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Sample records for aspartic proteinases based

  1. [Ulysses retrotransposon aspartate proteinase (Drosophila virilis)].

    PubMed

    Volkov, D A; Savvateeva, L V; Dergousova, N I; Rumsh, L D

    2002-01-01

    Retrotransposones are mobile genetic elements occurring in genomes of bacteria, plants or animals. Retrotransposones were found to contain nucleotide sequences encoding proteins which are homological to retroviral aspartic proteinases. Our research has been focused on Ulysses which is mobile genetic element found in Drosophila virilis. We suggested a primary structure of Ulysses proteinase using comparative analysis of amino acid sequences of retroviral proteinases and proteinases from retrotransposones. The appropriate cDNA fragment has been cloned and expressed in E. coli. The purification of recombinant protein (12 kD) has been carried out by affinity chromatography using pepstatine-agarose. The obtained protein has proteolytic activity at optimum pH 5.5 like the majority of aspartic proteinases.

  2. New aspartic proteinase of Ulysses retrotransposon from Drosophila virilis.

    PubMed

    Volkov, D A; Dergousova, N I; Rumsh, L D

    2004-06-01

    This work is focused on the investigation of a proteinase of Ulysses mobile genetic element from Drosophila virilis. The primary structure of this proteinase is suggested based on comparative analysis of amino acid sequences of aspartic proteinases from retroviruses and retrotransposons. The corresponding cDNA fragment has been cloned and expressed in E. coli. The protein accumulated in inclusion bodies. The recombinant protein (12 kD) was subjected to refolding and purified by affinity chromatography on pepstatin-agarose. Proteolytic activity of the protein was determined using oligopeptide substrates melittin and insulin B-chain. It was found that the maximum of the proteolytic activity is displayed at pH 5.5 as for the majority of aspartic proteinases. We observed that hydrolysis of B-chain of insulin was totally inhibited by pepstatin A in the micromolar concentration range. The molecular weight of the monomer of the Ulysses proteinase was determined by MALDI-TOF mass-spectrometry.

  3. Aspartic proteinases in the digestive tract of marine decapod crustaceans.

    PubMed

    Navarrete del Toro, María de Los Angeles; García-Carreño, Fernando; López, Manuel Díaz; Celis-Guerrero, Laura; Saborowski, Reinhard

    2006-08-01

    Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others. PMID:16788916

  4. Solution structure of the squash aspartic acid proteinase inhibitor (SQAPI) and mutational analysis of pepsin inhibition.

    PubMed

    Headey, Stephen J; Macaskill, Ursula K; Wright, Michele A; Claridge, Jolyon K; Edwards, Patrick J B; Farley, Peter C; Christeller, John T; Laing, William A; Pascal, Steven M

    2010-08-27

    The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel beta-sheet gripping an alpha-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting beta-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S' side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp(32)-Asp(215) diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.

  5. Characterization of recombinant CDR1, an Arabidopsis aspartic proteinase involved in disease resistance.

    PubMed

    Simões, Isaura; Faro, Rosário; Bur, Daniel; Faro, Carlos

    2007-10-26

    The Arabidopsis thaliana constitutive disease resistance 1 (CDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling (Xia, Y., Suzuki, H., Borevitz, J., Blount, J., Guo, Z., Patel, K., Dixon, R. A., and Lamb, C. (2004) EMBO J. 23, 980-988). This apoplastic enzyme is a member of the group of "atypical" plant aspartic proteinases. As for other enzymes of this subtype, CDR1 has remained elusive until recently as a result of its unusual properties and localization. Here we report on the heterologous expression and characterization of recombinant CDR1, which displays unique enzymatic properties among plant aspartic proteinases. The highly restricted specificity requirements, insensitivity toward the typical aspartic proteinase inhibitor pepstatin A, an unusually high optimal pH of 6.0-6.5, proteinase activity without irreversible prosegment removal, and dependence of catalytic activity on formation of a homo-dimer are some of the unusual properties observed for recombinant CDR1. These findings unveil a pattern of unprecedented functional complexity for Arabidopsis CDR1 and are consistent with a highly specific and regulated biological function. PMID:17650510

  6. Kinetic analysis of a general model of activation of aspartic proteinase zymogens.

    PubMed

    Varón, R; García-Moreno, M; Valera-Ruipérez, D; García-Molina, F; García-Cánovas, F; Ladrón-de Guevara, R G; Masiá-Pérez, J; Havsteen, B H

    2006-10-01

    Starting from a simple general reaction mechanism of activation of aspartic proteinase zymogens involving an uni- and a bimolecular simultaneous route, the time course equation of the concentration of the zymogen and of the activated enzyme have been derived. From these equations, an analysis quantifying the relative contribution to the global process of the two routes has been carried out for the first time. This analysis suggests a way to predict the time course of the relative contribution as well as the effect of the initial zymogen and activating enzyme concentrations, on the relative weight. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed. Finally, we apply some of our results to experimental data obtained by other authors in experimental studies of the activation of some aspartic proteinase zymogens.

