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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. Fecal Chymotrypsin and Trypsin Determinations

    Science.gov (United States)

    Smith, J. S.; Ediss, I.; Mullinger, M. A.; Bogoch, A.

    1971-01-01

    Trypsin and chymotrypsin concentrations were determined in 180 spot stool specimens from 110 control patients in hospital. The lower limit of normality for each enzyme was placed at the 5% level: 95% of this population excreted feces containing more than 100 μg. of chymotrypsin and 30 μg. of trypsin per g. of feces. Chymotrypsin concentrations appeared to be a more reliable guide to pancreatic function than trypsin concentrations. Fecal chymotrypsin concentrations were subnormal in five patients with chronic pancreatitis, borderline in one patient with relapsing pancreatitis, subnormal in one patient after pancreatectomy, and subnormal in five of nine with carcinoma of the pancreas. Subnormal concentrations of fecal chymotrypsin were found in seven of 21 patients with chronic liver disease related to alcoholism, eight of 32 with a partial gastrectomy, three of 10 with adult celiac disease and five of 16 with psoriasis. It appears that the determination of fecal chymotrypsin concentrations provides a valuable screening test for pancreatic exocrine deficiency. However, normal results may be found in some patients with pancreatic disease and subnormal values may occur in some patients with other conditions. PMID:5550376

  3. Chymotrypsin and trypsin sensitivities of avian reoviruses.

    OpenAIRE

    Drastini, Y; McKenna, P K; Kibenge, F S; Lopez, A

    1994-01-01

    Experiments were undertaken to examine the chymotrypsin sensitivity and trypsin sensitivity of 13 avian reoviruses, and to determine if there was any correlation with pathogenicity of some chicken reoviruses. A wide variation in the degree of sensitivity of avian reoviruses to chymotrypsin and trypsin was observed. Overall, the infectivity of the 13 avian reoviruses for Vero cells was markedly reduced by treatment with 0.01% chymotrypsin (the lowest concentration tested) while 0.5% trypsin si...

  4. 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

  5. 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

  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. Inhibition of chymotrypsin by heparin cofactor II.

    OpenAIRE

    CHURCH, F. C.; Noyes, C M; Griffith, M J

    1985-01-01

    Human heparin cofactor II is a plasma protein that is known to inhibit thrombin. The rate of thrombin inhibition by heparin cofactor II is accelerated (greater than or equal to 1000-fold) in the presence of the glycosaminoglycans, heparin and dermatan sulfate. We have found that chymotrypsin A alpha is also inhibited by heparin cofactor II with a second-order rate constant value of 1.8 X 10(6) M-1 X min-1 at pH 8.0 and 25 degrees C. However, there was no measurable effect of heparin or dermat...

  8. Benzoylsalireposide an antioxidant, lipoxygenase and chymotrypsin

    International Nuclear Information System (INIS)

    Two phenolic glycosides, benzoylsalireposide (1) and salireposide (2) were isolated from Symplocos racemosa Roxb, which showed DPPH radical scavenging activity, with the IC/sub 50/ values of 773 +- 11.83 micro meter and 757 +- 0.374 micro meter respectively. In addition to this, compound 1 also displayed in vitro inhibitory potential against lipoxygenase and chymotrypsin in a concentration-dependent fashion with the IC/sub 50/ values of 75.1 +- 0.5 micro meter and 65.07 +- 0.10 micro meter respectively, while 2 was inactive against these enzymes. (author)

  9. 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...

  10. 21 CFR 862.1180 - Chymotrypsin test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Chymotrypsin test system. 862.1180 Section 862.1180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  11. 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

  12. 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

    In sifts activity of intestinal chymotrypsin in sugar-salted whole herring during cold storage was evaluated by analysing changes in the low-molecular-weight nitrogen fraction when a specific inhibitor was added. Addition of chymostatin (0.01 mM) to sugar-salted herring gave 100% inhibition of...... chymotrypsin activity compared to sugar-salted herring without chymostatin. Inhibition of chymotrypsin did not affect the profile of low- molecular-weight peptides analysed by capillary zone electrophoresis, but resulted in 22% lower content after 26 weeks of storage. Inhibition of chymotrypsin resulted in 17...

  13. 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.

  14. 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...

  15. Alfa-glucosidase and chymotrypsin inhibiting lignans from commiphora mukul

    International Nuclear Information System (INIS)

    Phytochemical investigation of the whole plant of Commiphora mukul Engl. resulted in the isolation of two lignans, epiexcelsin (1) and 5 -demethoxyepiexcelsin (2) which are reported for the first time from this species. The structure elucidation of the isolated compounds was based on 1D and 2D-NMR analysis and by the comparison with the published data. The lignans 1 and 2 showed significant inhibitory activity against -glucosidase with the IC/sub 50/ of 59.8 +- 3.63455 micro meter and 75.2 +- 8.1616 micro meter respectively. They also showed weak inhibitory potential against chymotrypsin with the IC/sub 50/ of 110 +- 0.025 micro meter and 649 +- 0.013 micro meter respectively. (author)

  16. 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.

  17. 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

  18. Specificity of Trypsin and Chymotrypsin: Loop Motion Controlled Dynamic Correlation as a Determinant

    OpenAIRE

    Ma, Wenzhe; Tang, Chao; Lai, Luhua

    2005-01-01

    Trypsin and chymotrypsin are both serine proteases with high sequence and structural similarities, but with different substrate specificity. Previous experiments have demonstrated the critical role of the two loops outside the binding pocket in controlling the specificity of the two enzymes. To understand the mechanism of such a control of specificity by distant loops, we have used the Gaussian network model to study the dynamic properties of trypsin and chymotrypsin and the roles played by t...

  19. 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

  20. Magnetic bead cellulose as a suitable support for immobilization of .alpha.-chymotrypsin

    Czech Academy of Sciences Publication Activity Database

    Přikryl, P.; Lenfeld, Jiří; Horák, Daniel; Tichá, M.; Kučerová, Z.

    2012-01-01

    Roč. 168, č. 2 (2012), s. 295-305. ISSN 0273-2289 R&D Projects: GA ČR GA203/09/0857 Institutional research plan: CEZ:AV0Z40500505 Keywords : enzyme immobilization * human gastric juice * immobilized α-chymotrypsin Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 1.893, year: 2012

  1. 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

  2. Lucilia sericata Chymotrypsin Disrupts Protein Adhesin-Mediated Staphylococcal Biofilm Formation

    OpenAIRE

    Harris, Llinos G.; Nigam, Yamni; Sawyer, James; Mack, Dietrich; Pritchard, David I.

    2013-01-01

    Staphylococcus aureus and Staphylococcus epidermidis biofilms cause chronic infections due to their ability to form biofilms. The excretions/secretions of Lucilia sericata larvae (maggots) have effective activity for debridement and disruption of bacterial biofilms. In this paper, we demonstrate how chymotrypsin derived from maggot excretions/secretions disrupts protein-dependent bacterial biofilm formation mechanisms.

  3. MOLECULAR MODEL OF CYTOTOXIN-1 FROM NAJA MOSSAMBICA MOSSAMBICA VENOM IN COMPLEX WITH CHYMOTRYPSIN.

    Science.gov (United States)

    Munawar, Aisha; Akrem, Ahmed; Hussain, Ashiq; Spencer, Patrick; Betzel, Christian

    2015-01-01

    Snake venom is a myriad of biologically active proteins and peptides. Three finger toxins are highly conserved in their molecular structure, but interestingly possess diverse biological functions. During the course of evolution the introduction of subtle mutations in loop regions and slight variations in the three dimensional structure, has resulted in their functional versatility. Cytotoxin-1 (UniProt ID: P01467), isolated from Naja mossambica mossambica, showed the potential to inhibit chymotrypsin and the chymotryptic activity of the 20S proteasome. In the present work we describe a molecular model of cytotoxin-1 in complex with chymotrypsin, prepared by the online server ClusPro. Analysis of the molecular model shows that Cytotoxin-1 (P01467) binds to chymotrypsin through its loop I located near the N-terminus. The concave side of loop I of the toxin fits well in the substrate binding pocket of the protease. We propose Phe10 as the dedicated P1 site of the ligand. Being a potent inhibitor of the 20S proteasome, cytotoxin-1 (P01467) can serve as a potential antitumor agent. Already snake venom cytotoxins have been investigated for their ability as an anticancer agent. The molecular model of cytotoxin-1 in complex with chymotrypsin provides important information towards understanding the complex formation. PMID:27167912

  4. Lymphocyte Responses to Chymotrypsin- or Trypsin V-Digested β-Lactoglobulin in Patients with Cow's Milk Allergy

    Directory of Open Access Journals (Sweden)

    Kondo Masashi

    2007-03-01

    Full Text Available Chymotrypsin- or trypsin V- (a mixture of trypsin and chymotrypsin digested β-lactoglobulin (BLG peptides were prepared and were confirmed to have much less immunoglobulin (lgG and lgE reactivity compared with intact BLG by IgG inhibition enzymelinked immunosorbent assay and IgE dot blotting. The lymphocyte responses to intact BLG and these peptides were examined using peripheral blood mononuclear cells (PBMCs from 10 patients with cow's milk allergy. The PBMCs from most patients had lower lymphocyte responses to chymotrypsin- and trypsin V-digested BLG peptides than those to intact BLG. However, PBMCs from one and two patients retained significant proliferative responses to both peptides and to only the former peptide, respectively. Interferon-c production stimulated by chymotrypsin-digested peptides was still detectable in all five patients tested. Chymotrypsindigested BLG reduced lgE reactivity but still induced some lymphocyte responses.

  5. [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

  6. 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

  7. 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...

  8. Different expressions of trypsin and chymotrypsin in relation to growth in Atlantic salmon (Salmo salar L.)

    OpenAIRE

    Rungruangsak-Torrissen, K.; Moss, R.; Andresen, L. H.; A. Berg; Waagbø, R

    2006-01-01

    The expressions of trypsin and chymotrypsin in the pyloric caeca of Atlantic salmon (Salmo salar L.) were studied in three experiments. Two internal (trypsin phenotypes, life stages) and three common external factors (starvation, feeding, temperatures) influencing growth rates were varied. Growth was stimulated by increased temperature and higher feeding rate, and it was depressed during starvation. The interaction between trypsin phenotype and start-feeding temperature affected specific acti...

  9. Use of denatured radioalbumin for determination of trypsin and chymotrypsin inhibitors in different plant seeds

    International Nuclear Information System (INIS)

    The procedure for determination of trypsin and chymotrypsin inhibitors with urea-denatured albumin labeled by 125I is described. The content of both types of inhibitory activity has been determined in crude extracts of soybean, bean, lentil, pea, horse bean, maize, and 20 pea cultivars. The method is sufficiently sensitive, reliable, and particularly suitable when estimations must be done in crude plant extract with low inhibitory activity

  10. Quantitative structure-activity relationships for organophosphates binding to trypsin and chymotrypsin.

    Science.gov (United States)

    Ruark, Christopher D; Hack, C Eric; Robinson, Peter J; Gearhart, Jeffery M

    2011-01-01

    Organophosphate (OP) nerve agents such as sarin, soman, tabun, and O-ethyl S-[2-(diisopropylamino) ethyl] methylphosphonothioate (VX) do not react solely with acetylcholinesterase (AChE). Evidence suggests that cholinergic-independent pathways over a wide range are also targeted, including serine proteases. These proteases comprise nearly one-third of all known proteases and play major roles in synaptic plasticity, learning, memory, neuroprotection, wound healing, cell signaling, inflammation, blood coagulation, and protein processing. Inhibition of these proteases by OP was found to exert a wide range of noncholinergic effects depending on the type of OP, the dose, and the duration of exposure. Consequently, in order to understand these differences, in silico biologically based dose-response and quantitative structure-activity relationship (QSAR) methodologies need to be integrated. Here, QSAR were used to predict OP bimolecular rate constants for trypsin and α-chymotrypsin. A heuristic regression of over 500 topological/constitutional, geometric, thermodynamic, electrostatic, and quantum mechanical descriptors, using the software Ampac 8.0 and Codessa 2.51 (SemiChem, Inc., Shawnee, KS), was developed to obtain statistically verified equations for the models. General models, using all data subsets, resulted in R(2) values of .94 and .92 and leave-one-out Q(2) values of 0.9 and 0.87 for trypsin and α-chymotrypsin. To validate the general model, training sets were split into independent subsets for test set evaluation. A y-randomization procedure, used to estimate chance correlation, was performed 10,000 times, resulting in mean R(2) values of .24 and .3 for trypsin and α-chymotrypsin. The results show that these models are highly predictive and capable of delineating the complex mechanism of action between OP and serine proteases, and ultimately, by applying this approach to other OP enzyme reactions such as AChE, facilitate the development of biologically based

  11. 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

  12. 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.

  13. Determination of enzyme activity of trypsin and chymotrypsin using denatured 125J-labelled albumin

    International Nuclear Information System (INIS)

    The method of determining trypsin and chymotrypsin activity by means of modified 125I-labelled albumin is described. The radioalbumin, denatured with an alkaline urea solution is about 20 times more sensitive to enzyme hydrolysis than native radioalbumin. Owing to the higher sensitivity of the proposed procedure as compared with other available techniques, the method allows accurate determination of lower enzyme concentrations. The method is suitable for measuring the rate of proteolysis of raw enzyme extracts, and for exact kinetic measurements of purified enzymes. (author)

  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

    In sifts activity of intestinal chymotrypsin in sugar-salted whole herring during cold storage was evaluated by analysing changes in the low-molecular-weight nitrogen fraction when a specific inhibitor was added. Addition of chymostatin (0.01 mM) to sugar-salted herring gave 100% inhibition of......% 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...

  15. 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.

  16. Characterization and expression analysis of chymotrypsin after bacterial challenge in the mud crab, Scylla paramamosain

    Directory of Open Access Journals (Sweden)

    Jie Gong

    2014-06-01

    Full Text Available Chymotrypsin is one of the serine proteases families that have various biological functions. A chymotrypsin gene was isolated from hepatopancreas of the mud crab, Scylla paramamosain (designated SpCHY in this study. The full-length cDNA of SpCHY contained 942 nucleotides with a polyadenylation sequence and encoded a peptide of 270 amino acids with a signal peptide of 17 amino acids. The SpCHY gene contains seven exons, six introns, a TATA box and several transcription factor binding sites that were found in 5'-promoter region which is 1221 bp in length-time quantitative PCR analysis indicated that the expression level of SpCHY mRNA in hepatopancreas was significantly higher than that in other tissues. Immunocytochemistry and in situ hybridization exhibited the CHY-like reactivity presented in resorptive cells of the hepatopancreas. After bacterial challenge with Vibrio alginolyticus, the expression level of SpCHY mRNA was extremely up-regulated at 3 h in hepatopancreas. Our results suggest that SpCHY might play an important role in the mud crab's immune response.

  17. Using chymotrypsin and matrix-assisted laser desorption/ionization quadrupole ion trap mass spectrometer in proteomics of wheat gluten

    Czech Academy of Sciences Publication Activity Database

    Šalplachta, Jiří; Chmelík, Josef; Allmaier, G.

    2004, s. 50. ISBN 963 7067 108. [IMMS. Informal Meeting on Mass Spectrometry /22./. Tokaj (HU), 02.05.2004-06.05.2004] R&D Projects: GA MZe QD1023 Institutional research plan: CEZ:AV0Z4031919 Keywords : chymotrypsin * wheat gluten * MALDI Subject RIV: CB - Analytical Chemistry, Separation

  18. 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; Nedertoft, M M; Højrup, P

    1998-01-01

    catalytic 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...

  19. Time-of-flight neutron diffraction study of bovine γ-chymotrypsin at the Protein Crystallography Station

    International Nuclear Information System (INIS)

    In order to begin an exact determination of hydrogen positions in proteins, a neutron diffraction study of bovine gamma-chymotrypsin has been conducted. This paper details the data collection of the protein at pD (pH*) 7.1. The overarching goal of this research project is to determine, for a subset of proteins, exact hydrogen positions using neutron diffraction, thereby improving H-atom placement in proteins so that they may be better used in various computational methods that are critically dependent upon said placement. In order to be considered applicable for neutron diffraction studies, the protein of choice must be amenable to ultrahigh-resolution X-ray crystallography, be able to form large crystals (1 mm3 or greater) and have a modestly sized unit cell (no dimension longer than 100 Å). As such, γ-chymotrypsin is a perfect candidate for neutron diffraction. To understand and probe the role of specific active-site residues and hydrogen-bonding patterns in γ-chymotrypsin, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection. Time-of-flight neutron diffraction data were collected to 2.0 Å resolution at the PCS with ∼85% completeness. Here, the first time-of-flight neutron data collection from γ-chymotrypsin is reported

  20. 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. PMID:26773746

  1. 1H assignments and secondary structure determination of the soybean trypsin/chymotrypsin Bowman-Birk inhibitor

    International Nuclear Information System (INIS)

    The 1H resonance assignments and secondary structure of the trypsin/chymotrypsin Bowman-Birk inhibitor from soybeans were determined by nuclear magnetic resonance spectroscopy (NMR) at 600 MHz in an 18% acetonitrile-d3/aqueous cosolvent. Resonances from 69 to 71 amino acids were assigned sequence specifically. Residues Q11-T15 form an antiparallel β-sheet with residues Q21-S25 in the tryptic inhibitory domain and an analogous region of antiparallel sheet forms between residues S38-A42 and Q48-V52 in the chymotryptic inhibitory domain. The inhibitory sites of each fragment (K16-S17 for trypsin, L43-S44 for chymotrypsin) are each part of a type VI like turn at one end of their respective region of the antiparallel β-sheet. These structural elements are compared to those found in other Bowman-Birk inhibitors

  2. Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity.

    Science.gov (United States)

    Tedeschi, Francesca; Di Maro, Antimo; Facchiano, Angelo; Costantini, Susan; Chambery, Angela; Bruni, Natalia; Capuzzi, Valeria; Ficca, Anna Grazia; Poerio, Elia

    2012-10-30

    WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small β-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies. PMID:23090387

  3. Probing Immobilization Mechanism of alpha-chymotrypsin onto Carbon Nanotube in Organic Media by Molecular Dynamics Simulation

    OpenAIRE

    Liyun Zhang; Xiuchan Xiao; Yuan Yuan; Yanzhi Guo; Menglong Li; Xuemei Pu

    2015-01-01

    The enzyme immobilization has been adopted to enhance the activity and stability of enzymes in non-aqueous enzymatic catalysis. However, the activation and stabilization mechanism has been poorly understood on experiments. Thus, we used molecular dynamics simulation to study the adsorption of α-chymotrypsin (α-ChT) on carbon nanotube (CNT) in aqueous solution and heptane media. The results indicate that α-ChT has stronger affinity with CNT in aqueous solution than in heptane media, as confirm...

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. Bio-waste derived dialdehyde cellulose ethers as supports for α-chymotrypsin immobilization.

    Science.gov (United States)

    Kumari, Sapana; Chauhan, Ghanshyam S; Ahn, Jou-Hyeon; Reddy, N S

    2016-04-01

    Enzyme immobilization is an important technique to enhance stability, storability and reusability of enzymes. In the present work, pine needles, a forest bio-waste, were used as a feedstock of cellulose to synthesize new materials as supports for immobilization of α-chymotrypsin (CT) enzyme. The extracted cellulose from pine needles was etherified with different alkyl bromides (RBr) and etherified products were further modified to dialdehyde via oxidation with NaIO4 to get the desired products, dialdehyde cellulose ethers (ROcellCHO). CT was then covalently immobilized onto as-synthesized dialdehyde cellulose ethers via Schiff-base formation, i.e., imine linkage. The synthesized products and enzyme immobilization were confirmed by different characterization techniques and the activity assay of the free and the immobilized CT was carried out using standard protocol with variation of different parameters such as temperature, pH and substrate concentration. The storage stability and reusability of the immobilized CT were also investigated. CT activity was also studied in simulated physiological conditions in the artificial gastric fluid and artificial intestinal fluid. Artificial neural network (ANN) model was employed to correlate the relationship with% relative activity and time, temperature and pH affecting enzyme activity. A good correlation of experimental data was predicted by ANN model. PMID:26723248

  11. 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

  12. 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.

  13. 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.

  14. Effect of ethanol concentrations on temperature driven structural changes of chymotrypsin inhibitor 2

    Science.gov (United States)

    Mohanta, Dayanidhi; Jana, Madhurima

    2016-04-01

    A series of atomistic molecular dynamics (MD) simulations of a small enzymatic protein Chymotrypsin Inhibitor 2 (CI2) in water-ethanol mixed solutions were carried out to explore the underlying mechanism of ethanol driven conformational changes of the protein. Efforts have been made to probe the influence of ethanol concentrations ranging from 0% to 75% (v/v) at ambient condition (300 K (T1)) and at elevated temperatures (375 K (T2) and 450 K (T3)) to investigate the temperature induced conformational changes of the protein further. Our study showed that the effect of varying ethanol concentrations on protein's structure is almost insignificant at T1 and T2 temperatures whereas at T3 temperature, partial unfolding of CI2 in 10% ethanol solution followed by full unfolding of the protein at ethanol concentrations above 25% occurs. However, interestingly, at T3 temperature CI2's native structure was found to be retained in pure water (0% ethanol solution) indicating that the cosolvent ethanol do play an important role in thermal denaturation of CI2. Such observations were quantified in the light of root-mean-square deviations (RMSDs) and radius of gyration. Although higher RMSD values of β-sheet over α-helix indicate complete destruction of the β-structure of CI2 at high ethanol concentrations, the associated time scale showed that the faster melting of α-helix happens over β-sheet. Around 60%-80% of initial native contacts of the protein were found broken with the separation of hydrophobic core consisting eleven residues at ethanol concentrations greater than 25%. This leads protein to expand with the increase in solvent accessible surface area. The interactions between protein and solvent molecules showed that protein's solvation shell preferred to accommodate ethanol molecules as compared to water thereby excluded water molecules from CI2's surface. Further, concentration dependent differential self-aggregation behavior of ethanol is likely to regulate the

  15. 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

  16. 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.

  17. 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

  18. 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

  19. Extraction of lysozyme, alpha-chymotrypsin, and pepsin into reverse micelles formed using an anionic surfactant, isooctane, and water.

    Science.gov (United States)

    Chang, Q; Liu, H; Chen, J

    1994-11-01

    The extraction of lysozyme, alpha-chymotrypsin, and pepsin from buffered salt solutions into reverse micelles was examined at different pH values and surfactant concentrations. The reverse micelles was formed by mixing aqueous buffer supplemented with KCl and an organic phase of isooctane(2,2,4-trimethylpentane), containing the anionic surfactant, Aerosol O. T. (dioctyl ester of sodium sulfosuccinic acid). The technique of dynamic laser scattering was used to measure the size of reverse micelles which were in equilibrium with the aqueous phase. It was found that the size of the reverse micelles decreased with increasing ionic strength but increased with increasing AOT concentration. In the process of extraction, the reverse micelles might have rearranged themselves to host the protein. The sizes of protein-filled and -unfilled reverse micelles were different, and an open equilibrium could be reached between them. Under the extraction conditions, only a small number of micelles were found to contain protein. PMID:7522474

  20. Effect of dioxane on the binding of competitive inhibitor proflavin and catalytic activity of bovine pancreatic α-chymotrypsin

    Science.gov (United States)

    Sirotkin, V. A.; Mukhametzyanov, T. A.; Karmanova, Yu. V.

    2007-07-01

    The binding of competitive inhibitor proflavin by α-chymotrypsin in water-dioxane mixtures over the entire range of thermodynamic activities of water a w was studied. The data on the degree of binding of proflavin were compared to the results on the catalytic activity of the enzyme preliminary incubated in water-dioxane mixtures. An analysis of the behavior of the concentration dependences of these characteristics demonstrated that, at low a w values, the behavior of the interprotein contacts in the enzyme formed during its drying largely governs its functional properties, while at high a w values, they are determined by the interaction of the enzyme with the organic solvent. Interplay of these two factors is responsible for the observed complex shape of the isotherm of binding of proflavin, with the maximum degree of binding being attained at moderate a w values.

  1. 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)

  2. Twig extract of the apple mangrove affects the activities of trypsin, chymotrypsin and lipase in postlarval black tiger shrimp Penaeus monodon at varying feeding frequencies

    Directory of Open Access Journals (Sweden)

    Pedro Avenido

    2012-12-01

    Full Text Available This study aimed to determine the effect of the twig extract of the apple mangrove Sonneratia caseolaris on the activities of trypsin, chymotrypsin and lipase in the postlarval black tiger shrimpPenaeus monodon. Incorporating twig extracts of the apple mangrove S. caseolaris increased the activityof trypsin in juvenile P. monodon when fed at three to four times daily. When fed twice daily, trypsinactivity was at the same level as that in shrimp fed the control diet (without medication. The extractresulted in the enhanced chymotrypsin activity starting when shrimps were fed medicated diet twice dailyuntil four times daily. Lipase activity was not at all affected by the apple mangrove extract. Thesefindings demonstrated that the twig apple mangrove extract could be used as aprophylactic/therapeutant and was not deleterious to the nutrition of the black tiger shrimp; in fact, itstimulated protein digestion.

  3. A new approach in proteomics of wheat gluten: combining chymotrypsin cleavage and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron tandem mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Šalplachta, Jiří; Marchetti, M.; Chmelík, Josef; Allmaier, G.

    2005-01-01

    Roč. 19, č. 18 (2005), s. 2725-2728. ISSN 0951-4198 R&D Projects: GA MZe QD1023 Grant ostatní: Austrian Science Foundation(AT) P14181; Austrian Science Foundation;(AT) P15008 Institutional research plan: CEZ:AV0Z40310501 Keywords : wheat gluten * mass spectrometry * chymotrypsin Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.087, year: 2005

  4. The influence of dioxane on the hydration of bovine pancreatic α-chymotrypsin according to isothermal calorimetry and IR spectroscopy data

    Science.gov (United States)

    Sirotkin, V. A.; Korolev, D. V.

    2006-11-01

    The influence of dioxane on the thermochemical characteristics of the hydration of bovine pancreatic α-chymotrypsin enzyme over the whole range of water thermodynamic activities was studied by comparing the isothermal calorimetry data on the thermochemistry of interaction between the enzyme and water in the presence and absence of dioxane and using the IR spectral data on the adsorption of water and organic solvent vapors on the protein.

  5. A new method of testing pancreatin therapy in vivo by the use of a peroral chymotrypsin substrate 4-(N-acetyl-L-tyrosyl)aminobenzoic acid.

    Science.gov (United States)

    Fric, P; Malis, F; Kasafírek, E; Slabý, J

    1980-06-01

    The efficacy of pancreatin in vivo was determined in 14 patients with advanced pancreatic insufficiency using a peroral test with 2 g of chymotrypsin substrate, 4-(N-acetyl-L-tyrosyl)aminobenzoic acid, the Lundh test meal and 1000 ml tea. Chymotrypsin hydrolysis was quantified by 4-aminobenzoic acid excreted in 6-hr or 8-hr urine samples. After a control test without pancreatin, one or two tablets of Panpur (Nordmark-700 mg of pancreatin and 50 mg of bile per tablet) were applied simultaneously with the Lundh meal on repeated examinations. The urinary excretion of 4-aminobenzoic acid was restored to normal values in 5 subjects during both sampling periods. With this method, stimulated and substituted chymotrypsin is measured at the same time. The conditions of the tests, both with and without pancreatin replacement, are fully comparable and thus the significance of factors modifying the activity of enzymic components in the digestive tube is limited. The method appears appropriate for the institution of an effect pancreatin therapy and its control in vivo. PMID:6970159

  6. 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

  7. Probing immobilization mechanism of alpha-chymotrypsin onto carbon nanotube in organic media by molecular dynamics simulation.

    Science.gov (United States)

    Zhang, Liyun; Xiao, Xiuchan; Yuan, Yuan; Guo, Yanzhi; Li, Menglong; Pu, Xuemei

    2015-01-01

    The enzyme immobilization has been adopted to enhance the activity and stability of enzymes in non-aqueous enzymatic catalysis. However, the activation and stabilization mechanism has been poorly understood on experiments. Thus, we used molecular dynamics simulation to study the adsorption of α-chymotrypsin (α-ChT) on carbon nanotube (CNT) in aqueous solution and heptane media. The results indicate that α-ChT has stronger affinity with CNT in aqueous solution than in heptane media, as confirmed by more adsorption atoms, larger contact area and higher binding free energies. Although the immobilization causes significant structure deviations from the crystal one, no significant changes in secondary structure of the enzyme upon adsorption are observed in the two media. Different from aqueous solution, the stabilization effects on some local regions far from the surface of CNT were observed in heptane media, in particular for S1 pocket, which should contribute to the preservation of specificity reported by experiments. Also, CNT displays to some extent stabilization role in retaining the catalytic H-bond network of the active site in heptane media, which should be associated with the enhanced activity of enzymes. The observations from the work can provide valuable information for improving the catalytic properties of enzymes in non-aqueous media. PMID:25787884

  8. A New Approach for Characterizing the Intermediate Feature of α-Chymotrypsin Refolding by Hydrophobic Interaction Chromatography

    Directory of Open Access Journals (Sweden)

    Zhenling Liu

    2009-02-01

    Full Text Available A new approach for characterizing the intermediate of urea-denatured α-chymotrypsin (α-Chy by hydrophobic interaction chromatography (HIC is presented. The contact surface region (Z, S, affinity (logI, and the character of interaction force (j of the α-Chy to the stationary phase of HIC (STHIC between the intermediate (M and native (N states were found to be quite different as urea concentration (Curea changes. With the changes in Curea, a linear relationship between logI and Z was found to exist only for its N state, not for M state, indicating the interaction force between α-Chy in N state to the STHIC to be non-selective, but selective one for its M state. Also, the measured magnitude of both logI and Z in M state is only a fifth of that in N state. All three parameters were employed to distinguish protein in the N state from that in the M state. It would be expected that this result could be employed to distinguish any kind of non-functional protein having correct three-, or four-dimensional molecular structure from their stable M state of any kinds of proteins, and/or other proteins in proteome investigation, separation process of protein, and intensively understanding the intrinsic rule of protein folding in molecular biology.

  9. CHARACTERIZATION OF TRYPSIN-LIKE AND CHYMOTRYPSIN-LIKE SERINE PROTEASES FROM MIDGUT OF Mythimna separata Walker.

    Science.gov (United States)

    Zhou, Xiaoqun; Fan, Dong; Zhao, Kuijun

    2016-07-01

    Two cDNA sequences encoding a trypsin-like and a chymotrypsin-like serine protease (MsT and MsCT, GenBank accession Nos. KP730443 and KP730444, respectively) were cloned from midgut of oriental armyworm, Mythimna separata Walker. Multiple alignments revealed that the deduced amino acid sequences of MsT and MsCT contained a serine protease catalytic motif GDSGGPL and catalytic triads (His, Asp, and Ser). Analyses of tissue and developmental expression of MsT and MsCT showed that they were mainly expressed in midguts and could be detected in first to sixth instar larvae, prepupal and pupal stages. Expressions of both MsT and MsCT were downregulated after 24 h of starvation and upregulated by subsequent insect refeeding. MsT expression in response to 20-hydroxyecdysone (20E) was dose dependent and upregulated after 24 h. However, MsCT expression in response to 20E was downregulated compared with controls. MsCT, but not MsT, transcripts were upregulated after 24 h of Cry1Ac protoxin exposure. These results suggested that MsT was most likely involved in food protein digestion and molting in M. separata whereas MsCT was most likely involved in food protein digestion and Bacillus thuringiensis (Bt) protoxin activation. RNA interference indicated that MsT and MsCT expression levels decreased 76.7 and 86.2% after treated with MsT and MsCT dsRNA, respectively. This study showed that M. separata expressed midgut proteases in line with known lepidopteran counterparts and contributed valuable sequence resource information regarding insect proteases. PMID:26988941

  10. A murine ortholog of the human serpin SCCA2 maps to chromosome 1 and inhibits chymotrypsin-like serine proteinases.

    Science.gov (United States)

    Bartuski, A J; Kamachi, Y; Schick, C; Massa, H; Trask, B J; Silverman, G A

    1998-12-01

    Squamous cell carcinoma antigens (SCCA) 1 and 2 are inhibitory members of the high-molecular-weight serine proteinase inhibitor (serpin) family. The biological functions of SCCA1 and 2 are unknown. One approach to determining the function of human proteins is to study orthologs in other species, such as the mouse. The purpose of this study was to determine whether orthologs to human SCCA1 or 2 exist in the mouse. We report the identification and characterization of a novel serpin, sqn5 (now designated Scca2). Comparative amino acid sequence analysis suggests that Scca2 is a member of the ov-serpin subfamily of serpins with highest homology to SCCA1 and SCCA2. Fluorescence in situ hybridization revealed that the Scca2 mapped near Bcl2 on mouse chromosome 1. This region is syntenic with the human locus for SCCA1 and SCCA2 on 18q21.3. The tissue expression patterns as determined by RT-PCR showed a restricted distribution. Scca2 was detected in the lung, thymus, skin, and uterus, as are SCCA1 and SCCA2. Unlike the SCCAs, however, Scca2 was detected also in the gastrointestinal tract. Enzyme-inhibition assays using a GST-SCCA2 fusion protein revealed that SCCA2 inhibited chymotrypsin-like serine proteinases, but not papain-like cysteine proteinases. SCCA2 inhibited CTSG at 1:1 stoichiometry and with a second-order rate constant of kass = 1.7 x 10(5) M-1 s-1. SCCA2 also inhibited human mast cell chymase but the stoichiometry was 2:1, and the second-order rate constant was kass = 0.9 x 10(4) M-1 s-1. This inhibitory profile is identical to that observed for human SCCA2. Based on these findings, Scca2 appears to be the murine ortholog of human SCCA2. PMID:9828132

  11. 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).

