with the aim of reducing the number of human campylobacteriosis cases. A total of 1,150 LAB strains were screened for anti-Campylobacter activity. Six strains were selected: members of the species Lactobacillus reuteri, Lactobacillus agilis, Lactobacillus helveticus, Lactobacillus salivarius, Enterococcus faecalis and Enterococcus faecium. After treatment with catalase, proteinase K and a-chymotrypsin, anti-Campylobacter activity of cell-free culture supernatant fluid (CSF) for all six strains was retained, which indicated that activity was probably not exerted by bacteriocin production. Based on the activity found in CSF, the compounds produced by the selected strains are secreted and do not require presence of live bacterial producer cells for activity. During initial in vitro fermentation experiments, the E. faecalis strain exhibited the highest inhibitory activity for C. jejuni and was selected for further fermentation experiments. In these experiments we tested for therapeutic or protective effects of the E. faecalis strain against C. jejuni MB 4185 infection under simulated broiler caecal growth conditions. The best inhibition results were obtained when E. faecalis was inoculated before the C. jejuni strain, lowering C. jejuni counts at

  19. Yeast Pah1p phosphatidate phosphatase is regulated by proteasome-mediated degradation.

    Science.gov (United States)

    Pascual, Florencia; Hsieh, Lu-Sheng; Soto-Cardalda, Aníbal; Carman, George M

    2014-04-01

    Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation.

  20. Effect of proteasome inhibition on the ubiquitination of tau protein%蛋白酶体活性抑制对大鼠海马tau蛋白的泛素化影响

    Institute of Scientific and Technical Information of China (English)

    刘英华; 罗健东

    2011-01-01

    目的:探讨蛋白酶体活性抑制对大鼠海马tau蛋白的泛素化影响.方法:利用双侧海马注射蛋白酶体的特异性抑制剂--乳胞素分别进行蛋白酶体活性检测、免疫印迹、免疫共沉淀,以检测大鼠海马内tau蛋白的泛素化改变情况.结果:(1)乳胞素引起蛋白酶体活性在24和48 h较对照组明显下降;(2)免疫共沉淀结果显示,蛋白酶体活性抑制使tau蛋白发生了异常泛素化.结论:乳胞素抑制了蛋白酶体活性并导致了大鼠海马tau蛋白的异常泛素化.%Objective To investigate the effect of proteasome inhihition on the ubiquitination of tauprotein. Methods We injected bilaterally lactacystin, a specific inhihitor of proteasome , into the hippocampus of the rats and employed proteasome activity assay, Westem blotting and co-immunoprecipitation to observe the ubiquitination changes of tau protein in rats' hippocampus. Results We observed the following results in our experiment : (1) Lactacystin obviously inhibited the chymotrypsin-like activity of proteasome at the 24th h and the 48th h; (2) Proteasome inhibition increased the ubiquitination of tau. Conclusion Proteasome inhibition leads to ahnormal ubiquitination of tau protein in rats' hippocampus.

  1. In vitro fermentation studies for selection and evaluation of Bacillus strains as starter cultures for the production of okpehe, a traditional African fermented condiment.

    Science.gov (United States)

    Oguntoyinbo, Folarin A; Sanni, Abiodun I; Franz, Charles M A P; Holzapfel, Wilhelm H

    2007-01-25

    Selected Bacillus and Enterococcus strains, isolated from traditional okpehe fermentations, were studied for their suitability as starter cultures in laboratory-scale fermentations of Prosopis africana seeds for the production of okpehe, a traditional fermented vegetable product of Nigeria. The strains were selected on the basis of highest proteolytic activity, as determined with the APIZYM (BioMerieux) test. The choice of starter strains was narrowed to Bacillus subtilis strains BFE 5301 and BFE 5372. These were determined as the best starter combination because of rapid growth, high amylolytic and proteolytic activities, high levels of polyglutamic acid production by strain BFE 5372, as well as bacteriocin production by strain BFE 5301. Other mixed culture fermentations did not yield sensorically acceptable products. Although a monoculture fermentation, using only B. subtilis strain BFE 5372, produced okpehe with very good sensory characteristics, the growth of B. cereus could be detected after 48 h fermentation, indicating that this starter did not sufficiently contribute to product safety. Mixed culture fermentation with the combination of bacteriocin-producing starter B. subtilis BFE 5301 and the non-bacteriocin-producing B. subtilis BFE 5372, produced a product with good sensory characteristics, in which growth of B. cereus was delayed. The bacteriocin produced by B. subtilis strain BFE 5301 was identified as subtilisin, using subtilisin-specific primers and PCR amplification of the subtilisin gene. The bacteriocin was heat-stable at 100 degrees C for 10 min and exhibited highest activity at pH values lower or equal to pH 6.0. The bacteriocin was sensitive to the proteolytic enzymes trypsin and alpha-chymotrypsin at concentrations of 10 mg/ml.

  2. Tissue-Specific Transcriptome Profiling of Plutella Xylostella Third Instar Larval Midgut

    Directory of Open Access Journals (Sweden)

    Wen Xie, Yanyuan Lei, Wei Fu, Zhongxia Yang, Xun Zhu, Zhaojiang Guo, Qingjun Wu, Shaoli Wang, Baoyun Xu, Xuguo Zhou, Youjun Zhang

    2012-01-01

    Full Text Available The larval midgut of diamondback moth, Plutella xylostella, is a dynamic tissue that interfaces with a diverse array of physiological and toxicological processes, including nutrient digestion and allocation, xenobiotic detoxification, innate and adaptive immune response, and pathogen defense. Despite its enormous agricultural importance, the genomic resources for P. xylostella are surprisingly scarce. In this study, a Bt resistant P. xylostella strain was subjected to the in-depth transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes in the P. xylostella larval midgut.Using Illumina deep sequencing, we obtained roughly 40 million reads containing approximately 3.6 gigabases of sequence data. De novo assembly generated 63,312 ESTs with an average read length of 416bp, and approximately half of the P. xylostella sequences (45.4%, 28,768 showed similarity to the non-redundant database in GenBank with a cut-off E-value below 10-5. Among them, 11,092 unigenes were assigned to one or multiple GO terms and 16,732 unigenes were assigned to 226 specific pathways. In-depth analysis indentified genes putatively involved in insecticide resistance, nutrient digestion, and innate immune defense. Besides conventional detoxification enzymes and insecticide targets, novel genes, including 28 chymotrypsins and 53 ABC transporters, have been uncovered in the P. xylostella larval midgut transcriptome; which are potentially linked to the Bt toxicity and resistance. Furthermore, an unexpectedly high number of ESTs, including 46 serpins and 7 lysozymes, were predicted to be involved in the immune defense.As the first tissue-specific transcriptome analysis of P. xylostella, this study sheds light on the molecular understanding of insecticide resistance, especially Bt resistance in an agriculturally important insect pest, and lays the foundation for future functional genomics research. In

  3. Aii20J, a wide-spectrum thermostable N-acylhomoserine lactonase from the marine bacterium Tenacibaculum sp. 20J, can quench AHL-mediated acid resistance in Escherichia coli.

    Science.gov (United States)

    Mayer, C; Romero, M; Muras, A; Otero, A

    2015-11-01

    Acyl homoserine lactones (AHLs) are produced by many Gram-negative bacteria to coordinate gene expression in cellular density dependent mechanisms known as quorum sensing (QS). Since the disruption of the communication systems significantly reduces virulence, the inhibition of quorumsensing processes or quorum quenching (QQ) represents an interesting anti-pathogenic strategy to control bacterial infections. Escherichia coli does not produce AHLs but possesses an orphan AHL receptor, SdiA, which is thought to be able to sense the QS signals produced by other bacteria and controls important traits as the expression of glutamate-dependent acid resistance mechanism, therefore constituting a putative target for QQ. A novel AHL-lactonase, named Aii20J, has been identified, cloned and over expressed from the marine bacterium Tenacibaculum sp. strain 20 J presenting a wide-spectrum QQ activity. The enzyme, belonging to the metallo-β-lactamase family, shares less than 31 % identity with the lactonase AiiA from Bacillus spp. Aii20J presents a much higher specific activity than the Bacillus enzyme, maintains its activity after incubation at 100 ºC for 10 minutes, is resistant to protease K and α-chymotrypsin, and is unaffected by wide ranges of pH. The addition of Aii20J (20 μg/mL) to cultures of E. coli K-12 to which OC6-HSL was added resulted in a significant reduction in cell viability in comparison with the acidresistant cultures derived from the presence of the signal. Results confirm the interaction between AHLs and SdiA in E. coli for the expression of virulence-related genes and reveal the potential use of Aii20J as anti-virulence strategy against important bacterial pathogens and in other biotechnological applications.