  7. Characterization of a nucleotide stimulated aspartic proteinase in rat liver plasma membranes.

    PubMed

    Paule, C R; Larner, J

    1996-01-01

    Inositol phosphoglycan molecules are believed to mediate multiple intracellular actions of insulin. They are released from plasma membranes in response to insulin binding and are transported into the cell. Release of insulin mediators is stimulated by MnATP and MgATP and is inhibited by p-aminobenzamidine. Inositol phosphoglycan mediators may be released by a poorly characterized mechanism requiring proteolytic cleavage of an attached protein from the mediator and phospholipase cleavage of the mediator from its membrane anchor. We examined rat liver plasma membranes for proteinase activity stimulated by insulin and MnATP. Although we could not demonstrate insulin stimulation, we have found and characterized a nucleotide-stimulated aspartic proteinase bound to rat liver plasma membranes. We also detected and separated a soluble activating factor for the proteinase. The activating factor appears to be a protein with M(r) approximately 70 kDa. PMID:8876431

  8. Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.).

    PubMed

    Llorente, Berta E; Brutti, Cristina B; Caffini, Néstor O

    2004-12-29

    The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands.

  9. Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases.

    PubMed Central

    Loeb, D D; Hutchison, C A; Edgell, M H; Farmerie, W G; Swanstrom, R

    1989-01-01

    Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites. Images PMID:2642305

  10. Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16☆

    PubMed Central

    Barathy, Deivanayaga V.; Suguna, Kaza

    2013-01-01

    We report the crystal structure of the first prokaryotic aspartic proteinase-like domain identified in the genome of Mycobacterium tuberculosis. A search in the genomes of Mycobacterium species showed that the C-terminal domains of some of the PE family proteins contain two classic DT/SG motifs of aspartic proteinases with a low overall sequence similarity to HIV proteinase. The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsin-fold and catalytic site architecture. However, the active site was completely blocked by the N-terminal His-tag. Surprisingly, the enzyme was found to be inactive even after the removal of the N-terminal His-tag. A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977. PMID:23923105

  11. Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases.

    PubMed

    Athauda, Senarath B P; Matsumoto, Koji; Rajapakshe, Sanath; Kuribayashi, Masayuki; Kojima, Masaki; Kubomura-Yoshida, Nobuko; Iwamatsu, Akihiro; Shibata, Chiaki; Inoue, Hideshi; Takahashi, Kenji

    2004-07-01

    Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In the present study, we have purified, for the first time, to homogeneity two acid proteinases (nepenthesins I and II) from the pitcher fluid of Nepenthes distillatoria (a pitcher-plant known locally as badura) and investigated their enzymic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 towards acid-denatured haemoglobin; the specificity of nepenthesin I towards oxidized insulin B chain appears to be similar, but slightly wider than those of other APs (aspartic proteinases). Among the enzymic properties, however, the most notable is their unusual stability: both enzymes were remarkably stable at or below 50 degrees C, especially nepenthesin I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of nepenthesins I and II (437 and 438 residues respectively) from the pitcher tissue of N. gracilis. Although the corresponding mature enzymes (each 359 residues) are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues/molecule), which are assumed to form six unique disulphide bonds as suggested by computer modelling and are supposed to contribute towards the remarkable stability of nepenthesins. Moreover, the amino acid sequence identity of nepenthesins with ordinary APs, including plant vacuolar APs, is remarkably low (approx. 20%), and phylogenetic comparison shows that nepenthesins are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the nepenthesin-type AP-specific insertion', that includes a large number of novel, orthologous plant APs emerging in the gene/protein databases. PMID

  12. Nepenthesin, a unique member of a novel subfamily of aspartic proteinases: enzymatic and structural characteristics.

    PubMed

    Takahashi, Kenji; Athauda, Senarath B P; Matsumoto, Koji; Rajapakshe, Sanath; Kuribayashi, Masayuki; Kojima, Masaki; Kubomura-Yoshida, Nobuko; Iwamatsu, Akihiro; Shibata, Chiaki; Inoue, Hideshi

    2005-12-01

    Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In our recent study, we have purified, for the first time, to homogeneity two acid proteinases, nepenthesin I (Nep I) and nepenthesin II (Nep II) from the pitcher fluid of Nepenthes distillatoria and investigated their enzymatic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 toward acid-denatured hemoglobin; the specificity of Nep I toward oxidized insulin B chain appears to be similar, but slightly wider than those of other aspartic proteinases (APs). At or below 50 degrees C both enzymes were remarkably stable; especially Nep I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of Nep I and Nep II from the pitcher tissue of Nepenthes gracilis. Although the corresponding mature enzymes are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues per molecule), which are assumed to form six unique disulfide bonds as suggested by computer modeling and are thought to contribute toward the remarkable stability of Neps. Moreover, the amino acid sequence identity of Neps with ordinary APs, including plant vacuolar APs, are remarkably low (approx. 20%), and phylogenetic comparison shows that Neps are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the Nep-type AP (NAP)-specific insertion', including a large number of novel, orthologous plant APs emerging in the gene/protein databases. PMID:16381601

  13. Nepenthesin, a unique member of a novel subfamily of aspartic proteinases: enzymatic and structural characteristics.

    PubMed

    Takahashi, Kenji; Athauda, Senarath B P; Matsumoto, Koji; Rajapakshe, Sanath; Kuribayashi, Masayuki; Kojima, Masaki; Kubomura-Yoshida, Nobuko; Iwamatsu, Akihiro; Shibata, Chiaki; Inoue, Hideshi