  12. 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)

  13. Molecular cloning and expression analysis of chymotrypsin-like serine protease from the redclaw crayfish (Cherax quadricarinatus): a possible role in the junior and adult innate immune systems.

    Science.gov (United States)

    Fang, Di-An; Huang, Xian-Ming; Zhang, Zhi-Qin; Xu, Dong-Po; Zhou, Yan-Feng; Zhang, Min-Ying; Liu, Kai; Duan, Jin-Rong; Shi, Wei-Gang

    2013-06-01

    A novel chymotrypsin-like serine protease (CLSP) was isolated from the hepatopancreas of the redclaw crayfish Cherax quadricarinatus (Cq-chy). The full-length cDNA of Cq-chy contains 951 nucleotides encodes a peptide of 270 amino acids. The mature peptide comprising 223 amino acids contains the conserved catalytic triad (H, D, and S). Similarity analysis showed that Cq-chy shares high identity with chymotrypsins from the fiddler crab; Uca pugilator. Cq-chy mRNA expression in C. quadricarinatus was shown to be: (a) tissue-related with the highest expression in the hepatotpancreas and widely distributed, (b) highly responsive in the hepatopancreas to White Spot Syndrome Virus (WSSV) challenge, and (c) differently regulated in immature and adult crayfish. In this study we successfully isolated Cq-chy. Our observations indicate that Cq-chy is differently involved in the immature and adult innate immune reactions, thus suggesting a role for CLSPs in the invertebrate innate immune system. PMID:23541770

  14. 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.

  15. Thermodynamic studies of the interaction of alpha-chymotrypsin with water. II. Statistical analyses of the enthalpy-entropy compensation effect.

    Science.gov (United States)

    Lüscher, M; Rüegg, M; Schindler, P

    1978-09-26

    Differential enthalpies (deltaH) and entropies (deltaS) of the interaction of water with a high and low temperature conformer of alpha-chymotrypsin were determined previously by multitemperature sorption measurements. The changes in (deltaH) and (deltaS) with water content of the protein were found to show a pronounced compensation pattern. It is known that van 't Hoff data may exhibit enthalpy-entropy compensation, which is entirely due to statistical error propagation. To discriminate between artifactual and significant compensation, the experimental results are analyzed by statistical methods. The results of two different statistical analyses show that a linear, chemically caused compensation effect can be established for the interaction of water with both chymotrypsin conformers. The compensation temperature beta = deltaH/deltaS was found to be 433 +/- 22 K. The compensation effect is detectable only in the water content range above the monolayer volume (upsilonm), computed by the Brunauer, Emmett and Teller equation. This result is discussed in terms of a monolayer hydration mechanism, formulated on the basis of previous thermodynamic results: The interaction of the first water monolayer with the charged and polar surface area of the dry protein, largely stabilizes its tertiary structure. Further water addition then occurs to a practically invariable protein surface. According to this mechanism (which ensures a maximum of conformational stability with a minimum of hydration water), large conformational changes can be expected to occur mainly in the monolayer water content range. This expectation is confirmed by extra-thermodynamic data (infrared and X-ray measurements). The thermodynamic quantities of the sorption process are thus governed by conformational effects below upsilonm. Above the monolayer water content range, however, the water binding process per se strongly predominates. The deltaH/deltaS compensation effect established for this water content

  16. 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

  17. 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.

  18. 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

  19. The stereochemical resolution of the enantiomers of aspartame on an immobilized alpha-chymotrypsin HPLC chiral stationary phase: the effect of mobile-phase composition and enzyme activity.

    Science.gov (United States)

    Jadaud, P; Wainer, I W

    1990-01-01

    The enantioselective and diastereoselective resolutions of the stereoisomers of N alpha-aspartyl-phenylalanine 1-methyl ester (APME) have been accomplished on an HPLC chiral stationary phase based upon alpha-chymotrypsin (the ACHT-CSP) with observed enantioselectivities (alpha 1) for the DL-/LD-enantiomer of as high as 29.17 and for the DD-/LL-enantiomers of as high as 28.97. In addition, the effect on the chromatographic retention of the APME stereoisomers of the activity of the ACHT and the composition of the mobile phase--structure of the anionic component, molarity, and pH--have been studied. The results of this study suggest that the aspartyl moiety and/or the aspartyl-phenylalanine amide linkage play key roles in the observed enantioselectivity; the APME stereoisomers containing L-phenylalanine, i.e., DL- and LL-APME, bind at a different site in the ACHT molecule (the L-Phe site) than the APME stereoisomers containing D-phenylalanine (the D-Phe site); and the observed enantioselectivity is a measure of the difference in the binding affinities at the two sites rather than the consequence of differential affinities at a single site. PMID:2400637

  20. Hydrogen-deuterium exchange in the black-eyed pea trypsin and chymotrypsin inhibitar and its complex with β-trypsin

    International Nuclear Information System (INIS)

    The H-D exchange of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) in D2O was studied by an ultraviolet spectroscopic method recently proposed (J.J. Englander, D.B. Calhoun, and S.W. Englander (1979) Analytical Biochemistry, 92, 517-524). Isotopic exchange data are presented as plots of X (The fraction of unex-changed peptide hydrogen atoms at time t) versus log(k0t), where K0 is the pH dependent rate constant for peptide groups exposed to the solvent. In the range of pD2.25-6.9, at 200C, BTCI shows a continuous exchange curve which indicates that the exchange mechanism is of the EX2 type and no detectable conformational changes occur in the protein. Deviations from this exchange curve are found at pD 7.3 and 8.0. About 60% of the peptide hydrogens of BTCI are exchanged for ΔG00 < 6 kcal/mole. For reduced and carboxymethylated BTCI, exchange data suggest a much more open conformation in comparison with the unmodified protein. However, some residual structure appears to be maintained, after scission of the disulfide bonds. The exchange data indicate that, as a consequence of the formation of the β-trypsin-BTCI complex, part of the peptide groups of the enzyme and/or inhibitor become less accessible to the isotopic exchange. (Author)

  1. Effect of thermal denaturation, inhibition, and cleavage of disulfide bonds on the low-frequency Raman and FTIR spectra of chymotrypsin and albumin

    Science.gov (United States)

    Brandt, Nikolay N.; Chikishev, Andrey Yu; Mankova, Anna A.; Sakodynskaya, Inna K.

    2015-05-01

    The analysis of the structure-function relationship is extremely important in the study of proteins. The importance of function-related motions of large parts or subglobules of protein molecules stimulates the spectroscopic study in the low-frequency (terahertz) domain. However, only tentative assignments are available and the spectroscopic data are insufficiently discussed in terms of structural changes. This work is aimed at the analysis of regularities of changes in the low-frequency (100 to 600 cm-1) FTIR and Raman spectra of proteins related to their structural modifications. We study the spectra of two proteins with substantially different structures (albumin and chymotrypsin) and the spectra of samples in which the structures of protein molecules are modified using inhibition, thermal denaturation, and cleavage of disulfide bonds. The results indicate that the low-frequency spectral interval can be used to characterize protein conformations. Correlated variations in the intensities of several low-frequency bands are revealed in the spectra of the modified proteins. The strongest spectral changes are caused by thermal denaturation of proteins, and the effect of cleavage of disulfide bonds is generally weaker. It is demonstrated that the inhibitor binding in the active site causes spectral changes that can be compared to the changes induced by thermal denaturation.

  2. Crystallization and preliminary X-ray analysis of crinumin, a chymotrypsin-like glycosylated serine protease with thrombolytic and antiplatelet activity

    International Nuclear Information System (INIS)

    The crystallization and preliminary X-ray diffraction analysis of crinumin, a plant serine protease, is reported. Crinumin, a novel glycosylated serine protease with chymotrypsin-like catalytic specificity, was purified from the medicinally important plant Crinum asiaticum. Crinumin is a 67.7 kDa protease with an extraordinary stability and activity over a wide range of pH and temperature and is functional in aqueous, organic and chaotropic solutions. The purified protease has thrombolytic and antiplatelet activity. The use of C. asiaticum extracts has also been reported for the treatment of a variety of disorders such as injury, joint inflammation and arthritis. In order to understand its structure–function relationship, the enzyme was purified from the plant latex and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from a single crystal and processed to 2.8 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 121.61, b = 95.00, c = 72.10 Å, α = γ = 90, β = 114.19°. The Matthews coefficient was 2.81 Å3 Da−1, corresponding to a solvent content of 56%, assuming one molecule in the asymmetric unit. Structure determination of the enzyme is in progress

  3. NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics.

    Science.gov (United States)

    Gupta, Garvita; Lim, Liangzhong; Song, Jianxing

    2015-01-01

    Dengue genome encodes a two component protease complex (NS2B-NS3pro) essential for the viral maturation/infectivity, thus representing a key drug target. Previously, due to its "complete insolubility", the isolated NS3pro could not be experimentally studied and it remains elusive what structure it adopts without NS2B and why NS2B is indispensable. Here as facilitated by our previous discovery, the isolated NS3pro has been surprisingly deciphered by NMR to be the first intrinsically-disordered chymotrypsin-like fold, which exists in a loosely-packed state with non-native long-range interactions as revealed by paramagnetic relaxation enhancement (PRE). The disordered NS3pro appears to be needed for binding a human host factor to trigger the membrane remodeling. Moreover, we have in vitro refolded the NS3pro in complex with either NS2B (48-100) or the full-length NS2B (1-130) anchored into the LMPC micelle, and the two complexes have similar activities but different dynamics. We also performed molecular dynamics (MD) simulations and the results revealed that NS2B shows the highest structural fluctuations in the complex, thus providing the dynamic basis for the observation on its conformational exchange between open and closed states. Remarkably, the NS2B cofactor plays a central role in maintaining the correlated motion network required for the catalysis as we previously decoded for the SARS 3CL protease. Indeed, a truncated NS2B (48-100;Δ77-84) with the flexible loop deleted is able to trap the NS2B-NS3pro complex in a highly dynamic and catalytically-impotent state. Taken together, our study implies potential strategies to perturb the NS2B-NS3pro interface for design of inhibitors for treating dengue infection. PMID:26258523

  4. 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。

  5. New approach in proteomics of gluten: using chymotrypsin and MALDI hybrid multistage mass spectrometer

    Czech Academy of Sciences Publication Activity Database

    Šalplachta, Jiří; Chmelík, Josef; Allmaier, G.

    Olomouc: Palacký University, 2004 - (Frébort, I.; Walterová, D.), s. 20-21 ISBN 80-244-0882-1. [Biochemický sjezd /19./. Olomouc (CZ), 31.08.2004-03.09.2004] R&D Projects: GA MZe QD1023 Institutional research plan: CEZ:AV0Z4031919 Keywords : mass spectrometry * heat gluten * proteomics Subject RIV: CB - Analytical Chemistry, Separation

  6. Trypsin- and Chymotrypsin-Like Serine Proteases in Schistosoma mansoni - 'The Undiscovered Country'

    Czech Academy of Sciences Publication Activity Database

    Horn, Martin; Fajtová, Pavla; Arreola, L. R.; Ulrychová, Lenka; Bartošová-Sojková, Pavla; Franta, Zdeněk; Protasio, A. V.; Opavský, David; Vondrášek, Jiří; McKerrow, J. H.; Mareš, Michael; Caffrey, C. R.; Dvořák, Jan

    2014-01-01

    Roč. 8, č. 3 (2014), e2766/1-e2766/13. ISSN 1935-2735 R&D Projects: GA ČR(CZ) GAP302/11/1481; GA MŠk(CZ) ME10011 EU Projects: European Commission(XE) 248642 - SCHISTOSOMA PROTEASE Institutional support: RVO:61388963 ; RVO:68378050 ; RVO:60077344 Keywords : schistosomiasis * blood fluke * serine protease Subject RIV: CE - Biochemistry; EB - Genetics ; Molecular Biology (UMG-J); FN - Epidemiology, Contagious Diseases ; Clinical Immunology (BC-A) Impact factor: 4.446, year: 2014 http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0002766

  7. 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'…

  8. Chymotrypsin C (caldecrin) promotes degradation of human cationic trypsin: Identity with Rinderknecht's enzyme Y

    OpenAIRE

    Szmola, Richárd; Sahin-Tóth, Miklós

    2007-01-01

    Digestive trypsins undergo proteolytic breakdown during their transit in the human alimentary tract, which has been assumed to occur through trypsin-mediated cleavages, termed autolysis. Autolysis was also postulated to play a protective role against pancreatitis by eliminating prematurely activated intrapancreatic trypsin. However, autolysis of human cationic trypsin is very slow in vitro, which is inconsistent with the documented intestinal trypsin degradation or a putative protective role....

  9. 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.

  10. SwissProt search result: AK061101 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061101 006-207-A12 (P17538) Chymotrypsinogen B precursor (EC 3.4.21.1) [Contains: Chymotrypsin... B chain A; Chymotrypsin B chain B; Chymotrypsin B chain C] CTRB1_HUMAN 3e-30 ...

  11. SwissProt search result: AK112005 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112005 006-202-F10 (P17538) Chymotrypsinogen B precursor (EC 3.4.21.1) [Contains: Chymotrypsin... B chain A; Chymotrypsin B chain B; Chymotrypsin B chain C] CTRB1_HUMAN 3e-22 ...

  12. SwissProt search result: AK112119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112119 006-303-H09 (P17538) Chymotrypsinogen B precursor (EC 3.4.21.1) [Contains: Chymotrypsin... B chain A; Chymotrypsin B chain B; Chymotrypsin B chain C] CTRB1_HUMAN 1e-22 ...

  13. SwissProt search result: AK109059 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109059 002-154-F02 (P17538) Chymotrypsinogen B precursor (EC 3.4.21.1) [Contains: Chymotrypsin... B chain A; Chymotrypsin B chain B; Chymotrypsin B chain C] CTRB1_HUMAN 6e-27 ...

  14. 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 ...

  15. Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in Cry1Ab-susceptible and -resistant strains of sugarcane borer Diatraea saccharalis

    Science.gov (United States)

    Sugarcane borer, Diatraea saccharalis, is a major corn borer pest and a target of transgenic Bacillus thuringiensis (Bt) corn in South America and the U.S. mid-southern region. With a major role in dietary protein digestion, midgut serine proteinases are essential for insect growth and development. ...

  16. The Chymase, Mouse Mast Cell Protease 4, Constitutes the Major Chymotrypsin-like Activity in Peritoneum and Ear Tissue. A Role for Mouse Mast Cell Protease 4 in Thrombin Regulation and Fibronectin Turnover

    OpenAIRE

    Tchougounova, Elena; Pejler, Gunnar; Åbrink, Magnus

    2003-01-01

    To gain insight into the biological role of mast cell chymase we have generated a mouse strain with a targeted deletion in the gene for mast cell protease 4 (mMCP-4), the mouse chymase that has the closest relationship to the human chymase in terms of tissue localization and functional properties. The inactivation of mMCP-4 did not affect the storage of other mast cell proteases and did not affect the number of mast cells or the mast cell morphology. However, mMCP-4 inactivation resulted in c...

  17. Drug: D03484 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D03484 Drug Chymotrypsin (JAN/USP/INN); Catarase (TN) Enzyme [proteolytic] ATC code: B06AA04 S01 ... SENSORY ORGANS S01 OPHTHALMOLOGICALS S01K SURGICAL AIDS ... S01KX Other surgical aids ... S01KX01 Chymotrypsin D03 ...

  18. 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.

  19. 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).

  20. 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

  1. cDNA: 57843 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.1441 Mus musculus adult male testis cDNA, RIKEN full-length enriched library, c ... lone:4930519F16 product:hypothetical Serine proteases , trypsin family/Chymotrypsin serine protease famil ...

  2. 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...

  3. HYDROLYSIS OF CHEESEWHEY PROTEINSWITH TRYPSIN, CHYMOTRYPSINAND CARBOXYPEPTIDASEA

    OpenAIRE

    M. F. CUSTÓDIO; A. J. GOULART; D. P. MARQUES; R.C. Giordano; R. L. C. Giordano; R. MONTI

    2009-01-01

    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 te...

  4. 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.

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

    International Nuclear Information System (INIS)

    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

  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. Inhibition of serine proteases by oxygen, sulfur and selenium dialkyl phosphoryl derivatives: Biochemical and 31P NMR studies

    International Nuclear Information System (INIS)

    31P NMR spectroscopy was employed to investigate the interaction of chymotrypsin with various organophosphorus inhibitors. The 31P NMR spectrum of diethyl selenophosphoryl chymotrypsin revealed two resonances at approximately 71 ppm and a single resonance at 48 ppm attributed to the covalent and non-covalent enzyme-inhibitor complexes, respectively. The goal of this investigation was to elucidate the basis of the two peaks at ∼71 ppm in the 31P NMR spectrum of diethyl selenophosphosphoryl-α-chymotrypsin. The possibility that the selenium atom in the structure of the organophosphorus inhibitor might have been responsible for the two peaks was addressed by extending the 31P NMR studies to diethyl thiophosphoryl- and diethyl phosphoryl-α-chymotrypsin. The fact that the latter modified enzyme derivatives also yielded 31P NMR spectra that exhibited two resonances assigned to covalent species discounted this possibility. The effect of the alkyl group of the inhibitor on the 31P NMR spectrum of the enzyme-inhibitor complex was further investigated by examining diisopropyl phosphoryl-α-chymotrypsin by 31P NMR spectroscopy

  8. Screening of whey protein isolate hydrolysates for their dual functionality: influence of heat pre-treatment and enzyme specificity.

    Science.gov (United States)

    Adjonu, Randy; Doran, Gregory; Torley, Peter; Agboola, Samson

    2013-02-15

    Heat pre-treated and non heat pre-treated whey protein isolate (WPI) were hydrolysed using α-chymotrypsin (chymotrypsin), pepsin and trypsin. The in vitro antioxidant activity, ACE-inhibition activity and surface hydrophobicities of the hydrolysates were measured in order to determine if peptides with dual functionalities were present. Dual functional peptides have both biological (e.g. antioxidant, ACE-inhibition, opioid activities) and technological (e.g. nanoemulsification abilities) functions in food systems. Heat pre-treatment marginally enhanced the hydrolysis of WPI by pepsin and trypsin but had no effect on WPI hydrolysis with chymotrypsin. With the exception of the hydrolysis by trypsin, heat pre-treatment did not affect the peptide profile of the hydrolysates as analysed using size exclusion chromatography, or the antioxidant activity (P>0.05). Heat pre-treatment significantly affected the ACE-inhibition activities and the surface hydrophobicities of the hydrolysates (P0.05) but in some instances caused a reduction in the antioxidant activity of WPI hydrolysates. The chymotrypsin hydrolysate showed a broad MW size range, and was followed by pepsin and then trypsin. The bioactivities of the hydrolysates generally decreased in the order; chymotrypsin>trypsin>pepsin. This study showed that by manipulating protein conformation with pre-hydrolysis heat treatment, combined with careful enzyme selection, peptides with dual functionalities can be produced from WPI for use as functional ingredients in the manufacture of functional foods. PMID:23194546

  9. Ionizing radiation effect on enzymes. II

    International Nuclear Information System (INIS)

    The effects of gamma radiation on the efficacy of chymotrypsin in pancreatin prepared by the separation of enzymes from an activated pancreas extract, in the same sample in which the content of lipids was increased to 16.55%, and in pancreatin prepared by drying an incompletely activated ground pancreas were compared with the effect of radiation on crystaline lyophilized chymotrypsin. The working conditions were identical with those described in the previous communication, all samples possessed nearly identical humidity on irradiation. The efficacy of chymotrypsin was determined by the method of PhBs 3, ethyl ester L-tyrosine hydrochloride being used as the substrate. The results were statistically evaluated and after calculation for dried lipid-free substance represented in graphs. The sequence of the loss of efficacy in pancreatin corresponded to the sequence of the loss of the total proteolytic efficacy found in the previous communication. The lowest remaining efficacy was found in crystalline lyophilized chymotrypsin. Percent losses of chymotrypsin efficacy in pancreatin determined by the synthetic substrate were in good agreement with the loss of the total proteolytic efficacy of the same samples determined by casein. (author)

  10. Reduction of immunoreactivity of bovine beta-lactoglobulin upon combined physical and proteolytic treatment

    DEFF Research Database (Denmark)

    Bonomi, F.; Fiocchi, A.; Frøkiær, Hanne; Gaiaschi, A.; Iametti, S.; Poiesi, C.; Rasmussen, P.; Restani, P.; Rovere, P.

    2003-01-01

    Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6.8 for 10 min at 30, 37 and 44degreesC. The extent of beta-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when...... from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic...... fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact beta-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis...

  11. 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

  12. 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

  13. Irreversible inhibition of serine proteases by peptide derivatives of (α-aminoalkyl)phosphonate dephenyl esters

    International Nuclear Information System (INIS)

    Peptidyl derivatives of diphenyl (α-aminoalkyl)phosphonates have been synthesized and are effective and specific inhibitors of serine proteases at low concentration. Z-PheP(OPh)2 irreversibly reacts with chymotrypsin and does not react with two elastases. The best inhibitor for most chymotryspin-like enzymes including bovine chymotrypsin, cathespin G, and rat mast cell protease II is the tripeptide Suc-Val-Pro-PheP(OPh)2 which corresponds to the sequence of an excellent p-nitroanilide substrate for several chymases. The valine derivative Z-ValP(OPh)2 is specific for elastase and reacts with human leukocyte elastase but not with chymotrypsin. The tripeptide Boc-Val-Pro-ValP(OPh)2, which has a sequence found in a good trifluoromethyl ketone inhibitor of HLE, is the best inhibitor for HLE and porcine pancreatic elastase. The rates of inactivation of chymotrypsin by MeO-Suc-Ala-Ala-Pro-PheP(OPh)2 and PPE and HLE by MeO-Suc-Ala-Ala-Prov-ValP(OPh)2 were decreased 2-5 fold in the presence of the corresponding substrate, which demonstrates active site involvement. Only one of two diastereomers of Suc-Val-Pro-PehP(OPh)2 reacts with chymotrypsin and the enzyme-inhibitor complex had one broad signal at 25.98 ppm in the 31P NMR spectrum corresponding to the Ser-195 phosphonate ester. Phosphonylated serine proteases are extremely stable since the half-time for reactivation was ≥48 h for the inhibited elastases and 7.5-26 h for chymotrypsin. Peptidyl derivatives of diphenyl (α-aminoalkyl)phosphonates are relatively easy to synthesize, are chemically stable in buffer and in human plasma, form very stable derivatives with serine proteases, do not react with acetylcholinesterase, and thus should have considerable potential utility as therapeutic agents

  14. Adjustments of serine proteases of Daphnia pulex in response to temperature changes.

    Science.gov (United States)

    Dölling, Ramona; Becker, Dörthe; Hawat, Susan; Koch, Marita; Schwarzenberger, Anke; Zeis, Bettina

    2016-01-01

    Elevated temperatures considerably challenge aquatic invertebrates, and enhanced energy metabolism and protein turnover require adjustments of digestion. In Daphnia, the serine proteases chymotrypsin and trypsin represent the major proteolytic enzymes. Daphnia pulex acclimated to different temperature conditions or subjected to acute heat stress showed increased expression level of serine proteases with rising temperatures. Transcripts of trypsin isoforms were always present in higher amounts than observed for chymotrypsin. Additionally, trypsin isoform transcripts were induced by elevated temperatures to a larger extent. Correspondingly, trypsin activity dominated in cold-acclimated animals. However, the enzymatic activity of chymotrypsin increased at elevated temperatures, whereas trypsin activity slightly decreased, resulting in a shift to dominating chymotrypsin activity in warm-acclimated animals. Zymograms revealed eight bands with proteolytic activity in the range of 20 to 86kDa. The single bands were assigned to trypsin or chymotrypsin activity applying specific inhibitors or from casein cleavage products identified by mass spectrometric analysis. The total amount of proteolytic activity was elevated with acclimation temperature increase and showed a transient decrease under acute heat stress. The contribution of the different isoforms to protein digestion indicated induction of chymotrypsin with increasing acclimation temperature. For trypsin, the share of one isoform decreased with elevated temperature, while another isoform was enhanced. Thus differential expression of serine proteases was observed in response to chronic and acute temperature changes. The observed phenotypic plasticity adjusts the set of active proteases to the altered needs of protein metabolism optimizing protein digestion for the temperature conditions experienced in the habitat. PMID:26773656

  15. Partial characterization of hepatopancreatic and extracellular digestive proteinases of wild and cultivated Octopus maya

    OpenAIRE

    Martinez, Romain; R. Santos; A Alvarez; Cuzon, Gerard; L. Arena; M. Mascaro; Pascual, C; Rosas, C

    2011-01-01

    Proteinases from hepatopancreas (HP) and gastric juice (GJ) from wild and cultured red octopus (Octopus maya) were characterized. Hepatopancreas assays revealed optimal activity at pH 4, 9-10 and 10 for wild and pH 3, 8, and 9, for cultured octopuses, for total proteinases, trypsin and chymotrypsin, respectively. In the gastric juice, maximum activity was recorded at pH 6, 8, and 7 for total proteinases, trypsin, and chymotrypsin, respectively for both wild and cultured octopus. A reduction o...

  16. In vitro effects of gliotoxin, a natural proteasome inhibitor, on the infectivity and proteolytic activity of Toxoplasma gondii.

    Science.gov (United States)

    Paugam, André; Creuzet, Claudine; Dupouy-Camet, Jean; Roisin, Paule

    2002-08-01

    We examined the in vitro effect of increasing gliotoxin concentrations on the infectivity of Toxoplasma gondii for NIH-3T3 murine fibroblasts and on Toxoplasma chymotrypsin-like activity, which is specific to the proteasome. Parasite penetration of host cells was not modified by a high gliotoxin concentration (1 microM), but replication was markedly decreased (approximately 50% inhibition by 0.5 microM gliotoxin). Gliotoxin reduced the chymotrypsin-like activity of the Toxoplasma proteasome, but five times less potently than in HeLa cells. PMID:12122440

  17. 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.

  18. 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...

  19. 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.

  20. Proteomická identifikace glutenových bílkovin

    Czech Academy of Sciences Publication Activity Database

    Šalplachta, Jiří; Allmaier, G.; Chmelík, Josef

    2005-01-01

    Roč. 99, č. 12 (2005), s. 967-971. ISSN 0009-2770 R&D Projects: GA MZe 1B53002 Institutional research plan: CEZ:AV0Z40310501 Keywords : gluten * proteomic analysis * chymotrypsin Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 0.445, year: 2005

  1. Proteomic identification of celiac-related proteins of wheat flour

    Czech Academy of Sciences Publication Activity Database

    Šalplachta, Jiří; Chmelík, Josef; Allmaier, G.

    Padova: Servizi Grafici Editoriali, 2005. s. 36. ISBN 88-86281-96-X. [IMMS. Informal Meeting on Mass Spectrometry /23./. 15.05.2005-19.05.2005, Primiero] R&D Projects: GA MZe 1B53002 Keywords : mass spectrometry * wheat gluten * chymotrypsin Subject RIV: CB - Analytical Chemistry, Separation

  2. 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...bitor 2; Chain: B; Synonym: Chymotrypsin Inhibitor 2, Ci2; Engineered: Yes; Mutation: Yes; Other_detail

  3. Screening for anti-nutritional compounds in complementary foods and food aid products for infants and young children

    DEFF Research Database (Denmark)

    Roos, Nanna; Sørensen, Jens Christian; Sørensen, Hilmer;

    2013-01-01

    . The aim of this study was to screen complementary foods from developing countries for the anti-nutritional compounds, phytate, polyphenols, inhibitors of trypsin and chymotrypsin, and lectins. Commercial products based on whole grain cereals were included as a 'worst-case' scenario for anti...

  4. Main: 1CIR [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available are Molecule: Chymotrypsin Inhibitor 2; 1cir 5 Chain: A, B; 1cir 6 Domain: Residues 1 - 40, 41 - 64; 1cir 7 ...Synonym: Ci2; 1cir 8 Engineered: Yes; 1cir 9 Other_details: Cleaved Between Residues 40 And 41 1cir 10 Serin

  5. 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.

  6. Enzyme Profile of Lactobacillus Strain GG by a Rapid API ZYM System: A Comparison of Intestinal Bacterial Strains

    OpenAIRE

    Ling, W H; Saxelin, M.; Hanninen, O.; Salminen, S

    2011-01-01

    Lactobacillus GG and eight strains of lactobacilli (L. acidophilus, L. rhamnosus, L. bulgaricus and L. helviticus) and other clinical organisms (Escherichia coli, Peptostreptococcus anaerobius, Bacteroides fragilis and Clostridium difficile) were compared for their enzyme profiles. Specific activities of 19 hydrolytic enzymes for each strain were determined using the microenzyme API ZYM system. Lactobacillus GG enzyme profile showed high peptidase, chymotrypsin and phosphatase activities, and...

  7. 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

  8. 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.

  9. 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

  10. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Behrendt, N; Ploug, M; Patthy, L;

    1991-01-01

    The purified urokinase plasminogen activator receptor (u-PAR) was cleaved into two fragments by mild chymotrypsin treatment. The smaller fragment (apparent Mr 16,000) possessed the ligand-binding capability, as shown by chemical cross-linking analysis. This fragment constituted the NH2-terminal p...

  11. Bowman-Birk inhibitor affects pathways associated with energy metabolism in Drosophila melanogaster

    Science.gov (United States)

    Bowman-Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster. Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acid...

  12. 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.

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

    Czech Academy of Sciences Publication Activity Database

    Korecká, L.; Ježová, J.; Bílková, Z.; Beneš, Milan J.; Horák, Daniel; Hradcová, O.; Slováková, M.; Viovy, J.-L.

    2005-01-01

    Roč. 293, č. 1 (2005), s. 349-357. ISSN 0304-8853 R&D Projects: GA ČR(CZ) GA203/02/0023 Grant ostatní: EU project(XE) QLG-CT-2001-01903 Keywords : magnetic enzyme reactor * trypsin * chymotrypsin Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 0.985, year: 2005

  14. Serine and cysteine protease-like genes in the genome of a gall midge and their interactions with host plant genotypes

    Science.gov (United States)

    For plant-feeding insects, digestive proteases are targets for engineering protease inhibitors for pest control. In this study, we identified 105 putative serine- and cysteine-protease genes from Hessian fly genome. Among the genes, 31 encode putative trypsins, 18 encode putative chymotrypsins, se...

  15. cDNA: 57842 [ASTRA[Archive

    Lifescience Database Archive (English)

    Full Text Available M. musculus - Mm.1441 Mus musculus adult male spinal cord cDNA, RIKEN full-length enriched libra ... ry, clone:A330093C04 product:hypothetical Serine proteases , trypsin family/Chymotrypsin serine protease famil ...

  16. Main: 3CI2 [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 3CI2 大麦 Barley Hordeum vulgare l. Subtilisin-Chymotrypsin Inhibitor-2a Hordeum Vulgare Chymotr ... ucture Of Barley Serine Proteinase Inhibitor 2 And Comparison ... With The Structures In Crystals J.Mol.Biol. V. 222 ...

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

    Science.gov (United States)

    Korecká, Lucie; Ježová, Jana; Bílková, Zuzana; Beneš, Milan; Horák, Daniel; Hradcová, Olga; Slováková, Marcela; Viovy, Jean-Louis

    2005-05-01

    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.

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Safarik, I. Ivo E-mail: safarik@uek.cas.cz; Safarikova, Mirka

    2001-07-01

    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.

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

    International Nuclear Information System (INIS)

    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

  1. Fibrinogen degradation by two neutral granulocyte proteinases. Influence of calcium on the generation of fibrinogen degradation products with anticlotting properties.

    Science.gov (United States)

    Bingenhkeimer, C; Gramse, M; Egbring, R; Havemann, K

    1981-07-01

    Degradation of human fibrinogen by elastase-like proteinase, chymotrypsin-like proteinase and plasmin, was done in the presence and absence of calcium ions, respectively. The resulting fibrinogen degradation products were tested for their coagulant and anti-coagulant properties. The results show that 1. fibrinogenolysis is delayed in the presence of calcium ions. Higher enzyme concentrations are required to get unclottable split products when calcium ions are present. 2. The fibrinogen fragments obtained in the presence of calcium are different in their molecular weights and anticoagulant activities compared to those obtained in the absence of calcium ions. This effect of calcium is most striking during fibrinogen cleavage by chymotrypsin-like proteinase. Elastase and plasmin-induced fibrinogenolysis was substantially influenced by calcium only at a late degradation stage. PMID:6456216

  2. Main: 2CI2 [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 2CI 2 大麦 Barley Hordeum vulgare l. Subtilisin-Chymotrypsin Inhibitor-2a Hordeum Vulgare Chymotr ... ypsin Inhibitor 2 (/Ci $-2) Proteinase Inhibitor (Chymotrypsin) C.A.Mc*Pha ... ular Structure Of The Serine Proteinase Inhibitor /Ci $-2 From Barley Seeds Biochemistry V. 26 261 1987 E ... X05404; CAA28988.1; -.|PIR; A01292; EIBH2A.|PDB; 1CI Q; X-ray; A=20-59, B=60-83.|PDB; 1CI R; NMR; A=20-59 ... ; X-ray; I=20-83.|PDB; 1YPC; X-ray; I=20-83.|PDB; 2CI 2; X-ray; I=1-83.|PDB; 2SNI; X-ray; I=1-83.|PDB; 3C ...