  4. Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata.

    Science.gov (United States)

    Furuta, Tomoe; Miyabe, Yoshikatsu; Yasui, Hajime; Kinoshita, Yasunori; Kishimura, Hideki

    2016-02-01

    We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS) were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC50: 0.044 μmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477-17,638) and β-subunit (Mw: 17,455-18,407). The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and β-subunits. In addition, the LRY sequence was found in the β-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and β-subunits of PE (rPEα and rPEβ, respectively), and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEβ: 87.0%). Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins. PMID:26861357

  5. Liposomes containing glycocholate as potential oral insulin delivery systems: preparation, in vitro characterization, and improved protection against enzymatic degradation

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    Niu M

    2011-06-01

    Full Text Available Mengmeng Niu1, Yi Lu1, Lars Hovgaard2, Wei Wu11School of Pharmacy, Fudan University, Shanghai, People's Republic of China; 2Oral Formulation Development, Novo Nordisk A/S, Maalov, DenmarkBackground: Oral delivery of insulin is challenging and must overcome the barriers of gastric and enzymatic degradation as well as low permeation across the intestinal epithelium. The present study aimed to develop a liposomal delivery system containing glycocholate as an enzyme inhibitor and permeation enhancer for oral insulin delivery.Methods: Liposomes containing sodium glycocholate were prepared by a reversed-phase evaporation method followed by homogenization. The particle size and entrapment efficiency of recombinant human insulin (rhINS-loaded sodium glycocholate liposomes can be easily adjusted by tuning the homogenization parameters, phospholipid:sodium glycocholate ratio, insulin:phospholipid ratio, water:ether volume ratio, interior water phase pH, and the hydration buffer pH.Results: The optimal formulation showed an insulin entrapment efficiency of 30% ± 2% and a particle size of 154 ± 18 nm. A conformational study by circular dichroism spectroscopy and a bioactivity study confirmed the preserved integrity of rhINS against preparative stress. Transmission electron micrographs revealed a nearly spherical and deformed structure with discernable lamella for sodium glycocholate liposomes. Sodium glycocholate liposomes showed better protection of insulin against enzymatic degradation by pepsin, trypsin, and a-chymotrypsin than liposomes containing the bile salt counterparts of sodium taurocholate and sodium deoxycholate.Conclusion: Sodium glycocholate liposomes showed promising in vitro characteristics and have the potential to be able to deliver insulin orally.Keywords: liposomes, glycocholate, insulin, enzymatic degradation, oral

  6. Impairment of the ubiquitin-proteasome pathway by methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate leads to a potent cytotoxic effect in tumor cells: a novel antiproliferative agent with a potential therapeutic implication.

    Science.gov (United States)

    Dogra, Nilambra; Mukhopadhyay, Tapas

    2012-08-31

    In recent years, there has been a great deal of interest in proteasome inhibitors as a novel class of anticancer drugs. We report that fenbendazole (FZ) (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate) exhibits a potent growth-inhibitory activity against cancer cell lines but not normal cells. We show here, using fluorogenic substrates, that FZ treatment leads to the inhibition of proteasomal activity in the cells. Succinyl-Leu-Leu-Val-Tyr-methylcoumarinamide (MCA), benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-MCA, and t-butoxycarbonyl-Gln-Ala-Arg-7-amido-4-MCA fluorescent derivatives were used to assess chymotrypsin-like, post-glutamyl peptidyl-hydrolyzing, and trypsin-like protease activities, respectively. Non-small cell lung cancer cells transiently transfected with an expression plasmid encoding pd1EGFP and treated with FZ showed an accumulation of the green fluorescent protein in the cells due to an increase in its half-life. A number of apoptosis regulatory proteins that are normally degraded by the ubiquitin-proteasome pathway like cyclins, p53, and IκBα were found to be accumulated in FZ-treated cells. In addition, FZ induced distinct ER stress-associated genes like GRP78, GADD153, ATF3, IRE1α, and NOXA in these cells. Thus, treatment of human NSCLC cells with fenbendazole induced endoplasmic reticulum stress, reactive oxygen species production, decreased mitochondrial membrane potential, and cytochrome c release that eventually led to cancer cell death. This is the first report to demonstrate the inhibition of proteasome function and induction of endoplasmic reticulum stress/reactive oxygen species-dependent apoptosis in human lung cancer cell lines by fenbendazole, which may represent a new class of anticancer agents showing selective toxicity against cancer cells.

  7. Sweet potato cysteine proteases SPAE and SPCP2 participate in sporamin degradation during storage root sprouting.

    Science.gov (United States)

    Chen, Hsien-Jung; Liang, Shu-Hao; Huang, Guan-Jhong; Lin, Yaw-Huei

    2015-08-15

    Sweet potato sporamins are trypsin inhibitors and exhibit strong resistance to digestion by pepsin, trypsin and chymotrypsin. In addition, they constitute the major storage proteins in the sweet potato and, after degradation, provide nitrogen as a nutrient for seedling regrowth in sprouting storage roots. In this report, four cysteine proteases-one asparaginyl endopeptidase (SPAE), two papain-like cysteine proteases (SPCP1 and SPCP2), and one granulin-containing cysteine protease (SPCP3)-were studied to determine their association with sporamin degradation in sprouting storage roots. Sporamin degradation became significant in the flesh of storage roots starting from week 4 after sprouting and this correlated with expression levels of SPAE and SPCP2, but not of SPCP1 and SPCP3. In the outer flesh near the skin, sporamin degradation was more evident and occurred earlier than in the inner flesh of storage roots. Degradation of sporamins in the outer flesh was inversely correlated with the distance of the storage root from the sprout. Exogenous application of SPAE and SPCP2, but not SPCP3, fusion proteins to crude extracts of the outer flesh (i.e., extracted from a depth of 0.3cm and within 2cm of one-week-old sprouts) promoted in vitro sporamin degradation in a dose-dependent manner. Pre-treatment of SPAE and SPCP2 fusion proteins at 95°C for 5min prior to their application to the crude extracts reduced sporamin degradation. These data show that sweet potato asparaginyl endopeptidase SPAE and papain-like cysteine protease SPCP2 participate in sporamin degradation during storage root sprouting. PMID:26363719

  8. A Bowman-Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition.

    Science.gov (United States)

    Mehdad, A; Brumana, G; Souza, A A; Barbosa, Jarg; Ventura, M M; de Freitas, S M

    2016-01-01

    Proteasome inhibitors are emerging as a new class of chemopreventive agents and have gained huge importance as potential pharmacological tools in breast cancer treatment. Improved understanding of the role played by proteases and their specific inhibitors in humans offers novel and challenging opportunities for preventive and therapeutic intervention. In this study, we demonstrated that the Bowman-Birk protease inhibitor from Vigna unguiculata seeds, named black-eyed pea trypsin/chymotrypsin Inhibitor (BTCI), potently suppresses human breast adenocarcinoma cell viability by inhibiting the activity of proteasome 20S. BTCI induced a negative growth effect against a panel of breast cancer cells, with a concomitant cytostatic effect at the G2/M phase of the cell cycle and an increase in apoptosis, as observed by an augmented number of cells at the sub-G1 phase and annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining. In contrast, BTCI exhibited no cytotoxic effect on normal mammary epithelial cells. Moreover, the increased levels of intracellular reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in cells treated with BTCI indicated mitochondrial damage as a crucial cellular event responsible for the apoptotic process. The higher activity of caspase in tumoral cells treated with BTCI in comparison with untreated cells suggests that BTCI induces apoptosis in a caspase-dependent manner. BTCI affected NF-kB target gene expression in both non invasive and invasive breast cancer cell lines, with the effect highly pronounced in the invasive cells. An increased expression of interleukin-8 (IL-8) in both cell lines was also observed. Taken together, these results suggest that BTCI promotes apoptosis through ROS-induced mitochondrial damage following proteasome inhibition. These findings highlight the pharmacological potential and benefit of BTCI in breast cancer treatment. PMID:27551492