    2005-12-01

    Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In our recent study, we have purified, for the first time, to homogeneity two acid proteinases, nepenthesin I (Nep I) and nepenthesin II (Nep II) from the pitcher fluid of Nepenthes distillatoria and investigated their enzymatic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 toward acid-denatured hemoglobin; the specificity of Nep I toward oxidized insulin B chain appears to be similar, but slightly wider than those of other aspartic proteinases (APs). At or below 50 degrees C both enzymes were remarkably stable; especially Nep I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of Nep I and Nep II from the pitcher tissue of Nepenthes gracilis. Although the corresponding mature enzymes are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues per molecule), which are assumed to form six unique disulfide bonds as suggested by computer modeling and are thought to contribute toward the remarkable stability of Neps. Moreover, the amino acid sequence identity of Neps with ordinary APs, including plant vacuolar APs, are remarkably low (approx. 20%), and phylogenetic comparison shows that Neps are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the Nep-type AP (NAP)-specific insertion', including a large number of novel, orthologous plant APs emerging in the gene/protein databases.

  14. Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases.

    PubMed

    Athauda, Senarath B P; Matsumoto, Koji; Rajapakshe, Sanath; Kuribayashi, Masayuki; Kojima, Masaki; Kubomura-Yoshida, Nobuko; Iwamatsu, Akihiro; Shibata, Chiaki; Inoue, Hideshi; Takahashi, Kenji

    2004-07-01

    Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In the present study, we have purified, for the first time, to homogeneity two acid proteinases (nepenthesins I and II) from the pitcher fluid of Nepenthes distillatoria (a pitcher-plant known locally as badura) and investigated their enzymic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 towards acid-denatured haemoglobin; the specificity of nepenthesin I towards oxidized insulin B chain appears to be similar, but slightly wider than those of other APs (aspartic proteinases). Among the enzymic properties, however, the most notable is their unusual stability: both enzymes were remarkably stable at or below 50 degrees C, especially nepenthesin I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of nepenthesins I and II (437 and 438 residues respectively) from the pitcher tissue of N. gracilis. Although the corresponding mature enzymes (each 359 residues) are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues/molecule), which are assumed to form six unique disulphide bonds as suggested by computer modelling and are supposed to contribute towards the remarkable stability of nepenthesins. Moreover, the amino acid sequence identity of nepenthesins with ordinary APs, including plant vacuolar APs, is remarkably low (approx. 20%), and phylogenetic comparison shows that nepenthesins are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the nepenthesin-type AP-specific insertion', that includes a large number of novel, orthologous plant APs emerging in the gene/protein databases.

  15. Key features determining the specificity of aspartic proteinase inhibition by the helix-forming IA3 polypeptide.

    PubMed

    Winterburn, Tim J; Wyatt, David M; Phylip, Lowri H; Bur, Daniel; Harrison, Rebecca J; Berry, Colin; Kay, John

    2007-03-01

    The 68-residue IA(3) polypeptide from Saccharomyces cerevisiae is essentially unstructured. It inhibits its target aspartic proteinase through an unprecedented mechanism whereby residues 2-32 of the polypeptide adopt an amphipathic alpha-helical conformation upon contact with the active site of the enzyme. This potent inhibitor (K(i) < 0.1 nm) appears to be specific for a single target proteinase, saccharopepsin. Mutagenesis of IA(3) from S. cerevisiae and its ortholog from Saccharomyces castellii was coupled with quantitation of the interaction for each mutant polypeptide with saccharopepsin and closely related aspartic proteinases from Pichia pastoris and Aspergillus fumigatus. This identified the charged K18/D22 residues on the otherwise hydrophobic face of the amphipathic helix as key selectivity-determining residues within the inhibitor and implicated certain residues within saccharopepsin as being potentially crucial. Mutation of these amino acids established Ala-213 as the dominant specificity-governing feature in the proteinase. The side chain of Ala-213 in conjunction with valine 26 of the inhibitor marshals Tyr-189 of the enzyme precisely into a position in which its side-chain hydroxyl is interconnected via a series of water-mediated contacts to the key K18/D22 residues of the inhibitor. This extensive hydrogen bond network also connects K18/D22 directly to the catalytic Asp-32 and Tyr-75 residues of the enzyme, thus deadlocking the inhibitor in position. In most other aspartic proteinases, the amino acid at position 213 is a larger hydrophobic residue that prohibits this precise juxtaposition of residues and eliminates these enzymes as targets of IA(3). The exquisite specificity exhibited by this inhibitor in its interaction with its cognate folding partner proteinase can thus be readily explained. PMID:17145748

  16. Evolutionary fate of duplicate genes encoding aspartic proteinases. Nothepsin case study.

    PubMed

    Borrelli, Lucia; De Stasio, Roberta; Filosa, Silvana; Parisi, Elio; Riggio, Marilisa; Scudiero, Rosaria; Trinchella, Francesca