  3. Main: 2SNI [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 2SNI 大麦 Barley Hordeum vulgare l. Subtilisin-Chymotrypsin Inhibitor-2a Hordeum Vulgare Subtili ... 3.4.21.14) Complex With Chymotrypsin Inhibitor 2 (/Ci $-2) Complex (Proteinase/Inhibitor) C.A.Mc*Phalen, ... 2snia 3eglin-*C-*Subtilisin Carlsberg And 2snia 4/Ci $-2-*Subtilisin Novo 2snia 5biochemistry V. 27 6582 ... X05404; CAA28988.1; -.|PIR; A01292; EIBH2A.|PDB; 1CI Q; X-ray; A=20-59, B=60-83.|PDB; 1CI R; NMR; A=20-59 ... ; X-ray; I=20-83.|PDB; 1YPC; X-ray; I=20-83.|PDB; 2CI 2; X-ray; I=1-83.|PDB; 2SNI; X-ray; I=1-83.|PDB; 3C ...

  4. 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)

  5. 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.

  6. Irradiation inactivation of some antinutritional factors in plant seeds

    International Nuclear Information System (INIS)

    Effects of gamma-irradiation (1.0-10 kGy) on trypsin, chymotrypsin, and alpha-amylase inhibitors of soybean and Moringa peregrina seeds on tannin of sorghum, gossypol of cottonseed, and in vitro digestibility of soybean were investigated. A dose of 10.0 kGy caused decreases in trypsin (by 34.9%) and chymotrypsin (by 71.4%) inhibitor activities in soybean defatted flour, whereas its in vitro digestibility increased from 79.8 to 84.2%. The alpha-amylase inhibitor activity of Al-Yassar (M. peregrina) was decreased by 43.6 and 47.8% upon treatment of 7.0 and 10.0 kGy, respectively. Doses of 10.0 and 7.0 kGy significantly reduced the tannin content in Shahlla sorghum but not in Hemaira sorghum. Total and free gossypol contents were slightly reduced by irradiation

  7. 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...

  8. 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.

  9. Main: 1CIQ [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 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; 1ciq... 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 1ciq... 21 Towards The Complete Structural Characterization 1ciq 22 Of A Protein Folding Pathway. 2: Comparison Of The 1ciq

  10. Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities.

    Science.gov (United States)

    Qian, Ye-Qing; Li, Ye; Yang, Fan; Yu, Yan-Qin; Yang, Jin-Shu; Yang, Wei-Jun

    2012-03-01

    Kazal-type inhibitors (KPIs) play important roles in many biological and physiological processes, such as blood clotting, the immune response and reproduction. In the present study, two male reproductive tract KPIs, termed Man-KPI and Ers-KPI, were identified in Macrobrachium nipponense and Eriocheir sinensis, respectively. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The results showed that both of them strongly inhibit chymotrypsin and elastase. Kinetic studies were performed to elucidate their inhibition mechanism. Furthermore, individual domains were also expressed to learn further which domain contributes to the inhibitory activities of intact KPIs. Only Man-KPI_domain3 is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2 and 3 are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2, 3 and 4 are responsible for the inhibition of elastase. Meanwhile, the inhibitory activities of these two KPIs toward Macrobrachium rosenbergii, M. nipponense and E. sinensis sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that KPIs can completely inhibit the gelatinolytic activities of sperm proteases from their own species, while different levels of cross-inhibition were observed between KPI and proteases from different species. These results may provide new perspective to further clarify the mechanism of KPI-proteases interaction in the male reproductive system. PMID:22200638

  11. Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field

    OpenAIRE

    Dunse, K. M.; Stevens, J. A.; Lay, F. T.; Gaspar, Y. M.; Heath, R. L.; Anderson, M. A.

    2010-01-01

    Potato type I and II serine protease inhibitors are produced by solanaceous plants as a defense mechanism against insects and microbes. Nicotiana alata proteinase inhibitor (NaPI) is a multidomain potato type II inhibitor (pin II) that is produced at high levels in the female reproductive tissues of the ornamental tobacco, Nicotiana alata. The individual inhibitory domains of NaPI target the major classes of digestive enzymes, trypsin and chymotrypsin, in the gut of lepidopteran larval pests....

  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. Adaptation of digestive enzymes to dietary protein, carbohydrate and fibre levels and influence of protein and carbohydrate quality in Penaeus vannamei larvae (Crustacea, Decapoda)

    OpenAIRE

    Le Moullac, Gilles; Van Wormhoudt, Alain

    1994-01-01

    L'activité des enzymes digestives (trypsine, chymotrypsine et amylase) au cours des premiers stades larvaires de Penaeus vannamei est étudiée en relation avec l'alimentation. Les microparticules à base de caséine diminuent le contenu protéique soluble et les activités enzymatiques. L'activité spécifique de la trypsine est corrélée (p

  14. 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, ...

  15. 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

  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. 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...

  18. 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...

  19. 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.

  20. 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...

  1. Influence of Reactive Oxygen Species on the Enzyme Stability and Activity in the Presence of Ionic Liquids

    OpenAIRE

    Attri, Pankaj; Choi, Eun Ha

    2013-01-01

    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 {t...

  2. 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...

  3. Proteolytic cleavage of stingray phospholipase A2: Isolation and biochemical characterization of an active N-terminal form

    OpenAIRE

    Mejdoub Hafedh; Ben Bacha Abir G

    2011-01-01

    Abstract Background Mammalian GIB-PLA2 are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. The aim of this study was to check some biochemical and structural properties of a marine stingray phospholipase A2 (SPLA2). Results The effect of some proteolytic enzymes on SPLA2 was checked. Chymotrypsin and trypsin were able to hydrolyze S...

  4. 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...

  5. Sequencing of folding events in Go-like proteins

    OpenAIRE

    Hoang, Trinh Xuan; Cieplak, Marek

    2000-01-01

    We have studied folding mechanisms of three small globular proteins: crambin (CRN), chymotrypsin inhibitor 2 (CI2) and the fyn Src Homology 3 domain (SH3) which are modelled by a Go-like Hamiltonian with the Lennard-Jones interactions. It is shown that folding is dominated by a well-defined sequencing of events as determined by establishment of particular contacts. The order of events depends primarily on the geometry of the native state. Variations in temperature, coupling strengths and visc...

  6. Quantifying tetrahedral adduct formation and stabilization in the cysteine and the serine proteases.

    Science.gov (United States)

    Cleary, Jennifer A; Doherty, William; Evans, Paul; Malthouse, J Paul G

    2015-10-01

    Two new papain inhibitors have been synthesized where the terminal α-carboxyl groups of Z-Phe-Ala-COOH and Ac-Phe-Gly-COOH have been replaced by a proton to give Z-Phe-Ala-H and Ac-Phe-Gly-H. We show that for papain, replacing the terminal carboxylate group of a peptide inhibitor with a hydrogen atom decreases binding 3-4 fold while replacing an aldehyde or glyoxal group with a hydrogen atom decreases binding by 300,000-1,000,000 fold. Thiohemiacetal formation by papain with aldehyde or glyoxal inhibitors is shown to be ~10,000 times more effective than hemiacetal or hemiketal formation with chymotrypsin. It is shown using effective molarities, that for papain, thiohemiacetal stabilization is more effective with aldehyde inhibitors than with glyoxal inhibitors. The effective molarity obtained when papain is inhibited by an aldehyde inhibitor is similar to the effective molarity obtained when chymotrypsin is inhibited by glyoxal inhibitors showing that both enzymes can stabilize tetrahedral adducts by similar amounts. Therefore the greater potency of aldehyde and glyoxal inhibitors with papain is not due to greater thiohemiacetal stabilization by papain compared to the hemiketal and hemiacetal stabilization by chymotrypsin, instead it reflects the greater intrinsic reactivity of the catalytic thiol group of papain compared to the catalytic hydroxyl group of chymotrypsin. It is argued that while the hemiacetals and thiohemiacetals formed with the serine and cysteine proteases respectively can mimic the catalytic tetrahedral intermediate they are also analogues of the productive and non-productive acyl intermediates that can be formed with the cysteine and serine proteases. PMID:26169698

  7. Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme

    OpenAIRE

    RATIA, Kiira; Saikatendu, Kumar Singh; Bernard D. Santarsiero; Barretto, Naina; Baker, Susan C.; Stevens, Raymond C.; MESECAR, Andrew D.

    2006-01-01

    Replication of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro). These proteases are important targets for development of antiviral drugs that would inhibit viral replication and reduce mortality associated with outbreaks of SARS-CoV. In this work, we describe the 1.85-Å crystal structure of the catalytic core of ...

  8. Thioamide-Based Fluorescent Protease Sensors

    OpenAIRE

    Goldberg, Jacob M.; Chen, Xing; Meinhardt, Nataline; Greenbaum, Doron C.; Petersson, E. James

    2014-01-01

    Thioamide quenchers can be paired with compact fluorophores to design “turn-on” fluorescent protease substrates. We have used this method to study a variety of serine-, cysteine-, carboxyl-, and metallo-proteases, including trypsin, chymotrypsin, pepsin, thermolysin, papain, and calpain. Since thioamides quench some fluorophores red-shifted from those naturally occurring in proteins, this technique can be used for real time monitoring of protease activity in crude preparations of virtually an...

  9. Heat, Cold and Pressure Induced Denaturation of Proteins

    OpenAIRE

    Panick, G.; Herberhold, H.; Z. Sun; Winter, R

    2003-01-01

    We studied the pressureinduced unfolding and refolding of monomeric proteins, such as SNase, αchymotrypsin and ubiquitin, by using synchrotron Xray smallangle scattering and Fouriertransform infrared spectroscopy, which monitor changes in the tertiary and secondary structural properties of the proteins upon pressurization. Furthermore, by using the pressurejump relaxation technique in combination with timeresolved Xray diffraction and infrared spectroscopy, the kinetics of the unfolding/refol...

  10. Production of secretory leucocyte protease inhibitor (SLPI) in human pancreatic beta-cells.

    OpenAIRE

    Nyström, M; Bergenfeldt, M; Ljungcrantz, I.; Lindeheim, A; Ohlsson, K.

    1999-01-01

    Secretory leucocyte protease inhibitor (SLPI) is a potent inhibitor of granulocyte elastase and cathepsin G, and also an inhibitor of pancreatic enzymes like trypsin, chymotrypsin and pancreatic elastase. SLPI has also been shown to inhibit HIV-1 infections by blocking viral DNA synthesis. Since SLPI is an inhibitor of pancreatic proteases we wished to investigate whether SLPI was also actually produced in the pancreas. M-RNA from human pancreatic tissue showed evidence of SLPI production usi...

  11. Production of Secretory Leucocyte Protease Inhibitor (SLPI) in Human Pancreatic β-Cells

    OpenAIRE

    Max Nyström; Magnus Bergenfeldt; Irena Ljungcrantz; Èsa Lindeheim; Kjell Ohlsson

    1999-01-01

    Secretory leucocyte protease inhibitor (SLPI) is a potent inhibitor of granulocyte elastase and cathepsin G, and also an inhibitor of pancreatic enzymes like trypsin, chymotrypsin and pancreatic elastase. SLPI has also been shown to inhibit HIV-1 infections by blocking viral DNA synthesis. Since SLPI is an inhibitor of pancreatic proteases we wished to investigate whether SLPI was also actually produced in the pancreas. M-RNA from human pancreatic tissue showed evidence of SLPI production usi...

  12. Isolation, purification, and amino acid sequence of lactobin A, one of the two bacteriocins produced by Lactobacillus amylovorus LMG P-13139.

    OpenAIRE

    Contreras, B G; De Vuyst, L.; Devreese, B.; Busanyova, K; Raymaeckers, J; Bosman, F; Sablon, E; Vandamme, E. J.

    1997-01-01

    Lactobacillus amylovorus LMG P-13139, isolated from corn steep liquor, produces two bactericidal peptides with respective estimated molecular masses of 4.5 and 6.0 kDa upon denaturing sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The antimicrobial activity detected in the fermentation supernatant fraction of L. amylovorus LMG P-13139 was heat stable (20 min, 121 degrees C), displayed a narrow inhibitory spectrum, and was sensitive to proteinase K, trypsin, and alpha-chymotrypsin b...

  13. 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...

  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. 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...

  16. 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...

  17. Mapping the major antigenic domains of the native flagellar antigen of Borrelia burgdorferi.

    OpenAIRE

    Jiang, W.; Luft, B J; Schubach, W; Dattwyler, R J; Gorevic, P D

    1992-01-01

    Purified flagellar protein (p41) of Borrelia burgdorferi (strain B31) was subjected to chemical cleavage with hydroxylamine or proteolysis with V8 protease, endoproteinase Asp-N, or alpha-chymotrypsin. The resulting polypeptides were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their positions in the published DNA sequence of the p41 protein were determined by amino-terminal sequencing and amino acid analysis. Epitope specificities of antibody binding by a mono...

  18. Responses of digestive enzymes of tambaqui (Colossoma macropomum) to dietary cornstarch changes and metabolic inferences.

    Science.gov (United States)

    Corrêa, Cristina Ferro; de Aguiar, Lúcia Helena; Lundstedt, Lícia Maria; Moraes, Gilberto

    2007-08-01

    Digestive enzyme responses plus metabolic implications were studied in tambaqui (Colossoma macropomum) fed isoproteic diets containing 28% crude protein, 3300 kcal of gross energy/kg and different amounts of cornstarch (30, 40 and 50%). Amylase, maltase, acid protease, trypsin and chymotrypsin from the alimentary tract were assayed. Plasma, liver and white muscle metabolites were gauged to profile metabolism of the fish. The alimentary tract of tambaqui is compartmentalized morphologically and enzymatically. Amylase was present through the gut; acid protease was detected in stomach; maltase, trypsin and chymotrypsin were found in pyloric caeca and proximal and distal intestine sections. Increase of cornstarch levels from 40 to 50% in the diet resulted in an increase in amylase and maltase. Trypsin and chymotrypsin were unresponsive to starch levels. Acid protease follows the protein/carbohydrate ratio decrease. The increase of dietary cornstarch resulted in liver glycogenesis and the increase in plasma triglycerides is suggestive of lipogenesis. Digestive biochemical responses of tambaqui correlated with changes of feeding plus the analyses of metabolic profile are assumed as a tool for optimizing diet formulation and are a clue to other feeding optimizations for freshwater tropical species. PMID:17490905

  19. 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.

  20. 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

  1. 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.

  2. Susceptibility of sweet potato (Ipomoea batatas) peel proteins to digestive enzymes.

    Science.gov (United States)

    Maloney, Katherine P; Truong, Van-Den; Allen, Jonathan C

    2014-07-01

    Sweet potato proteins have been shown to possess antioxidant and antidiabetic properties in vivo. The ability of a protein to exhibit systemic effects is somewhat unusual as proteins are typically susceptible to digestive enzymes. This study was undertaken to better understand how digestive enzymes affect sweet potato proteins. Two fractions of industrially processed sweet potato peel, containing 6.8% and 8.5% protein and 80.5% and 83.3% carbohydrate, were used as a source of protein. Sweet potato proteins were incubated with pepsin, trypsin, and chymotrypsin and protein breakdown was visualized with SDS-PAGE. After pepsin digestion, samples were assayed for amylase inhibitory activity. Sporamin, the major storage protein in sweet potatoes, which functions as a trypsin inhibitor as well, exhibited resistance to pepsin, trypsin, and chymotrypsin. Sporamin from blanched peel of orange sweet potatoes was less resistant to pepsin digestion than sporamin from outer peel and from extract of the white-skinned Caiapo sweet potato. Trypsin inhibitory activity remained after simulated gastric digestion, with the Caiapo potato protein and peel samples exhibiting higher inhibitory activity compared to the blanched peel sample. Amylase and chymotrypsin inhibitory activity was not present in any of the samples after digestion. PMID:25473492

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

    International Nuclear Information System (INIS)

    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

  4. 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.

  5. 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.

  6. Resistance of structure and antigenic determinations of native hexon of type 1 adenovirus to protease

    Energy Technology Data Exchange (ETDEWEB)

    Kiseleva, E.K.; Khil' ko, S.N.; Grigor' ev, V.G.; Dyachenko, N.S.; Vantsak, N.P.; Tikhonenko, T.I.

    1987-02-10

    Native hexon capsomers (trimers) of human type 1 adenovirus (Ad hl) were labeled with /sup 125/I and subjected to cleavage by trypsin, chymotrypsin, and papain. The hydrolysis of the hexon of Ad 1 by these enzymes is limited and in each case a set of relatively large fragments, which are not cleaved during prolonged hydrolysis, is formed. The degree of hydrolysis of the Ad hl hexon increases in the order trypsin < chymotrypsin < papain, the molecular weight of the largest fragments constituting 80,000, 40,000, and 32,000, respectively. At a decreased temperature all the large fragments of the hydrolysates obtained are confined in aggregates (the cores of the hexon), similar in structure to the original hexon trimers, the papain core of the Ad hl hexon being the most stable during electrophoresis in polyacrylamide gel and the chymotrypsin core being the least. A radioimmunoprecipitation analysis showed that denatured fragments of the tryptic, chymotryptic, and papain hydrolysates do not possess antigenic activity.

  7. Resistance of structure and antigenic determinations of native hexon of type 1 adenovirus to protease

    International Nuclear Information System (INIS)

    Native hexon capsomers (trimers) of human type 1 adenovirus (Ad hl) were labeled with 125I and subjected to cleavage by trypsin, chymotrypsin, and papain. The hydrolysis of the hexon of Ad 1 by these enzymes is limited and in each case a set of relatively large fragments, which are not cleaved during prolonged hydrolysis, is formed. The degree of hydrolysis of the Ad hl hexon increases in the order trypsin < chymotrypsin < papain, the molecular weight of the largest fragments constituting 80,000, 40,000, and 32,000, respectively. At a decreased temperature all the large fragments of the hydrolysates obtained are confined in aggregates (the cores of the hexon), similar in structure to the original hexon trimers, the papain core of the Ad hl hexon being the most stable during electrophoresis in polyacrylamide gel and the chymotrypsin core being the least. A radioimmunoprecipitation analysis showed that denatured fragments of the tryptic, chymotryptic, and papain hydrolysates do not possess antigenic activity

  8. The impact of ingested potato type II inhibitors on the production of the major serine proteases in the gut of Helicoverpa armigera.

    Science.gov (United States)

    Stevens, J A; Dunse, K M; Guarino, R F; Barbeta, B L; Evans, S C; West, J A; Anderson, M A

    2013-02-01

    The flowers of the ornamental tobacco produce high levels of a series of 6 kDa serine protease inhibitors (NaPIs) that are effective inhibitors of trypsins and chymotrypsins from lepidopteran species. These inhibitors have a negative impact on the growth and development of lepidopteran larvae and have a potential role in plant protection. Here we investigate the effect of NaPIs on the activity and levels of serine proteases in the gut of Helicoverpa armigera larvae and explore the adaptive mechanisms larvae employ to overcome the negative effects of NaPIs in the diet. Polyclonal antibodies were raised against a Helicoverpa punctigera trypsin that is a target for NaPIs and two H. punctigera chymotrypsins; one that is resistant and one that is susceptible to inhibition by NaPIs. The antibodies were used to optimize procedures for extraction of proteases for immunoblot analysis and to assess the effect of NaPIs on the relative levels of the proteases in the gut and frass. We discovered that consumption of NaPIs did not lead to over-production of trypsins or chymotrypsins but did result in excessive loss of proteases to the frass. PMID:23247047

  9. Ionizing radiation effect on enzymes. III

    International Nuclear Information System (INIS)

    A decrease in the efficacy of trypsin (determination according to PhBs 3 with the use of L-lysine ethyl ester chloride) was investigated in pancreatin obtained by enzyme precipitation from a pancreas extraction after autolysis, in the identical sample with an additionally increased content of lipids, in pancreatin containing parts of the pancreatic tissue, in crystalline trypsin, and in crystalline salt-free and lyophilized trypsine after irradiation with gamma rays from 60Co, doses ranging from 1x104 Gy to 12x104 Gy. The results were statistically evaluated and after the conversion to dried or lipid-free substance expressed in graphs. The dependence of the efficacy on the radiation dose has a linear course in semi-logarithmic arrangement, similarly as it occurred in chymotrypsin and in the total proteolytic efficacy. The decrease in the efficacy of trypsin in the samples of pancreatin in percentage maintains the same sequence in the samples under study as it was in the decrease in the efficacy of chymotrypsin and the total proteolytic efficacy, but it is smaller. The decrease in the efficacy of pure enzyme is, similarly to chymotrypsin, greater than the decrease in the efficacy of the enzyme in pancreatin. The present ballast substances thus significantly influence stability. (author)

  10. Protein-mediated adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to hydrous ferric oxide

    Energy Technology Data Exchange (ETDEWEB)

    Caccavo, F. Jr.

    1999-11-01

    The rate and extent of bacterial Fe(III) mineral reduction are governed by molecular-scale interactions between the bacterial cell surface and the mineral surface. These interactions are poorly understood. This study examined the role of surface proteins in the adhesion of Shewanella alga BrY to hydrous ferric oxide (HFO). Enzymatic degradation of cell surface polysaccharides had no effect on cell adhesion to HFO. The proteolytic enzymes Streptomyces griseus protease and chymotrypsin inhibited the adhesion of S. alga BrY cells to HFO through catalytic degradation of surface proteins. Trypsin inhibited S. alga BrY adhesion solely through surface-coating effects. Protease and chymotrypsin also mediated desorption of adhered S. alga BrY cells from HFO while trypsin did not mediate cell desorption. Protease removed a single peptide band that represented a protein with an apparent molecular mass of 50 kDa. Chymotrypsin removed two peptide bands that represented proteins with apparent molecular masses of 60 and 31 kDa. These proteins represent putative HGO adhesion molecules. A. alga BrY adhesion was inhibited by up to 46% when cells were cultured at sub-MICs of chloramphenicol, suggesting that protein synthesis is necessary for adhesion. Proteins extracted from the surface of S. alga BrY cells inhibited adhesion to HFO by up to 41%. A number of these proteins bound specifically to HFO, suggesting that a complex system of surface proteins mediates S. alga BrY adhesion to HFO.

  11. Crystallization and preliminary X-ray analysis of the ligand-binding domain of cAMP receptor protein

    International Nuclear Information System (INIS)

    N-terminal fragments of the cyclic AMP receptor protein from E. coli created by two different proteases, subtilisin and chymotrypsin, have been crystallized and diffracted to 2.0 and 2.8 Å resolution, respectively. The cyclic AMP receptor protein (CRP) from Escherichia coli regulates the expression of a large number of genes. In this work, CRP has been overexpressed, purified and digested by subtilisin and chymotrypsin. The fragments S-CRP (digested by subtilisin) and CH-CRP (digested by chymotrypsin) have been purified and crystallized. Crystals of S-CRP diffracted to 2.0 Å resolution and belonged to space group P21, with unit-cell parameters a = 59.7, b = 75.1, c = 128.3 Å, β = 91.5°. Crystals of CH-CRP diffracted to 2.8 Å resolution and belonged to space group P222, with unit-cell parameters a = 45.8, b = 60.9, c = 205.6 Å

  12. 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.

  13. 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

  14. Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

    Energy Technology Data Exchange (ETDEWEB)

    Vu, N.D.; Wagner, P.D.

    1987-07-28

    Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of /sup 32/P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca/sup 2 +/- and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1. Although this subfragment 1 contained intact light chains, its actin-activated ATPase activity was not affected by light chain phosphorylation.

  15. On the structure, function and biosynthesis of human inter-. alpha. inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Swaim, M.W.

    1989-01-01

    Human inter-{alpha} inhibitor (I{alpha}I) is a {approx}200-kD serum glycoprotein with serine proteinase-inhibitory activity whose physiologic role remains unclear. I{alpha}I is related to smaller inhibitors found in physiologic fluids and is a complex of {approx}40-kD light and {approx}90-kD heavy chains. I{alpha}I proteinase-inhibitory activity resides exclusively in the light chain, which has tandem Kunitz inhibitory domains with methionine and arginine residues, respectively, at position P{sub 1}. The inhibitory activity of the reactive centers was heretofore uncharacterized. Cis-dichlorodiammineplatinum (II) (cis-DDP) reacts with sulfur containing residues in a limited and selective fashion. In preliminary studies, cis-DDP was evaluated as a reagent to modify the methionine reactive centers of two other plasma proteinase inhibitors, {alpha}{sub 1}-antitrypsin and {alpha}{sub 2}-antiplasmin. Cis-DDP readily abolished the proteinase-inhibitory activity of both proteins. Methionine oxidation, papain digestion, and platinum binding assays showed that cis-DDP inactivates {alpha}-antitrypsin by binding exclusively to its reactive-center methionine. Cis-DDP partially eliminated I{alpha}I inhibitory activity against cathepsin G and neutrophil elastase but did not affect inhibition of trypsin or chymotrypsin. Conversely, reaction with the arginine-modifying reagent 2,3-butanedione afforded complete loss of activity against trypsin and chymotrypsin but partial loss of activity against cathepsin G and elastase. Employment of both reagents eliminated inhibition of cathepsin G and elastase. Thus eathepsin G and elastase are apparently inhibited at either reactive center. Trypsin and chymotrypsin are inhibited exclusively at the arginine reactive center.

  16. 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.

  17. Determination of digestive proteolytic profile in the larvae of Dyspessa palidata (Staudinger (Lepidoptera: Cossidae

    Directory of Open Access Journals (Sweden)

    M. Mardani-Talaee

    2015-12-01

    Full Text Available Digestive proteolytic profile was determined in the larvae of Dyspessa palidata (Staudinger, which is the most important pest of Alliaceae in Europe and Iran. Compartmentalisation of the proteolytic activities by considering soluble and membrane-bound fractions revealed that soluble fractions of the whole midgut preparations had higher general proteolytic activity than membrane-bound fractions. Also, four proteolytic bands were observed in the soluble fraction of the total midgut preparation in electrophoresis. Compartmentalisation of the specific proteases revealed presence of trypsin, elastase, aminoand carboxy peptidases in posterior midgut but the highest activities of other proteases were found in anterior midgut. The highest activity of general protease was found at pHs of 6 and 8. Also, pH dependency of trypsin, chymotrypsin and elastase were found at values of 8, 7-8 and 9 but cathepsins had the optimal pH at 6. Exopeptidases showed the optimal value at pH of 7 although carboxypeptidase showed same activity at values of 6 and 7. The inhibitory concentrations 50% (IC50 of AEBSF.HCL on trypsin, chymotrypsin and elastase proteases were found to be 3.69, 3.31 and 4.09 mM, respectively. IC50 concentrations of TLCK, SBTI and TPCK significantly inhibited trypsin and chymotrypsin activities. IC50 of E-64 were 3.67 and 4.16 mM on cathepsin B and L but cystatin revealed 5.22 and 4.48 mM concentrations on cathepsin B and L, respectively. EDTA and phenathroline as metalloproteinase inhibitors had IC50 of 3.25 and 3.91 mM on general proteolytic activity. Results of the current study revealed larvae of D. palidata utilised different proteases to increase digestive efficiency when they fed on the host plants containing several toxic molecules.

  18. 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.

  19. Main: 1JXC [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1JXC シロイヌナズナ Arabidopsis Arabidopsis thaliana (L.) Heynh. Trypsin Inhibitor Atti-2 Precur ... i-2; Chain: A; Engineered: Yes Hydrolase Inhibitor Q .Zhao, Y.K.Chae, J.L.Markley Q .Zhao, Y.K.Chae, J.L. ... n Inhibitor, Chymotrypsin Inhibitor SWS:ITI2_ARATH,Q 42328|EMBL; Z46816; CAA86849.1; ALT_FRAME.|EMBL; AJ ... lecular weight: 9885 Da MAKAIVSIFVVFFIFFLVISDVPEIEAQ GNECLKEYGGDVGFGFCAPRIFPTICYTRCRENKGAKGGRCRWGQ GSNVKC ... LCDFCDDTPQ ... arabi_1JXC.jpg ...

  20. A recombinant wheat serpin with inhibitory activity

    DEFF Research Database (Denmark)

    Rasmussen, Søren K; Dahl, Søren Weis; Nørgård, Anette;

    1996-01-01

    A full-length clone encoding the wheat (Triticum aestivum L.) serpin WSZ1 was isolated from a cDNA library based on mRNA from immature grain. The 398 amino acid sequence deduced from the cDNA was corroborated by sequencing CNBr peptides of WSZ1 purified from resting grain. WSZ1 belongs to the...... sequencing indicated that only few serpins are encoded by wheat, but at least three distinct genes are expressed in the grain. Cleavage experiments on a chymotrypsin column suggested a Gln-Gln reactive site bond not previously observed in inhibitory serpins....

  1. 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.

  2. 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

  3. 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...... 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....

  4. 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.

  5. Inhibitors of human heart chymase based on a peptide library.

    OpenAIRE

    Bastos, M; Maeji, N J; Abeles, R H

    1995-01-01

    We have synthesized two sets of noncleavable peptide-inhibitor libraries to map the S and S' subsites of human heart chymase. Human heart chymase is a chymotrypsin-like enzyme that converts angiotensin I to angiotensin II. The first library consists of peptides with 3-fluorobenzylpyruvamides in the P1 position. (Amino acid residues of substrates numbered P1, P2, etc., are toward the N-terminal direction, and P'1, P'2, etc., are toward the C-terminal direction from the scissile bond.) The P'1 ...

  6. 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

  7. 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)

  8. Synthesis and renin inhibitory activity of novel angiotensinogen transition state analogues modified at the P(2)-histidine position.

    Science.gov (United States)

    Salimbeni, A; Paleari, F; Poma, D; Criscuoli, M; Scolastico, C

    1996-01-01

    With the aim of finding new renin inhibitors with improved bioavailability properties, two angiotensinogen transition state analogues 1a and 1b, containing a novel unnatural amino acid at the P(2) position, namely the (2R,3S)- and (2S,3S)-2-amino-3-(1,3-dithiolan-2-yl)-3-hydroxypropanoic acid (ADHPA), have been synthesized and tested for human renin inhibitory activity and for chemical and enzymatic stability. Only compound 1a (the S-isomer) possessed a significant activity, which was lower than that of the corresponding histidyl derivative KRI-1314, and combined with a low stability to the gut enzyme chymotrypsin. PMID:22026939

  9. 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...

  10. FK506 Binding Protein from the Hyperthermophilic Archaeon Pyrococcus horikoshii Suppresses the Aggregation of Proteins in Escherichia coli

    OpenAIRE

    Ideno, Akira; Furutani, Masahiro; Iba, Yoshitaka; Kurosawa, Yoshikazu; Maruyama, Tadashi

    2002-01-01

    The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (kcat/Km) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15°C. Besides t...

  11. The molecular basis for the chemical denaturation of proteins by urea

    OpenAIRE

    Bennion, Brian J.; DAGGETT, VALERIE

    2003-01-01

    Molecular dynamics simulations of the protein chymotrypsin inhibitor 2 in 8 M urea at 60°C were undertaken to investigate the molecular basis of chemical denaturation. The protein unfolded rapidly under these conditions, but it retained its native structure in a control simulation in water at the same temperature. The overall process of unfolding in urea was similar to that observed in thermal denaturation simulations above the protein's Tm of 75°C. The first step in unfolding was expansion o...

  12. 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....

  13. 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.

  14. 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

  15. 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...

  16. 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...

  17. On the structural features of the substrates of protein kinase

    International Nuclear Information System (INIS)

    Structural integrity of case in and phosvitin as substrates of a mitochondrial protein kinase preparation has been examined with reference to maximal phosphate incorporation with AT32P. These proteins subjected to degradative treatments with trypsin and chymotrypsin gave rise to peptides which could still be phosphorylated by the kinase to the extent of 30.80% as compared to the parent proteins. The more active peptides from both casein and phosvitin contained high proportion of serine residue along with certain other amino acids. The hexosamine content in phosvitin did not determine its function as substrate of protein kinase. (author)

  18. 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...

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

    Directory of Open Access Journals (Sweden)

    Jorge E Suarez

    2000-08-01

    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.

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

    Science.gov (United States)

    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

  1. Reduction of major peanut allergens Ara h 1 and Ara h 2, in roasted peanuts by ultrasound assisted enzymatic treatment.