  9. Chemical and organic fertilizers affect physiological performance and antioxidant

    Directory of Open Access Journals (Sweden)

    M Mardani-Talaee1

    2016-04-01

    Full Text Available Myzus persicae is a widespread and polyphagous insect that causes severe damages to hundreds of host plants. In the current study, zinc sulfate and vermicompost as chemical and organic fertilizers, were added into cultural soil of Capsicum annuum to determine their effects on physiology and antioxidant activities of M. persicae. The aphids reared on zinc sulfate-treated culture showed the highest activities of general protease, trypsin, cathepsins, carboxypeptidase and lipase but activities of chymotrypsin and aminopeptidase were the highest in vermicompost-treated culture. Although activities of α-amylase in the fertilizer-treated cultures were higher than control but activities of α- and β-glucosideases showed the highest values in zinc sulfate and vermicompost treatments, respectively. Aspartate aminotransferase and γ-glutamyl transferase showed the highest activity in the aphids reared on the vermicompost-treated culture but alanine aminotransferase activity got the lowest value in fertilizer-treated cultures. Activities of aldolase and lactate dehydrogenase in the fertilizer-treated aphids were higher than those of control and vermicompost-treated aphids, but alkaline phosphatase showed the lower activity although activity of acid phosphatase decreased in vermicompost- treated aphids compared to other treatments. Activities of antioxidant enzymes were found to be the highest in the aphids fed on vermicompost-treated culture including glucose-6-phosphate dehydrogenase, superoxide dismutase, peroxidase and ascorbate oxidase but catalase in vermicompost treatment had lower activity than control and zinc-sulfate treatments. Also, malondialdehyde and RSSR/RSH ratio demonstrated higher values in the aphids fed on zinc sulfate- and vermicompost-treated plants than control, respectively. Finally, the amounts of glycogen and triglyceride revealed the highest values in zinc sulfate-treated plants compared to other treatments. These results

  10. Purification and characterization of a Bowman-Birk proteinase inhibitor from the seeds of black gram (Vigna mungo).

    Science.gov (United States)

    Prasad, E R; Dutta-Gupta, A; Padmasree, K

    2010-03-01

    A proteinase inhibitor (BgPI) was purified from black gram, Vigna mungo (cv. TAU-1) seeds by using ammonium sulfate fractionation, followed by ion-exchange, affinity and gel-filtration chromatography. BgPI showed a single band in SDS-PAGE under non-reducing condition with an apparent molecular mass of approximately 8kDa correlating to the peak 8041.5Da in matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum. BgPI existed in different isoinhibitor forms with pI values ranging from 4.3 to 6.0. The internal sequence "SIPPQCHCADIR" of a peak 1453.7 m/z, obtained from MALDI-TOF-TOF showed 100% similarity with Bowman-Birk inhibitor (BBI) family. BgPI exhibited non-competitive-type inhibitory activity against both bovine pancreatic trypsin (K(i) of 309.8nM) and chymotrypsin (K(i) of 10.7muM), however, with a molar ratio of 1:2 with trypsin. BgPI was stable up to a temperature of 80 degrees C and active over a wide pH range between 2 and 12. The temperature-induced conformational changes in secondary structure are reversed when BgPI was cooled from 90 to 25 degrees C. Further, upon reduction with dithiothreitol, BgPI lost both its inhibitory activity as well as secondary structural conformation. Lysine residue(s) present in the reactive site of BgPI play an important role in inhibiting the bovine trypsin activity. The present study provides detailed biochemical characteristic features of a BBI type serine proteinase inhibitor isolated from V. mungo.

  11. Studies on prekallikrein of bovine plasma. II. Activation of prekallikrein with proteinases and properties of kallikrein activated by bovine Hageman factor.

    Science.gov (United States)

    Takahashi, H; Nagasawa, S; Suzuki, T

    1980-01-01

    Activation of bovine plasma prekallikrein was investigated with several proteinases. Highly purified bovine plasma prekallikrein was rapidly activated to kallikrein [EC 3.4.21.8] by bovine activated Hageman factor, trypsin [EC 3.4.21.4] and Pronase P (proteinases from Streptomyces griseus) and more gradually by papain [EC 3.4.22.2] and ficin [EC 3.4.22.3]. Activation of prekallikrein was also observed with bovine plasmin [EC 3.4.21.7], but not with bovine clotting factors Xa (Stuart factor) [EC 3.4.21.6] and IXa (Christmas factor) or thrombin [EC 3.4.21.5]. Urokinase [EC 3.4.99.26], Reptilase, collagenase [EC 3.4.24.3], elastase [EC 3.4.21.11], alpha-chymotrypsin [EC 3.4.21.1], Nagarse [EC 3.4.21.14], and stem bromelain [EC 3.4.22 4] did not convert prekallikrein to kallikrein. Plasma kallikrein activated to Hageman factor released kinin rapidly from bovine high molecular weight (HMW) kininogen. However, from bovine low molecular weight (LMW) kininogen, liberation of kinin was extremely slow. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI), Trasylol, diisopropylfluorophosphate (DFP), and N-alpha-tosyl-L-lysine chloromethylketone (TLCK), but not by egg-white trypsin inhibitor (EWTI), lima bean trypsin inhibitor (LBTI), heparin or hexadimethrine bromide (Polybrene). The kallikrein formed an enzyme-inhibitor complex with SBTI and Trasylol, but not with LBTI. Prekallikrein did not react with SBTI. Prekallikrein consists of a single polypeptide chain of molecular weight about 90,000, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Activation of prekallikrein by Hageman factor was found to involve cleavage of the single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation. The peptide bond cleaved in prekallikrein by the activation was an Arg-X peptide bond on a disulfide-bridged polypeptide chain.

  12. A Bowman–Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition

    Science.gov (United States)

    Mehdad, A; Brumana, G; Souza, AA; Barbosa, JARG; Ventura, MM; de Freitas, SM

    2016-01-01

    Proteasome inhibitors are emerging as a new class of chemopreventive agents and have gained huge importance as potential pharmacological tools in breast cancer treatment. Improved understanding of the role played by proteases and their specific inhibitors in humans offers novel and challenging opportunities for preventive and therapeutic intervention. In this study, we demonstrated that the Bowman–Birk protease inhibitor from Vigna unguiculata seeds, named black-eyed pea trypsin/chymotrypsin Inhibitor (BTCI), potently suppresses human breast adenocarcinoma cell viability by inhibiting the activity of proteasome 20S. BTCI induced a negative growth effect against a panel of breast cancer cells, with a concomitant cytostatic effect at the G2/M phase of the cell cycle and an increase in apoptosis, as observed by an augmented number of cells at the sub-G1 phase and annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining. In contrast, BTCI exhibited no cytotoxic effect on normal mammary epithelial cells. Moreover, the increased levels of intracellular reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in cells treated with BTCI indicated mitochondrial damage as a crucial cellular event responsible for the apoptotic process. The higher activity of caspase in tumoral cells treated with BTCI in comparison with untreated cells suggests that BTCI induces apoptosis in a caspase-dependent manner. BTCI affected NF-kB target gene expression in both non invasive and invasive breast cancer cell lines, with the effect highly pronounced in the invasive cells. An increased expression of interleukin-8 (IL-8) in both cell lines was also observed. Taken together, these results suggest that BTCI promotes apoptosis through ROS-induced mitochondrial damage following proteasome inhibition. These findings highlight the pharmacological potential and benefit of BTCI in breast cancer treatment. PMID:27551492

  13. Aspartic proteinases in the digestive tract of marine decapod crustaceans.

    Science.gov (United States)

    Navarrete del Toro, María de Los Angeles; García-Carreño, Fernando; López, Manuel Díaz; Celis-Guerrero, Laura; Saborowski, Reinhard

    2006-08-01

    Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others. PMID:16788916

  14. Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion.