    2006-03-01

    Gene duplication is considered an important evolutionary mechanism leading to new gene functions. According to the classical model, one gene copy arising from gene duplication retains the ancestral function, whilst the other becomes subject to directional selection for some novel functions. Hence, according to this model, long-term persistence of two paralogous genes is possible only with the acquisition of functional innovation. In the absence of neofunctionalization, one of the duplicate genes may be lost following accumulation of deleterious mutations, ultimately leading to the loss of function. Recently, new mechanisms have been proposed according to which both paralogs are maintained without apparent neofunctionalization. In this paper we describe the molecular evolution of the aspartic proteinase gene family, with particular regard for the nothepsin gene, a sex- and tissue-specific form of aspartic proteinase active in fish. The finding of nothepsin in a reptile is indicative of the presence of this gene in organisms other than fish. However, the failure to find any nothepsin-like gene in avian, murine and human genome suggests that the gene has been lost in certain lineages during evolution. At variance with piscine nothepsin expressed exclusively in female liver under the estrogens action, the reptilian counterpart lacks both tissue and sex specificity, as it is constitutively expressed in different tissues of male and female specimens. The expression of the nothepsin gene in fish and lizard is accompanied by the expression of a paralogous gene encoding for cathepsin D. Functional divergence analysis indicates that cathepsin D accumulated amino acid substitutions, whereas nothepsin retained most of the ancestral functions. Phylogenetic analysis shows a preponderance of replacement substitutions compared to silent substitutions in the branch leading to the cathepsin D clade, whilst nothepsin evolves under negative selection. To explain the loss of the

  17. Adaptation of the behaviour of an aspartic proteinase inhibitor by relocation of a lysine residue by one helical turn.

    PubMed

    Winterburn, Tim J; Wyatt, David M; Phylip, Lowri H; Berry, Colin; Bur, Daniel; Kay, John

    2006-08-01

    In addition to self-inhibition of aspartic proteinase zymogens by their intrinsic proparts, the activity of certain members of this enzyme family can be modulated through active-site occupation by extrinsic polypeptides such as the small IA3 protein from Saccharomyces cerevisiae. The unprecedented mechanism by which IA3 helicates to inhibit its sole target aspartic proteinase locates an i, i+4 pair of charged residues (Lys18+Asp22) on an otherwise-hydrophobic face of the amphipathic helix. The nature of these residues is not crucial for effective inhibition, but re-location of the lysine residue by one turn (+4 residues) in the helical IA3 positions its side chain in the mutant IA3-proteinase complex in an orientation essentially identical to that of the key lysine residue in zymogen proparts. The binding of the extrinsic mutant IA3 shows pH dependence reminiscent of that required for the release of intrinsic zymogen proparts so that activation can occur. PMID:16895485

  18. N-terminal extension of the yeast IA3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity.

    PubMed

    Winterburn, Tim J; Phylip, Lowri H; Bur, Daniel; Wyatt, David M; Berry, Colin; Kay, John

    2007-07-01

    Yeast IA(3) aspartic proteinase inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme, saccharopepsin. Even aspartic proteinases that are very closely similar to saccharopepsin (e.g. the vacuolar enzyme from Pichia pastoris) are not susceptible to significant inhibition. The Pichia proteinase was selected as the target for initial attempts to engineer IA(3) to re-design the specificity. The IA(3) polypeptides from Saccharomyces cerevisiae and Saccharomyces castellii differ considerably in sequence. Alterations made by deletion or exchange of the residues in the C-terminal segment of these polypeptides had only minor effects. By contrast, extension of each of these wild-type and chimaeric polypeptides at its N-terminus by an MK(H)(7)MQ sequence generated inhibitors that displayed subnanomolar potency towards the Pichia enzyme. This gain-in-function was completely reversed upon removal of the extension sequence by exopeptidase trimming. Capture of the potentially positively charged aromatic histidine residues of the extension by remote, negatively charged side-chains, which were identified in the Pichia enzyme by modelling, may increase the local IA(3) concentration and create an anchor that enables the N-terminal segment residues to be harboured in closer proximity to the enzyme active site, thus promoting their interaction. In saccharopepsin, some of the counterpart residues are different and, consistent with this, the N-terminal extension of each IA(3) polypeptide was without major effect on the potency of interaction with saccharopepsin. In this way, it is possible to convert IA(3) polypeptides that display little affinity for the Pichia enzyme into potent inhibitors of this proteinase and thus broaden the target selectivity of this remarkable small protein. PMID:17608726

  19. Kinetic analysis of a general model of activation of aspartic proteinase zymogens involving a reversible inhibitor. I. Kinetic analysis.

    PubMed

    Muñoz-López, A; Sotos-Lomas, A; Arribas, E; Masia-Perez, J; Garcia-Molina, F; García-Moreno, M; Varon, R

    2007-04-01

    Starting from a simple general reaction mechanism of activation of aspartic proteinases zymogens involving a uni- and a bimolecular simultaneous activation route and a reversible inhibition step, the time course equation of the zymogen, inhibitor and activated enzyme concentrations have been derived. Likewise, expressions for the time required for any reaction progress and the corresponding mean activation rates as well as the half-life of the global zymogen activation have been derived. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed.

  20. A thermolabile aspartic proteinase from Mucor mucedo DSM 809: gene identification, cloning, and functional expression in Pichia pastoris.