    Science.gov (United States)

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

    2013-11-15

    This study investigated the effects of ultrasound, enzyme concentration and enzyme treatment time on soluble protein and major allergenic proteins (Ara h 1 and Ara h 2) of roasted peanut kernels. A 3-factor, five-level orthogonal experimental design was implemented with various ultrasonication times, concentrations of trypsin or α-chymotrypsin and treatment times. The total soluble proteins were determined by the Bicinchoninic acid (BCA) method, Ara h 1 and Ara h 2 were evaluated by SDS-PAGE and sandwich ELISA. The IgE-binding of peanut extracts was analysed by a competitive inhibition ELISA. Results indicate that ultrasound treatment, followed by protease digestion of peanuts, significantly increased the solubility of peanut protein and decreased the concentrations of Ara h 1 and Ara h 2. The sequential treatment of peanuts by ultrasonication-trypsin-alpha chymotrypsin, resulted in maximum reductions of Ara h 1/Ara h 2, and lowest IgE-binding. This study provides an approach to significantly reduce allergenic proteins in peanut product. PMID:23790845

  2. Specific proteolysis of native alanine racemases from Salmonella typhimurium: identification of the cleavage site and characterization of the clipped two-domain proteins

    International Nuclear Information System (INIS)

    Native DadB and Alr alanine racemases (M/sub r/ 39,000) from Salmonella typhimurium are proteolyzed at homologous positions by α-chymotrypsin, trypsin, and subtilisin to generate in all cases two nonoverlapping polypeptides of M/sub r/ 28,000 and 11,000. Under nondenaturing conditions, chymotryptic digest results in an associated form of the two fragments which possesses 3% of the original catalytic activity, incorporates 0.76 equiv of the mechanism-based inactivator β-chloro-[14C]-D-alanine, and exhibits a UV circular dichroism profile identical with that of native enzyme. Protein sequence analysis of the denatured chymotryptic fragments indicates the presence of a tetrapeptide interdomain hinge (DadB, residues 254-257; Alr, residues 256-259) that is attacked at both ends during proteolysis. Under the previously employed digest conditions, NaB3H4-reduced DadB holoenzyme is resistant to α-chymotrypsin and trypsin and is labile only toward subtilisin. These data suggest that the hinge structure is essential for a catalytically efficient enzyme species and is sensitive to active site geometry. The sequence at the hinge region is also conserved in alanine racemases from Gram-positive bacteria

  3. 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

  4. Focal dysfunction of the proteasome: a pathogenic factor in a mouse model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Kabashi, Edor; Agar, Jeffrey N; Taylor, David M; Minotti, Sandra; Durham, Heather D

    2004-06-01

    Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis. PMID:15189335

  5. Studies on the thermal inactivation of immobilized enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Ulbrich, R.; Schellenberger, A.; Damerau, W.

    1986-04-01

    The thermal inactivation of a great number of immobilized enzymes shows a biphasic kinetics, which distinctly differs from the first-order inactivation kinetics of the corresponding soluble enzymes. As shown for ..cap alpha..-amylase, chymotrypsin, and trypsin covalently bound to silica, polystyrene, or polyacrylamide, the dependence of the remaining activities on the heating time can be well described by the sum of two exponential terms. To interpret this mathematical model function, the catalytic properties of immobilized enzymes (number of active sites in silica-bound trypsin, Km and Ea values in silica-bound ..cap alpha..-amylase and chymotrypsin) at different stages of inactivation and the influence of various factors (coupling conditions, addition of denaturants or stabilizers, etc.) on the thermal inactivation of silica-bound ..cap alpha..-amylase were studied. Furthermore, conformational alterations in the thermal denaturation of spin-labeled soluble and silica-bound ..beta..-amylase were compared by electron spin resonance (ESR) studies. The results suggest that the biphasic inactivation kinetics reflects two different pathways according to which catalytically identical enzyme molecules are predominantly inactivated. 45 references.

  6. Nutritional and antinutritional evaluation of raw and processed Australian wattle (Acacia saligna) seeds.

    Science.gov (United States)

    Ee, K Y; Yates, P

    2013-06-01

    Raw and processed (soaked, soaked/boiled, roasted) wattle, Acacia saligna subspecies (subsp.) saligna, pruinescens, stolonifera and lindleyi, seeds were analysed for nutritional and antinutritional qualities. Whole wattle seeds mainly comprised proteins (27.6-32.6%) and carbohydrates (30.2-36.4%), which had approximately 12.0-14.0% fat and 13.0-15.0% crude fibre. Palmitic (9.6%), stearic (2.0%), oleic (20.0%) and linoleic (64.3%) acids were identified by gas chromatography (GC) analysis. Phenolic (∼0.2%), oxalate (2.2-3.4%) and saponin (2.6-3.0%) contents were fairly high; phytate content was low. All untreated samples contained a high level of trypsin inhibitor (2474.3-3271.4 trypsin inhibitor units per gramme (TIU/g) of flour) and low level of α-chymotrypsin inhibitor (120.4-150.6 CIU/g). Soaking overnight following with 2-min boiling led to a significant reduction of protease inhibitor activity. Roasting at 2 min or longer was sufficient to reduce both trypsin and α-chymotrypsin inhibitors to negligible values, also to reduce phytate, oxalate and saponin contents, simultaneously enhanced the nutritional values of wattle seeds. PMID:23411173

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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; Ferranti, P.; Addeo, F.; Bonomi, F.

    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...

  12. 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. PMID:25313365

  13. Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Activity kinetics, conformation, and energetics.

    Science.gov (United States)

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

    2016-05-01

    This study seeks to examine the ability of non-ionic/non-polar Colloidial Liquid Aphrons (CLAs) to preserve enzyme functionality upon immobilization and release. CLAs consisting of micron-sized oil droplets surrounded by a thin aqueous layer stabilized by a mixture of surfactants, were formulated by direct addition (pre-manufacture addition) using 1% Tween 80/mineral oil and 1% Tween 20 and the enzymes lipase, aprotinin and α-chymotrypsin. The results of activity assays for both lipase and α-chymotrypsin showed that kinetic activity increased upon immobilization by factors of 7 and 5.5, respectively, while aprotinin retained approximately 85% of its native activity. The conformation of the enzymes released through desorption showed no significant alterations compared to their native state. Changes in pH and temperature showed that optimum conditions did not change after immobilization, while analysis of activation energy for the immobilized enzyme showed an increase in activity at higher temperatures. Furthermore, the effect of bound water within the aphron structure allowed for some degree of enzyme hydration, and this hydration was needed for an active conformation with results showing a decrease in ΔH* for the immobilized system compared to its native counterpart. Biotechnol. Bioeng. 2016;113: 970-978. © 2015 Wiley Periodicals, Inc. PMID:26497856

  14. 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.

  15. 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.

  16. Purification and characterization of a novel angiotensin I-converting enzyme inhibitory peptide derived from an enzymatic hydrolysate of duck skin byproducts.

    Science.gov (United States)

    Lee, Seung-Jae; Kim, Yon-Suk; Kim, Seong-Eun; Kim, Eun-Kyung; Hwang, Jin-Woo; Park, Tae-Kyu; Kim, Bo Kyung; Moon, Sang-Ho; Jeon, Byong-Tae; Jeon, You-Jin; Ahn, Chang-Bum; Je, Jae-Young; Park, Pyo-Jam

    2012-10-10

    An angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated and identified from hydrolysates of duck skin byproducts. Duck skin byproducts were hydrolyzed using nine proteases (Alcalase, Collagenase, Flavourzyme, Neutrase, papain, pepsin, Protamex, trypsin, and α-chymotrypsin) to produce an antihypertensive peptide. Of the various hydrolysates produced, the α-chymotrypsin hydrolysate exhibited the highest ACE inhibitory activity. The hydrolysate was purified using fast protein liquid chromatography (FPLC) and high-performance liquid chromatography (HPLC). The amino acid sequence of the ACE inhibitory peptide was identified as a hexapeptide Trp-Tyr-Pro-Ala-Ala-Pro, with a molecular weight of 693.90 Da. The peptide had an IC50 value of 137 μM, and the inhibitory pattern of the purified ACE inhibitor from duck skin byproducts was determined to be competitive by Lineweaver-Burk plots. In addition, the peptide was synthesized and the ACE inhibitory activity was verified in vivo. Spontaneously hypertensive rats (SHR) exhibited significantly decreased blood pressure and heart rate after peptide injection. Taken together, the results suggest that Trp-Tyr-Pro-Ala-Ala-Pro may be useful as a new antihypertensive agent. PMID:22994628

  17. 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.

  18. Ionizing radiation effect on enzymes. V

    International Nuclear Information System (INIS)

    A decrease in proteolytic efficacy of crystalline lyophilized chymotrypsin, crystalline trypsin, and crystalline trypsin free of salts and lyophilized was observed after gamma irradiation, the source being a 60Co, doses ranging from 1x104 to 12x104 Gy. Enzyme efficacy was determined with the use of casein as the substrate by the method used in PhBs 3 for the determination of proteolytic efficacy of pancreatin. The results are shown and statistically evaluated in tables and after calculation to the dried substance presented in diagrams. It was shown that after irradiation with a dose of 12x104 Gy there was no statistically significant difference between the percentage of residual efficacy of the samples. The comparison of the percentage of residual proteolytic efficacy with the results obtained in the investigation of esterolytic efficacy of the same enzymes indicates that no statistically significant difference can be demonstrated either between the decrease in the proteolytic efficacy and the decrease in the esterolytic efficacy determined with the use of the substrates and methods prescribed for the determination of efficacy of chymotrypsin and trypsin in PhBs 3. (author)

  19. 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

  20. 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

  1. 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.

  2. Loss of fibrinogen receptors from the platelet surface during simulated extracorporeal circulation

    International Nuclear Information System (INIS)

    In vitro recirculation of fresh human heparinized blood in an extracorporeal circuit with a membrane oxygenator decreased fibrinogen- induced platelet aggregation and diminished the number of fibrinogen receptors and glycoprotein IIb/IIIa (GPIIb/GPIIIa) antigenic sites on the platelet surface. In seven experiments, the mean +/- SD Km value for fibrinogen (i.e., molar concentration of fibrinogen required to cause 50% of the maximal rate of aggregation) was 1.58 x 10(-7) mol/L +/- 0.68 x 10(-7) mol/L. After recirculation, this value increased to 3.8 x 10(-7) mol/L +/- 1.94 x 10(-7) mol/L. The maximal aggregation rate of chymotrypsin-treated platelets decreased by 40% after 2 hours of recirculation. The number of fibrinogen receptors on platelets, which were treated with chymotrypsin after a recirculation, decreased from 41,370 +/- 24,000 to 13,230 +/- 10,230/platelet under the same conditions. The number of antigenic sites for monoclonal antibody reacting with GPIIb/GPIIIa complex of adenosine diphosphate-stimulated platelets decreased from 34,200 +/- 5,940 to 19,500 +/- 9,680/platelet after recirculation. Prostaglandin E1 (0. 3 mumol/L) in the perfusion circuit preserved the ability of platelets to react with fibrinogen. In conclusion, the loss of fibrinogen receptors from the surface of platelet membranes results from the interaction of platelets with the surfaces of perfusion circuits

  3. Cytoplasmic Trafficking of Minute Virus of Mice: Low-pH Requirement, Routing to Late Endosomes, and Proteasome Interaction

    Science.gov (United States)

    Ros, Carlos; Burckhardt, Christoph J.; Kempf, Christoph

    2002-01-01

    The cytoplasmic trafficking of the prototype strain of minute virus of mice (MVMp) was investigated by analyzing and quantifying the effect of drugs that reduce or abolish specific cellular functions on the accumulation of viral macromolecules. With this strategy, it was found that a low endosomal pH is required for the infection, since bafilomycin A1 and chloroquine, two pH-interfering drugs, were similarly active against MVMp. Disruption of the endosomal network by brefeldin A interfered with MVMp infection, indicating that viral particles are routed farther than the early endocytic compartment. Pulse experiments with endosome-interfering drugs showed that the bulk of MVMp particles remained in the endosomal compartment for several hours before its release to the cytosol. Drugs that block the activity of the proteasome by different mechanisms, such as MG132, lactacystin, and epoxomicin, all strongly blocked MVMp infection. Pulse experiments with the proteasome inhibitor MG132 indicated that MVMp interacts with cellular proteasomes after endosomal escape. The chymotrypsin-like but not the trypsin-like activity of the proteasome is required for the infection, since the chymotrypsin inhibitors N-tosyl-l-phenylalanine chloromethyl ketone and aclarubicin were both effective in blocking MVMp infection. However, the trypsin inhibitor Nα-p-tosyl-l-lysine chloromethyl ketone had no effect. These results suggest that the ubiquitin-proteasome pathway plays an essential role in the MVMp life cycle, probably assisting at the stages of capsid disassembly and/or nuclear translocation. PMID:12438589

  4. 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

  5. Proteolytic processing and activation of Clostridium perfringens epsilon toxin by caprine small intestinal contents.

    Science.gov (United States)

    Freedman, John C; Li, Jihong; Uzal, Francisco A; McClane, Bruce A

    2014-01-01

    Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. Importance: Processing and activation by intestinal proteases is a prerequisite for ETX-induced toxicity. Previous studies had characterized the activation of ETX using only arbitrarily chosen amounts of purified trypsin and/or chymotrypsin. Therefore, the current study examined ETX activation ex vivo by natural

  6. Effects of proteinase inhibitor from Adenanthera pavonina seeds on short- and long term larval development of Aedes aegypti.

    Science.gov (United States)

    Sasaki, Daniele Yumi; Jacobowski, Ana Cristina; de Souza, Antônio Pancrácio; Cardoso, Marlon Henrique; Franco, Octávio Luiz; Macedo, Maria Lígia Rodrigues

    2015-05-01

    Currently, one of the major global public health concerns is related to the transmission of dengue/yellow fever virus by the vector Aedes aegypti. The most abundant digestive enzymes in Ae. aegypti midgut larvae are trypsin and chymotrypsin. Since protease inhibitors have the capacity to bind to and inhibit the action of insect digestive proteinases, we investigated the short- and long-term effects of Adenanthera pavonina seed proteinase inhibitor (ApTI) on Ae. aegypti larvae, as well as a possible mechanism of adaptation. ApTI had a significant effect on Ae. aegypti larvae exposed to a non-lethal concentration of ApTI during short- and long-duration assays, decreasing survival, weight and proteinase activities of midgut extracts of larvae. The zymographic profile of ApTI demonstrated seven bands; three bands apparently have trypsin-like activity. Moreover, the peritrophic membrane was not disrupted. The enzymes of ApTI-fed larvae were found to be sensitive to ApTI and to have a normal feedback mechanism; also, the larval digestive enzymes were not able to degrade the inhibitor. In addition, ApTI delayed larval development time. Histological studies demonstrated a degeneration of the microvilli of the posterior midgut region epithelium cells, hypertrophy of the gastric caeca cells and an augmented ectoperitrophic space in larvae. Moreover, Ae. aegypti larvae were incapable of overcoming the negative effects of ApTI, indicating that this inhibitor might be used as a promising agent against Ae. aegypti. In addition, molecular modeling and molecular docking studies were also performed in order to construct three-dimensional theoretical models for ApTI, trypsin and chymotrypsin from Ae. aegypti, as well as to predict the possible interactions and affinity values for the complexes ApTI/trypsin and ApTI/chymotrypsin. In this context, this study broadens the base of our understanding about the modes of action of proteinase inhibitors in insects, as well as the way insects

  7. Protein chips for detection of mite allergens using Kunitz-type protease inhibitors.

    Science.gov (United States)

    Poltronieri, Palmiro; Cimaglia, Fabio; Santino, Angelo; De Blasi, Marialuce Daniela; Krizkova-Kudlikova, Iva; Liu, Shaoyang; Wang, Yuhong; Wang, Yifen

    2010-06-01

    Stored-food and house-dust arthropods include many species of mites and beetles that affect human health. For diagnostic tests proteases such as trypsin are utilized as they are indicators of the presence of allergen contaminants in food. We recently characterized Kunitz-type protease inhibitors (KPIs) from Solanum palustre. Here we studied biotechnological applications of KPI-B1 and -B4. We manufactured a protein chip with immobilized KPI-B1 and -B4 and showed trypsin/chymotrypsin-binding specificity, indicating that the recombinant proteins have protease selectivity. We employed the protein chip to capture mite proteins belonging to the protease family with polyclonal anti-mite antibodies. The mite diagnostic chip can be useful for detecting mite allergens. PMID:20518061

  8. 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

  9. ¬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.

  10. Studies of Protein Solution Properties Using Osmotic Pressure Measurements

    Science.gov (United States)

    Agena, S.; Bogle, David; Pusey, Marc; Agena, S.

    1998-01-01

    Examination of the protein crystallization process involves investigation of the liquid and solid state and a protein's properties in these states. Liquid state studies such as protein self association in solution by light scattering methods or other methods have been used to examine a protein Is properties and therefore its crystallization process and conditions. Likewise can osmotic pressure data be used to examine protein properties and various published osmotic pressure studies were examined by us to correlate osmotic pressure to protein solution properties. The solution behavior of serum albumin, alpha - chymotrypsin, beta - lactoglobulin and ovalbumin was examined over a range of temperatures, pH values and different salt types and concentrations. Using virial expansion and a local composition model the non ideal solution behavior in form of the activity coefficients (thermodynamic) was described for the systems. This protein activity coefficient data was related to a protein's solubility behavior and this process and the results will be presented.

  11. Purification of charybdotoxine, a specific inhibitor of the high-conductance Ca2+-activated K+ channel

    International Nuclear Information System (INIS)

    Charybdotoxim is a high-affinity specific inhibitor of the high-conductance Ca2+-activated K+ channel found in the plasma membranes of many vertebrate cell types. Using Ca2+-activated K+ channels reconstituted into planar lipid bilayer membranes as an assay, the authors have purified the toxin from the venom of the scorpion Leiurus quinquestriatus by a two-step procedure involving chromatofocusing on SP-Sephadex, followed by reversed-phase high-performance liquid chromatography. Charybdotoxin is shown to be a highly basic protein with a mass of 10 kDa. Under the standard assay conditions, the purified toxin inhibits the Ca2+-activated K+ channel with an apparent dissociation constant of 3.5 nM. The protein is unusually stable, with inhibitory potency being insensitive to boiling or exposure to organic solvents. The toxin's activity is sensitive to chymotrypsin treatment and to acylation of lysine groups. The protein may be radioiodinated without loss of activity

  12. Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity

    DEFF Research Database (Denmark)

    Hejgaard, Jørn; Hauge, S.

    2002-01-01

    Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct...... chymotrypsin at the putative reactive centre bond P-1 -P-1 ' Tyrdown arrowSer, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins...... compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens....

  13. Digestive Enzymes in the Crayfish Cherax albidus: Polymorphism and Partial Characterization

    Directory of Open Access Journals (Sweden)

    Elena Coccia

    2011-01-01

    Full Text Available We will deal with the partial characterization and the activity of the following digestive enzymes: amylase, pectinase, alginase, lipase, and protease present in the digestive tract of juvenile freshwater crayfish Cherax albidus. Gastric juices, the hepatopancreas, and the intestine were sampled for enzyme analysis. Among carbohydratases, amylase activity was the highest. It was significantly higher in the intestine than in the gastric juice and hepatopancreas. Lipase activity was significantly higher in the hepatopancreas and the intestine compared to the gastric juice. Both alkaline and acid proteases were found. Alkaline proteases were characterized by employing specific protease inhibitors. Both trypsin and chymotrypsin activity was detected. The majority of alkaline protease activity was ascribable to trypsin. Several isoforms of digestive enzymes were identified by gel electrophoresis. This work provides basic information to study the digestive abilities of crayfish.

  14. 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.

  15. 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.

  16. AcEST: DK953905 [AcEST

    Lifescience Database Archive (English)

    Full Text Available o... 30 4.9 sp|P35003|CTRL_HALRU Chymotrypsin-like serine proteinase OS=Hali... 30 4.9 sp|Q28478|ADAM2_MACFA Disintegrin...ER7|B0DER7_LACBS Predicted protein OS=Laccaria bicolor (st... 33 9.3 >tr|Q4TG08|Q4TG08_TETNG Chromosom...VL3|PRIC3_MOUSE Prickle-like protein 3 OS=Mus musculus GN=... 31 1.7 sp|Q8GU88|PDR7_ORYSJ Putative pleiotropic drug resistance prot... FLJ46204 ... 30 3.8 sp|Q9H2D6|TARA_HUMAN TRIO and F-actin-binding protein OS=Homo sa... 3... and metalloproteinase domain-c... 30 4.9 sp|P36795|VE2_HPV49 Regulatory protein E2 OS=Human papillom

  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. Purification, crystallization and preliminary crystallographic analysis of the adhesion domain of Epf from Streptococcus pyogenes

    International Nuclear Information System (INIS)

    The putative adhesion domain of the multidomain protein Epf from S. pyogenes has been crystallized in space groups P21 and P212121. The crystals diffracted to 2.0 and 1.6 Å resolution, respectively, at the Australian Synchrotron. The extracellular protein Epf from Streptococcus pyogenes is important for streptococcal adhesion to human epithelial cells. However, Epf has no sequence identity to any protein of known structure or function. Thus, several predicted domains of the 205 kDa protein Epf were cloned separately and expressed in Escherichia coli. The N-terminal domain of Epf was crystallized in space groups P21 and P212121 in the presence of the protease chymotrypsin. Mass spectrometry showed that the species crystallized corresponded to a fragment comprising residues 52–357 of Epf. Complete data sets were collected to 2.0 and 1.6 Å resolution, respectively, at the Australian Synchrotron

  19. /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.

  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. Primary structure and pathological study of phospholipase A2-I from Agkistrodon bilineatus (common cantil) venom.

    Science.gov (United States)

    Komori, Yumiko; Ohara, Akihito; Nikai, Toshiaki

    2002-05-01

    Phospholipase A2-I (PLA2-I) was isolated from Agkistrodon bilineatus venom by Nikai (Nikai et al., 1993). The amino acid sequence of the phospholipase A2-I was determined by the Edman sequencing procedure of peptides derived from digests utilizing cyanogen bromide, clostripain, metalloendopeptidase, chymotrypsin, and Staphylococcus aureus V8 protease. In the reduced state, purified phospholipase A2's molecular weight was determined to be 14,000 as demonstrated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Purified PLA2-I also contained 1 mol of Ca per mol of protein and consists of 123 amino acid residues resulting in a calculated molecular weight of 14,133. Both phospholipase and lethal activities were found to be inhibited by bromophenacyl bromide, suggesting that the histidine residue is involved in this activity. Also there was an increase in the creatine kinase activity of mice serum, which is an indicator that PLA2-I is involved in muscle damage. PMID:12009114

  2. Effect of zinc bacitracin and salinomycin on intestinal microflora and performance of broilers

    DEFF Research Database (Denmark)

    Engberg, R. M.; Hedemann, M.S.; Leser, T.D.; Jensen, B. B.

    2000-01-01

    , enterococci, and Clostridium perfringens were counted. In homogenates of pancreas obtained from four animals, the activities of amylase, lipase, trypsin, and chymotrypsin were measured. A significant growth-promoting effect was observed in the group receiving zinc bacitracin in combination with salinomycin......A feeding experiment was carried out over 42 d with four groups of broiler chickens fed experimental diets formulated to provide no supplementation, 20 mg zinc bacitracin, 60 mg salinomycin, or both feed additives in combination. During the fifth week of the experiment, four chickens from each pen....... Zinc bacitracin significantly reduced the number of coliform bacteria in the ileum and increased the activities of amylase and lipase in pancreas homogenates. Supplementation with salinomycin and zinc bacitracin, alone or in combination, resulted in significantly lower counts of C. perfringens as well...

  3. 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.

  4. 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

  5. Changes in digestive enzyme activity, intestine morphology, mucin characteristics and tocopherol status in mink kits (Mustela neovision) during the weaning period

    DEFF Research Database (Denmark)

    Hedemann, Mette Skou; Clausen, T.N.; Jensen, Søren Krogh

    2011-01-01

    . Twenty-four mink kits from eight litters were included in the experiment. The dams and their litters were kept under standard farm conditions. The dams and the kits were fed a diet consisting of 48.1% protein, 40.7% fat and 11.1% carbohydrate of metabolizable energy. The mink kits were weaned at 42 days...... of age. At 34, 47 and 59 days of age, one male mink kit from each litter was euthanized. The activity of amylase, trypsin, chymotrypsin and lipase in the pancreatic tissue increased during the experimental period, whereas the activity of carboxyl ester hydrolase remained constant. The vitamin E......, whereas the biologically less active 2S isomers showed a clear increase. The concentration of bile salts did not change during the experimental period. The villous height increased in the proximal part of the small intestine and decreased in the distal part, whereas the crypt depth was doubled in both the...

  6. 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.

  7. 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.

  8. Development of a radioimmunoassay for pig pancreatic kallikrein

    International Nuclear Information System (INIS)

    A radioimmunoassay for the determination of pig pancreatic kallikrein was developed. The chloramine-T method was employed for the labelling of the antigen with 125I. The assay allows the determination of kallikrein in concentrations as low as 0.4 μg/l. Pig urinary and pig submandibular kallikreins are indistinguishable from pig pancreatic kallikrein by the assay. No cross reactivity was observed for bovine trypsin and chymotrypsin, porcine trypsin and kallikreins of guinea pig submandibular glands and guinea pig coagulation glands. Because of the high specificity of the assay, which is not attainable with conventional assays based on the enzymatic activity, the radioimmunoassay is highly suited for investigations into the physiological role and the pharmacological mechanism of action of pig glandular kallikreins. (orig.)

  9. 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. PMID:25903181

  10. Evidence for the existence of a proteasome in Toxoplasma gondii: intracellular localization and specific peptidase activities.

    Science.gov (United States)

    Paugam, A; Creuzet, C; Dupouy-Camet, J; Roisin, M P

    2001-12-01

    The proteasome is a large intracellular protein complex whose main function is proteolytic removal of damaged proteins. It has recently been shown that the proteasome has a crucial role in the pathogenesis of protozoan parasites. We attempted to characterize the proteasome of T. gondii (RH strain). In immunoblot experiments, we showed that MCP231 monoclonal antibody, directed against the human 20S proteasome, labelled homologous proteins in T. gondii with a pattern similar to that observed in mammalian cells. The study of in vitro proteolytic activities showed that chymotrypsin-like activity (the only activity obtained with archaebacteria) was present in Toxoplasma, with Km and specific activity values close to those observed with eukaryotic cells. Immunofluorescence studies showed that the Toxoplasma proteasome predominated in the cytosol. PMID:11802263

  11. Protease activity in the larval stage of the parasitoid wasp, Eulophus pennicornis (Nees) (Hymenoptera: Eulophidae); effects of protease inhibitors.

    Science.gov (United States)

    Down, R E; Ford, L; Mosson, H J; Fitches, E; Gatehouse, J A; Gatehouse, A M

    1999-08-01

    Hymenopteran, parasitoid wasps have good potential for use in integrated pest management (IPM); for example, the gregarious ectoparasitoid, Eulophus pennicornis, has been suggested as a biological control agent for larvae of the tomato moth (Lacanobia oleracea L.). However, the processes by which such parasitic larvae are able to utilize the nutritional resource provided by the host have been little studied. Protease activity was present in E. pennicornis larvae, and characterization of the enzymes responsible for proteolysis was performed using a range of synthetic substrates and specific inhibitors. Serine protease enzymes was both trypsin- and chymotrypsin-like activities were present. A range of plant-derived serine protease inhibitors was tested for activity against these enzymes. Certain inhibitors, notably soybean Kunitz inhibitor (SKTI), inhibited enzyme activity by > 80% at pests are to form a component of IPM systems, possible adverse effects, whether direct or indirect, of transgene expression on parasitoids like E. pennicornis should be considered. PMID:10466123

  12. Dyeing of wool fibres with natural dyes: effect of proteolytic enzymes.

    Science.gov (United States)

    Doğru, Mehmet; Baysal, Zübeyde; Aytekin, Cetin

    2006-01-01

    In spite of the widespread use of proteins (casein, peptone, etc.) and protein fragments as a substrate for the proteolytic enzymes, a substrate prepared from dyes that adsorb onto appropriate materials, such as wool and cotton, are also used for enzyme activity determination. In the point of view of this thought, it was our aim to develop the substrates which are easily and economically obtainable and also environmentally safer for the frequently used proteolytic enzymes, such as subtilisin carlsberg, trypsin, chymotrypsin, and protease type XVI and, if possible, to prepare the specific substrate at least for one of these enzymes. For this aim, wool was dyed with natural dyes such as juglone, lawsone, berberine, and quercetin. The optimum pH, incubation time, and agitation rate were determinated. The results indicate that, of all the tested enzymes on wool-dye complex as an insoluble substrate, the most appropriate complex was found to be wool-lawsone complex. PMID:16707332

  13. PHARMACOLOGICAL REGULATION OF DIGESTION IN THE ANAUTOGENOUS FLESH FLY, Sarcophaga crassipalpis, BY SIMPLE INJECTION OF 6-HYDROXYDOPAMINE.

    Science.gov (United States)

    Bil, Magdalena; Huybrechts, Roger

    2016-03-01

    Female anautogenous Sarcophaga flesh flies need a protein meal to start large-scale yolk polypeptides (YPs) production and oocyte maturation. Protein meal rapidly elicits a brain-dependent increase in midgut proteolytic activity. Trypsin and chymotrypsin together represent over 80% of protease activity in liver-fed flies. Abdominal injection of 6-hydroxydopamine (6-OHDA) dose-dependently prohibits this increase in proteolytic activity at translational level in a similar way as post liver feeding decapitation. Delayed injection of 6-OHDA later than 6 h post liver meal has no effect. In flesh flies, chemical decapitation by 6-OHDA, by interrupting the brain-gut dopaminergic signaling, can be used as tool for the controlled inhibition of midgut proteolytic activity and subsequent ovarial development. Inhibition of ovarial development is probably indirect due to a deficit in circulating amino acids needed for YPs synthesis. PMID:26728276

  14. [The use of enzymatic hydrolysis for isolation of barbituric acid derivatives from blood (as exemplified by phenobarbital and barbamyl)].

    Science.gov (United States)

    Chuvina, N A; Kolupaeva, A S; Strelova, O Iu; Zabolotskaia, I V; Gorbacheva, T V

    2010-01-01

    Modern isolation techniques by direct extraction with organic solvents or after protein precipitation by various sedimenting or salting-out agents are characterized by low efficiency and do not permit to liberate derivatives of barbituric acid from their complexes with blood proteins. The use of enzymatic hydrolysis makes it possible to break bonds between barbiturates and protein and thereby improve the efficiency of isolation. We performed enzymatic hydrolysis of the model phenobarbital-blood and barbamyl-blood complexes with the use of trypsin, pepsin, chymotrypsin, and papain. The degree of phenobarbital extraction with trypsin and barbamyl was estimated at 62.1 +/- 1.2% and 75.1 +/- 1.6% respectively; in other words, it was 32.7 +/- 1.0% and 51.1 +/- 1.0% higher than that achieved by traditional methods. Certain validation characteristics of the new method are presented. PMID:21265178

  15. 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.

  16. Digestive response to restricted feeding in migratory yellow-rumped warblers.

    Science.gov (United States)

    Lee, Kelly A; Karasov, William H; Caviedes-Vidal, Enrique

    2002-01-01

    Smaller guts and slow initial mass gains at stopover sites have led to the idea that digestive physiology limits refueling rates in migrating birds. We tested the digestive-limitation hypothesis in yellow-rumped warblers using food restriction to simulate infrequent feeding during migration, which may cause a reduction in alimentary tract mass. Restricted birds had small intestine, pancreas, and liver masses 18%-22% lower than ad lib.-fed controls. Total activities of sucrase, maltase, aminopeptidase, and amylase were significantly lower in restricted birds, while those of trypsin and chymotrypsin were not. Only aminopeptidase mass-specific activity was significantly lower in restricted birds. Previously restricted birds were able to feed and digest at a high rate immediately following return to ad lib. feeding. Digestive efficiency did not differ between groups. These results suggest that before migration yellow-rumped warblers have some spare digestive capacity to compensate for declines in their digestive organ masses during migration. PMID:12177834

  17. 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

  18. 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

  19. Biocatalytic Pathway Selection in Transient Tripeptide Nanostructures.

    Science.gov (United States)

    Pappas, Charalampos G; Sasselli, Ivan R; Ulijn, Rein V

    2015-07-01

    Structural adaption in living systems is achieved by competing catalytic pathways that drive assembly and disassembly of molecular components under the influence of chemical fuels. We report on a simple mimic of such a system that displays transient, sequence-dependent formation of supramolecular nanostructures based on biocatalytic formation and hydrolysis of self-assembling tripeptides. The systems are catalyzed by α-chymotrypsin and driven by hydrolysis of dipeptide aspartyl-phenylalanine-methyl ester (the sweetener aspartame, DF-OMe). We observed switch-like pathway selection, with the kinetics and consequent lifetime of transient nanostructures controlled by the peptide sequence. In direct competition, kinetic (rather than thermodynamic) component selection is observed. PMID:26014441

  20. A novel angiotensin-І converting enzyme (ACE) inhibitory peptide from gastrointestinal protease hydrolysate of silkworm pupa (Bombyx mori) protein: Biochemical characterization and molecular docking study.