    Directory of Open Access Journals (Sweden)

    Yanan Hou

    Full Text Available The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1. Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.

  15. Purification and characterization of a trypsin inhibitor from the seeds of Artocarpus heterophyllus Lam.

    Science.gov (United States)

    Lyu, Junchen; Liu, Yuan; An, Tianchen; Liu, Yujun; Wang, Manchuriga; Song, Yanting; Zheng, Feifei; Wu, Dan; Zhang, Yingxia; Deng, Shiming

    2015-05-01

    A proteinaceous inhibitor against trypsin was isolated from the seeds of Artocarpus heterophyllus Lam. by successive ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatography. The trypsin inhibitor, named as AHLTI (A. heterophyllus Lam. trypsin inhibitor), consisted of a single polypeptide chain with a molecular weight of 28.5 kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration chromatography. The N-terminal sequence of AHLTI was DEPPSELDAS, which showed no similarity to other known trypsin inhibitor sequence. AHLTI completely inhibited bovine trypsin at a molar ratio of 1:2 (AHLTI:trypsin) analyzed by native polyacrylamide gel electrophoresis, inhibition activity assay, and gel-filtration chromatography. Moreover, kinetic enzymatic studies were carried out to understand the inhibition mechanism of AHLTI against trypsin. Results showed that AHLTI was a competitive inhibitor with an equilibrium dissociation constant (Ki) of 3.7 × 10(-8) M. However, AHLTI showed weak inhibitory activity toward chymotrypsin and elastase. AHLTI was stable over a broad range of pH 4-8 and temperature 20-80°C. The reduction agent, dithiothreitol, had no obvious effect on AHLTI. The trypsin inhibition assays of AHLTI toward digestive enzymes from insect pest guts in vitro demonstrated that AHLTI was effective against enzymes from Locusta migratoria manilensis (Meyen). These results suggested that AHLTI might be a novel trypsin inhibitor from A. heterophyllus Lam. belonging to Kunitz family, and play an important role in protecting from insect pest. PMID:25851516

  16. Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus.

    Science.gov (United States)

    Ranasinghe, Shiwanthi L; Fischer, Katja; Zhang, Wenbao; Gobert, Geoffrey N; McManus, Donald P

    2015-12-01

    The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic.

  17. Isolation and Characterization of Gut Bacterial Proteases Involved in Inducing Pathogenicity of Bacillus thuringiensis Toxin in Cotton Bollworm, Helicoverpa armigera

    Science.gov (United States)

    Regode, Visweshwar; Kuruba, Sreeramulu; Mohammad, Akbar S.; Sharma, Hari C.

    2016-01-01

    Bacillus thuringiensis toxin proteins are deployed in transgenic plants for pest management. The present studies were aimed at characterization of gut bacterial proteases involved in activation of inactive Cry1Ac protoxin (pro-Cry1Ac) to active toxin in Helicoverpa armigera. Bacterial strains were isolated from H. armigera midgut and screened for their proteolytic activation toward pro-Cry1Ac. Among 12 gut bacterial isolates seven isolates showed proteolytic activity, and proteases from three isolates (IVS1, IVS2, and IVS3) were found to be involved in the proteolytic conversion of pro-Cry1Ac into active toxin. The proteases from IVS1, IVS2, and IVS3 isolates were purified to 11.90-, 15.50-, and 17.20-fold, respectively. The optimum pH and temperature for gut bacterial protease activity was 8.0 and 40°C. Maximum inhibition of total proteolytic activity was exerted by phenylmethane sulfonyl fluoride followed by EDTA. Fluorescence zymography revealed that proteases from IVS1, IVS2, and IVS3 were chymotrypsin-like and showing protease band at ~15, 65, and 15 kDa, respectively. Active Cry1Ac formed from processing pro-Cry1Ac by gut bacterial proteases exhibited toxicity toward H. armigera. The gut bacterial isolates IVS1, IVS2, and IVS3 showed homology with B. thuringiensis (CP003763.1), Vibrio fischeri (CP000020.2), and Escherichia coli (CP011342.1), respectively. Proteases produced by midgut bacteria are involved in proteolytic processing of B. thuringiensis protoxin and play a major role in inducing pathogenicity of B. thuringiensis toxins in H. armigera. PMID:27766093

  18. Increasing intracellular bioavailable copper selectively targets prostate cancer cells.

    Science.gov (United States)

    Cater, Michael A; Pearson, Helen B; Wolyniec, Kamil; Klaver, Paul; Bilandzic, Maree; Paterson, Brett M; Bush, Ashley I; Humbert, Patrick O; La Fontaine, Sharon; Donnelly, Paul S; Haupt, Ygal

    2013-07-19

    The therapeutic efficacy of two bis(thiosemicarbazonato) copper complexes, glyoxalbis[N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(gtsm)] and diacetylbis[N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(atsm)], for the treatment of prostate cancer was assessed in cell culture and animal models. Distinctively, copper dissociates intracellularly from Cu(II)(gtsm) but is retained by Cu(II)(atsm). We further demonstrated that intracellular H2gtsm [reduced Cu(II)(gtsm)] continues to redistribute copper into a bioavailable (exchangeable) pool. Both Cu(II)(gtsm) and Cu(II)(atsm) selectively kill transformed (hyperplastic and carcinoma) prostate cell lines but, importantly, do not affect the viability of primary prostate epithelial cells. Increasing extracellular copper concentrations enhanced the therapeutic capacity of both Cu(II)(gtsm) and Cu(II)(atsm), and their ligands (H2gtsm and H2atsm) were toxic only toward cancerous prostate cells when combined with copper. Treatment of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model with Cu(II)(gtsm) (2.5 mg/kg) significantly reduced prostate cancer burden (∼70%) and severity (grade), while treatment with Cu(II)(atsm) (30 mg/kg) was ineffective at the given dose. However, Cu(II)(gtsm) caused mild kidney toxicity in the mice, associated primarily with interstitial nephritis and luminal distention. Mechanistically, we demonstrated that Cu(II)(gtsm) inhibits proteasomal chymotrypsin-like activity, a feature further established as being common to copper-ionophores that increase intracellular bioavailable copper. We have demonstrated that increasing intracellular bioavailable copper can selectively kill cancerous prostate cells in vitro and in vivo and have revealed the potential for bis(thiosemicarbazone) copper complexes to be developed as therapeutics for prostate cancer.

  19. Advances in the etiology of chronic pancreatitis.

    Science.gov (United States)

    Lerch, Markus M; Mayerle, Julia; Aghdassi, Ali A; Budde, Christoph; Nitsche, Claudia; Sauter, Gabriele; Persike, Maria; Günther, Annett; Simon, Peter; Weiss, F Ulrich

    2010-01-01

    In the past, chronic pancreatitis has been regarded as a fairly uniform and largely untreatable disorder that most commonly affects patients who both lack gainful employment or adequate insurance coverage and have a tendency to smoke and drink. Large clinical trials suggest that this perception is not only misguided and discriminatory but also not based on facts. We forgot that the perception of chronic liver disease was similar before World War II, and just like liver cirrhosis the fibrosis and cirrhosis of the pancreas--i.e. chronic pancreatitis--is the end result of a range of environmental, inflammatory, infectious and genetic disorders. A growing number of these have only recently been recognized as a distinct entity and several of which are becoming truly treatable. A large proportion of the risk for developing pancreatitis is conveyed by genetic risk factors, and we estimate that less than half of those have been identified so far. The same holds true for protective factors that can prevent pancreatitis, even in the face of excessive alcohol abuse. Various gene mutations and polymorphisms appear to determine an individual's susceptibility for developing pancreatic disease, for the severity of the disease, and for the disease progression. The spectrum of genotype/phenotype associations ranges from straightforward autosomal dominant traits with near-complete penetrance, as for the most common mutations in the cationic trypsinogen gene (PRSS1), to moderate risks factors without mendelian inheritance patterns, as for SPINK1 and CFTR mutations, to very subtle risk associations and disease modifiers that can only be identified in large cohort studies, as for the chymotrypsin C, calcium-sensing receptor and the anionic trypsin (PRSS2) mutations. Only a better understanding of the disease mechanisms that underlie these changes will make an individualized therapy of pancreatic disorders a realistic option. PMID:20814206