    PubMed

    Yegin, Sirma; Fernandez-Lahore, Marcelo

    2013-06-01

    In this study, the cDNA encoding the aspartic proteinase of Mucor mucedo DSM 809 has been identified by RNA ligased-mediated and oligo-capping rapid amplification of cDNA ends (RACE) technique. The gene contained an open reading frame of 1,200 bp and encoded for a signal peptide of 21 amino acid residues. Two N-glycosylation sites were observed within the identified sequence. The proteinase gene was cloned into the vector pGAPZαA and expressed in Pichia pastoris X-33 for the first time. The protein has been secreted in functionally active form into the culture medium. The expression system does not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. Maximum enzyme production was observed with an initial medium pH of 3.5 at 20 °C and 220 rpm shaking speed utilizing 4 % glucose as a carbon and energy source. The enzyme was purified with cation exchange chromatography and further studies revealed that the enzyme was secreted in glycosylated form. The purified enzyme exhibited remarkable sensitivity to thermal treatment and became completely inactivated after incubation at 55 °C for 10 min. These results indicated that the recombinant proteinase could be considered as a potential rennet candidate for the cheese-making industry.

  1. Rapid detection of Candida albicans in clinical samples by DNA amplification of common regions from C. albicans-secreted aspartic proteinase genes.

    PubMed

    Flahaut, M; Sanglard, D; Monod, M; Bille, J; Rossier, M

    1998-02-01

    Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increasing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a silica membrane was developed. The targets used for DNA amplification were the Candida albicans-secreted aspartic proteinase (SAP) genes, a multiple-gene family of at least seven members in C. albicans. A single pair of primers was designed in order to detect six of these SAP genes and, subsequently, to increase the sensitivity of the test. Detection of the PCR product by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an SAP-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted Candida cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed blindly with 156 clinical samples and were 100 and 98%, respectively, compared with the results of culture. For the subset of blood culture samples (n = 124), the sensitivity and the specificity were 100%. The two false-positive PCR samples came from patients treated with azole antifungal agents, indicating that PCR was probably able to detect damaged organisms that could not be recovered by culture.

  2. The squash aspartic proteinase inhibitor SQAPI is widely present in the cucurbitales, comprises a small multigene family, and is a member of the phytocystatin family.

    PubMed

    Christeller, John T; Farley, Peter C; Marshall, Richelle K; Anandan, Ananda; Wright, Michele M; Newcomb, Richard D; Laing, William A

    2006-12-01

    The squash (Cucurbita maxima) phloem exudate-expressed aspartic proteinase inhibitor (SQAPI) is a novel aspartic acid proteinase inhibitor, constituting a fifth family of aspartic proteinase inhibitors. However, a comparison of the SQAPI sequence to the phytocystatin (a cysteine proteinase inhibitor) family sequences showed approximately 30% identity. Modeling SQAPI onto the structure of oryzacystatin gave an excellent fit; regions identified as proteinase binding loops in cystatin coincided with regions of SQAPI identified as hypervariable, and tryptophan fluorescence changes were also consistent with a cystatin structure. We show that SQAPI exists as a small gene family. Characterization of mRNA and clone walking of genomic DNA (gDNA) produced 10 different but highly homologous SQAPI genes from Cucurbita maxima and the small family size was confirmed by Southern blotting, where evidence for at least five loci was obtained. Using primers designed from squash sequences, PCR of gDNA showed the presence of SQAPI genes in other members of the Cucurbitaceae and in representative members of Coriariaceae, Corynocarpaceae, and Begoniaceae. Thus, at least four of seven families of the order Cucurbitales possess member species with SQAPI genes, covering approximately 99% of the species in this order. A phylogenetic analysis of these Cucurbitales SQAPI genes indicated not only that SQAPI was present in the Cucurbitales ancestor but also that gene duplication has occurred during evolution of the order. Phytocystatins are widespread throughout the plant kingdom, suggesting that SQAPI has evolved recently from a phytocystatin ancestor. This appears to be the first instance of a cystatin being recruited as a proteinase inhibitor of another proteinase family.

  3. The diversity and evolutionary relationships of the pregnancy-associated glycoproteins, an aspartic proteinase subfamily consisting of many trophoblast-expressed genes

    PubMed Central

    Xie, Sancai; Green, Jonathan; Bixby, James B.; Szafranska, Bozena; DeMartini, James C.; Hecht, Steven; Roberts, R. Michael

    1997-01-01

    The pregnancy-associated glycoproteins (PAGs) are structurally related to the pepsins, thought to be restricted to the hooved (ungulate) mammals and characterized by being expressed specifically in the outer epithelial cell layer (chorion/trophectoderm) of the placenta. At least some PAGs are catalytically inactive as proteinases, although each appears to possess a cleft capable of binding peptides. By cloning expressed genes from ovine and bovine placental cDNA libraries, by Southern genomic blotting, by screening genomic libraries, and by using PCR to amplify portions of PAG genes from genomic DNA, we estimate that cattle, sheep, and most probably all ruminant Artiodactyla possess many, possibly 100 or more, PAG genes, many of which are placentally expressed. The PAGs are highly diverse in sequence, with regions of hypervariability confined largely to surface-exposed loops. Nonsynonymous (replacement) mutations in the regions of the genes coding for these hypervariable loop segments have accumulated at a higher rate than synonymous (silent) mutations. Construction of distance phylograms, based on comparisons of PAG and related aspartic proteinase amino acid sequences, suggests that much diversification of the PAG genes occurred after the divergence of the Artiodactyla and Perissodactyla, but that at least one gene is represented outside the hooved species. The results also suggest that positive selection of duplicated genes has acted to provide considerable functional diversity among the PAGs, whose presence at the interface between the placenta and endometrium and in the maternal circulation indicates involvement in fetal–maternal interactions. PMID:9371757

  4. Purification and molecular cloning of aspartic proteinases from the stomach of adult Japanese fire belly newts, Cynops pyrrhogaster.