    Science.gov (United States)

    Wu, Qiongying; Jia, Junqiang; Yan, Hui; Du, Jinjuan; Gui, Zhongzheng

    2015-06-01

    Silkworm pupa (Bombyx mori) protein was hydrolyzed using gastrointestinal endopeptidases (pepsin, trypsin and α-chymotrypsin). Then, the hydrolysate was purified sequentially by ultrafiltration, gel filtration chromatography and RP-HPLC. A novel ACE inhibitory peptide, Ala-Ser-Leu, with the IC50 value of 102.15μM, was identified by IT-MS/MS. This is the first report of Ala-Ser-Leu from natural protein. Lineweaver-Burk plots suggest that the peptide is a competitive inhibitor against ACE. The molecular docking studies revealed that the ACE inhibition of Ala-Ser-Leu is mainly attributed to forming very strong hydrogen bonds with the S1 pocket (Ala354) and the S2 pocket (Gln281 and His353). The results indicate that silkworm pupa (B. mori) protein or its gastrointestinal protease hydrolysate could be used as a functional ingredient in auxiliary therapeutic foods against hypertension. PMID:25111373

  1. Experimental and clinical studies on dynamic circulation of intervertebral discs by radiation clearance method

    International Nuclear Information System (INIS)

    Few studies on the mechanism of dynamic circulation in intervertebral discs are reported. The purpose of this study is to clarify the dynamic mechanism of circulation in the lumbar intervertebra discs with radioisotope clearance, experimentally and clinically. Rabbits were used as experimental animals and were divided into two groups, normal and artificially injured. I131-sodium was injected into them and the clearance was followed. Injected I131-sodium was cleared in the order of; 1) discs with injured annulus fibrosus, 2) discs with injection of α-chymotrypsin, 3) discs with injuries in the border between vertebral body and annulus fibrosus, 4) and normal discs. Human degenerated discs were also used for this study. In the clinical cases, I131-sodium was cleared faster in the degenerated discs than in the normal. The pathway for I131-sodium to be cleared is supposed to be extravascular and interstitial spaces in the disc. (author)

  2. 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

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

    International Nuclear Information System (INIS)

    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 Å3 Da−1 and 50.61%, respectively, for one molecule in the asymmetric unit

  4. 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

  5. Mechanism of trypsin inactivation by intact Hymenolepis diminuta (Cestoda)

    International Nuclear Information System (INIS)

    The mechanism of trypsin inactivation by intact Hymenolepis diminuta has been investigated by biochemical and autoradiographic methods. Although worms inactivate trypsin and chymotrypsin in vitro, no inactivation of other endoproteases (subtilisin, pepsin and papain) could be demonstrated. Trypsin inactivation, as demonstrated by macromolecular substrates (azoalbumin, hemoglobin and casein), could not be detected using low molecular weight synthetic substrates such as N-p-benzoyl-DL-arginine-p-nitroanilide (BAPA) or N-p-tosyl-L-arginine methyl ester (TAME). In addition, the kinetic parameters (K/sub m/ and k3) for H. diminuta-inactivated trypsin, using BAPA as the substrate, were not different from those of the native enzyme. The number of active sites for both native and inactivated trypsin were determined by titration with p-nitro-phenyl-p'-guanidinobenzoate. Absorbance values for both titrations were found to be identical. Dialysis, heating to 50 C, or repeated freezing and thawing of the inactivated trypsin did not reverse inactivation

  6. 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

  7. Porcine myofibrillar proteins as potential precursors of bioactive peptides - an in silico study.

    Science.gov (United States)

    Kęska, Paulina; Stadnik, Joanna

    2016-06-15

    Selected porcine myofibrillar proteins have been assessed as potential precursors of bioactive peptides based on in silico analysis. The potential of protein sequences for releasing peptides was evaluated by determining the profile of their potential biological activity and the frequency of occurrence of fragments with a given activity using the BIOPEP database. Digestive enzymes: pepsin, trypsin and chymotrypsin have been used for the in silico proteolysis with the use of the "Enzyme(s) action" tool in BIOPEP. After simulated gastrointestinal digestion the tested sequences of pig myofibrillar proteins are a potential source of a total of 399 peptides with activities such as enzyme inhibition, antioxidative, hypotensive, stimulating or regulating various body functions and antiamnestic activities. Within the intact proteins and after simulated gastrointestinal digestion, dipeptidyl peptidase IV inhibitory peptide sequences were the most frequently observed. The results indicate that pork myofibrillar proteins are a promising source of peptides with biological activity. PMID:27247979

  8. 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

  9. 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...

  10. Free radical scavenging and angiotensin-I converting enzyme inhibitory peptides from Pacific cod (Gadus macrocephalus) skin gelatin.

    Science.gov (United States)

    Ngo, Dai-Hung; Ryu, Bomi; Vo, Thanh-Sang; Himaya, S W A; Wijesekara, Isuru; Kim, Se-Kwon

    2011-12-01

    Potent antioxidative peptides were purified from Pacific cod (Gadus macrocephalus) skin gelatin using alcalase, neutrase, papain, trypsin, pepsin, and α-chymotrypsin. Among them, the papain hydrolysate exhibited the highest antioxidant activity. Therefore, it was further purified and obtained two peptides with amino acid sequences of Thr-Cys-Ser-Pro (388 Da) and Thr-Gly-Gly-Gly-Asn-Val (485.5 Da). The antioxidant activity of the purified peptides was performed by electron spin resonance technique. Moreover, their intracellular free radical scavenging activity using 2',7'-dichlorofluorescin diacetate and the protective effect against oxidation-induced DNA damage were evaluated in mouse macrophages (RAW 264.7 cells). Furthermore, both peptides have shown potential angiotensin-I converting enzyme inhibitory effect. The present study demonstrated that the peptides derived from Pacific cod (G. macrocephalus) skin gelatin could be used in the food industry as functional ingredients with potent antioxidative and antihypertensive benefits. PMID:21945677

  11. Synthesis, characterization and biological evaluation of m-phenetidine derivatives

    International Nuclear Information System (INIS)

    The synthesis of compounds bearing sulfamoyl group and amide linkage is pharmacologically important due to their splendid biological activities. In the presented research work N-(3-ethoxyphenyl)-4-methylbenzenesulfonamide (3) was synthesized by the coupling of m-phenetidine (1) with 4-methylbenzenesulfonyl chloride (2) in basic aqueous media. Compound 3 on reaction with 2-bromo-N-aryl/aralkyl substituted acetamides, 5a-d, synthesized the products, 6a-d. The bromination of compound 3 yielded N-(2,4-dibromo-5-ethoxyphenyl)-4-methylbenzenesulfonamide (7) which on further treatment with different electrophiles, 8a-d yielded compound 9a-d. Enzyme inhibition activities of all the synthesized compounds were evaluated against acetylcholinesterase, butyrylcholinesterase, lipoxygenase, urease, chymotrypsin and tyrosinase enzymes; and found to be the most prominent inhibitor of tyrosinase enzyme. (author)

  12. Denatured state is critical in determining the properties of model proteins designed on different folds

    DEFF Research Database (Denmark)

    Amatori, Andrea; Ferkinghoff-Borg, Jesper; Tiana, Guido; Broglia, Richardo A.

    2008-01-01

    The thermodynamics of proteins designed on three common folds (SH3, chymotrypsin inhibitor 2 [CI2], and protein G) is studied with a simplified C alpha, model and compared with the thermodynamics of proteins designed on random-generated folds. The model allows to design sequences to fold within a d......RMSD ranging from 1.2 to 4.2 angstrom from the crystallographic native conformation and to study properties that are hard to be measured experimentally. It is found that the denatured state of all of them is not random but is, to different extents, partially structured. The degree of structure is more abundant...... for SH3 and protein G, giving rise to a weaker stability but a more efficient folding kinetics than CI2 and, even more, than the random-generated folds. Consequently, the features of the unfolded state seem to be as important in the determination of the thermodynamic properties of these proteins as...

  13. Interaction of the chaperone calreticulin with proteins and peptides of different structural classes

    DEFF Research Database (Denmark)

    Duus, K; Sandhu, N; Jørgensen, C S; Hansen, Paul Robert; Steinø, A; Thaysen-Andersen, M; Højrup, P; Houen, G

    2009-01-01

    The interaction of calreticulin with native and denatured forms and polypeptides in proteolytic digests of proteins representing structural classes of all-alpha-helix (hemoglobin, serum albumin), all-beta-sheet (IgG) and alpha-helix + beta-sheets (lysozyme, ovalbumin) was investigated. The binding...... of calreticulin to denatured proteins was found to depend on conformation and structural class of the protein. No interaction was observed with the native proteins, whereas binding was seen for the denatured proteins, the order of interaction being lysozyme = IgG > ovalbumin >> hemoglobin = serum...... albumin. Moreover, the interaction between calreticulin and the heat-denatured proteins depended on the temperature and time used for denaturation and the degree of proteolytic fragmentation. Calreticulin bound well to peptides in proteolytic digests from protease K or chymotrypsin treatment of lysozyme...

  14. Modulation of enzyme-substrate selectivity using tetraethylene glycol functionalized gold nanoparticles

    International Nuclear Information System (INIS)

    Tetraethylene glycol (TEG) functionalized gold nanoparticles with 2 nm core diameters (AuTEG) enhance α-chymotrypsin (ChT) enzyme activity in a substrate-selective fashion. We explored the hydrolysis of four different substrates and observed a marked increase in activity with the most hydrophobic substrate N-succinyl-alanine-alanine-proline-phenylalanine- p-nitroanilide (TP), while the other substrates remain virtually unaffected by the AuTEG 'crowding effect' in solution. The enhancement in catalysis is indicated by an increase in Kcat/Km as obtained from Lineweaver-Burk analysis and we hypothesize it to arise from a macromolecular crowding effect analogous to that observed with high molecular weight poly(ethylene glycol) (PEG) polymers.

  15. 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.

  16. The use of combined radiation methods for decreasing the bacterial dissemination of enzyme preparations

    International Nuclear Information System (INIS)

    A study was made on possibility of using ionizing radiation in combination with alternative magnetic field (AMF) and heating for decreasing the bacterial dissemination of proteolytic enzymes. Papain, trypsin, chymotrypsin and amylorysin (the preparation possessing proteolytic and amylolytic activities) were subjected to gamma irradiation at 10-25 kGy dose range, the effect of AMF with 750 oe and heating at 50 deg during 60 min. Model tests conducted with the use of Escherichia Coli cells and Bacillus anthracoides spores showed that survival rate of bacteria irradiated in protective medium was lower in the case of combined magnetoradiation and thermoradiation effect. The use of 10 kGy dose of ionizing radiation in combination with treatment in alternative magnetic field or with heating provided the required decrease of dissemination of irradiated enzyme samples with complete conservation of proteolytic activity by them

  17. Enhanced sensitivity to conformation in various proteins. Vibrational circular dichroism results

    International Nuclear Information System (INIS)

    Vibrational circular dichroism (VCD) spectra of several globular proteins dissolved in D2O are presented and compared to conventional UV-CD results. It can be seen that, for the alpha, beta, and alpha + beta categories of Levitt and Chothia, VCD evidences much larger band shape variations, including sign alteration, than does UV-CD. A direct parallel is seen between the VCD of the alpha-helix found in model polypeptides and the amide I' VCD of myoglobin. Since all structural aspects of the protein contribute to the VCD on a roughly equal footing, a similar correlation of the chymotrypsin amide I' VCD with that of beta-sheet models is not as clear. In addition, the VCD of random-coil-type proteins is found to be clearly related to VCD results from random-coil polypeptides. Finally, simulations are presented to postulate the expected VCD for protein structures having conformations that lie between the limiting cases discussed here

  18. Trichoderma harzianum L1 as a potential source for lytic enzymes and elicitor of defense responses in chickpea (Cicer arietinum L. against wilt disease caused by Fusarium oxysporum f. sp. ciceri.

    Directory of Open Access Journals (Sweden)

    Sreeramulu K

    2009-01-01

    Full Text Available The effect of some natural lignocellulosic substances on the production of ß-glucanase, chitinase, protease and xylanase from Trichoderma harzianum L1 has been studied under solid state fermentation conditions. Maximum activities of all these enzymes were observed in the fermentation medium containing the mixture of 1% rice bran, neem cake and 0.1% crab shell powder. The induction of plant defense response was investigated by inoculating the roots of chickpea cv JG62 with the biocontrol agent, T. harzianum L1. A root extract of chickpea inoculated with T. harzianum L1 showed increased activities of phenylalanine ammonia lyase and polyphenol oxidase, as well as induction of new trypsin and chymotrypsin inhibitors. The Fusarium oxysporum protease-2 was inhibited completely by root extract of chickpea inoculated with T. harzianum L1 and showed maximum resistance to rotting of roots caused by wilt disease

  19. Synthesis of enantiomerically pure α-[14C]methyl-L-tryptophan

    International Nuclear Information System (INIS)

    A practical method for the preparation of large amounts of enantiomerically pure α-[14C]methyl-L-tryptophan using the enzymatic resolution of the corresponding D,L-methyl ester is reported. The radiolabelled α-methyl group was introduced using the α-methylation of the lithium enolate of the Schiff base of L-tryptophan methyl ester. Hydrolysis of the Schiff base with 1 N HCl provided the D,L-methyl ester of α-[14C]methyl tryptophan. Enantioselective enzymatic hydrolysis of the L-methyl ester by α-chymotrypsin gave the enantiometrically pure α-[14C]methyl-L-tryptophan. The overall yield of this preparation was 33%. (author)

  20. Biodegradable Epoxy Networks Cured with Polypeptides

    Science.gov (United States)

    Nakamura, Shigeo; Kramer, Edward J.

    2006-03-01

    Epoxy resins are used widely for adhesives as well as coatings. However, once cured they are usually highly cross-linked and are not biodegradable. To obtain potentially biodegradable polypeptides that can cure with epoxy resins and achieve as good properties as the conventional phenol novolac hardeners, poly(succinimide-co-tyrosine) was synthesized by thermal polycondensation of L-aspartic acid and L-tyrosine with phosphoric acid under reduced pressure. The tyrosine/succinimide ratio in the polypeptide was always lower than the tyrosine/(aspartic acid) feed ratio and was influenced by the synthesis conditions. Poly(succinimide-tyrosine- phenylalanine) was also synthesized from L-aspartic acid, L- tyrosine and L-phenylalanine. The thermal and mechanical properties of epoxy resins cured with these polypeptides are comparable to those of similar resins cured with conventional hardeners. In addition, enzymatic degradability tests showed that Chymotrypsin or Subtilisin A could cleave cured films in an alkaline borate buffer.

  1. Bioactive protein-based nanofibers interact with intestinal biological components resulting in transepithelial permeation of a therapeutic protein

    DEFF Research Database (Denmark)

    Boutrup Stephansen, Karen; García-Díaz, María; Jessen, Flemming;

    2015-01-01

    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......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...

  2. Primary structure of human pancreatic elastase 2 determined by sequence analysis of the cloned mRNA

    International Nuclear Information System (INIS)

    A cDNA encoding elastase 2 has been cloned from a human pancreatic cDNA library. The cDNA contains a translation initiation site and a poly(A) recognition site and encodes a protein of 269 amino acids, including a proposed 16-residue signal peptide. The amino acid sequence of the deduced mature protein contains a 12-residue activation peptide containing a cysteine at residue 1 similar to that of chymotryspin. The proposed active enzyme contains all of the characteristic active-site amino acids, including His-57, Asp-102, and Ser-195. The S1 binding pocket is bounded by Gly-216 and Ser-226, making this pocket intermediate in size between chymotrypsins and elastase 1 or protease E, consistent with the substrate specificity of elastase 2 for long-chain aliphatic or aromatic amino acids. Computer modeling studies using the amino acid sequence of elastase 2 superimposed on the X-ray structure of porcine elastase 1 suggest that a change of Gln-192 in elastase 1 to Asn-192 in elastase 2 may account for the lower catalytic efficiency of the latter enzyme. Several basic residues appear to be near the ends of the extended binding pocket of elastases which might serve to anchor the enzyme to the elastin substrate. These studies indicate that elastases 2 and elastase 1 both contain an Arg-65A as well as a basic dipeptide at 223/224 which is not present in chymotrypsins. In addition, Arg-217A is present in humaan elastase 2 but absent in rat pancreatic protein which has been proposed to be an elastase 2 on the basis of sequence homology, but which was not isolated during screening of rat pancreatic tissue extracts for elastolytic activity

  3. 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.

  4. 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

  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. [Impact of energy related pollutants on chromosome structure]: Progress report

    International Nuclear Information System (INIS)

    Studies of the structure of nucleosome cores using chymotrypsin as a probe of hydrophobic residues showed that only leu-20 of H3 was readily accessible. Primary sites of cleavage of H2a, H2b, and H4 were identified. Chymotrypsin removal of specific histone termini showed that removal of the amino-terminal plus the carboxy-terminal 13 residues of H2a caused little unfolding. Using carbon-13 NMR spectroscopic, about 10% of histone amino acid residues were found to be in termini that are highly mobile. The major mobile segments were amino terminal regions of H3 and H2a, plus a carboxy-terminal region of H2a. The histone variants and developmental changes during embryogenesis of sea urchin were characterized. The early histone gene organization in L. variegatus was characterized, a genomic library was cloned in lambda phage, and several histone gene clones were selected. The nucleosome core length DNA forms crystalline phases at physiological concentrations. Microscopic and NMR spectroscopic methods were used to identify crystalline phases and to establish phase diagrams for transitions between phases as functions of DNA concentration and temperature. The sequence specificities of binding of several polycyclic aromatic chemicals to early H3 and H2a genes were examined. Chemicals studied were the bis-(o-phenanthroline) Cu(I) complex, AAAF, benzopyrene dihydrodiol epoxide, methylene blue, and acridine orange A preliminary map of binding sites of CuOP, AAAF and BPDE in and near the H3 gene showed that several sequence regions were bound preferentially by two or more of these chemicals. CuOP appeared to exhibit the most specificity. 80 refs., 4 figs

  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. Reactive oxygen species play a role in muscle wasting during thyrotoxicosis.

    Science.gov (United States)

    Bernardes, Sara Santos; Guarnier, Flávia Alessandra; Marinello, Poliana Camila; Armani, André; Simão, Andréa Name Colado; Cecchini, Rubens; Cecchini, Alessandra Lourenço

    2014-09-01

    The role of reactive oxygen species (ROS) in muscle protein hydrolysis and protein oxidation in thyrotoxicosis has not been explored. This study indicates that ROS play a role in skeletal muscle wasting pathways in thyrotoxicosis. Two experimental groups (rats) were treated for 5 days with either 3,3',5-triiodothyronine (HT) or HT with α-tocopherol (HT + αT). Two controls were used, vehicle (Control) and control treated with αT (Control + αT). Serum T3, peritoneal fat, serum glycerol, muscle and body weight, temperature, mitochondrial metabolism (cytochrome c oxidase activity), oxidative stress parameters and proteolytic activities were examined. High body temperature induced by HT returned to normal when animals were treated with αT, although total body and muscle weight did not. An increase in lipolysis was observed in the HT + αT group, as peritoneal fat decreased significantly together with an increase in serum glycerol. GSH, GSSG and total radical-trapping antioxidant parameter (TRAP) decreased and catalase activity increased in the HT group. The glutathione redox ratio was higher in HT + αT than in both HT and Control + αT groups. Carbonyl proteins, AOPP, mitochondrial and chymotrypsin-like proteolytic activities were higher in the HT group than in the Control. HT treatment with αT restored mitochondrial metabolism, TRAP, carbonyl protein, chymotrypsin-like activity and AOPP to the level as that of the Control + αT. Calpain activity was lower in the HT + αT group than in HT and Control + αT and superoxide dismutase (SOD) activity was higher in the HT + αT group than in the Control + αT. Although αT did not reverse muscle loss, ROS was involved in proteolysis to some degree. PMID:24842047

  9. The action of enzymes on rhodopsin.

    Science.gov (United States)

    RADDING, C M; WALD, G

    1958-11-20

    The effects have been examined of chymotrypsin, pepsin, trypsin, and pancreatic lipase on cattle rhodopsin in digitonin solution. The digestion of rhodopsin by chymotrypsin was measured by the hydrolysis of peptide bonds (formol titration), changes in pH, and bleaching. The digestion proceeds in two stages: an initial rapid hydrolysis which exposes about 30 amino groups per molecule, without bleaching; superimposed on a slower hydrolysis which exposes about 50 additional amino groups, with proportionate bleaching. The chymotryptic action begins at pH about 6.0 and increases logarithmically in rate to pH 9.2. Trypsin and pepsin also bleach rhodopsin in solution. A preparation of pancreatic lipase bleached it slightly, but no more than could be explained by contamination with proteases. In digitonin solution each rhodopsin molecule is associated in a micelle with about 200 molecules of digitonin; yet the latter do not appear to hinder enzyme action. It is suggested that the digitonin sheath is sufficiently fluid to be penetrated on collision with an enzyme molecule; and that once together the enzyme and substrate are held together by intermolecular attractive forces, and by the "cage effect" of bombardment by surrounding solvent molecules. The two stages of chymotryptic digestion of rhodopsin may correspond to an initial rapid fragmentation, such as has been observed with many proteinases and substrates; superimposed upon a slower digestion of the fragments. Since the first phase involves no bleaching, this may mean that rhodopsin can be broken into considerably smaller fragments without loss of optical properties. PMID:13587919

  10. 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

  11. Multiple IgE-mediated sensitizations to enzymes after occupational exposure: evaluation by skin prick test, RAST, and immunoblot.

    Science.gov (United States)

    Zentner, A; Jeep, S; Wahl, R; Kunkel, G; Kleine-Tebbe, J

    1997-09-01

    We investigated 10 sensitized and 10 nonsensitized workers from a pharmaceutic factory who had been exposed to powdered trypsin, chymotrypsin, bromelain, papain, amylase, and lipase. Ten nonallergic subjects served as a control group. Titrated skin prick tests (SPT), RAST, and immunoblot studies were performed with all six enzymes. SPT reactivity revealed multiple sensitizations to proteolytic enzymes, i.e., papain (specifically sensitized/total number of sensitizations: 9/10), trypsin (8/10), chymotrypsin (8/10), and bromelain (7/10) and appeared to be more frequent and more pronounced than sensitizations to amylase (3/10) or lipase (3/10). The low molecular weight of proteolytic enzymes (20-30 kDa) and their biologic activity might facilitate mucosal penetration more easily and thus-compared to amylase and lipase-permit an immune response and induction of allergic hypersensitivity. Immunoblot studies demonstrated IgG-binding bands in both SPT-positive and -negative workers, indicating exposure to the enzymes, but not in 10 unexposed control subjects. IgE-binding bands of the enzymes were detected only in workers with a positive SPT reaction and/or a positive RAST result. IgG bands were more frequent and the IgG/IgE ratio was increased in workers without allergic complaints compared to symptomatic workers. This might indicate that high levels of specific IgG antibodies to enzymes are associated with an immune response lacking allergic manifestations in spite of IgE-mediated sensitizations to the enzymes. Atopic subjects were at greater risk of developing IgE-mediated sensitization (7/10) and allergic symptoms to enzymes (5/7). However, even without risk of atopy, IgE-mediated hypersensitivity occurred in a few subjects (3/13) exposed to enzymes by inhalation for prolonged periods of time. PMID:9298178

  12. Aspergillus Oryzae S2 α-Amylase Domain C Involvement in Activity and Specificity: In Vivo Proteolysis, Molecular and Docking Studies.

    Science.gov (United States)

    Sahnoun, Mouna; Jemli, Sonia; Trabelsi, Sahar; Ayadi, Leila; Bejar, Samir

    2016-01-01

    We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α-amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α-amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino-acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α-amylases termed AmyB1 and AmyB2 reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (β/α)8 barrel domain and the domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility. PMID:27101008

  13. Structural and functional analysis of a bacterial cellulase by proteolysis.

    Science.gov (United States)

    Gilkes, N R; Kilburn, D G; Miller, R C; Warren, R A

    1989-10-25

    CenA is an endo-beta 1,4-glucanase from the cellulolytic bacterium Cellulomonas fimi. It is a bifunctional enzyme comprising an amino-terminal cellulose-binding domain and a carboxyl-terminal catalytic domain joined by a short sequence of prolyl and threonyl residues (the Pro-Thr box). Additional structural and functional information was revealed by a detailed analysis of the products generated by proteolytic cleavage of a nonglycosylated form of CenA. An extracellular C. fimi protease attacked nonglycosylated CenA at the junctions between the Pro-Thr box and the two functional domains. A stable "core" peptide (p30), corresponding to the catalytic domain, remained after extensive proteolysis. p30 was resistant to further attack even in the presence of 2-mercaptoethanol plus urea or dithiothreitol, but treatment in the presence of sodium dodecyl sulfate allowed complete fragmentation to small peptides. Stable peptides, identical, or closely related to p30, were generated by alpha-chymotrypsin or papain. These results indicated that the catalytic domain adopts a tightly folded conformation affording protection from proteolytic attack. In contrast, the cellulose-binding domain showed a relatively loose conformation. Progressive proteolytic truncation from the amino terminus was apparent during incubation with alpha-chymotrypsin or papain, or with C. fimi protease under reducing conditions. Affinity for cellulose was retained by products missing up to 64 amino-terminal amino acids. The remaining carboxyl-proximal region of the cellulose-binding domain with affinity (47 amino acids) contained sequences highly conserved in analogous domains from other bacterial endo-beta 1,4-glucanases. By analogy with other systems, the properties of the Pro-Thr box are consistent with an elongated conformation. The results of this investigation suggest that CenA has a tertiary structure which resembles that of certain fungal cellulases. PMID:2681184

  14. 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.

  15. Prospeção de inibidores de serinoproteinases em folhas de leguminosas arbóreas da floresta Amazônica Prospecting serine proteinase inhibitors in leaves from leguminous trees of the Amazon forest

    Directory of Open Access Journals (Sweden)

    Larissa Ramos Chevreuil

    2011-03-01

    , Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata and S. polyphylla. Leaves were collected, dried at 30ºC for 48 h, crushed and subjected to extraction with NaCl (0.15 M, 10% w/v, resulting in the total extract. Tests were performed to determine the concentration of proteins and to detect of inhibitory activity against bovine trypsin and chymotrypsin. The content of crude and soluble protein in leaf extracts varied from 7.9 to 31.2% and 1.3 to 14.8%, respectively. The inhibitory activity on trypsin and chymotrypsin was observed in all leaf extracts. However, in extracts of E. maximum, L. leucocephala, P. pendula, S. corrugata and S. polyphylla, the inhibition was greater on trypsin, while extract of P. multijuga was more effective against chymotrypsin. We conclude that leaf extracts of leguminous trees have serine proteinase inhibitors and show potential biotecnological applications.

  16. 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

  17. Determination of Digestive Enzyme Activity in the Digesta from the Small Intestinal of Growing Pigs and Development of in vitro Evaluation System for Feed Bioavailability Using Artificial Small Intestinal Juice

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    Junjun Wang

    2012-01-01

    Full Text Available The objective of this study was to determine enzyme activity in the digesta from the small intestine of growing pigs and develope an in vitro evaluation system for feed bioavailability using artificial small intestinal juice. Ten pigs (22.77±0.89 kg and fitted with a simple T-cannula at the jejunum were used in a doubly 5x5 Latin square design. In each period, pigs were offered one of five diets differing in nutrient level for 14 days. The Standard diet (ST contained 3400 kcal kg-1 of Digestible Energy value (DE and 17% Crude Protein (CP. The DE for the other four diets was 3600, 3200, 3600 and 3200 kcal kg-1, respectively while the CP level was 21, 13, 13 and 21%, respectively. The small intestinal digesta was collected for determining digestive enzyme activity. Meanwhile, feces were collected for determining in vivo feed digestibility. The results showed that the range of amylase, trypsin, chymotrypsin and lipase activities in the intestinal fluid of growing pigs was 15.52-251.43, 21.24-67.39, 3.45-19.17 and 0.02-3.59 U mL-1, respectively. To establish an in vitro evaluation system for feed bioavailability, Artificial Small Intestinal Juice (ASIJ was prepared with mixed enzyme reagents based on the mean activities of amylase, trypsin, chymotrypsin and lipase in the digesta used to evaluate the five diets by three-stage enzymatic incubation. By comparing the DE of the five feedstuffs from the in vivo method and the digestibility of the feedstuffs from the in vitro ASIJ analysis, mathematical models for predicting in vivo DE of In Vitro Dry Matter (IVDM, Organic Matter (IVOM and DE (IVDE was established (DE = 0.1076xIVDM+0.3741, R2 = 0.34; DE = 0.1276xIVOM+1.6486, R2 = 0.31; DE = 0.4625xIVDE+7.2065, R2 = 0.71. There were no significant differences between the in vitro evaluation results and the developed in vitro method. Therefore, the system in this study based on ASIJ is a convinent and reasonably accurate method for in vitro evaluation of

  18. 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

  19. 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.

  20. N-hydroxypropyltrimethylammonium polydimethylaminoethylmethacrylate sub-microparticles for oral delivery of insulin--an in vitro evaluation.

    Science.gov (United States)

    Sonia, T A; Sharma, Chandra P

    2013-07-01

    The present study describes the synthesis and in vitro evaluation of quaternised polydimethylaminoethylmethacrylate for oral delivery of insulin. Quaternisation of the polymer was carried out by conjugating N-hyroxypropyltrimethylammonium chloride to aminoterminated polydimethylaminoethylmethacrylate. Quaternised particles were characterised by particle size, zeta potential measurements, nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), differential scanning calorimetry (DSC) and atomic force microscopy (AFM). In addition, in vitro insulin release experiments, cytotoxic evaluation on L929 & Caco-2 cells, mucoadhesion, enzymatic degradation and tight junction visualisation studies were also performed to evaluate the potential of this matrix for oral delivery of insulin. Results suggest that the quaternised particles exhibited positive zeta potential with a particle size of 513.6±17 nm. Dose-dependent cytotoxic evaluation of quaternised particles on L929 & Caco-2 cells confirmed the nontoxic nature of the matrix. Quaternised particles were more mucoadhesive compared to parent polymer. Adhesive behaviour of mucin with quaternised particles were confirmed by DSC. Moreover these particles exhibited calcium chelating ability and displayed significant inhibitory effect towards trypsin and chymotrypsin. These particles also helped in the opening of tight junctions by disruption of actin filaments and binding to Zona Occludens (ZO-1) proteins. Preliminary studies suggest that the quaternised particles can act as suitable candidates for oral delivery of insulin. PMID:23500732

  1. Myonase is localized in skeletal muscle myofibrils.

    Science.gov (United States)

    Hori, Shinichiro; Yamada, Makoto; Ohtani, Sachiko; Hori, Chiyo; Yokomizo, Tadahiro; Webb, Timothy; Shimokawa, Teruhiko

    2002-09-01

    A novel chymotrypsin-like proteinase termed myonase was previously purified from MDX-mouse skeletal muscle [Hori et al. (1998) J. Biochem. 123, 650-658]. Western blots and immunohistochemical analyses showed that myonase was present within myocytes of both MDX-mouse and control mouse, and subcellular fractionation showed that it was associated with myofibrils. No significant difference was observed on Western blots between the amounts of myonase in myofibrils of MDX-mouse and control mouse, but the amount of myonase recoverable as a pure protein was 5-10-fold more when MDX-mouse was the source of the skeletal muscle. Myofibrils also possessed an endogenous inhibitor of myonase, whose inhibitory activity at physiological pH (pH 7.4) depended on salt concentration, stronger inhibition being observed at a low salt concentration. Inhibition at alkaline pH (pH 9) was weak and independent of salt concentration. Myonase in myofibrils was partially released at neutral pH by a high salt concentration (>0.6 M NaCl). However, even at 4 M NaCl, more than 80% of myonase remained within the myofibrils. Under alkaline conditions, release of myonase from myofibril was more extensive. At pH 12, myonase was almost completely present in the soluble fraction. Release of myonase under these conditions coincided with the solubilization of other myofibrillar proteins. PMID:12204111

  2. Click-Based Libraries of SFTI-1 Peptides: New Methods Using Reversed-Phase Silica.

    Science.gov (United States)

    Cistrone, Philip A; Dawson, Philip E

    2016-03-14

    Performing sequential reactions for the orthogonal derivatization of peptides in solution often requires intermediate handling and purification steps. To solve these problems, we have exploited the distinct adsorption kinetics of peptides toward particulate reversed-phase (RP) C18 silica material, enabling consecutive reactions to be performed without intermediate elution. To illustrate this approach, sequential CuAAC/click reactions were used to modify an analog of the bicyclic peptide sunflower trypsin inhibitor 1 (SFTI-1), a potent scaffold for trypsin and chymotrypsin-like enzyme inhibitors. The SFTI-1 scaffold was synthesized containing both β-azido alanine and propargyl glycine residues. Despite the mutual reactivity of these groups, site isolation on RP silica enabled consecutive click reactions and associated washing steps to be performed while the peptide remained immobilized. Importantly, this approach eliminated side products that could form between two peptides or within a single peptide. These studies suggest a broad utility for RP silica in solving both peptide handling problems and in improving synthetic workflows. PMID:26914614

  3. Structure of the catalytic domain of the hepatitis C virus NS2-3 protease

    Energy Technology Data Exchange (ETDEWEB)

    Lorenz,I.; Marcotrigiano, J.; Dentzer, T.; Rice, C.