  20. The Kunitz-like modulatory protein haemangin is vital for hard tick blood-feeding success.

    Directory of Open Access Journals (Sweden)

    M Khyrul Islam

    2009-07-01

    Full Text Available Ticks are serious haematophagus arthropod pests and are only second to mosquitoes as vectors of diseases of humans and animals. The salivary glands of the slower feeding hard ticks such as Haemaphysalis longicornis are a rich source of bioactive molecules and are critical to their biologic success, yet distinct molecules that help prolong parasitism on robust mammalian hosts and achieve blood-meals remain unidentified. Here, we report on the molecular and biochemical features and precise functions of a novel Kunitz inhibitor from H. longicornis salivary glands, termed Haemangin, in the modulation of angiogenesis and in persistent blood-feeding. Haemangin was shown to disrupt angiogenesis and wound healing via inhibition of vascular endothelial cell proliferation and induction of apoptosis. Further, this compound potently inactivated trypsin, chymotrypsin, and plasmin, indicating its antiproteolytic potential on angiogenic cascades. Analysis of Haemangin-specific gene expression kinetics at different blood-feeding stages of adult ticks revealed a dramatic up-regulation prior to complete feeding, which appears to be functionally linked to the acquisition of blood-meals. Notably, disruption of Haemangin-specific mRNA by a reverse genetic tool significantly diminished engorgement of adult H. longicornis, while the knock-down ticks failed to impair angiogenesis in vivo. To our knowledge, we have provided the first insights into transcriptional responses of human microvascular endothelial cells to Haemangin. DNA microarray data revealed that Haemangin altered the expression of 3,267 genes, including those of angiogenic significance, further substantiating the antiangiogenic function of Haemangin. We establish the vital roles of Haemangin in the hard tick blood-feeding process. Moreover, our results provide novel insights into the blood-feeding strategies that enable hard ticks to persistently feed and ensure full blood-meals through the modulation of

  1. Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases.

    Science.gov (United States)

    Bougatef, Ali; Nedjar-Arroume, Naima; Ravallec-Plé, Rozenn; Leroy, Yves; Guillochon, Didier; Barkia, Ahmed; Nasri, Moncef

    2008-11-15

    The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from heads and viscera of sardinelle (Sardinella aurita) by treatment with various proteases were investigated. Protein hydrolysates were obtained by treatment with Alcalase(®), chymotrypsin, crude enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina pilchardus) viscera. All hydrolysates exhibited inhibitory activity towards ACE. The alkaline protease extract from the viscera of sardine produced hydrolysate with the highest ACE inhibitory activity (63.2±1.5% at 2mg/ml). Further, the degrees of hydrolysis and the inhibitory activities of ACE increased with increasing proteolysis time. The protein hydrolysate generated with alkaline proteases from the viscera of sardine was then fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P1-P8). Biological functions of all fractions were assayed, and P4 was found to display a high ACE inhibitory activity. The IC50 values for ACE inhibitory activities of sardinelle by-products protein hydrolysates and fraction P4 were 1.2±0.09 and 0.81±0.013mg/ml, respectively. Further, P4 showed resistance to in vitro digestion by gastrointestinal proteases. The amino acid analysis by GC/MS showed that P4 was rich in phenylalanine, arginine, glycine, leucine, methionine, histidine and tyrosine. The added-value of sardinelle by-products may be improved by enzymatic treatment with visceral serine proteases from sardine. PMID:26047434

  2. Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs.

    Science.gov (United States)

    Chen, Xiaole; Wang, He; Shen, Yue; Wang, Lei; Zhou, Mei; Chen, Tianbao; Shaw, Chris

    2016-08-19

    Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to "shotgun" clone the cDNAs encoding two novel but structurally-related peptides from the lyophilised skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin. PMID:27311856

  3. Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb, Cry3Ca, and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentidae)

    Science.gov (United States)

    Hernández-Martínez, Patricia; Vera-Velasco, Natalia Mara; Martínez-Solís, María; Ghislain, Marc; Ferré, Juan

    2014-01-01

    Bacillus thuringiensis Cry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genus Cylas, which are important pests of sweet potato worldwide and particularly in sub-Saharan Africa. However, relatively little is known about the processing and binding interactions of these coleopteran-specific Cry proteins. The aim of the present study was to determine whether Cry3Bb, Cry3Ca, and Cry7Aa proteins have shared binding sites in Cylas puncticollis to orient the pest resistance strategy by genetic transformation. Interestingly, processing of the 129-kDa Cry7Aa protoxin using commercial trypsin or chymotrypsin rendered two fragments of about 70 kDa and 65 kDa. N-terminal sequencing of the trypsin-activated Cry7Aa fragments revealed that processing occurs at Glu47 for the 70-kDa form or Ile88 for the 65-kDa form. Homologous binding assays showed specific binding of the two Cry3 proteins and the 65-kDa Cry7Aa fragment to brush border membrane vesicles (BBMV) from C. puncticollis larvae. The 70-kDa fragment did not bind to BBMV. Heterologous-competition assays showed that Cry3Bb, Cry3Ca, and Cry7Aa (65-kDa fragment) competed for the same binding sites. Hence, our results suggest that pest resistance mediated by the alteration of a shared Cry receptor binding site might render all three Cry toxins ineffective. PMID:25261517

  4. New poly(ester urea) derived from L-leucine: Electrospun scaffolds loaded with antibacterial drugs and enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Díaz, Angélica; Valle, Luis J. del [Departament d' Enginyeria Química, Universitat Politècnica de Catalunya, Av. Diagonal 647, Barcelona E-08028 (Spain); Tugushi, David; Katsarava, Ramaz [Institute of Chemistry and Molecular Engineering, Agricultural University of Georgia, 13 km. David Aghmashenebeli Alley, Tblisi 0131, Georgia (United States); Puiggalí, Jordi, E-mail: Jordi.Puiggali@upc.edu [Departament d' Enginyeria Química, Universitat Politècnica de Catalunya, Av. Diagonal 647, Barcelona E-08028 (Spain)

    2015-01-01

    Electrospun scaffolds from an amino acid containing poly(ester urea) (PEU) were developed as promising materials in the biomedical field and specifically in tissue engineering applications. The selected poly(ester urea) was obtained with a high yield and molecular weight by reaction of phosgene with a bis(α-aminoacyl)-α,ω-diol-diester monomer. The polymer having L-leucine, 1,6-hexanediol and carbonic acid units had a semicrystalline character and relatively high glass transition and melting temperatures. Furthermore it was highly soluble in most organic solvents, an interesting feature that facilitated the electrospinning process and the effective incorporation of drugs with bactericidal activity (e.g. biguanide derivatives such as clorhexidine and polyhexamethylenebiguanide) and enzymes (e.g. α-chymotrypsin) that accelerated the degradation process. Continuous micro/nanofibers were obtained under a wide range of processing conditions, being diameters of electrospun fibers dependent on the drug and solvent used. Poly(ester urea) samples were degradable in media containing lipases and proteinases but the degradation rate was highly dependent on the surface area, being specifically greater for scaffolds with respect to films. The high hydrophobicity of new scaffolds had repercussions on enzymatic degradability since different weight loss rates were found depending on how samples were exposed to the medium (e.g. forced or non-forced immersion). New scaffolds were biocompatible, as demonstrated by adhesion and proliferation assays performed with fibroblast and epithelial cells. - Highlights: • Electrospun scaffolds from a biodegradable poly(ester urea) have been prepared. • Scaffolds were effectively loaded with bactericide agents. • Enzymatic degradability of the L-leucine derived poly(ester urea) was demonstrated. • Enzymes that accelerate degradation were incorporated in the electrospun fibers. • Cell adhesion/proliferation assays demonstrated