    PubMed

    Nagasawa, Tatsuki; Sano, Kaori; Kawaguchi, Mari; Kobayashi, Ken-Ichiro; Yasumasu, Shigeki; Inokuchi, Tomofumi

    2016-04-01

    Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians. PMID:26711235

  5. Purification and molecular cloning of aspartic proteinases from the stomach of adult Japanese fire belly newts, Cynops pyrrhogaster.

    PubMed

    Nagasawa, Tatsuki; Sano, Kaori; Kawaguchi, Mari; Kobayashi, Ken-Ichiro; Yasumasu, Shigeki; Inokuchi, Tomofumi

    2016-04-01

    Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians.

  6. Cauliflower mosaic virus produces an aspartic proteinase to cleave its polyproteins.

    PubMed

    Torruella, M; Gordon, K; Hohn, T

    1989-10-01

    Cauliflower mosaic virus (CaMV), a plant pararetrovirus, produces polyproteins from its adjacent genes for the coat protein (ORF IV) and for enzymatic functions (ORF V). The N-terminal domain of the latter gene includes a sequence showing homology to the active site of other retroviral and acid proteases. We have now shown that this domain does indeed produce a functional aspartic protease that can process both the polyproteins. Mutations in the putative active site abolished virus infectivity. In transient expression studies in protoplasts, the N-terminal domain of ORF V was able to free active CAT enzyme from a precursor containing an N-terminal fusion of a portion of ORF IV. The junction between the two domains of this artificial polyprotein comprised sequences from the ORF IV product that had previously been shown to include a proteolytic processing site. The protease mutants were not able to free active CAT enzyme from this precursor. Direct analysis of cleavage at the same site in the ORF IV product using proteins expressed in Escherichia coli revealed the expected products. In vitro translation of a synthetic transcript covering ORF V was used to study the autocatalytic cleavage of the ORF product. Pulse-chase experiments showed that the 80 kd initial translation product was processed to yield a N-terminal doublet of polypeptides of 22 and 20 kd apparent mol. wt, which cover the protease domain. The mutants in the active site were not processed. PMID:2684630

  7. Engineering the substrate specificity of rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.

    PubMed Central

    Lowther, W. T.; Majer, P.; Dunn, B. M.

    1995-01-01

    Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients. PMID:7613467

  8. Inactivation of α1-proteinase inhibitor by Candida albicans aspartic proteases favors the epithelial and endothelial cell colonization in the presence of neutrophil extracellular traps.

    PubMed

    Gogol, Mariusz; Ostrowska, Dominika; Klaga, Kinga; Bochenska, Oliwia; Wolak, Natalia; Aoki, Wataru; Ueda, Mitsuyoshi; Kozik, Andrzej; Rapala-Kozik, Maria

    2016-01-01

    Candida albicans, a causative agent of opportunistic fungal infections in immunocompromised patients, uses ten secreted aspartic proteases (SAPs) to deregulate the homeostasis of the host organism on many levels. One of these deregulation mechanisms involves a SAP-dependent disturbance of the control over proteolytic enzymes of the host by a system of dedicated proteinase inhibitors, with one important example being the neutrophil elastase and alpha1-proteinase inhibitor (A1PI). In this study, we found that soluble SAPs 1-4 and the cell membrane-anchored SAP9 efficiently cleaved A1PI, with the major cleavage points located at the C-terminal part of A1PI in a close vicinity to the reactive-site loop that plays a critical role in the inhibition mechanism. Elastase is released by neutrophils to the environment during fungal infection through two major processes, a degranulation or formation of neutrophil extracellular traps (NET). Both, free and NET-embedded elastase forms, were found to be controlled by A1PI. A local acidosis, resulting from the neutrophil activity at the infection sites, favors A1PI degradation by SAPs. The deregulation of NET-connected elastase affected a NET-dependent damage of epithelial and endothelial cells, resulting in the increased susceptibility of these host cells to candidal colonization. Moreover, the SAP-catalyzed cleavage of A1PI was found to decrease its binding affinity to a proinflammatory cytokine, interleukin-8. The findings presented here suggest a novel strategy used by C. albicans for the colonization of host tissues and overcoming the host defense. PMID:26641639

  9. Preliminary neutron and ultrahigh-resolution X-ray diffraction studies of the aspartic proteinase endothiapepsin cocrystallized with a gem-diol inhibitor