    2006-01-01

    Hepatitis C virus is a major global health problem affecting an estimated 170 million people worldwide. Chronic infection is common and can lead to cirrhosis and liver cancer. There is no vaccine available and current therapies have met with limited success. The viral RNA genome encodes a polyprotein that includes two proteases essential for virus replication. The NS2-3 protease mediates a single cleavage at the NS2/NS3 junction, whereas the NS3-4A protease cleaves at four downstream sites in the polyprotein. NS3-4A is characterized as a serine protease with a chymotrypsin-like fold, but the enzymatic mechanism of the NS2-3 protease remains unresolved. Here we report the crystal structure of the catalytic domain of the NS2-3 protease at 2.3 Angstroms resolution. The structure reveals a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer, and the nucleophilic cysteine by the other. The carboxy-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. Proteolysis through formation of a composite active site occurs in the context of the viral polyprotein expressed in mammalian cells. These features offer unexpected insights into polyprotein processing by hepatitis C virus and new opportunities for antiviral drug design.

  4. Data of in vitro synthesized dsRNAs on growth and development of Helicoverpa armigera.

    Science.gov (United States)

    Chikate, Yojana R; Dawkar, Vishal V; Barbole, Ranjit S; Tilak, Priyadarshini V; Gupta, Vidya S; Giri, Ashok P

    2016-06-01

    The data presented in this article is related to the research article "RNAi of selected candidate genes interrupts growth and development of Helicoverpa armigera" (Chikate et al., 2016) [1]. RNA interference (RNAi) is emerging as a potent insect pest control strategy over current methods and their resistance by pest. In this study we tested 15 different in vitro synthesized dsRNAs for gene silencing in Helicoverpa armigera. These dsRNAs were specific against H. armigera enzymes/proteins such as proteases like trypsins (HaTry2, 3, 4 and 6), chymotrypsin (HaChy4) and cysteine proteases such as cathepsin (HaCATHL); glutathione S-transferases (HaGST1a, 6 and 8); esterases (HaAce4, HaJHE); catalase (HaCAT); super-oxide-dismutase (HaCu/ZnSOD); fatty acid binding protein (HaFabp) and chitin deacetylase (HaCda5b). These dsRNAs were fed to second instar larvae at an optimized dose (60 µg/day) for 3 days separately. Effects of dsRNA feeding were observed in terms of larval mass gain, percentage mortality and phenotypic abnormalities in later developmental stages of H. armigera. These findings might provide potential new candidates for designing sequence-specific dsRNA as pesticide in crop protection. PMID:27222861

  5. 43-kilodalton protein of Torpedo nicotinic postsynaptic membranes: purification and determination of primary structure

    International Nuclear Information System (INIS)

    The primary structure of the 43-kilodalton peripheral membrane protein (43-kDa protein) of Torpedo nicotinic postsynaptic membrane has been determined. The 14C-labelled 43-kDa protein, which was isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has an amino terminus resistant to Edman degradation, while the sequence at the carboxyl terminus is Tyr-Val. An amino acid sequence of 405 residues was obtained by NH2-terminal sequence analysis of complementary peptides generated by digestion with trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and endoproteinase Lys-C, as well as by chemical cleavage at methionine. This sequence of molecular mass 45,618 daltons lacks the amino terminus but extends to the carboxyl terminus of the 43-kDa protein. Unusual structural features of the 43-kDa protein include two regions of ∼ 80 residues, each containing 10% cysteine, as well as stretches predicted to exist as amphipathic α-helices. Other than the group blocking the amino terminus, no evidence was found for posttranslational modification of amino acids. The 43-kDa protein may represent a novel protein family because a computer search of this sequence with the National Biomedical Research Foundation data base (Release 12.0) did not reveal any significant homology to known protein sequences

  6. Proteinase inhibitory activities of two two-domain Kazal proteinase inhibitors from the freshwater crayfish Pacifastacus leniusculus and the importance of the P(2) position in proteinase inhibitory activity.

    Science.gov (United States)

    Donpudsa, Suchao; Söderhäll, Irene; Rimphanitchayakit, Vichien; Cerenius, Lage; Tassanakajon, Anchalee; Söderhäll, Kenneth

    2010-11-01

    Serine proteinase inhibitors are found ubiquitously in living organisms and involved in homeostasis of processes using proteinases as well as innate immune defense. Two two-domain Kazal-type serine proteinase inhibitors (KPIs), KPI2 and KPI8, have been identified from the hemocyte cDNA library of the crayfish Pacifastacus leniusculus. Unlike other KPIs from P. leniusculus, they are found specific to the hemocytes and contain an uncommon P(2) amino acid residue, Gly. To unveil their inhibitory activities, the two KPIs and their domains were over-expressed. By testing against subtilisin, trypsin, chymotrypsin and elastase, the KPI2 was found to inhibit strongly against subtilisin and weakly against trypsin, while the KPI8 was strongly active against only trypsin. With their P(1) Ser and Lys residues, the KPI2_domain2 and KPI8_domain2 were responsible for strong inhibition against subtilisin and trypsin, respectively. Mutagenesis of KPI8_domain1 at P(2) amino acid residue from Gly to Pro, mimicking the P(2) residue of KPI8_domain2, rendered the KPI8_domain1 strongly active against trypsin, indicating the important role of P(2) residue in inhibitory activities of the Kazal-type serine proteinase inhibitors. Only the KPI2 was found to inhibit against the extracellular serine proteinases from the pathogenic oomycete of the freshwater crayfish, Aphanomyces astaci. PMID:20621193

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein.

    Science.gov (United States)

    Oliva, M L; Santomauro-Vaz, E M; Andrade, S A; Juliano, M A; Pott, V J; Sampaio, M U; Sampaio, C A

    2001-01-01

    We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates. PMID:11258660

  16. 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.

  17. 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

  18. 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.

  19. Purification and characterization of a novel extracellular alkaline protease from Cellulomonas bogoriensis.

    Science.gov (United States)

    Li, Fan; Yang, Liyuan; Lv, Xue; Liu, Dongbo; Xia, Hongmei; Chen, Shan

    2016-05-01

    An extracellular alkaline protease produced by the alkali-tolerant Cellulomonas bogoriensis was purified by a combination of ammonium sulfate precipitation and cation exchange chromatography. The purity of the protease was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was confirmed to be 18.3 kDa. The enzyme showed optimum activity at 60 °C and pH 11. The stability of the protease was maintained at a wide temperature range of 4-60 °C and pH range of 3-12. Irreversible inhibition of the enzyme activity by phenylmethylsulfonyl fluoride and tosyl-l-phenylalanine chloromethyl ketone demonstrated that the purified enzyme is a chymotrypsin of the serine protease family. The Km and Vmax of the protease activity on casein were 19.2 mg/mL and 25000 μg/min/mg, respectively. The broad substrate specificity and remarkable stability in the presence of organic solvents, salt, and commercial detergents, as well as its excellent stain removal and dehairing capability, make the purified alkaline protease a promising candidate for industrial applications. PMID:26849962

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. Radiolabeling and pharmacokinetics of Bowman-Byrk inhibitor from Macrotyloma axillare

    International Nuclear Information System (INIS)

    There are several evidences that dietary factors have a tight correlation with different kinds of cancer development. Among the compounds of the diet related with cancer development prevention, we could mention the Bowman-Birk Inhibitors (BBI). We isolated preparations of BBI from Macrotyloma axillare showing high specific inhibitory activities over bovine trypsin and chymotrypsin enzymes, specially the germinated seeds preparation. These inhibitors had their pharmacokinetics parameters determined and compared with soybeans inhibitors parameters, which are by the way, the most studied inhibitors of this class. Macrotyloma axillare inhibitors are widely distributed over mice organs, but they show an important difference in comparison with soybean inhibitor biodistribution, which is a meaning bigger affinity for the stomach. Macrotyloma axillare inhibitors when injected intravenously have a fast distribution, with distribution volumes of approximately four folds the plasmatic volume, but they are eliminated slowly, with plasmatic half-lives between 7 and 15 hours. When applied in isolated intestinal loops of Swiss mice, soybean inhibitors get to almost 50% of bioavailability, while Macrotyloma axillare inhibitors from seeds and cotyledons get to 30 and 40% respectively. Meanwhile, the biggest potency of cotyledons inhibitors of Macrotyloma makes available a bigger activity (approximately nine folds) then soybean inhibitors do. (author)

  5. 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.

  6. 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

  7. 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.

  8. The Characterization of SaPIN2b, a Plant Trichome-Localized Proteinase Inhibitor from Solanum americanum

    Directory of Open Access Journals (Sweden)

    Zeng-Fu Xu

    2012-11-01

    Full Text Available Proteinase inhibitors play an important role in plant resistance of insects and pathogens. In this study, we characterized the serine proteinase inhibitor SaPIN2b, which is constitutively expressed in Solanum americanum trichomes and contains two conserved motifs of the proteinase inhibitor II (PIN2 family. The recombinant SaPIN2b (rSaPIN2b, which was expressed in Escherichia coli, was demonstrated to be a potent proteinase inhibitor against a panel of serine proteinases, including subtilisin A, chymotrypsin and trypsin. Moreover, rSaPIN2b also effectively inhibited the proteinase activities of midgut trypsin-like proteinases that were extracted from the devastating pest Helicoverpa armigera. Furthermore, the overexpression of SaPIN2b in transgenic tobacco plants resulted in enhanced resistance against H. armigera. Taken together, our results demonstrated that SaPIN2b is a potent serine proteinase inhibitor that may act as a protective protein in plant defense against insect attacks.

  9. 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

  10. Preparation of antioxidant enzymatic hydrolysates from alpha-lactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/MS.

    Science.gov (United States)

    Hernández-Ledesma, Blanca; Dávalos, Alberto; Bartolomé, Begoña; Amigo, Lourdes

    2005-02-01

    We have investigated the antioxidant activity of hydrolysates from whey proteins bovine alpha-lactalbumin (alpha-La) and beta-lactoglobulin A (beta-Lg A) by commercial proteases (pepsin, trypsin, chymotrypsin, thermolysin, and Corolase PP). Corolase PP was the most appropriate enzyme to obtain antioxidant hydrolysates from alpha-La and beta-Lg A (ORAC-FL values of 2.315 and 2.151 micromol of Trolox equivalent/mg of protein, respectively). A total of 42 peptide fragments were identified by HPLC-MS/MS in the beta-Lg A hydrolysate by Corolase PP. One of the sequences (Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile) possessed radical scavenging (ORAC-FL value of 2.621 micromol of Trolox equivalent/micromol of peptide) higher than that of butylated hydroxyanisole (BHA). Our results suggest that whey protein hydrolysates could be suitable as natural ingredients in enhancing antioxidant properties of functional foods and in preventing oxidation reaction in food processing. PMID:15686406

  11. Buffalo Cheese Whey Proteins, Identification of a 24 kDa Protein and Characterization of Their Hydrolysates: In Vitro Gastrointestinal Digestion

    Science.gov (United States)

    Bassan, Juliana C.; Goulart, Antonio J.; Nasser, Ana L. M.; Bezerra, Thaís M. S.; Garrido, Saulo S.; Rustiguel, Cynthia B.; Guimarães, Luis H. S.; Monti, Rubens

    2015-01-01

    Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey. PMID:26465145

  12. Antioxidative activity of whey protein hydrolysates in a liposomal system.

    Science.gov (United States)

    Peña-Ramos, E A; Xiong, Y L

    2001-12-01

    Whey protein isolate (WPI) with or without preheating (90 degrees C for 5 min) was hydrolyzed for 0.5 to 6 h using four pure enzymes (pepsin, papain, trypsin, and chymotrypsin) and three commercial crude proteases. After determining the degree of hydrolysis, the hydrolysates were incubated (37 degrees C, 1 h) with a liposome oxidizing system (50 mM FeCl3/0.1 mM ascorbate, pH 7.0). Lipid oxidation was measured by determining the concentrations of TBA-reactive substances (TBARS). The degree of hydrolysis of WPI ranged from 4 to 37% depending on the enzymes used and whether the substrate was heated or not. WPI hydrolysates prepared by pure enzyme treatments did not prevent TBARS formation in the oxidative model system, but WPI hydrolyzed by the commercial crude enzymes, especially protease F, exhibited antioxidant activity. The antioxidative potential of hydrolyzed WPI was not affected by the degree of hydrolysis, and it was improved by preheat treatment in only some samples. PMID:11814013

  13. β-Lactoglobulin as nanotransporter--Part II: Characterization of the covalent protein modification by allicin and diallyl disulfide.

    Science.gov (United States)

    Wilde, Sandra Catharina; Treitz, Christian; Keppler, Julia Katharina; Koudelka, Tomas; Palani, Kalpana; Tholey, Andreas; Rawel, Harshadrai M; Schwarz, Karin

    2016-04-15

    The whey protein β-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified β-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys(121) turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified β-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to β-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein. PMID:26617049

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. iCVD growth of poly(N-vinylimidazole) and poly(N-vinylimidazole-co-N-vinylpyrrolidone)

    International Nuclear Information System (INIS)

    The imidazole group plays an important role in α-chymotrypsin catalysis, metal-ion complexation, counterion or dye binding. Poly(N-vinylimidazole), PVI, is also a good model polymer interacting with neutral salts. The poly(N-vinylimidazole-co-N-vinylpyrrolidone) copolymer P(VI-co-VP), can be used to produce highly functionalized polymers. PVI and P(VI-co-VP) thins films were achieved via initiated chemical vapor deposition (iCVD), a solvent-free process to form films under mild conditions. The polymerization was initiated by hot wire heated tert-butyl peroxide (TBPO). The chemical structure and compositions of the polymers were analyzed using FTIR and XPS. The growth rate of PVI as a function of the pressure inside the iCVD reactor was measured to be 1 nm/h mTorr. The XPS results show that the functional groups were retained in the polymer deposited. For the P(VI-co-VP) deposition, there are more VI groups found in the co-polymer chain even when the reacting monomers were fed in the same ratio.

  19. iCVD growth of poly(N-vinylimidazole) and poly(N-vinylimidazole-co-N-vinylpyrrolidone)

    Energy Technology Data Exchange (ETDEWEB)

    Chen Guohua, E-mail: kechengh@ust.h [Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 (United States); Lau, Kenneth K.S., E-mail: klau@drexel.ed [Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 (United States); Gleason, Karen K., E-mail: kkg@mit.ed [Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 (United States)

    2009-04-30

    The imidazole group plays an important role in {alpha}-chymotrypsin catalysis, metal-ion complexation, counterion or dye binding. Poly(N-vinylimidazole), PVI, is also a good model polymer interacting with neutral salts. The poly(N-vinylimidazole-co-N-vinylpyrrolidone) copolymer P(VI-co-VP), can be used to produce highly functionalized polymers. PVI and P(VI-co-VP) thins films were achieved via initiated chemical vapor deposition (iCVD), a solvent-free process to form films under mild conditions. The polymerization was initiated by hot wire heated tert-butyl peroxide (TBPO). The chemical structure and compositions of the polymers were analyzed using FTIR and XPS. The growth rate of PVI as a function of the pressure inside the iCVD reactor was measured to be 1 nm/h mTorr. The XPS results show that the functional groups were retained in the polymer deposited. For the P(VI-co-VP) deposition, there are more VI groups found in the co-polymer chain even when the reacting monomers were fed in the same ratio.

  20. 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.

  1. A trypsin inhibitor from rambutan seeds with antitumor, anti-HIV-1 reverse transcriptase, and nitric oxide-inducing properties.

    Science.gov (United States)

    Fang, Evandro Fei; Ng, Tzi Bun

    2015-04-01

    Nephelium lappaceum L., commonly known as "rambutan," is a typical tropical tree and is well known for its juicy and sweet fruit which has an exotic flavor. Chemical studies on rambutan have led to the identification of various components such as monoterpene lactones and volatile compounds. Here, a 22.5-kDa trypsin inhibitor (N . lappaceum trypsin inhibitor (NLTI)) was isolated from fresh rambutan seeds using liquid chromatographical techniques. NLTI reduced the proteolytic activities of both trypsin and α-chymotrypsin. Dithiothreitol reduced the trypsin inhibitory activity of NLTI at a concentration of 1 mM, indicating that an intact disulfide bond is essential to the activity. NLTI inhibited HIV-1 reverse transcriptase with an IC50 of 0.73 μM. In addition, NLTI manifested a time- and dose-dependent inhibitory effect on growth in many tumor cells. NLTI is one of the few trypsin inhibitors with nitric oxide-inducing activity and may find application in tumor therapy. PMID:25820360

  2. 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, Madan L.B.; Mishra, Durga Prasad

    2015-01-01

    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

  3. 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.

  4. 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.

  5. 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

  6. Primary structure of a coagulant enzyme, bilineobin, from Agkistrodon bilineatus venom.

    Science.gov (United States)

    Nikai, T; Ohara, A; Komori, Y; Fox, J W; Sugihara, H

    1995-04-01

    The amino acid sequence and disulfide bridge location of the coagulant enzyme, named bilineobin, isolated from the venom of Agkistrodon bilineatus was determined by Edman sequencing of the peptides derived from digests with cyanogen bromide, clostripain, Staphylococcus aureus V8 protease, trypsin, and chymotrypsin. This enzyme has a molecular weight of 57,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, bilineobin consists of 235 amino acids and has a calculated molecular weight of 26,481. The enzyme contains fucose, GlcNAc, galactose, mannose and NeuAc and six N-linked glycosylation consensus sites. The carboxyterminal amino acid, proline, was determined using carboxypeptidase Y. The six disulfide bonds of bilineobin link Cys78 to Cys234, Cys120 to Cys188, Cys178 to Cys203, Cys7 to Cys141, Cys152 to Cys167, and Cys28 to Cys44. The amino acid sequence similarity to flavoxobin (T.C. Shieh et al., 1988, J. Biochem (Tokyo) 103, 596-605) and batroxobin (N. Itoh et al., 1987, J. Biol. Chem. 262, 3132-3135) was 67%. The deglycosylated enzyme more rapidly generated fibrinopeptide A than native bilineobin. PMID:7726578

  7. 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 (72mg McLTP1 from 100g of noni seeds). McLTP1 exhibited molecular masses of 9.450 and 9.466kDa, 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 8mg/kg) and orally (8mg/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 30min incubation at 80°C. Pretreatment of mice with McLTP1 (8mg/kg, i.p. and p.o.) also decreased neurogenic and inflammatory phases of nociception in the formalin test. Naloxone (2mg/kg, i.p.) antagonised the antinociceptive effect of McLTP1 suggesting that the opioid mechanisms mediate the analgesic properties of this protein. PMID:26783638

  8. Isolation and partial characterization of Alzheimer neurofibrillary tangles

    International Nuclear Information System (INIS)

    Neurofibrillary tangles (NFT) were isolated from cerebral cortex of three cases of Alzheimer's disease (AD) by SDS-βME treatment followed by sucrose gradient ultracentrifugation. This material was predominantly NFT by electron microscopy and was excluded from all pore-sized polyacrylamide gels. It remained insoluble in strong acid and basic conditions, chaotropic and reducing agents. It resisted digestion by trypsin, chymotrypsin, subtilisin, urea-pepsin, collagenase, pronase, hyaluronidase, lipases and phospholipases but yielded a consistent amino acid analysis showing the presence of cysteine and methionine, more than 20% hydrophobic residues and 12% basic residues. Subjected to automated Edman degradation presented a non-reactive amino terminus. Under electron microscopy NFT appeared to be composed mainly by single and double filaments. Single filaments can turn and intertwine with themselves to make the regular arrangement of the double filaments. Purified NFT have been used to raise high titered polyclonal antisera for immunohistological studies. It specifically reacted with isolated NFT, affected neurons in cases of AD, aging brains, postencephalitic Parkinson's disease, Down's syndrome and dementia pugilistica but no reaction was observed with normal brain, cerebrovascular amyloid angiopathy, or the amyloid core from neuritic plaques

  9. Impaired proteasome function in sporadic amyotrophic lateral sclerosis.

    Science.gov (United States)

    Kabashi, Edor; Agar, Jeffrey N; Strong, Michael J; Durham, Heather D

    2012-06-01

    Abstract The ubiquitin-proteasome system, important for maintaining protein quality control, is compromised in experimental models of familial ALS. The objective of this study was to determine if proteasome function is impaired in sporadic ALS. Proteasomal activities and subunit composition were evaluated in homogenates of spinal cord samples obtained at autopsy from sporadic ALS and non-neurological control cases, compared to cerebellum as a clinically spared tissue. The level of 20S α structural proteasome subunits was assessed in motor neurons by immunohistochemistry. Catalysis of peptide substrates of the three major proteasomal activities was substantially reduced in ALS thoracic spinal cord, but not in cerebellum, accompanied by alterations in the constitutive proteasome machinery. Chymotrypsin-like activity was decreased to 60% and 65% of control in ventral and dorsal spinal cord, respectively, concomitant with reduction in the β5 subunit with this catalytic activity. Caspase- and trypsin-like activities were reduced to a similar extent (46% - 68% of control). Proteasome levels, although generally maintained, appeared reduced specifically in motor neurons by immunolabelling. In conclusion, there are commonalities of findings in sporadic ALS patients and presymptomatic SOD1-G93A transgenic mice and these implicate inadequate proteasome function in the pathogenesis of both familial and sporadic ALS. PMID:22632443

  10. Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts.

    Science.gov (United States)

    Taylor, David M; Kabashi, Edor; Agar, Jeffrey N; Minotti, Sandra; Durham, Heather D

    2005-01-01

    Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes. PMID:16184768

  11. Proteasomes remain intact, but show early focal alteration in their composition in a mouse model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Kabashi, Edor; Agar, Jeffrey N; Hong, Yu; Taylor, David M; Minotti, Sandra; Figlewicz, Denise A; Durham, Heather D

    2008-06-01

    In amyotrophic lateral sclerosis caused by mutations in Cu/Zn-superoxide dismutase (SOD1), altered solubility and aggregation of the mutant protein implicates failure of pathways for detecting and catabolizing misfolded proteins. Our previous studies demonstrated early reduction of proteasome-mediated proteolytic activity in lumbar spinal cord of SOD1(G93A) transgenic mice, tissue particularly vulnerable to disease. The purpose of this study was to identify any underlying abnormalities in proteasomal structure. In lumbar spinal cord of pre-symptomatic mice [postnatal day 45 (P45) and P75], normal levels of structural 20S alpha subunits were incorporated into 20S/26S proteasomes; however, proteasomal complexes separated by native gel electrophoresis showed decreased immunoreactivity with antibodies to beta3, a structural subunit of the 20S proteasome core, and beta5, the subunit with chymotrypsin-like activity. This occurred prior to increase in beta5i immunoproteasomal subunit. mRNA levels were maintained and no association of mutant SOD1 with proteasomes was identified, implicating post-transcriptional mechanisms. mRNAs also were maintained in laser captured motor neurons at a later stage of disease (P100) in which multiple 20S proteins are reduced relative to the surrounding neuropil. Increase in detergent-insoluble, ubiquitinated proteins at P75 provided further evidence of stress on mechanisms of protein quality control in multiple cell types prior to significant motor neuron death. PMID:18315558

  12. 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

  13. Isolation of selenium organic species from antarctic krill after enzymatic hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Siwek, Mariana; Galunsky, Boris [Technical University Hamburg-Harburg, Institute of Biotechnology II, Hamburg (Germany); Niemeyer, Bernd [GKSS Research Centre, Institute for Coastal Research, Geesthacht (Germany); University of the Federal Armed Forces Hamburg, Institute of Thermodynamics, Hamburg (Germany)

    2005-02-01

    Total selenium content and its distribution in the soluble and insoluble protein-bound fractions obtained after aqueous extraction of antarctic krill samples were determined. About 26% of the total selenium (2.4 {mu}g g{sup -1} dry weight) was found in the supernatant; the rest was in the pellet. Isolation of low molecular selenium-containing fractions was also performed by enzymatic digestion of the protein, followed by size-exclusion chromatography in conjunction with atomic absorption spectrometry. From the applied various proteinases (pronase E, subtilisin Carlsberg, trypsin, chymotrypsin, proteinase and proteinase N from Bacillus subtilis and Novo 0.6 MPX enzyme), the treatment with pronase E led to best recovery of selenium. About 96% of the total Se was found in the hydrolysate, mainly in low molecular weight fractions. Eighty percent of the Se species were in fractions with molecular weights in the range of amino acids and short peptides. High-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC-ICP-MS) allowed the identification of selenomethionine and the assumption that selenocystine or its derivatives were the main species in these fractions. (orig.)

  14. 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

  15. Photochemical labeling of membrane hydrophobic core of human erythrocytes using a new photoactivable reagent 2-(/sup 3/H)diazofluorene

    Energy Technology Data Exchange (ETDEWEB)

    Pradhan, D.; Lala, A.K.

    1987-06-15

    Photoactivable reagents have been useful for studying the structural aspects of membrane hydrophobic core. We have reported earlier the use of diazofluorene as a probe for fluorescent photochemical labeling of hydrophobic core in artificial membranes. To quantitate and enhance the monitoring ability of this probe, we have synthesized 2-(/sup 3/H)diazofluorene of high specific activity. This reagent rapidly partitions into phosphatidylcholine vesicles and selectively labels the fatty acyl chains of phosphatidylcholine. The insertion yield (13%) is not affected by the presence of scavengers like reduced glutathione. 2-(/sup 3/H)Diazofluorene also readily partitions into erythrocyte membranes and on photolysis labels the membrane. The overall insertion was 48% with 9.7% in protein fraction and the rest in lipids. The distribution of radioactivity in labeled protein fraction was restricted to integral membrane proteins with Band 3 being the major protein labeled. There is little or no labeling associated with extrinsic proteins like spectrin. Further analysis of labeled Band 3 by treatment with chymotrypsin indicated that the labeling was restricted to the membrane spanning CH-17 and CH-35 fragments. No labeling of the cytoplasmic fragment of Band 3 could be observed. 2-(/sup 3/H)Diazofluorene should prove useful for studying integral membrane proteins and their membrane-spanning regions.

  16. Photochemical labeling of membrane hydrophobic core of human erythrocytes using a new photoactivable reagent 2-[3H]diazofluorene

    International Nuclear Information System (INIS)

    Photoactivable reagents have been useful for studying the structural aspects of membrane hydrophobic core. We have reported earlier the use of diazofluorene as a probe for fluorescent photochemical labeling of hydrophobic core in artificial membranes. To quantitate and enhance the monitoring ability of this probe, we have synthesized 2-[3H]diazofluorene of high specific activity. This reagent rapidly partitions into phosphatidylcholine vesicles and selectively labels the fatty acyl chains of phosphatidylcholine. The insertion yield (13%) is not affected by the presence of scavengers like reduced glutathione. 2-[3H]Diazofluorene also readily partitions into erythrocyte membranes and on photolysis labels the membrane. The overall insertion was 48% with 9.7% in protein fraction and the rest in lipids. The distribution of radioactivity in labeled protein fraction was restricted to integral membrane proteins with Band 3 being the major protein labeled. There is little or no labeling associated with extrinsic proteins like spectrin. Further analysis of labeled Band 3 by treatment with chymotrypsin indicated that the labeling was restricted to the membrane spanning CH-17 and CH-35 fragments. No labeling of the cytoplasmic fragment of Band 3 could be observed. 2-[3H]Diazofluorene should prove useful for studying integral membrane proteins and their membrane-spanning regions

  17. 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.

  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. 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

  20. A novel coagulation inhibitor from Schistosoma japonicum.

    Science.gov (United States)

    Ranasinghe, Shiwanthi L; Fischer, Katja; Gobert, Geoffrey N; McManus, Donald P

    2015-12-01

    Little is known about the molecular mechanisms whereby the human blood fluke Schistosoma japonicum is able to survive in the host venous blood system. Protease inhibitors are likely released by the parasite enabling it to avoid attack by host proteolytic enzymes and coagulation factors. Interrogation of the S. japonicum genomic sequence identified a gene, SjKI-1, homologous to that encoding a single domain Kunitz protein (Sjp_0020270) which we expressed in recombinant form in Escherichia coli and purified. SjKI-1 is highly transcribed in adult worms and eggs but its expression was very low in cercariae and schistosomula. In situ immunolocalization with anti-SjKI-1 rabbit antibodies showed the protein was present in eggs trapped in the infected mouse intestinal wall. In functional assays, SjKI-1 inhibited trypsin in the picomolar range and chymotrypsin, neutrophil elastase, FXa and plasma kallikrein in the nanomolar range. Furthermore, SjKI-1, at a concentration of 7·5 µ m, prolonged 2-fold activated partial thromboplastin time of human blood coagulation. We also demonstrate that SjKI-1 has the ability to bind Ca(++). We present, therefore, characterization of the first Kunitz protein from S. japonicum which we show has an anti-coagulant properties. In addition, its inhibition of neutrophil elastase indicates SjKI-1 have an anti-inflammatory role. Having anti-thrombotic properties, SjKI-1 may point the way towards novel treatment for hemostatic disorders. PMID:26463744

  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. Effects of plant protease inhibitors, oryzacystatin I and soybean Bowman-Birk inhibitor, on the aphid Macrosiphum euphorbiae (Homoptera, Aphididae) and its parasitoid Aphelinus abdominalis (Hymenoptera, Aphelinidae).

    Science.gov (United States)

    Azzouz, H; Cherqui, A; Campan, E D M; Rahbé, Y; Duport, G; Jouanin, L; Kaiser, L; Giordanengo, P

    2005-01-01

    Transgenic plants expressing protease inhibitors (PIs) have emerged in recent years as an alternative strategy for pest control. Beneficial insects such as parasitoids may therefore be exposed to these entomotoxins either via the host or by direct exposure to the plant itself. With the objective of assessing the effects of PIs towards aphid parasitoids, bioassays using soybean Bowman-Birk inhibitor (SbBBI) or oryzacystatin I (OCI) on artificial diet were performed on Macrosiphum euphorbiae-Aphelinus abdominalis system. OCI significantly reduced nymphal survival of the potato aphid M. euphorbiae and prevented aphids from reproducing. This negative effect was much more pronounced than with other aphid species. On the contrary, SbBBI did not affect nymphal viability but significantly altered adult demographic parameters. Enzymatic inhibition assays showed that digestive proteolytic activity of larvae and adults of Aphelinus abdominalis predominantly relies on serine proteases and especially on chymotrypsin-like activity. Immunoassays suggested that OCI bound to aphid proteins and accumulated in aphid tissues, whereas SbBBI remained unbound in the gut. Bioassays using M. euphorbiae reared on artificial diets supplemented with both OCI and SbBBI showed a fitness impairment of Aphelinus abdominalis that developed on intoxicated aphids. However, only SbBBI was detected in parasitoid larvae, while no PI could be detected in adult parasitoids that emerged from PI-intoxicated aphids. The potential impact of PI-expressing plants on aphid parasitoids and their combined efficiency for aphid control are discussed. PMID:15686649

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

    Science.gov (United States)

    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 healing defects. Topical application of recombinant α1-ACT to wounds of diabetic mice rescued the impaired healing phenotype. LC-MS analysis of α1-ACT cleavage fragments identified a novel cleavage site within the reactive center loop and showed that neutrophil elastase was the predominant protease involved in unusual α1-ACT cleavage and inactivation in nonhealing human wounds. These results reveal critical functions for locally acting α1-ACT in the acute phase response following skin injury, provide mechanistic insight into its function during the repair response, and raise novel perspectives for its potential therapeutic value in inflammation-mediated tissue damage. PMID:21693707

  4. Pivotal role for alpha1-antichymotrypsin in skin repair.

    Science.gov (United States)

    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

    2011-08-19

    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 healing defects. Topical application of recombinant α1-ACT to wounds of diabetic mice rescued the impaired healing phenotype. LC-MS analysis of α1-ACT cleavage fragments identified a novel cleavage site within the reactive center loop and showed that neutrophil elastase was the predominant protease involved in unusual α1-ACT cleavage and inactivation in nonhealing human wounds. These results reveal critical functions for locally acting α1-ACT in the acute phase response following skin injury, provide mechanistic insight into its function during the repair response, and raise novel perspectives for its potential therapeutic value in inflammation-mediated tissue damage. PMID:21693707

  5. 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.

  6. Postnatal development of the exocrine pancreas in suckling goat kids.

    Science.gov (United States)

    Lopez, V; Martínez-Victoria, E; Yago, M D; Lupiani, M J; Mañas, M

    1997-04-01

    A total of 25 preruminant Granadina breed goats were used. They were bottle-fed goat milk ad libitum from postnatal day 3 to 28. Until the age of 3 d, kids were fed colostrum. Body weight, pancreas weight, total protein concentration, enzyme activities in pancreatic tissue and hormone concentrations (cortisol, gastrin, T3 and T4) were determined at 3, 7, 14, 21 and 28 d of age. Our results show that the rates of pancreatic synthesis and secretion of chymotrypsin are well developed at birth in the kid, and may compensate for possible deficiencies in gastric and/or enterocytes intracellular proteolysis. In week 4, there was a marked increase in amylase activity, change that can be attributed to the beginning of the transitional period known as weaning. The significant increase in circulating concentration of cortisol during week 4 suggests the involvement of corticosteroid as a mediator of pancreatic development at weaning. Changes in blood levels of this hormone are believed to be important in the expression of amylase in the neonatal period. However, T3-T4 blood levels remained unchanged from d 3 to 28, suggesting that, in the kid, these hormones appear to have no clear influence upon the postnatal development of the exocrine pancreas. PMID:9255407

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. Characterization of a high affinity cocaine binding site in rat brain

    International Nuclear Information System (INIS)

    Binding of [3H]cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of [3H]cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 950C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of [3H]cocaine (15 nM) was inhibited by increasing concentrations of Na+ ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific [3H]cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC50 = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting [3H]cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC50 values below μM concentrations

  13. 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.