  5. Three genes expressing Kunitz domains in the epididymis are related to genes of WFDC-type protease inhibitors and semen coagulum proteins in spite of lacking similarity between their protein products

    Directory of Open Access Journals (Sweden)

    Lilja Hans

    2011-10-01

    Full Text Available Abstract Background We have previously identified a locus on human chromosome 20q13.1, encompassing related genes of postulated WFDC-type protease inhibitors and semen coagulum proteins. Three of the genes with WFDC motif also coded for the Kunitz-type protease inhibitor motif. In this report, we have reinvestigated the locus for homologous genes encoding Kunitz motif only. The identified genes have been analyzed with respect to structure, expression and function. Results We identified three novel genes; SPINT3, SPINT4 and SPINT5, and the structure of their transcripts were determined by sequencing of DNA generated by rapid amplification of cDNA ends. Each gene encodes a Kunitz domain preceded by a typical signal peptide sequence, which indicates that the proteins of 7.6, 8.7, and 9.7 kDa are secreted. Analysis of transcripts in 26 tissues showed that the genes predominantly are expressed in the epididymis. The recombinantly produced proteins could not inhibit the amidolytic activity of trypsin, chymotrypsin, plasmin, thrombin, coagulation factor Xa, elastase, urokinase and prostate specific antigen, whereas similarly made bovine pancreatic trypsin inhibitor (BPTI had the same bioactivity as the protein isolated from bovine pancreas. Conclusions The similar organization, chromosomal location and site of expression, suggests that the novel genes are homologous with the genes of WFDC-type protease inhibitors and semen coagulum proteins, despite the lack of similarity in primary structure of their protein products. Their restricted expression to the epididymis suggests that they could be important for male reproduction. The recombinantly produced proteins are presumably bioactive, as demonstrated with similarly made BPTI, but may have a narrower spectrum of inhibition, as indicated by the lacking activity against eight proteases with differing specificity. Another possibility is that they have lost the protease inhibiting properties, which is

  6. Nanomolar Inhibitors of AmpC [beta]-Lactamase

    Energy Technology Data Exchange (ETDEWEB)

    Morandi, Federica; Caselli, Emilia; Morandi, Stefania; Focia, Pamela J.; Blazquez, Jesus; Shoichet, Brian K.; Prati, Fabio (Degali); (NIH); (NWU); (UCSF)

    2010-03-08

    {beta}-lactamases are the most widespread resistance mechanism to {beta}-lactam antibiotics, such as the penicillins and the cephalosporins. In an effort to combat these enzymes, a combination of stereoselective organic synthesis, enzymology, microbiology, and X-ray crystallography was used to design and evaluate new carboxyphenyl-glycylboronic acid transition-state analogue inhibitors of the class C {beta}-lactamase AmpC. The new compounds improve inhibition by over 2 orders of magnitude compared to analogous glycylboronic acids, with K{sub i} values as low as 1 nM. On the basis of the differential binding of different analogues, the introduced carboxylate alone contributes about 2.1 kcal/mol in affinity. This carboxylate corresponds to the ubiquitous C3(4)' carboxylate of {beta}-lactams, and this energy represents the first thermodynamic measurement of the importance of this group in molecular recognition by class C {beta}-lactamases. The structures of AmpC in complex with two of these inhibitors were determined by X-ray crystallography at 1.72 and 1.83 {angstrom} resolution. These structures suggest a structural basis for the high affinity of the new compounds and provide templates for further design. The highest affinity inhibitor was 5 orders of magnitude more selective for AmpC than for characteristic serine proteases, such as chymotrypsin. This inhibitor reversed the resistance of clinical pathogens to the third generation cephalosporin ceftazidime; it may serve as a lead compound for drug discovery to combat bacterial resistance to {beta}-lactam antibiotics.

  7. Mapping the structural domains of E. coli carbamoyl phosphate synthetase using limited proteolysis.

    Science.gov (United States)

    Mareya, S M; Raushel, F M

    1995-05-01

    The structural and functional domains of Escherichia coli carbamoyl phosphate synthetase (CPS) have been identified by limited proteolysis. Incubation of CPS with several proteases, including trypsin, chymotrypsin, subtilisin and endoproteinase Asp-N, under native conditions, causes a time-dependent loss of enzymatic activity and the generation of a common fragmentation pattern. Amino-terminal sequencing studies demonstrated that the initial cleavage event by trypsin occurred at the carboxy-terminal end of the large subunit. The ultimate fragments produced in most of the proteolysis studies, 35- and 45-kDa peptides, were derived from areas corresponding to the putative ATP binding regions. Substrate protection studies showed that the addition of ligands did not affect the final fragmentation pattern of the protein. However, ornithine and UMP were found to significantly reduce the rate of inactivation by inhibition of proteolytic cleavage. MgATP and IMP provided modest protection whereas bicarbonate and glutamine showed no overall effect on proteolysis. Limited proteolysis by endoproteinase Asp-N resulted in the production of a fragment (or multiple fragments) which contained enzymatic activity but had lost all regulation by the allosteric ligands, UMP and ornithine. The small subunit has been shown to be protected from proteolysis by the large subunit. Proteolysis of the isolated small subunit resulted in the generation of a stable 31-kDa species which contained 10% of the original glutaminase activity. These studies demonstrate that a portion of the C-terminal end of the large subunit can be excised without entirely destroying the ability of CPS to catalyze the formation of carbamoyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7648201

  8. Transamidation of gluten proteins during the bread-making process of wheat flour to produce breads with less immunoreactive gluten.

    Science.gov (United States)

    Heredia-Sandoval, Nina Gisella; Islas-Rubio, Alma Rosa; Cabrera-Chávez, Francisco; Calderón de la Barca, Ana María

    2014-08-01

    Due to an increasing incidence of celiac disease (CD) and other gluten-related disorders, different gluten-free breads have been developed using starches and additives as a substitute for gluten. Thus, patients miss not only the taste and aroma of wheat bread but also risk their sensitive intestines. Therefore, modifying gluten to avoid an immune response in CD and its application to baking is in progress. The aim of the study was to enzymatically modify gluten on wheat flour, during bread-making avoiding the use of additives, to reduce immunoreactivity, preserving its properties. Microbial transglutaminase (mTG) or chymotrypsin (ChT) was used to bind lysine or valine to gluten proteins in a model system. The best conditions were directly applied to wheat flour for bread-making with and without punching at 45 min. Subsequently, the rheological properties of the doughs, specific volume of the loaves, immunoreactive gluten content and modification of the extracted proteins were evaluated. ChT-treated breads presented a better appearance with a more homogeneous crumb, higher specific volume values (3.34-4.25 cm(3) g(-1)) and higher reactive gluten reduction (up to 71%) than the mTG-treated ones (1.23-2.66 cm(3) g(-1)) with only a 42% reactive gluten reduction. Thus, transpeptidation during bread-making is a promising technology, although it is necessary to improve the modification process to obtain the reactive gluten reduction required in breads for the treatment of CD patients and other gluten-related disorders. PMID:24917417

  9. Selective anticancer copper(II)-mixed ligand complexes: targeting of ROS and proteasomes.

    Science.gov (United States)

    Ng, Chew Hee; Kong, Siew Ming; Tiong, Yee Lian; Maah, Mohd Jamil; Sukram, Nurhazwani; Ahmad, Munirah; Khoo, Alan Soo Beng