    SciTech Connect

    Tuan, Han-Fang; Erskine, Peter; Langan, Paul; Cooper, Jon; Coates, Leighton

    2007-12-01

    Three data sets have been collected on endothiapepsin complexed with the gem-diol inhibitor PD-135,040: a high-resolution synchrotron X-ray data set, a room-temperature X-ray data set and a neutron diffraction data set. Until recently, it has been impossible to grow large protein crystals of endothiapepsin with any gem-diol inhibitor that are suitable for neutron diffraction. Endothiapepsin has been cocrystallized with the gem-diol inhibitor PD-135,040 in a low solvent-content (39%) unit cell, which is unprecedented for this enzyme–inhibitor complex and enables ultrahigh-resolution (1.0 Å) X-ray diffraction data to be collected. This atomic resolution X-ray data set will be used to deduce the protonation states of the catalytic aspartate residues. A room-temperature neutron data set has also been collected for joint refinement with a room-temperature X-ray data set in order to locate the H/D atoms at the active site.

  10. [Evalution of activity of acid aspartic proteinase in Candida strains isolated from oral cavity of patients with increased risk of mycosis].

    PubMed

    Rózga, A; Kurnatowska, A J; Raczyńsak-Witońska, G; Loga, G

    2001-01-01

    We have evaluated the activity of acid aspartic protease in 195 strains of Candida isolated from the oral cavity of three groups of patients. The first group comprised patients with cancer of the larynx qualified for surgery, the second- patients with neoplastic disease ( Hodgkin s disease, lymphoma, acute granulocytic leukaemia, lymphatic leukaemia, lung cancer, multiple myeloma, stomach cancer, breast cancer) who were not treated, the third group- patients with neoplastic diseases treated by chemotherapy. The strains of fungi were differentiated using API 20C and Api 20C AUX tests according to the protocol adopted at the Department of Medical Parasitology and Biology, Medical University of Lódz. The activity of acid protease was studied by Staib method in Rózga modification. Almost all strains showed high and very high proteolytic activity. The rang of proteolysis zone of Candida strains from the three groups of patients varied from 2,5 to 12,5 mm. We have found the mean proteolytic zones of strains isolated from groups I and III differed statistically significantly (p<0,001). Similarly, statisticall sihnificant difference was seen between these parameters for groups II and III (p<0,05), while there was no difference between strains from group I and II.

  11. Enhanced response of a proteinase K-based conductometric biosensor using nanoparticles.

    PubMed

    Nouira, Wided; Maaref, Abderrazak; Elaissari, Hamid; Vocanson, Francis; Siadat, Maryam; Jaffrezic-Renault, Nicole

    2014-07-23

    Proteinases are involved in a multitude of important physiological processes, such as protein metabolism. For this reason, a conductometric enzyme biosensor based on proteinase K was developed using two types of nanoparticles (gold and magnetic). The enzyme was directly adsorbed on negatively charged nanoparticles and then deposited and cross-linked on a planar interdigitated electrode (IDE). The biosensor was characterized with bovine serum albumin (BSA) as a standard protein. Higher sensitivity was obtained using gold nanoparticles. The linear range for BSA determination was then from 0.5 to 10 mg/L with a maximum response of 154 µs. These results are greater than that found without any nanoparticles (maximum response of 10 µs). The limit of detection (LOD) was 0.3 mg/L. An inter-sensor reproducibility of 3.5% was obtained.

  12. Enhanced Response of a Proteinase K-Based Conductometric Biosensor Using Nanoparticles

    PubMed Central

    Nouira, Wided; Maaref, Abderrazak; Elaissari, Abdelhamid; Vocanson, Francis; Siadat, Maryam; Jaffrezic-Renault, Nicole

    2014-01-01

    Proteinases are involved in a multitude of important physiological processes, such as protein metabolism. For this reason, a conductometric enzyme biosensor based on proteinase K was developed using two types of nanoparticles (gold and magnetic). The enzyme was directly adsorbed on negatively charged nanoparticles and then deposited and cross-linked on a planar interdigitated electrode (IDE). The biosensor was characterized with bovine serum albumin (BSA) as a standard protein. Higher sensitivity was obtained using gold nanoparticles. The linear range for BSA determination was then from 0.5 to 10 mg/L with a maximum response of 154 μs. These results are greater than that found without any nanoparticles (maximum response of 10 μs). The limit of detection (LOD) was 0.3 mg/L. An inter-sensor reproducibility of 3.5% was obtained. PMID:25057139

  13. Concordance of collagen-based radiocarbon and aspartic-acid racemization ages.

    PubMed

    Bada, J L; Schroeder, R A; Protsch, R; Berger, R

    1974-03-01

    By determining the extent of racemization of aspartic acid in a well-dated bone, it is possible to calculate the in situ first-order rate constant for the interconversion of the L and D enantiomers of aspartic acid. Collagen-based radiocarbon-dated bones are shown to be suitable samples for use in "calibrating" the racemization reaction. Once the aspartic-acid racemization reaction has been "calibrated" for a site, the reaction can be used to date other bones from the deposit. Ages deduced by this method are in good agreement with radiocarbon ages. These results provide evidence that the aspartic-acid racemization reaction is an important chronological tool for dating bones either too old or too small for radiocarbon dating. As an example of the potential application of the technique for dating fossil man, a piece of Rhodesian Man from Broken Hill, Zambia, was analyzed and tentatively assigned an age of about 110,000 years.