  14. Crystallization and preliminary X-ray analysis of a protease inhibitor from the latex of Carica papaya

    International Nuclear Information System (INIS)

    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 P31 or P32. 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 Co2+, diffracts to a resolution of 1.7 Å and belongs to space group P212121, with unit-cell parameters a = 44.26, b = 81.99, c = 140.89 Å

  15. 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.

  16. 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

  17. Single-cell characterization of autotransporter-mediated Escherichia coli surface display of disulfide bond-containing proteins.

    Science.gov (United States)

    Ramesh, Balakrishnan; Sendra, Victor G; Cirino, Patrick C; Varadarajan, Navin

    2012-11-01

    Autotransporters (ATs) are a family of bacterial proteins containing a C-terminal β-barrel-forming domain that facilitates the translocation of N-terminal passenger domain whose functions range from adhesion to proteolysis. Genetic replacement of the native passenger domain with heterologous proteins is an attractive strategy not only for applications such as biocatalysis, live-cell vaccines, and protein engineering but also for gaining mechanistic insights toward understanding AT translocation. The ability of ATs to efficiently display functional recombinant proteins containing multiple disulfides has remained largely controversial. By employing high-throughput single-cell flow cytometry, we have systematically investigated the ability of the Escherichia coli AT Antigen 43 (Ag43) to display two different recombinant reporter proteins, a single-chain antibody (M18 scFv) that contains two disulfides and chymotrypsin that contains four disulfides, by varying the signal peptide and deleting the different domains of the native protein. Our results indicate that only the C-terminal β-barrel and the threaded α-helix are essential for efficient surface display of functional recombinant proteins containing multiple disulfides. These results imply that there are no inherent constraints for functional translocation and display of disulfide bond-containing proteins mediated by the AT system and should open new avenues for protein display and engineering. PMID:23019324

  18. A Trypsin Inhibitor from Clitoria fairchildiana Cotyledons is Active Against Digestive Enzymes of Aedes aegypti Larvae.

    Science.gov (United States)

    de Oliveira, Lucilene O; Fernandes, Kátia V S; Pádua, Dayanni de Souza; Carvalho, André de O; Lemos, Francisco J A; Gomes, Valdirene M; Oliveira, Antônia E A; Ferreira, André T da Silva; Perales, Jonas

    2015-01-01

    Aedes aegypti, the principal mosquito vector of yellow fever, dengue fever and chikungunya fever virus-transmitted diseases, is an insect closely associated with humans and their housing habitats. As there is no commercially available vaccine, prevention is the most suggested form of avoiding disease spreading and a number of studies are being developed in order to give support to vector control operations. The present study reports on the identification of a trypsin inhibitor isolated from cotyledons of the Clitoria fairchildiana amazonic tree seeds, which was able to reduce by 87.93 % the activity of digestive enzymes of fourth instar A. aegypti larva. A partial amino acid sequence showed strong similarity with sequences from several trypsin inhibitors already reported in the literature. The 13,000 Da isolated inhibitor was seen to be active solely against trypsin-like enzymes, neither acting on papain, α-amylase nor on other serine proteases, such as elastase, chymotrypsin or subtilisin. At least six from seven active digestive proteases from A. aegypti larvae, visualized by zymography, were severely affected soon after exposed to the inhibitor. The strong and specific action of the isolated inhibitor against trypsin digestive enzymes of this insect vector led us to believe that this protein may be a good candidate for a prospective alternative biocontrol method. PMID:26156641

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. Label-Free Quantitation and Mapping of the ErbB2 Tumor Receptor by Multiple Protease Digestion with Data-Dependent (MS1 and Data-Independent (MS2 Acquisitions

    Directory of Open Access Journals (Sweden)

    Jason M. Held

    2013-01-01

    Full Text Available The receptor tyrosine kinase ErbB2 is a breast cancer biomarker whose posttranslational modifications (PTMs are a key indicator of its activation. Quantifying the expression and PTMs of biomarkers such as ErbB2 by selected reaction monitoring (SRM mass spectrometry has several limitations, including minimal coverage and extensive assay development time. Therefore, we assessed the utility of two high resolution, full scan mass spectrometry approaches, MS1 Filtering and SWATH MS2, for targeted ErbB2 proteomics. Endogenous ErbB2 immunoprecipitated from SK-BR-3 cells was in-gel digested with trypsin, chymotrypsin, Asp-N, or trypsin plus Asp-N in triplicate. Data-dependent acquisition with an AB SCIEX TripleTOF 5600 and MS1 Filtering data processing was used to assess peptide and PTM coverage as well as the reproducibility of enzyme digestion. Data-independent acquisition (SWATH was also performed for MS2 quantitation. MS1 Filtering and SWATH MS2 allow quantitation of all detected analytes after acquisition, enabling the use of multiple proteases for quantitative assessment of target proteins. Combining high resolution proteomics with multiprotease digestion enabled quantitative mapping of ErbB2 with excellent reproducibility, improved amino acid sequence and PTM coverage, and decreased assay development time compared to typical SRM assays. These results demonstrate that high resolution quantitative proteomic approaches are an effective tool for targeted biomarker quantitation.

  4. 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

  5. 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

  6. An experimental test of dietary enzyme modulation in pine warblers Dendroica pinus.

    Science.gov (United States)

    Levey, D J; Place, A R; Rey, P J; Martínez Del Rio, C

    1999-01-01

    Modulation of gut function is important in an ecological and evolutionary context because it likely determines what food items an animal can and cannot eat. We examined how diet affects activity of digestive enzymes in an omnivorous bird, the pine warbler (Dendroica pinus). Pine warblers were fed insect-based, fruit-based, and seed-based diets for approximately 54 d. We then measured activity of amylase, maltase, sucrase, aminopeptidase-N, trypsin, chymotrypsin, carboxypeptidase A, carboxypeptidase B, pancreatic lipase, and carboxyl ester lipase. We predicted that carbohydrase activities would be highest in birds fed the diet highest in carbohydrates (fruit based), protease activities would be highest in those fed the diet highest in protein (insect based), and lipase activities would be highest in those fed the diets highest in lipid (insect based and seed based). Also, we predicted that pine warblers would exhibit greater dietary modulation of enzyme activity than reported for a less omnivorous congener, the yellow-rumped warbler (Dendroica coronata). All predictions were upheld, supporting the hypothesis that pine warblers modulate the activity of digestive enzymes in proportion to demand from substrates in the diet. PMID:10521325

  7. 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.

  8. Antihypertensive effect of angiotensin i converting enzyme-inhibitory peptide from hydrolysates of Bigeye tuna dark muscle, Thunnus obesus.

    Science.gov (United States)

    Qian, Zhong-Ji; Je, Jae-Young; Kim, Se-Kwon

    2007-10-17

    Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from tuna dark muscle hydrolysate prepared by alcalase, neutrase, pepsin, papain, alpha-chymotrypsin, and trypsin, respectively. Among hydrolysates, the pepsin-derived hydrolysate exhibited the highest ACE I inhibitory activity versus those of other enzyme hydrolysates. The structure of the peptide was identified to be Trp-Pro-Glu-Ala-Ala-Glu-Leu-Met-Met-Glu-Val-Asp-Pro (molecular weight 1581 Da) by time of flight mass spectrometry/mass spectrometry analysis, and the IC 50 value of the peptide was 21.6 microM. The Lineweaver-Burk plots revealed that the peptide acts as a noncompetitive inhibitor, and the inhibitor constant ( K i) was calculated as 26.6 microM using the secondary plots. The peptide had an antihypertensive effect according to the time-course measurement after oral administration to spontaneously hypertensive rats. Maximal reduction was detected 3 h after oral administration at a dose of 10 mg/kg of body weight. These results suggest that the peptide derived from tuna dark muscle would be a beneficial ingredient for functional food or pharmaceuticals against hypertension and its related diseases. PMID:17894458

  9. 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

  10. 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

  11. A lepidopteran pacifastin member: cloning, gene structure, recombinant production, transcript profiling and in vitro activity.

    Science.gov (United States)

    Breugelmans, Bert; Simonet, Gert; van Hoef, Vincent; Van Soest, Sofie; Smagghe, Guy; Vanden Broeck, Jozef

    2009-07-01

    Members of the pacifastin family have been characterized as serine peptidase inhibitors (PI), but their target enzyme(s) are unknown in insects. So far, the structural and biochemical characteristics of pacifastin-like PI have only been studied in locusts. Here we report the molecular identification and functional characterization of a pacifastin-like precursor in a lepidopteran insect, i.e. the silkworm Bombyx mori. The bmpp-1 gene contains 17 exons and codes for two pacifastin-related precursors of different length. The longest splice variant encodes 13 inhibitor domains, more than any other pacifastin-like precursor in arthropods. The second transcript lacks two exons and codes for 11 inhibitor domains. By studying the expression profile of the Bombyx pacifastin-like gene a different expression pattern for the two variants was observed suggesting functional diversification. Next, several PI domains of BMPP-1 were produced and, contrary to locust pacifastin peptides, they were found to be potent inhibitors of both bovine trypsin and chymotrypsin. Surprisingly, the same Bombyx PI are only weak inhibitors of endogenous digestive peptidases, indicating that other peptidases are the in vivo targets. Interestingly, the Bombyx PI inhibit a fungal trypsin-like cuticle degrading enzyme, suggesting a protective function for BMPP-1 against entomopathogenic fungi. PMID:19364530

  12. Effect of Confinement on the Properties of Sequestered Mixed Polar Solvents: Enzymatic Catalysis in Nonaqueous 1,4-Bis-2-ethylhexylsulfosuccinate Reverse Micelles.

    Science.gov (United States)

    Durantini, Andres M; Falcone, R Dario; Silber, Juana J; Correa, N Mariano

    2016-06-01

    The influence of different glycerol, N,N-dimethylformamide (DMF) and water mixtures encapsulated in 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane reverse micelles (RMs) on the enzymatic hydrolysis of 2-naphthyl acetate by α-chymotrypsin is demonstrated. In the case of the mixtures with DMF and protic solvents it has been previously shown, using absorption, emission and dynamic light-scattering techniques, that solvents are segregated inside the polar core of the RMs. Protic solvents anchor to the AOT, whereas DMF locates to the polar core of the aggregate. Thus, DMF not only helps to solubilize the hydrophobic substrate, increasing its effective concentrations but surprisingly, it does not affect the enzyme activity. The importance of ensuring the presence of RMs, encapsulation of the polar solvents and the corrections by substrate partitioning in order to obtain reliable conclusions is highlighted. Moreover, the effect of a constrained environment on solvent-solvent interactions in homogenous media and its impact on the use of RMs as nanoreactors is stressed. PMID:26891863

  13. 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.)

  14. 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

  15. 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

  16. Protein cleavage strategies for an improved analysis of the membrane proteome

    Directory of Open Access Journals (Sweden)

    Poetsch Ansgar

    2006-03-01

    Full Text Available Abstract Background Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria. Results For the membrane proteomes from all three analyzed organisms, we identified cleavage conditions that achieve better sequence and proteome coverage than trypsin. Greater improvement was obtained for bacteria than for yeast, which was attributed to differences in protein size and GRAVY. It was demonstrated for bacteriorhodopsin that the in silico predictions agree well with the experimental observations. Conclusion For all three examined organisms, it was found that a combination of chymotrypsin and staphylococcal peptidase I gave significantly better results than trypsin. As some of the improved cleavage conditions are not more elaborate than trypsin digestion and have been proven useful in practice, we suppose that the cleavage at both hydrophilic and hydrophobic amino acids should facilitate in general the analysis of membrane proteins for all organisms.

  17. Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula.

    Science.gov (United States)

    Lomate, Purushottam R; Bonning, Bryony C

    2016-01-01

    Stink bugs negatively impact numerous plant species of agricultural and horticultural importance. While efforts to develop effective control measures are underway, the unique digestive physiology of these pests presents a significant hurdle for either protein- or nucleotide-based management options. Here we report the comparative biochemical and proteomic characterization of proteases and nucleases from the gut, salivary gland and saliva of the southern green stink bug, Nezara viridula. The pH optimum for protease activity was acidic (5 to 6) in the gut with the primary proteases being cysteine proteases, and alkaline (8 to 9) in the saliva and salivary gland with the primary proteases being serine proteases. The serine proteases in saliva differ biochemically from trypsin and chymotrypsin, and the cathepsins in the gut and saliva showed distinct properties in inhibitor assays. Nuclease activity (DNase, RNase, dsRNase) was concentrated in the salivary gland and saliva with negligible activity in the gut. The most abundant proteins of the gut (530) and salivary gland (631) identified by proteomic analysis included four gut proteases along with eight proteases and one nuclease from the salivary gland. Understanding of N. viridula digestive physiology will facilitate the design of new strategies for management of this significant pest. PMID:27282882

  18. Purification and reconstitution of the human platelet α2-adrenergic receptor

    International Nuclear Information System (INIS)

    Human platelet α2-adrenergic receptors have been purified ∼80,000 fold to apparent homogeneity by a five step chromatographic procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of M/sub r/ 64,000. The competitive binding of ligands to the purified receptor protein shows the proper α2-adrenergic specificity. The α2-adrenergic receptor contains an essential sulfhydryl residues. Thus, exposure of the purified receptor to the sulfhydryl specific reagent, phenylmercuric chloride (PMC), resulted in a 80% loss of binding activity. This loss of binding activity was prevented when exposure to PMC was done in the presence of α2-adrenergic ligands and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified α2-adrenergic receptors was obtained with S. aureus V-8 protease, α-chymotrypsin and papain. In a comparison with purified β2-adrenergic receptors no common partial proteolytic products were found. Partially purified preparations of the α2-adrenergic receptor were successfully reconstituted into phospholipid vesicles with the inhibitory guanyl nucleotide-binding regulatory protein, N/sub i/. In these reconstituted preparations, epinephrine could stimulate, and phentolamine could block, the GTPase activity of N/sub i/

  19. Chemistry in a microenvironment of low pH, generated with the aid of an immobilized proteinase.

    Science.gov (United States)

    Silver, M S; Haskell, J H

    1990-05-31

    alpha-Chymotrypsin, when immobilized in a collodion membrane, exhibits high activity and remarkable stability. When the immobilized proteinase is exposed to 15 mM ethyl N-acetyl-L-tyrosinate in dilute pH 8.5 buffer it generates a microenvironment which, indicator studies suggest, has an effective pH of approximately 4. The presence of this locally highly acidic region produces a marked increase in the rate of hydrolysis of BzPheal = Ala dissolved in the buffer solution (BzPheal = Ala is the acylhydrazide obtained from the reaction between N-benzoyl-L-phenylalaninal and N-acetyl-L-alanine hydrazide). The observed rate is 10-times greater than in comparable control experiments incorporating a concentrated buffer solution, in which a pH-gradient does not form. The enhanced hydrolysis rate is quantitatively explained if it is attributed to the approximately 20 microliters of pH 4 solution within the membrane. Other experimental data are also consistent with this hypothesis. PMID:2354198

  20. Four-photon microwave laser spectroscopy of aqueous solutions of biopolymers

    International Nuclear Information System (INIS)

    The four-photon laser radiation scattering spectra are obtained in the submillimetre range (75-95 cm-1) for deionised water, aqueous solutions of DNA and α-chymotrypsin protein. Narrow resonances are recorded whose frequencies coincide (within the resolution power of a spectrometer) with rotational frequencies in the ground electronic state and vibrational state of ortho and para isomers of H2O molecule in a gas phase and with the frequencies of the lines of H2O2 and OH- molecules. It is shown that the resonance contribution of the rotational lines of ortho isomers of H2O to the signal of four-photon scattering of native solutions of biopolymers increases by a factor of at least 8 compared to their contribution to the scattering signal in water, and becomes considerably larger than the contribution from the paraisomer lines. Denaturation of DNA after heating and cooling of the solution leads to the disappearance of such selectivity. (special issue devoted to the 25th anniversary of the a.m. prokhorov general physics institute)

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli.

    Science.gov (United States)

    Ideno, Akira; Furutani, Masahiro; Iba, Yoshitaka; Kurosawa, Yoshikazu; Maruyama, Tadashi

    2002-02-01

    The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k(cat)/K(m)) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15 degrees C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100 degrees C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells. PMID:11823179

  10. Clinical potential of carfilzomib in the treatment of relapsed and refractory multiple myeloma

    Directory of Open Access Journals (Sweden)

    Gupta VA

    2013-05-01

    Full Text Available Vikas A Gupta, Ajay K Nooka, Sagar Lonial, Lawrence H BoiseDepartment of Hematology and Medical Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USAAbstract: Treatment of refractory and/or relapsed multiple myeloma has been a challenging problem for over 20 years. However, we have made significant progress addressing this disease with the use of bortezomib, the first in class proteasome inhibitor, and the immunomodulatory agents, thalidomide and lenalidomide. Carfilzomib, the second-generation proteasome inhibitor, has also been approved for treatment of relapsed/refractory multiple myeloma. Carfilzomib is a highly selective and potent inhibitor of proteasome chymotrypsin-like activity. Phase I and II clinical trials have reported an acceptable toxicity profile, with manageable thrombocytopenia and anemia being the most common side effects. Peripheral neuropathy, a frequent dose-limiting side effect of bortezomib, was rare. Further, carfilzomib demonstrated encouraging single-agent activity and appeared to be effective even in patients refractory to bortezomib. Based on these promising data, carfilzomib is moving forward into Phase III trials for relapsed multiple myeloma and is also being investigated as front-line combination therapy for patients with newly diagnosed myeloma.Keywords: proteasome inhibitor, bortezomib, pharmacology, safety, efficacy

  11. SPR imaging biosensor for the 20S proteasome: Sensor development and application to measurement of proteasomes in human blood plasma

    International Nuclear Information System (INIS)

    The 20S proteasome is a multicatalytic enzyme complex responsible for intracellular protein degradation in mammalian cells. Its antigen level or enzymatic activity in blood plasma are potentially useful markers for various malignant and nonmalignant diseases. We have developed a method for highly selective determination of the 20S proteasome using a Surface Plasmon Resonance Imaging (SPRI) technique. It is based on the highly selective interaction between the proteasome's catalytic β5 subunit and immobilized inhibitors (the synthetic peptide PSI and epoxomicin). Inhibitor concentration and pH were optimized. Analytical responses, linear ranges, accuracy, precision and interferences were investigated. Biosensors based on either PSI and epoxomicin were found to be suitable for quantitative determination of the proteasome, with a precision of ±10% for each, and recoveries of 102% and 113%, respectively, and with little interference by albumin, trypsin, chymotrypsin, cathepsin B and papain. The proteasome also was determined in plasma of healthy subjects and of patients suffering from acute leukemia. Both biosensors gave comparable results (2860 ng.mL-1 on average for control, and 42300 ng.mL-1 on average for leukemia patients). (author)

  12. Antiproliferative activity of Humulus lupulus extracts on human hepatoma (Hep3B), colon (HT-29) cancer cells and proteases, tyrosinase, β-lactamase enzyme inhibition studies.

    Science.gov (United States)

    Cömert Önder, Ferah; Ay, Mehmet; Aydoğan Türkoğlu, Sümeyye; Tura Köçkar, Feray; Çelik, Ayhan

    2016-01-01

    The aims of this study were to examine the antiproliferation of Humulus lupulus extracts on human hepatoma carcinoma (Hep3B) and human colon carcinoma (HT-29) cell lines along with enzyme inhibitory effects of the crude extracts. Potential cell cytotoxicity of six different H. lupulus extracts were assayed on various cancer cells using MTT assay at 24, 48 and 72 h intervals. Methanol-1 extract has inhibited the cell proliferation with doses of 0.6-1 mg/mL in a time dependent (48 and 72 hours) manner in Hep3B cells with 70% inhibition, while inhibitory effect was not seen in colon cancer cells. Acetone extract has increased the cell proliferation at low doses of 0.1 mg/mL for 72 h in Hep3B cells and 0.1-0.2 mg/mL for 48 and 72 h in HT29 cells. The inhibitory effects of the extracts were compared by relative maximum activity values (V(max)) using proteases such as α-chymotrypsin, trypsin and papain, tyrosinase and β-lactamase (penicillinase). PMID:25683080

  13. Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV.

    Science.gov (United States)

    Park, Ji-Young; Ko, Jin-A; Kim, Dae Wook; Kim, Young Min; Kwon, Hyung-Jun; Jeong, Hyung Jae; Kim, Cha Young; Park, Ki Hun; Lee, Woo Song; Ryu, Young Bae

    2016-01-01

    Two viral proteases of severe acute respiratory syndrome coronavirus (SARS-CoV), a chymotrypsin-like protease (3CL(pro)) and a papain-like protease (PL(pro)) are attractive targets for the development of anti-SARS drugs. In this study, nine alkylated chalcones (1-9) and four coumarins (10-13) were isolated from Angelica keiskei, and the inhibitory activities of these constituents against SARS-CoV proteases (3CL(pro) and PL(pro)) were determined (cell-free/based). Of the isolated alkylated chalcones, chalcone 6, containing the perhydroxyl group, exhibited the most potent 3CL(pro) and PL(pro) inhibitory activity with IC50 values of 11.4 and 1.2 µM. Our detailed protein-inhibitor mechanistic analysis of these species indicated that the chalcones exhibited competitive inhibition characteristics to the SARS-CoV 3CL(pro), whereas noncompetitive inhibition was observed with the SARS-CoV PL(pro). PMID:25683083

  14. The immobilization of enzymes onto poly(ethylene)-g. co-methacrylic acid, (poly(ethylene)-g. co-hydroxyethyl methacrylate)-g. co-methacrylic acid and (poly(ethylene)-g. co-methacrylic acid)-g. co-hydroxyethyl methacrylate

    Energy Technology Data Exchange (ETDEWEB)

    Da Silva, M.A.; Gil, M.H.; Guiomar, J.; Lapa, E.; Machado, E.; Moreira, M. (Coimbra Univ. (Portugal). Dept. of Chemistry); Guthrie, J.T. (Leeds Univ. (UK). Dept. of Colour Chemistry); Kotov, S. (Higher Inst. of Chemical Technology, Burgas (Bulgaria))

    1990-01-01

    A series of graft copolymers has been prepared on the poly(ethylene) backbone. These carry functional groups which are effective in coupling and provide a level of hydrophilicity which is thought to be consistent with generating a suitable micro-environment for enzyme immobilization and subsequent enhanced biocatalyst stability. Four enzymes have been immobilized. These are papain, trypsin, glucose oxidase and {alpha}-chymotrypsin. The parent copolymers were assembled via radiation-induced grafting. Secondary grafting was achieved in two ways. The first involved grafting methacrylic acid onto poly(ethylene)-g.co-hydroxyethyl methacrylate, while the second involved grafting hydroxyethyl methacrylate onto poly(ethylene)-g.co-methacrylic acid. The results suggest that a high degree of specificity arises in the systems examined with regard to the enzymes, the type of copolymers and the coupling procedures. Generally, relatively large amounts of enzyme become covalently attached to the copolymers, though the overall level of activity is low. In this work it has been observed that the most satisfactory results were obtained when the partly hydrolyzed poly(ethylene)-g.co-hydroxyethyl methacrylate was used in the immobilization of the biocatalysts. (author).

  15. The immobilization of enzymes onto poly(ethylene)-g.co-methacrylic acid, [poly(ethylene)-g.co-hydroxyethyl methacrylate]-g.co-methacrylic acid and [poly(ethylene)-g.co-methacrylic acid]-g.co-hydroxyethyl methacrylate

    International Nuclear Information System (INIS)

    A series of graft copolymers has been prepared on the poly(ethylene) backbone. These carry functional groups which are effective in coupling and provide a level of hydrophilicity which is thought to be consistent with generating a suitable micro-environment for enzyme immobilization and subsequent enhanced biocatalyst stability. Four enzymes have been immobilized. These are papain, trypsin, glucose oxidase and α-chymotrypsin. The parent copolymers were assembled via radiation-induced grafting. Secondary grafting was achieved in two ways. The first involved grafting methacrylic acid onto poly(ethylene)-g.co-hydroxyethyl methacrylate, while the second involved grafting hydroxyethyl methacrylate onto poly(ethylene)-g.co-methacrylic acid. The results suggest that a high degree of specificity arises in the systems examined with regard to the enzymes, the type of copolymers and the coupling procedures. Generally, relatively large amounts of enzyme become covalently attached to the copolymers, though the overall level of activity is low. In this work it has been observed that the most satisfactory results were obtained when the partly hydrolyzed poly(ethylene)-g.co-hydroxyethyl methacrylate was used in the immobilization of the biocatalysts. (author)

  16. Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane

    International Nuclear Information System (INIS)

    The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki=3.3 nM) and baupain (Ki=2.1x10-8 M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125 nM), cathepsin K (Ki=0.76 nM), cathepsin L (Ki=0.6 nM), and cathepsin V (Ki=1.0 nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases

  17. The immobilization of enzymes onto poly(ethylene)—g.co—methacrylic acid, [poly(ethylene)—g.co—hydroxyethyl methacrylate]—g.co—methacrylic acid and [poly(ethylene)—g.co—methacrylic acid]—g.co—hydroxyethyl methacrylate

    Science.gov (United States)

    Da Silva, M. Alves; Gil, M. H.; Guiomar, J.; Lapa, E.; Machado, E.; Moreira, M.; Guthrie, J. T.; Kotov, S.

    A series of graft copolymers has been prepared based on the poly(ethylene) backbone. These carry functional groups which are effective in coupling and provide a level of hydrophilicity which is thought to be consistent with generating a suitable micro-environment for enzyme immobilization and subsequent enhanced biocatalyst stability. Four enzymes have been immobilized. These are papain, trypsin, glucose oxidase and α-chymotrypsin. The parent copolymers were assembled via radiation-induced grafting. Secondary grafting was achieved in two ways. The first involved grafting methacrylic acid onto poly(ethylene)—g.co—hydroxyethyl methacrylate, while the second involved grafting hydroxyethyl methacrylate onto poly(ethylene)—g.co—methacrylic acid. The results suggest that a high degree of specificity arises in the systems examined with regard to the enzymes, the type of copolymers and the coupling procedures. Generally, relatively large amounts of enzyme become covalently attached to the copolymers, though the overall level of activity is low. In this work it has been observed that the most satisfactory results were obtained when the partly hydrolyzed poly(ethylene)—g.co—hydroxyethyl methacrylate was used in the immobilization of the biocatalysts.

  18. The D2O effect on catalysis by triose phosphate isomerase requires isotope exchange on the enzyme

    International Nuclear Information System (INIS)

    The present work shows the value of measuring the rate of onset or loss of a D2O effect to distinguish between enzyme-bound protons and protons of the medium as the cause of the effect. This alternative was examined in the important studies of Bender on deacylation of chymotrypsin and papain in which the isotope effect in D2O was lost within 10 s of dilution into H2O and therefore was concluded to result from D2O rather than from a deuterium-substituted enzyme. However, 10 s is a long interval for all but the hydrogen-bonded backbone hydrogens. With the ususal rapid mixing device this interval can be shortened by a least 103, putting it in the range of many group ionizations. The results suggest that H/D exchange on the enzyme, occurring more slowly than catalysis, is required for the isotope effect to be seen. Because the isotope effect implies that a proton transfer within the enzyme occurs at some step of the reaction and because exchange is much slower than catalysis, the transfer must be reversed without exchange with the medium during product formation. It is proposed that a conformational change of E-G3P prior to the enolization is the step requiring such a proton transfer

  19. Broad specificity alkaline proteases efficiently reduce the visual scaling associated with soap-induced xerosis.

    Science.gov (United States)

    El-Kadi, K N; Rawlings, A V; Feinberg, C; Watkinson, A; Nunn, C C; Battaglia, A; Chandar, P; Richardson, N; Pocalyko, D J

    2001-11-01

    In xerotic skin, the proteolysis of desmosomes is reduced leading to the accumulation of corneocytes on the surface of the skin. The effect of proteases applied topically to soap-induced xerotic skin was evaluated using a five-point visual scale. The visual scaling associated with soap-induced xerosis could be ameliorated by the topical application of exogenous protease. Bovine pancreatic chymotrypsin, papain, and a bacterial protease from Bacillus licheniformis were all capable of facilitating the reduction in visual scaling in a short time. Alcalase and Optimase, both broad specificity alkaline bacterial proteases, were the most weight-efficient at delivering this clinical effect. The reduction in scaling could be achieved either by occluded application of an aqueous enzyme solution or by a two-step unoccluded application first of an aqueous enzyme solution followed by a commercial moisturizer. Morphological and immunological analysis of bacterial enzyme-treated skin revealed that topically applied protease specifically induced the degradation of the desmosomes thereby promoting desquamation. These results indicate that topical application of protease can significantly and rapidly reduce the visual scaling associated with soap-induced xerosis by promoting desmosome degradation within the corneocyte clumps. PMID:11820726

  20. Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw.