    2014-04-01

    Copper compounds can be alternatives to platinum-based anticancer drugs. This study investigated the effects of a series of ternary copper(II) complexes, [Cu(phen)(aa)(H2O)]NO3·xH2O 1-4 (phen = 1,10-phenanthroline; aa = gly (1), DL-ala (2), sar (3), C-dmg (4)), on metastatic and cisplatin-resistant MDA-MB-231 breast cancer cells and MCF10A non-cancerous breast cells, and some aspects of the mechanisms. These complexes were distinctively more antiproliferative towards and induced greater apoptotic cell death in MDA-MB-231 than in MCF10A cells. 2 and 4 could induce cell cycle arrest only in cancer cells. Further evidence from DCFH-DA assay showed higher induction of reactive oxygen species (ROS) in treated cancer cells but minimal ROS increase in normal cells. DNA double-strand breaks, via a γ-H2AX assay, were only detected in cancer cells treated with 5 μM of the complexes. These complexes poorly inhibited chymotrypsin-like activity in the 20S rabbit proteasome while they did not inhibit the three proteolytic sites of MDA-MB-231 cells at 10 μM. However, the complexes could inhibit degradation of ubiquinated proteins of MDA-MB-231 cells. In addition, compound 4 was found to be effective against cervical (Hela), ovarian (SKOV3), lung (A549, PC9), NPC (Hone1, HK1, C666-1), breast (MCF7, T47D), lymphoma and leukemia (Nalmawa, HL60) and colorectal (SW480, SW48, HCT118) cancer cell lines with IC50 values (24 h) in the 1.7-19.0 μM range. Single dose NCI60 screening of 4 showed the complex to be highly cytotoxic to most cancer cell types and more effective than cisplatin.

  10. Serine Protease-mediated Host Invasion by the Parasitic Nematode Steinernema carpocapsae*

    Science.gov (United States)

    Toubarro, Duarte; Lucena-Robles, Miguel; Nascimento, Gisela; Santos, Romana; Montiel, Rafael; Veríssimo, Paula; Pires, Euclides; Faro, Carlos; Coelho, Ana V.; Simões, Nelson

    2010-01-01

    Steinernema carpocapsae is an insect parasitic nematode used in biological control, which infects insects penetrating by mouth and anus and invading the hemocoelium through the midgut wall. Invasion has been described as a key factor in nematode virulence and suggested to be mediated by proteases. A serine protease cDNA from the parasitic stage was sequenced (sc-sp-1); the recombinant protein was produced in an Escherichia coli system, and a native protein was purified from the secreted products. Both proteins were confirmed by mass spectrometry to be encoded by the sc-sp-1 gene. Sc-SP-1 has a pI of 8.7, a molecular mass of 27.3 kDa, a catalytic efficiency of 22.2 × 104 s−1 m−1 against N-succinyl-Ala-Ala-Pro-Phe-pNA, and is inhibited by chymostatin (IC 0.07) and PMSF (IC 0.73). Sc-SP-1 belongs to the chymotrypsin family, based on sequence and biochemical analysis. Only the nematode parasitic stage expressed sc-sp-1. These nematodes in the midgut lumen, prepared to invade the insect hemocoelium, expressed higher levels than those already in the hemocoelium. Moreover, parasitic nematode sense insect peritrophic membrane and hemolymph more quickly than they do other tissues, which initiates sc-sp-1 expression. Ex vivo, Sc-SP-1 was able to bind to insect midgut epithelium and to cause cell detachment from basal lamina. In vitro, Sc-SP-1 formed holes in an artificial membrane model (Matrigel), whereas Sc-SP-1 treated with PMSF did not, very likely because it hydrolyzes matrix glycoproteins. These findings highlight the S. carpocapsae-invasive process that is a key step in the parasitism thus opening new perspectives for improving nematode virulence to use in biological control. PMID:20656686

  11. Molecular Cloning and Functional Studies of Two Kazal-Type Serine Protease Inhibitors Specifically Expressed by Nasonia vitripennis Venom Apparatus

    Directory of Open Access Journals (Sweden)

    Cen Qian

    2015-08-01

    Full Text Available Two cDNA sequences of Kazal-type serine protease inhibitors (KSPIs in Nasonia vitripennis, NvKSPI-1 and NvKSPI-2, were characterized and their open reading frames (ORFs were 198 and 264 bp, respectively. Both NvKSPI-1 and NvKSPI-2 contained a typical Kazal-type domain. Real-time quantitative PCR (RT-qPCR results revealed that NvKSPI-1 and NvKSPI-2 mRNAs were mostly detected specifically in the venom apparatus, while they were expressed at lower levels in the ovary and much lower levels in other tissues tested. In the venom apparatus, both NvKSPI-1 and NvKSPI-2 transcripts were highly expressed on the fourth day post eclosion and then declined gradually. The NvKSPI-1 and NvKSPI-2 genes were recombinantly expressed utilizing a pGEX-4T-2 vector, and the recombinant products fused with glutathione S-transferase were purified. Inhibition of recombinant GST-NvKSPI-1 and GST-NvKSPI-2 to three serine protease inhibitors (trypsin, chymotrypsin, and proteinase K were tested and results showed that only NvKSPI-1 could inhibit the activity of trypsin. Meanwhile, we evaluated the influence of the recombinant GST-NvKSPI-1 and GST-NvKSPI-2 on the phenoloxidase (PO activity and prophenoloxidase (PPO activation of hemolymph from a host pupa, Musca domestica. Results showed PPO activation in host hemolymph was inhibited by both recombinant proteins; however, there was no significant inhibition on the PO activity. Our results suggested that NvKSPI-1 and NvKSPI-2 could inhibit PPO activation in host hemolymph and trypsin activity in vitro.

  12. Colicin type 7 produced by majority of Shigella sonnei isolated from Thai patients with diarrhoea

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    Siriporn Kaewklom

    2013-09-01

    Full Text Available Thirty one out of 153 strains of Shigella sonnei isolated from Thai patients with diarrhoea showed antibacterial activity against S. sonnei by agar well diffusion method. All of them harbor plasmids with the genetic determination of colicin type 7 (Js gene but without colicin E and colicin U gene. The PCR product obtained from strain 35/44 was shown to be the gene for colicin type 7 lytic protein (cja. The partially purified bacteriocin (PPB containing colicin type 7 of strain 35/44 was prepared and used for characterization. The antibacterial activity of PPB against a total of 17 selected Gram-positive and Gram-negative bacteria was tested. It was found that PPB of strain 35/44 was active against E. coli O157, S. sonnei and S. boydii. The sensitivity of PPB from this strain to proteinase K, trypsin and α-chymotrypsin suggests the proteinaceous nature of these antimicrobial substances. Therefore, this isolated bacterium can be regarded as bacteriocin producing bacteria. The bacteriocin produced by this isolated S. sonnei was heat stable as evidenced by its ability to maintain the activity at 80 °C for 60 min. In addition, it was stable within a wide range of pH (3-9. The molecular weight of colicin type 7 from isolated S. sonnei strain 35/44 analyzed by SDS-PAGE was 54.4 kDa composing of at least five subunits. It is to our knowledge; the first report of Thai patients with diarrhoea that S. sonnei isolated from them contained colicin type 7.

  13. Isolation and Characterization of a Broad Spectrum Bacteriocin from Bacillus amyloliquefaciens RX7

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    Kong Boon Lim

    2016-01-01

    Full Text Available We isolated a Bacillus strain, RX7, with inhibitory activity against Listeria monocytogenes from soil and identified it as Bacillus amyloliquefaciens based on 16S rRNA gene sequencing. The inhibitory activity was stable over a wide range of pH and was fully retained after 30 min at 80°C, after which it decreased gradually at higher temperatures. The activity was sensitive to the proteolytic action of α-chymotrypsin, proteinase-K, and trypsin, indicating its proteinaceous nature. This bacteriocin was active against a broad spectrum of bacteria and the fungus Candida albicans. Direct detection of antimicrobial activity on a sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of approximately 5 kDa. Ammonium sulfate precipitation and anion-exchange and gel permeation chromatography integrated with reverse phase-high-performance liquid chromatography were used for bacteriocin purification. Automated N-terminal Edman degradation of the purified RX7 bacteriocin recognized the first 15 amino acids as NH2-X-Ala-Trp-Tyr-Asp-Ile-Arg-Lys-Leu-Gly-Asn-Lys-Gly-Ala, where the letter X in the sequence indicates an unknown or nonstandard amino acid. Based on BLAST similarity search and multiple alignment analysis, the obtained partial sequence showed high homology with the two-peptide lantibiotic haloduracin (HalA1 from Bacillus halodurans, although at least two amino acids differed between the sequences. A time-kill study demonstrated a bactericidal mode of action of RX7 bacteriocin.