    Science.gov (United States)

    Qian, Zhong-Ji; Jung, Won-Kyo; Kim, Se-Kwon

    2008-04-01

    In the present study, a peptide having antioxidant properties was isolated from bullfrog skin protein, Rana catesbeiana Shaw. Bullfrog skin protein was hydrolyzed using alcalase, neutrase, pepsin, papain, alpha-chymotrypsin and trypsin. Antioxidant activities of respective hydrolysates were evaluated using lipid peroxidation inhibition assay and direct free radical scavenging activity by using electron spin resonance (ESR) spectrometer. Among hydrolysates, alcalase derived hydrolysate exhibited the highest antioxidant activities than those of other enzyme hydrolysates. In order to purity a peptide having potent antioxidant properties, alcalase hydrolysate was separated using consecutive chromatographic methods on a Hiprep 16/10 DEAE FF anion exchange column, Superdex Peptide 10/300 GL gel filtration column and highan octadecylsilane (ODS) C18 reversed phase column. Finally, a potent antioxidative peptide was isolated and its sequence was identified to be LEELEEELEGCE (1487 Da) by Q-TOF ESI mass spectroscopy. This antioxidant peptide from bullfrog skin protein (APBSP) inhibited lipid peroxidation higher than that of alpha-tocopherol as positive control and efficiently quenched different sources of free radicals: DPPH radical (IC(50)=16.1 microM), hydroxyl radical (IC(50)=12.8 microM), superoxide radical (IC(50)=34.0 microM) and peroxyl radical (IC(50)=32.6 microM). Moreover, MTT assay showed that this peptide does not exert any cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5). PMID:17512726

  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. Relationships between cell surface insulin binding and endocytosis in adipocytes

    International Nuclear Information System (INIS)

    Chymotrypsin substrate analogues, such as N-acetyl-Tyr ethyl ester, have recently been demonstrated to inhibit the endocytic uptake of insulin in isolated rat adipocytes. In this study, the effect of N-acetyl-Tyr ethyl ester on cell surface insulin binding and dissociation were examined. Surface-bound 125I-insulin was distinguished from intracellular 125I-insulin by the sensitivity of the former to rapid dissociation with an acidic buffer. Plateau levels of surface-bound insulin at 37 degree C were increased 70% by inhibiting the internalization pathway. This increase was temperature and insulin concentration dependent. Thus differences in surface binding were small at 12 degree C and also at high insulin concentrations. Inhibition of internalization with N-acetyl-Tyr ethyl ester markedly slowed the loss of surface-bound insulin observed during dissociation the loss of surface-bound insulin observed during dissociation studies. After 20-30 min of dissociation, the remaining levels of surface-bound insulin were three- to fourfold higher in treated adipocytes compared with control adipocytes. Added unlabeled insulin retained its ability to accelerate the dissociation of insulin in N-acetyl-Tyr ethyl ester-treated cells. These observations indicate that the internalization pathway is a quantitatively important factor in determining levels of surface binding at 37 degree C and in determining the rat of deactivation of insulin binding

  3. Characterization of a novel Kazal-type serine proteinase inhibitor of Arabidopsis thaliana.

    Science.gov (United States)

    Pariani, Sebastián; Contreras, Marisol; Rossi, Franco R; Sander, Valeria; Corigliano, Mariana G; Simón, Francisco; Busi, María V; Gomez-Casati, Diego F; Pieckenstain, Fernando L; Duschak, Vilma G; Clemente, Marina

    2016-04-01

    Many different types of serine proteinase inhibitors have been involved in several kinds of plant physiological processes, including defense mechanisms against phytopathogens. Kazal-type serine proteinase inhibitors, which are included in the serine proteinase inhibitor family, are present in several organisms. These proteins play a regulatory role in processes that involve serine proteinases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. In the present work, we characterized two putative Kazal-type serine proteinase inhibitors from Arabidopsis thaliana, which have a single putative Kazal-type domain. The expression of these inhibitors is transiently induced in response to leaf infection by Botrytis cinerea, suggesting that they play some role in defense against pathogens. We also evaluated the inhibitory specificity of one of the Kazal-type serine proteinase inhibitors, which resulted to be induced during the local response to B. cinerea infection. The recombinant Kazal-type serine proteinase inhibitor displayed high specificity for elastase and subtilisin, but low specificity for trypsin, suggesting differences in its selectivity. In addition, this inhibitor exhibited a strong antifungal activity inhibiting the germination rate of B. cinerea conidia in vitro. Due to the important role of proteinase inhibitors in plant protection against pathogens and pests, the information about Kazal-type proteinase inhibitors described in the present work could contribute to improving current methods for plant protection against pathogens. PMID:26853817

  4. Radiolabeled enzyme inhibitors as pancreatic scanning agents

    International Nuclear Information System (INIS)

    Radiolabeled inhibitors of the pancreatic protease enzymes trypsin and chymotrypsin: I-125-soybean inhibitor (SBI), I-125-human antitrypsin (HAT), and 3H-diisopropylfluorophosphate (DFP) were studied in rats for their uptake in the pancreas. The protease enzyme inhibitor, SBI, was found to retain its enzymatic activity after radio-iodine labeling by iodine-monochloride method. Assay of tissues radioactivity at 5, 15, 30, 60 min and 24 hr after iv injection in rats showed pancreas:liver (P:L) ratios of < 1.4 and pancreas:blood (P:B) ratios of < 1 at all time intervals for each compound. However, ip injection of I-125 SBI showed preferential uptake in pancreas, P:L mean ratios [anti x (range)] of 6.0 (5.5 to 6.3) and P:B mean ratios of [anti x (range)] 2.60 (1.8 to 3.4), up to 60 minutes. These ratios resemble those of Se-75-selenomethionine except that kidney values are higher. Our data suggest that radiolabeled proteins can also show similar good early pancreatic uptake when given ip by escaping immediate liver metabolism. Proteins can readily be tagged with desirable gamma-emitting radionuclides (viz. I-131, I-123, Tc-99m) compared to amino acids and may potentially be suitable for imaging the pancreas. It may be important to study both iv and ip routes of administration in evaluating the specificity/affinity of potential pancreatic radiopharmaceuticals

  5. Studies on porcine pancreatic elastase activity, 1

    International Nuclear Information System (INIS)

    An improved method of radioimmunoassay was devised to offer a successful formula for determining blood concentration of elastase. With porcine pancreatic elastase as the antigen, rabbits were immunized to obtain antiserum. Iodinated elastase labeled by the chloramine-T procedure using 131I (or 125I) had a specific activity of 200 - 300 mCi/mg. The double antibody method was used for BF separation. While the usual method of radioimmunoassay was not always successful in obtaining accurate serum concentration of elastase, the use of diisopropyl fluorophosphate (DFP) was able to eliminate the disturbing influence of intra-serous inhibitors, α1-AT and α2-M, eventually producing satisfactory results. With the use of DFP, the elastase standard curve and the porcine serum dilution curve had a statistically significant correlation; precision and recovery were both satisfactory; cross-reactivity of the antiserum with trypsin and chymotrypsin was less than 0.001%. The minimal detectable concentration of elastase was 5 ng/ml, and the range of normal fasting porcine serum level was 70 - 100 ng/ml. (author)

  6. Evidence of a new serine protease in the rat pure pancreatic juice that degrades somatostatin

    International Nuclear Information System (INIS)

    Somatostatin (SS) is found in the endocrine pancreas and has been reported in the pure pancreatic juice (PPJ) of different species. Characterization by gel filtration of immunoreactive SS (irSS) in the rat PPJ (rPPJ) results in a single peak corresponding to 23kDa molecular weight. Incubation of the 23kDa fraction with labeled or synthetic SS results in time dependent degradation of both peptides. This degradation is inhibitable by PMSF, calcium and by heat, whereas specific inhibitors of trypsin and chymotrypsin are without effect. These data suggest that irSS previously measured in rPPJ samples by RIA without confirmation of radioactive tracer stability may lead to false positive results. Indeed, our study indicates the presence of a 23kDa enzyme in the rPPJ degrading radiolabeled somatostatin during the RIA procedure. This putative new enzyme found into the rPPJ may thus be partially responsible for the apparent irSS presence

  7. Acute effects of whole body gamma irradiation on exocrine pancreatic secretion in the pig

    International Nuclear Information System (INIS)

    Reports on radiation damage to the pancreas deal essentially with long-term morphological changes with few data on pancreatic exocrine function. The aim of this work was to study the acute effects of whole body irradiation on volume and enzyme activities in the pancreatic juice. A whole body gamma irradiation (6 Gy) was investigated in pigs with continuous sampling of pancreatic juice before and after exposure via an indwelling catheter in the pancreatic duct. For each sample collected, total protein concentration and enzyme activities of trypsin, chymotrypsin, elastase, lipase and amylase were determined. Pancreatic juice volume was monitored during all periods of collection. The volume of pancreatic juice secreted daily decreased one day after irradiation and remained lower than the control values over the experimental period. Total proteins secreted in the pancreatic juice and total activities of pancreatic enzymes were reduced similarly. On the other hand, only specific activities of elastase and lipase were affected by irradiation. Whole body gamma irradiation resulted in a rapid and marked decrease of exocrine pancreatic secretion, in terms of volume as well as secreted enzymes. This may contribute in part to the intestinal manifestations of the acute and/or late radiation syndrome. (author)

  8. 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.

  9. Autosomal ichthyosis with hypotrichosis syndrome displays low matriptase proteolytic activity and is phenocopied in ST14 hypomorphic mice.

    Science.gov (United States)

    List, Karin; Currie, Brooke; Scharschmidt, Tiffany C; Szabo, Roman; Shireman, Jessica; Molinolo, Alfredo; Cravatt, Benjamin F; Segre, Julia; Bugge, Thomas H

    2007-12-14

    Human autosomal recessive ichthyosis with hypotrichosis (ARIH) is an inherited disorder recently linked to homozygosity for a point mutation in the ST14 gene that causes a G827R mutation in the matriptase serine protease domain (G216 in chymotrypsin numbering). Here we show that human G827R matriptase has strongly reduced proteolytic activity toward small molecule substrates, as well as toward its candidate epidermal target, prostasin. To further investigate the possible contribution of low matriptase activity to ARIH, we generated an ST14 hypomorphic mouse strain that displays a 100-fold reduction in epidermal matriptase mRNA levels. Interestingly, unlike ST14 null mice, ST14 hypomorphic mice were viable and fertile but displayed a spectrum of abnormalities that strikingly resembled ARIH. Thus, ST14 hypomorphic mice developed hyperproliferative and retention ichthyosis with impaired desquamation, hypotrichosis with brittle, thin, uneven, and sparse hair, and tooth defects. Biochemical analysis of ST14 hypomorphic epidermis revealed reduced prostasin proteolytic activation and profilaggrin proteolytic processing, compatible with a primary role of matriptase in this process. This work strongly indicates that reduced activity of a matriptase-prostasin proteolytic cascade is the etiological origin of human ARIH and provides an important mouse model for the exploration of matriptase function in ARIH, as well as multiple other physiological and pathological processes. PMID:17940283

  10. 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

  11. 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.

  12. 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.

  13. The conditions for the modification of radiation transformation in vitro by a tumor promoter and protease inhibitors

    International Nuclear Information System (INIS)

    These experiments were designed to define the conditions necessary for the modification of radiation-induced transformation in C3H/10T1/2 cells by TPA and protease inhibitors. The results show that: (i) the lowest effective dose of various protease inhibitors to suppress transformation in vitro varies over several orders of magnitude; on a molar basis, the inhibitors of chymotrypsin appear to be the most effective protease inhibitors at suppression of radiation-induced transformation in vitro, (ii) the protease inhibitors antipain and the Bowman-Birk (soybean) protease inhibitor has no effect on radiation transformation when present only during irradiation, (iii) the protease inhibitor antipain can suppress radiation transformation in vitro when applied to proliferating 'initiated' cells as late as 10 days and 13 cell divisions post-irradiation, and (iv) TPA treatment following a 10-day protease inhibitor (antipain) exposure of X-irradiated 'initiated' cells does not lead to promotion in vitro. These results suggest that protease inhibitor treatment of the initiated cells has irreversibly reverted cells to their original or 'uninitiated' condition which existed before irradiation. (author)

  14. A cluster of hematopoietic serine protease genes is found on the same chromosomal band as the human α/δ T-cell receptor locus

    International Nuclear Information System (INIS)

    The chymotrypsin-like family of serine protease genes includes several members that are expressed exclusively in subsets of hematopoietic cells. For example, human neutrophil elastase and cathepsin G are expressed only in myelomonocytic precursors, and cytotoxic-T-cell serine proteases are found only in cytotoxic lymphocytes. The authors have used a cathepsin G cDNA probe to clone two cathepsin G-like genes (designated CGL-1 and CGL-2) from a human genomic library. They have determined that CGL-1 is identical to a previously identified gene (known as CCPI, CTLA I, or cytotoxic serine protease B) that is expressed only in activated cytotoxic T lymphocytes. They show here that cathepsin G, CGL-1, and CGL-2 are linked on an ∼50-kilobase locus found on human chromosome 14 at band q11.2. This gene cluster maps to the same chromosomal band as the α and δ T-cell receptor genes; this region is involved in most chromosomal translocations and inversions that are specifically associated with T-cell malignancies

  15. Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme

    International Nuclear Information System (INIS)

    Proton NMR spectra of serine proteases in 1H2O solutions typically show a single resonance at very low magnetic field i.e., 14-18 ppm from dimethylsilylapentanesulfonate. This resonance has been assigned to the proton hydrogen bonded between aspartic acid-102 and histidine-57 (chymotrypsin numbering system) of the charge-relay system or catalytic triad of serine proteases. There have been a number of reports that have cast doubt on its correctness. In the present work the authors have tested this assignment using α-lytic protease, a bacterial serine protease homologous to elastase, which is specifically labeled with nitrogen-15 at N/sup delta1/ of its single histidine residue. The low-field region of the proton spectra of this labeled enzyme shows a single resonance having the properties reported which, in addition, exhibits spin-spin splitting to the nitrogen-15 label. The observation of this 15N-/sup delta1/-H coupling makes the assignment of this resonance to the charge-relay proton unequivocal

  16. Conditions for the modification of radiation transformation in vitro by a tumor promoter and protease inhibitors

    International Nuclear Information System (INIS)

    These experiments were designed to define the conditions necessary for the modification of radiation-induced transformation in C3H/10T1/2 cells by TPA and protease inhibitors. The results show that: (i) the lowest effective dose of various protease inhibitors to suppress transformation in vitro varies over several orders of magnitude; on a molar basis, the inhibitors of chymotrypsin appear to be the most effective protease inhibitors at suppression of radiation-induced transformation in vitro, (ii) the protease inhibitors antipain and the Bowman-Birk (soybean) protease inhibitor have no effect on radiation transformation when present only during irradiation, (iii) the protease inhibitor antipain can suppress radiation transformation in vitro when applied to proliferating initiated cells as late as 10 days and 13 cell divisions post-irradiation, and (iv) TPA treatment following a 10-day protease inhibitor (anti-pain) exposure of X-irradiated initiated cells does not lead to promotion in vitro. These results suggest that protease inhibitor treatment of the initiated cells has irreversibly reverted cells to their original or uninitiated condition which existed before irradiation

  17. Earthworm Protease

    International Nuclear Information System (INIS)

    The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibrinolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP). The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate pro enzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

  18. Assessment of IgE and IgG4 Binding Capacities of Cow's Milk Proteins Selectively Altered by Proteases.

    Science.gov (United States)

    Charcosset, Alexandre; Adel-Patient, Karine; Dupont, Christophe; Bernard, Hervé

    2016-05-01

    Specific IgE and IgG4 have been reported to play key roles in the context of IgE-mediated cow's milk allergy (CMA), but the persistence of their epitopes in milk hydrolysates has not been evaluated. Using sera from 32 CMA patients, 6 CMA patients treated by epicutaneous therapy (CM-treated), and 4 CM-tolerant peanut allergic patients, we analyzed the IgE and IgG4 binding capacities related to major milk allergens in processed milk. Different proteases (plasmin, chymosin, α-chymotrypsin, or pepsin) were used progressively and selectively to hydrolyze β-lactoglobulin (β-LG) and casein (CN) in milk. We then showed that proteases differentially affect IgE or IgG4 immunoreactivities of CN and β-LG and also that we could not relate IgE and/or IgG4 levels or specificities to milk hydrolysates to the clinical status of the patients. PMID:27015440

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. Elimination of turbidity interference in serum iron colorimetric assay by enzymatic proteolysis

    Directory of Open Access Journals (Sweden)

    Cardoso Leonardo M.

    2003-01-01

    Full Text Available We describe a modification in the commercial colorimetric method for the determination of serum iron by using Ferrozine® . The modification was proposed because during the conventional procedure, turbidity observed when the serum of animals submitted to surgery was used interfered with the assay. We added to the original method, a previous treatment of the serum with proteolytic enzymes. This modification was also tested using plasma samples, although this was not recommended when the original method was used. The results demonstrated that: a the treatment with a mixture of trypsin and chymotrypsin was effective in order to eliminate turbidity; b there was no difference between the standard curves obtained by the conventional and the modified method for control assays; c the absorbencies of the samples of serum and plasma submitted to proteolysis, estimated by the addition of different concentrations of iron, were directly proportional to iron concentrations; d the pre-treatment with enzymes allowed the utilization of plasma; e the pre-treatment with guanidine. HCl was not effective.

  4. 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.

  5. [Experiment study on solubilization of cholesteatoma debris].

    Science.gov (United States)

    Gyo, K; Sasaki, Y; Kobayashi, T

    1991-01-01

    A variety of solutions were tested in vitro to find a suitable solvent of cholesteatoma debris for use in clinical practice. The specimens were taken during surgery from the patients of otitis media with cholesteatoma. They were divided in pieces and put in test tubes. Each tube was then admixed with one of the test solutions and incubated at 37 degrees C for 48 hours. Hydrochloric acid (1N) and sodium hydroxide (1N) had no substantial effect to solve the debris. Urea (10N), acetylcysteine (20%) and chymotrypsin (1%) had a weak such effect. Proteolytic agents such as diiodosalitylic acid (0.1N), sodium dodecyl sulfate (0.1N) and cholic acid (0.1N) showed a stronger effect but not enough for clinical use. In contrast, a detergent which contains interfacial active agents and a proteolytic enzyme (alkaline cellulase), such as Attack and Hi-Top, proved to be more effective to solve the debris. However, biological effect of such detergent on the ear is not clear. Further study will be necessary before actual application in the patients. PMID:2019913

  6. Partial characterization of a novel endogenous opioid in human cerebrospinal fluid

    International Nuclear Information System (INIS)

    Human cerebrospinal fluid (CSF) contains many uncharacterized endogenous opioids, in addition to the known enkephalins, endorphins, and dynorphins. These opioids may be separated by gel filtration chromatography and identified by radioreceptor assay for opioid activity. One region of the chromatographic elution profile, designated Peak B has previously been shown to be related to the pain status of chronic pain patients. The authors now report that human Peak B isolated from the CSF of pain-free elective surgery patients is present at a typical concentration equivalent in activity to 1.4 pmol of morphine sulfate per ml of CSF measured by radioreceptor assay. At a dose of 0.06 and 0.12 pmol morphine sulfate equivalents of CSF (MSE), injected into the cerebroventricular system of the mouse, Peak B produced an antinociceptive effect, the intensity and duration of which was dose-dependent and which was antagonized by naloxone. The mouse vas deferens (MVD) preparation was inhibited by Peak B in a manner that was sensitive to antagonism by naloxone only at low (6.0 μM) concentrations of the antagonist. Peak B activity in the MVD assay was unaffected by treatment with trypsin or α-chymotrypsin. 32 references, 4 figures, 1 table

  7. Partial characterization of a novel endogenous opioid in human cerebrospinal fluid

    Energy Technology Data Exchange (ETDEWEB)

    Miller, B.E.; Lipman, J.J.; Byrne, W.L.

    1987-12-07

    Human cerebrospinal fluid (CSF) contains many uncharacterized endogenous opioids, in addition to the known enkephalins, endorphins, and dynorphins. These opioids may be separated by gel filtration chromatography and identified by radioreceptor assay for opioid activity. One region of the chromatographic elution profile, designated Peak B has previously been shown to be related to the pain status of chronic pain patients. The authors now report that human Peak B isolated from the CSF of pain-free elective surgery patients is present at a typical concentration equivalent in activity to 1.4 pmol of morphine sulfate per ml of CSF measured by radioreceptor assay. At a dose of 0.06 and 0.12 pmol morphine sulfate equivalents of CSF (MSE), injected into the cerebroventricular system of the mouse, Peak B produced an antinociceptive effect, the intensity and duration of which was dose-dependent and which was antagonized by naloxone. The mouse vas deferens (MVD) preparation was inhibited by Peak B in a manner that was sensitive to antagonism by naloxone only at low (< 1.0 ..mu..M) but not at higher (>6.0 ..mu..M) concentrations of the antagonist. Peak B activity in the MVD assay was unaffected by treatment with trypsin or ..cap alpha..-chymotrypsin. 32 references, 4 figures, 1 table.

  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. Characterization of grain-specific peptide markers for the detection of gluten by mass spectrometry.

    Science.gov (United States)

    Fiedler, Katherine L; McGrath, Sara C; Callahan, John H; Ross, Mark M

    2014-06-25

    Global and targeted mass spectrometry-based proteomic approaches were developed to discover, evaluate, and apply gluten peptide markers to detect low parts per million (ppm) wheat contamination of oats. Prolamins were extracted from wheat, barley, rye, and oat flours and then reduced, alkylated, and digested with chymotrypsin. The resulting peptides were subjected to LC-MS/MS analysis and database matching. No peptide markers common to wheat, barley, and rye were identified that could be used for global gluten detection. However, many grain-specific peptide markers were identified, and a set of these markers was selected for gluten detection and grain differentiation. Wheat flour was spiked into gluten-free oat flour at concentrations of 1-100,000 ppm and analyzed to determine the lowest concentration at which the wheat "contaminant" could be confidently detected in the mixture. The same 2D ion trap instrument that was used for the global proteomics approach was used for the targeted proteomics approach, providing a seamless transition from target discovery to application. A powerful, targeted MS/MS method enabled detection of two wheat peptide markers at the 10 ppm wheat flour-in-oat flour concentration. Because gluten comprises approximately 10% of wheat flour protein, the reported wheat gluten-specific peptides can enable detection of approximately 1 ppm of wheat gluten in oats. PMID:24866027

  10. 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.

  11. Six alternative proteases for mass spectrometry-based proteomics beyond trypsin.

    Science.gov (United States)

    Giansanti, Piero; Tsiatsiani, Liana; Low, Teck Yew; Heck, Albert J R

    2016-05-01

    Protein digestion using a dedicated protease represents a key element in a typical mass spectrometry (MS)-based shotgun proteomics experiment. Up to now, digestion has been predominantly performed with trypsin, mainly because of its high specificity, widespread availability and ease of use. Lately, it has become apparent that the sole use of trypsin in bottom-up proteomics may impose certain limits in our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments or even subsets of proteins. To overcome this problem, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated from these alternative proteases, have not been systematically documented. Therefore, here we provide an optimized protocol for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, LysC, LysN, AspN, GluC and ArgC. This protocol is formulated to promote ease of use and robustness, which enable parallel digestion with each of the six tested proteases. We present data on protease availability and usage including recommendations for reagent preparation. We additionally describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in ∼2 d. PMID:27123950

  12. 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.)

  13. 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

  14. Interactive effects of CO2 and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria

    International Nuclear Information System (INIS)

    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 at all CO2 levels. In contrast, trypsin- and caspase-like activities of the oyster proteasome

  15. 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

  16. 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

  17. Incorporação de farinhas de resíduos de Jundiá na dieta: bioquímica plasmática, parâmetros hepáticos e digestivos Addition of Jundiá waste meal in diet: plasmatic biochemistry, hepatic and digestive parameters

    Directory of Open Access Journals (Sweden)

    Suzete Rossato

    2013-01-01

    full Jundiá meal (FJI. In experiment II (EXP II, different FCJCV levels were tested (0, 3.75, 7.5, 15, and 30%. Total circulating proteins, cholesterol, triglyceride, glucose, albumin and amino acids were quantified in plasma. Glucose, ammonia, total protein, glycogen, amino acids and transaminases were analyzed in the liver. The acid protease activity was determined in the stomach, and trypsin and chymotrypsin activity were established in the intestine. EXP I, found a lower glucose concentration in plasma and alanine aminotransferase (ALT in the liver for FCJSV treatment and higher levels of these parameters for FCO treatment. The activity of acid protease and chymotrypsin were lower for FCO and higher for FCJCV. In EXP II, rates of free amino acids and total cholesterol in plasma increased with increasing levels FCJCV dietary. The incorporation of Jundiá waste meal diet modified plasmatic biochemistry, liver and digestive parameters of Jundiá juveniles, but these changes did not influence the fish health, demonstrating its nutritional quality and efficiency in fish diet.

  18. Incorporação de farinhas de resíduos de Jundiá na dieta: bioquímica plasmática, parâmetros hepáticos e digestivos Addition of Jundiá waste meal in diet: plasmatic biochemistry, hepatic and digestive parameters

    Directory of Open Access Journals (Sweden)

    Suzete Rossato

    2013-06-01

    full Jundiá meal (FJI. In experiment II (EXP II, different FCJCV levels were tested (0, 3.75, 7.5, 15, and 30%. Total circulating proteins, cholesterol, triglyceride, glucose, albumin and amino acids were quantified in plasma. Glucose, ammonia, total protein, glycogen, amino acids and transaminases were analyzed in the liver. The acid protease activity was determined in the stomach, and trypsin and chymotrypsin activity were established in the intestine. EXP I, found a lower glucose concentration in plasma and alanine aminotransferase (ALT in the liver for FCJSV treatment and higher levels of these parameters for FCO treatment. The activity of acid protease and chymotrypsin were lower for FCO and higher for FCJCV. In EXP II, rates of free amino acids and total cholesterol in plasma increased with increasing levels FCJCV dietary. The incorporation of Jundiá waste meal diet modified plasmatic biochemistry, liver and digestive parameters of Jundiá juveniles, but these changes did not influence the fish health, demonstrating its nutritional quality and efficiency in fish diet.

  19. 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

  20. 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.

  1. Amelioration of neuronal cell death in a spontaneous obese rat model by dietary restriction through modulation of ubiquitin proteasome system.

    Science.gov (United States)

    Shruthi, Karnam; Reddy, S Sreenivasa; Reddy, P Yadagiri; Shivalingam, Potula; Harishankar, Nemani; Reddy, G Bhanuprakash

    2016-07-01

    Dietary restriction (DR) has been shown to increase longevity, delay onset of aging, reduce DNA damage and oxidative stress and prevent age-related decline of neuronal activity. We previously reported the role of altered ubiquitin proteasome system (UPS) in the neuronal cell death in a spontaneous obese rat model (WNIN/Ob rat). In this study, we investigated the effect of DR on obesity-induced neuronal cell death in a rat model. Two groups of 40-day-old WNIN/Ob rats were either fed ad libitum (Ob) or pair-fed with lean. The lean phenotype of WNIN/Ob rats served as ad libitum control. These animals were maintained for 6.5months on their respective diet regime. At the end of the study, cerebral cortex was collected and markers of UPS, endoplasmic reticulum (ER) stress and autophagy were analyzed by quantitative real-time polymerase chain reaction, immunoblotting and immunohistochemistry. Chymotrypsin-like activity of proteasome was assayed by the fluorimetric method. Apoptotic cells were analyzed by TUNEL assay. DR improved metabolic abnormalities in obese rats. Alterations in UPS (up-regulation of UCHL1, down-regulation of UCHL5, declined proteasomal activity), increased ER stress, declined autophagy and increased expression of α-synuclein, p53 and BAX were observed in obese rats and DR alleviated these changes in obese rats. Further, DR decreased TUNEL-positive cells. In conclusion, DR in obese rats could not only restore the metabolic abnormalities but also preserved neuronal health in the cerebral cortex by preventing alterations in the UPS. PMID:27260470

  2. Relationship of protein phosphorylation to the activation of the respiratory burst in human neutrophils. Defects in the phosphorylation of a group of closely related 48-kDa proteins in two forms of chronic granulomatous disease

    International Nuclear Information System (INIS)

    When 32P-labeled human neutrophils were activated by exposure to phorbol myristate acetate, three 48-kDa proteins (designated pp48/6.8, pp48/7.3, and pp48/7.8, from their isoelectric points) were found to have become labeled. With maximal stimulation, labeling was complete by 30 s. With lesser degrees of stimulation, the extent of labeling at 2 min correlated with rates of production by the phorbol-treated cells. Increased labeling of these 48-kDa proteins was also seen in cells exposed to f-Met-Leu-Phe. In phorbol-treated neutrophils from patients with X-linked cytochrome b558-negative chronic granulomatous disease, pp48/7.8 was labeled in a normal fashion, but pp48/6.8 and pp48/7.3 failed to take up 32P. In cells from patients with autosomal recessive cytochrome b558-positive chronic granulomatous disease, however, none of the three proteins took up 32P in response to phorbol. The three proteins appear to be very closely related, as indicated by the findings that phosphoserine was the only phosphoamino acid found in any of the three, and all three yielded identical one-dimensional phosphopeptide maps after digestion with either chymotrypsin or staphylococcal proteinase V8. These results reconcile earlier observations on protein phosphorylation in chronic granulomatous disease and provide further evidence for a relationship between the phosphorylation of this group of 48-kDa proteins and the activation of the respiratory burst oxidase

  3. 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

  4. The structure of MBL-associated serine protease-2 reveals that identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions.

    Science.gov (United States)

    Harmat, Veronika; Gál, Péter; Kardos, József; Szilágyi, Katalin; Ambrus, Géza; Végh, Barbara; Náray-Szabó, Gábor; Závodszky, Péter

    2004-10-01

    A family of serine proteases mediates the proteolytic cascades of several defense mechanisms in vertebrates, such as the complement system, blood coagulation and fibrinolysis. These proteases usually form large complexes with other glycoproteins. Their common features are their modular structures and restricted substrate specificities. The lectin pathway of complement, where mannose-binding lectin (MBL) recognizes the carbohydrate structures on pathogens, is activated by mannose-binding lectin-associated serine protease-2 (MASP-2). We present the 2.25A resolution structure of the catalytic fragment of MASP-2 encompassing the second complement control protein module (CCP2) and the serine protease (SP) domain. The CCP2 module stabilizes the structure of the SP domain as demonstrated by differential scanning calorimetry measurements. The asymmetric unit contains two molecules with different CCP-SP domain orientations, reflecting increased modular flexibility at the CCP2/SP joint. This flexibility may partly explain the ability of the MASP-2 dimer to perform all of its functions alone, whereas the same functions are mediated by the much larger C1r2-C1s2 tetramer in the C1 complex of the classical pathway. The main scaffold of the MASP-2 SP domain is chymotrypsin-like. Eight surface loops determine the S1 and other subsite specificities. Surprisingly, some surface loops of MASP-2, e.g. loop 1 and loop 2, which form the S1 pocket are similar to those of trypsin, and show significant differences if compared with those of C1s, indicating that the nearly identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions. PMID:15364579

  5. 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

  6. Relationship of protein phosphorylation to the activation of the respiratory burst in human neutrophils. Defects in the phosphorylation of a group of closely related 48-kDa proteins in two forms of chronic granulomatous disease

    Energy Technology Data Exchange (ETDEWEB)

    Okamura, N.; Curnutte, J.T.; Roberts, R.L.; Babior, B.M.

    1988-05-15

    When 32P-labeled human neutrophils were activated by exposure to phorbol myristate acetate, three 48-kDa proteins (designated pp48/6.8, pp48/7.3, and pp48/7.8, from their isoelectric points) were found to have become labeled. With maximal stimulation, labeling was complete by 30 s. With lesser degrees of stimulation, the extent of labeling at 2 min correlated with rates of production by the phorbol-treated cells. Increased labeling of these 48-kDa proteins was also seen in cells exposed to f-Met-Leu-Phe. In phorbol-treated neutrophils from patients with X-linked cytochrome b558-negative chronic granulomatous disease, pp48/7.8 was labeled in a normal fashion, but pp48/6.8 and pp48/7.3 failed to take up 32P. In cells from patients with autosomal recessive cytochrome b558-positive chronic granulomatous disease, however, none of the three proteins took up 32P in response to phorbol. The three proteins appear to be very closely related, as indicated by the findings that phosphoserine was the only phosphoamino acid found in any of the three, and all three yielded identical one-dimensional phosphopeptide maps after digestion with either chymotrypsin or staphylococcal proteinase V8. These results reconcile earlier observations on protein phosphorylation in chronic granulomatous disease and provide further evidence for a relationship between the phosphorylation of this group of 48-kDa proteins and the activation of the respiratory burst oxidase.

  7. [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

  8. Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

    Science.gov (United States)

    Oliva, M L V; Andrade, S A; Juliano, M A; Sallai, R C; Torquato, R J; Sampaio, M U; Pott, V J; Sampaio, C A M

    2003-07-01

    The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays. PMID:12678803

  9. Degradation of human anaphylatoxin C3a by rat peritoneal mast cells: a role for the secretory granule enzyme chymase and heparin proteoglycan

    International Nuclear Information System (INIS)

    Purified human C3a was iodinated (125I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of 125I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of 125I-C3a and the rate of dissociation from the cell were temperature dependent. At 00C, the binding of 125I-C3a was increased and the rate of dissociation reduced, as compared to 370C. Once 125I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of 125I precipitable by TCA and the appearance of 125I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of 125I-C3a. Treatment of RMC bearing 125I-C3a with Bis (sulfosuccinimidyl) suberate (BS3) covalently crosslinked the 125I-Ca to chymase, the predominant enzyme found in the secretory granules. Indirect immunofluorescence of RMC using the IgG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. The results indicate that 125I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan. In addition, this proteolysis of 125I-C3a by chymase must be blocked in order to detect plasma membrane C3a binding components on RMC

  10. Cardiac-specific catalase overexpression rescues anthrax lethal toxin-induced cardiac contractile dysfunction: role of oxidative stress and autophagy

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

  12. 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; Escriche, Baltasar

    2014-12-01

    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 Glu(47) for the 70-kDa form or Ile(88) 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

  13. Increased 4-hydroxy-2-nonenal-induced proteasome dysfunction is correlated with cardiac damage in streptozotocin-injected rats with isoproterenol infusion.

    Science.gov (United States)

    Deshpande, Mandar; Mali, Vishal R; Pan, Guodong; Xu, Jiang; Yang, Xiao-Ping; Thandavarayan, Rajarajan A; Palaniyandi, Suresh Selvaraj

    2016-07-01

    Increase in 4-hydroxy-2-nonenal (4HNE) due to oxidative stress has been observed in a variety of cardiac diseases such as diabetic cardiomyopathy. 4HNE exerts a damaging effect in the myocardium by interfering with subcellular organelles like mitochondria by forming adducts. Therefore, we hypothesized that increased 4HNE adduct formation in the heart results in proteasome inactivation in isoproterenol (ISO)-infused type 1 diabetes mellitus (DM) rats. Eight-week-old male Sprague Dawley rats were injected with streptozotocin (STZ, 65 mg kg(-1) ). The rats were infused with ISO (5 mg kg(-1) ) for 2 weeks by mini pumps, after 8 weeks of STZ injection. We studied normal control (n = 8) and DM + ISO (n = 10) groups. Cardiac performance was assessed by echocardiography and Millar catheter at the end of the protocol at 20 weeks. Initially, we found an increase in 4HNE adducts in the hearts of the DM + ISO group. There was also a decrease in myocardial proteasomal peptidase (chymotrypsin and trypsin-like) activity. Increases in cardiomyocyte area (446 ± 32·7 vs 221 ± 10·83) (µm(2) ), per cent area of cardiac fibrosis (7·4 ± 0·7 vs 2·7 ± 0·5) and cardiac dysfunction were also found in DM + ISO (P pathology in STZ-injected rats infused with ISO. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27273517

  14. Partial characterization of murine migration inhibitory factor (MIF).

    Science.gov (United States)

    Kühner, A L; David, J R

    1976-01-01

    These studies describe the production of murine migration inhibitory factor (MIF)3 in sufficient quantities to allow its partial characterization by physiochemical and enzymatic methods. MIF was obtained from murine spleen cell cultures (C57BL/6 strain) stimulated with concanavalin A (Con A). Characterization of murine MIF was performed using Sephadex G-100 gel chromatography, isopycnic centrifugation in a CsCl density gradient, polyacrylamide disc electrophoresis, heat stability, and enzymatic treatment. MIF-containing and control fractions were assayed on normal C57BL/6 peritoneal exudate cells by using a microcapillary tube assay. Peak MIF activity was found in a Sephadex G-100 fraction containing molecules the size of albumin and slightly smaller, molecular weight 67,000 to 48,000. Murine MIF was stable to heating at 56 degrees C for 30 min but lost its activity at 80 degrees C for 30 min. Incubation of G-100 fractions containing MIF with water insoluble chymotrypsin destroyed the activity of MIF, indicating its protein nature. CsCl density gradient centrifugation revealed that murine MIF had a buoyand density greater than protein, consistent with its being a glycoprotein. Further, when subjected to disc electrophoresis on polyacylamide gels, murine MIF migrated in a region cathodal to albumin. Thus, mitogen stimulation of murine spleen cells produced MIF in quantities which allowed its partial characterization and purification, and its comparison with human and guinea pig MIF; this makes it feasible to analyze the role of murine MIF in cellular immunity and in its relationship to lymphocyte mediators which regulate humoral immune responses. PMID:1107423

  15. 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

  16. 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

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  19. 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

  20. 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.