  14. Absolute Quantification of Prion Protein (90-231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow

    Science.gov (United States)

    Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M.; Pedersen, Joel A.; Li, Lingjun

    2012-09-01

    Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

  15. Anticancer Effect of a Novel Proteasome Inhibitor, YSY01A, via G2/M Arrest in PC-3M Cells in vitro and in vivo.

    Science.gov (United States)

    Zhang, Mei; Yuan, Xia; Xu, Bo; Guo, Wei; Ran, Fu-Xiang; Li, Run-Tao; Cui, Jing-Rong

    2015-01-01

    YSY01A is a new tripeptideboronic acid and an analog of PS341. However, YSY01A's antitumor effects and mechanism have not yet been elucidated. This study demonstrates that YSY01A inhibited proteasome activity by combining with the chymotrypsin-like (CT-L) site (β5i/β5), the post-glutamyl peptide hydrolase (PGPH) site (β1i/β1) and the trypsin-like (T-L) site (β2i/β2) in special fluorgonic substrates and proteasome probe tests. We explored the anticancer effect using methyl thiazolyltetrazolium (MTT) or sulforhodamine B (SRB), and PC-3M cells were sensitive to YSY01A among the four cancer cell types tested. The YSY01A antiproliferative effect was stronger than that of PS341. In vivo, YSY01A (1.25, 2.25, and 3.25 mg/kg) inhibited PC-3M cell xenograft tumor growth, and the tumor volume inhibition rate was approximately 40% to 60%. YSY01A arrested PC-3M cells in the G2/M phase of the cell cycle by flow cytometry (FCM). Many proteins related to the cell cycle were analyzed using western blot, and YSY01A was shown to increase p21, p27, cyclinB1, P-cdc2 (tyr15) and wee1 protein expression in both cells and tumor tissue in a concentration-dependent manner. YSY01A, a proteasome inhibitor, exerts anticancer effects on PC-3M cells in vitro and in vivo. The mechanism of the YSY01A-mediated antitumor effect is that the cell cycle is arrested at the G2/M stage. This study suggests that YSY01A may be a novel therapeutic agent for prostate cancer. PMID:26185531

  16. Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis.

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    Nora Semren

    Full Text Available Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ, provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ. OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution.

  17. Higher insulin sensitivity in EDL muscle of rats fed a low-protein, high-carbohydrate diet inhibits the caspase-3 and ubiquitin-proteasome proteolytic systems but does not increase protein synthesis.

    Science.gov (United States)

    Dos Santos, Maísa Pavani; Batistela, Emanuele; Pereira, Mayara Peron; Paula-Gomes, Silvia; Zanon, Neusa Maria; Kettelhut, Isis do Carmo; Karatzaferi, Christina; Andrade, Claudia Marlise Balbinotti; de França, Suélem Aparecida; Baviera, Amanda Martins; Kawashita, Nair Honda

    2016-08-01

    Compared with the extensor digitorum longus (EDL) muscle of control rats (C), the EDL muscle of rats fed a low-protein, high-carbohydrate diet (LPHC) showed a 36% reduction in mass. Muscle mass is determined by the balance between protein synthesis and proteolysis; thus, the aim of this work was to evaluate the components involved in these processes. Compared with the muscle from C rats, the EDL muscle from LPHC diet-fed rats showed a reduction (34%) in the in vitro basal protein synthesis and a 22% reduction in the in vitro basal proteolysis suggesting that the reduction in the mass can be associated with a change in the rate of the two processes. Soon after euthanasia, in the EDL muscles of the rats fed the LPHC diet for 15days, the activity of caspase-3 and that of components of the ubiquitin-proteasome system (atrogin-1 content and chymotrypsin-like activity) were decreased. The phosphorylation of p70(S6K) and 4E-BP1, proteins involved in protein synthesis, was also decreased. We observed an increase in the insulin-stimulated protein content of p-Akt. Thus, the higher insulin sensitivity in the EDL muscle of LPHC rats seemed to contribute to the lower proteolysis in LPHC rats. However, even with the higher insulin sensitivity, the reduction in p-E4-BP1 and p70(S6K) indicates a reduction in protein synthesis, showing that factors other than insulin can have a greater effect on the control of protein synthesis. PMID:27239756

  18. An Ethylene-Protected Achilles’ Heel of Etiolated Seedlings for Arthropod Deterrence

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    Boex-Fontvieille, Edouard; Rustgi, Sachin; von Wettstein, Diter; Pollmann, Stephan; Reinbothe, Steffen; Reinbothe, Christiane

    2016-01-01

    A small family of Kunitz protease inhibitors exists in Arabidopsis thaliana, a member of which (encoded by At1g72290) accomplishes highly specific roles during plant development. Arabidopsis Kunitz-protease inhibitor 1 (Kunitz-PI;1), as we dubbed this protein here, is operative as cysteine PI. Activity measurements revealed that despite the presence of the conserved Kunitz-motif the bacterially expressed Kunitz-PI;1 was unable to inhibit serine proteases such as trypsin and chymotrypsin, but very efficiently inhibited the cysteine protease RESPONSIVE TO DESICCATION 21. Western blotting and cytolocalization studies using mono-specific antibodies recalled Kunitz-PI;1 protein expression in flowers, young siliques and etiolated seedlings. In dark-grown seedlings, maximum Kunitz-PI;1 promoter activity was detected in the apical hook region and apical parts of the hypocotyls. Immunolocalization confirmed Kunitz-PI;1 expression in these organs and tissues. No transmitting tract (NTT) and HECATE 1 (HEC1), two transcription factors previously implicated in the formation of the female reproductive tract in flowers of Arabidopsis, were identified to regulate Kunitz-PI;1 expression in the dark and during greening, with NTT acting negatively and HEC1 acting positively. Laboratory feeding experiments with isopod crustaceans such as Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug) pinpointed the apical hook as ethylene-protected Achilles’ heel of etiolated seedlings. Because exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and mechanical stress (wounding) strongly up-regulated HEC1-dependent Kunitz-PI;1 gene expression, our results identify a new circuit controlling herbivore deterrence of etiolated plants in which Kunitz-PI;1 is involved. PMID:27625656

  19. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.; (Monash); (Queensland Inst. of Med. Rsrch.)

    2009-08-07

    The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

  20. Effects of non-starch polysaccharides enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley

    Institute of Scientific and Technical Information of China (English)

    Wei-Fen Li; Jie Feng; Zi-Rong Xu; Cai-Mei Yang

    2004-01-01

    AIM: To investigate effects of non-starch polysaccharides(NSP) enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley. METHODS: Sixty crossbred piglets averaging 13.5 kg were randomly assigned to two treatment groups with three replications (pens) based on sex and mass. Each group was fed on the diet based on barley with or without added NSP enzymes (0.15%) for a 40-d period. At the end of the experiment the pigs were weighed. Three piglets of each group were chosen and slaughtered. Pancreas, digesta from the distal end of the duodenum and jejunal mucosa were collected for determination. Activities of the digestive enzymes trypsin, chymotrypsin, amylase and lipase were determined in the small intestinal sections as well as in homogenates of pancreatic tissue. Maltase, sucrase, lactase and γ-glutamyltranspeptidase (γ-GT) activities were analyzed in jejunal mucosa. RESULTS: Supplementation with NSP enzymes improved growth performance of piglets. It showed that NSP enzymes had no effect on digestive enzyme activities in pancreas, but decreased the activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents by 57.56%, 76.08%, 69.03% and 40.22%(P<0.05) compared with control, and increased y-GT activities in jejunal mucosa by 118.75%(P<0.05).CONCLUSION: Supplementation with NSP enzymes in barley based diets could improve piglets' growth performance, decrease activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents and increase γ-GT activities in jejunal mucosa.