  14. Digestive proteinases of the larger black flour beetle, Cynaeus angustus (Coleoptera: Tenebrionidae).

    PubMed

    Oppert, B; Walters, P; Zuercher, M

    2006-04-01

    Digestion in the larger black flour beetle, Cynaeus angustus (LeConte), was studied to identify new control methods for this pest of stored grains and grain products. The physiological pH of the larval gut, as measured with extracts in water, was approximately 6.1, and the pH for optimal hydrolysis of casein by gut extracts was 6.2 when buffers were reducing. However, under non-reducing conditions, hydrolysis of casein and synthetic serine proteinase substrates was optimal in alkaline buffer. Three major proteinase activities were observed in zymograms using casein or gelatin. Caseinolytic activity of C. angustus gut extracts was inhibited by inhibitors that target aspartic and serine proteinase classes, with minor inhibition by a cysteine proteinase inhibitor. In particular, soybean trypsin and trypsin/chymotrypsin inhibitors were most effective in reducing the in vitro caseinolytic activity of gut extracts. Based on these data, further studies are suggested on the effects of dietary soybean inhibitors of serine proteinases, singly and in combination with aspartic and cysteine proteinase inhibitors, on C. angustus larvae. Results from these studies can be used to develop new control strategies to prevent damage to grains and stored products by C. angustus and similar coleopteran pests.

  15. Biodegradation and Osteosarcoma Cell Cultivation on Poly(aspartic acid) Based Hydrogels.

    PubMed

    Juriga, Dávid; Nagy, Krisztina; Jedlovszky-Hajdú, Angéla; Perczel-Kovách, Katalin; Chen, Yong Mei; Varga, Gábor; Zrínyi, Miklós

    2016-09-14

    Development of novel biodegradable and biocompatible scaffold materials with optimal characteristics is important for both preclinical and clinical applications. The aim of the present study was to analyze the biodegradability of poly(aspartic acid)-based hydrogels, and to test their usability as scaffolds for MG-63 osteoblast-like cells. Poly(aspartic acid) was fabricated from poly(succinimide) and hydrogels were prepared using natural amines as cross-linkers (diaminobutane and cystamine). Disulfide bridges were cleaved to thiol groups and the polymer backbone was further modified with RGD sequence. Biodegradability of the hydrogels was evaluated by experiments on the base of enzymes and cell culture medium. Poly(aspartic acid) hydrogels possessing only disulfide bridges as cross-links proved to be degradable by collagenase I. The MG-63 cells showed healthy, fibroblast-like morphology on the double cross-linked and RGD modified hydrogels. Thiolated poly(aspartic acid) based hydrogels provide ideal conditions for adhesion, survival, proliferation, and migration of osteoblast-like cells. The highest viability was found on the thiolated PASP gels while the RGD motif had influence on compacted cluster formation of the cells. These biodegradable and biocompatible poly(aspartic acid)-based hydrogels are promising scaffolds for cell cultivation. PMID:27541725

  16. Efficient aspartic acid production by a psychrophile-based simple biocatalyst.

    PubMed

    Tajima, Takahisa; Hamada, Mai; Nakashimada, Yutaka; Kato, Junichi

    2015-10-01

    We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds. PMID:26254042

  17. DNA interaction with octahedral and square planar Ni(II) complexes of aspartic-acid Schiff-bases

    NASA Astrophysics Data System (ADS)

    Sallam, S. A.; Orabi, A. S.; Abbas, A. M.

    2011-12-01

    Ni(II) complexes of (S,E)-2-(2-OHbenzilydene)aspartic acid; (S,E)-2-(2,3-diOHbenzilydene)aspartic acid-; (S,E)-2-(2,4-diOH-benzilydene)aspartic acid; (S,E)-2-(2,5-diOHbenzilydene)aspartic acid and (S,E)-2-((2-OHnaphthalene-1-yl)methylene)aspartic acid Schiff-bases have been synthesized by template method in ethanol or ammonia media. They were characterized by elemental analyses, conductivity measurements, magnetic moment, UV, IR and 1H nmr spectra as well as thermal analysis (TG, DTG, DTA). The Schiff-bases are dibasic tridentate or tetradentate donors and the complexes have square planar and octahedral structures. The complexes decompose in two or three steps where kinetic and thermodynamic parameters of the decomposition steps were computed. The interactions of the formed complexes with FM-DNA were monitored by UV and fluorescence spectroscopy.

  18. Acid-Base Titration of (S)-Aspartic Acid: A Circular Dichroism Spectrophotometry Experiment

    NASA Astrophysics Data System (ADS)

    Cavaleiro, Ana M. V.; Pedrosa de Jesus, Júlio D.

    2000-09-01

    The magnitude of the circular dichroism of (S)-aspartic acid in aqueous solutions at a fixed wavelength varies with the addition of strong base. This laboratory experiment consists of the circular dichroism spectrophotometric acid-base titration of (S)-aspartic acid in dilute aqueous solutions, and the use of the resulting data to determine the ionization constant of the protonated amino group. The work familiarizes students with circular dichroism and illustrates the possibility of performing titrations using a less usual instrumental method of following the course of a reaction. It shows the use of a chiroptical property in the determination of the concentration in solution of an optically active molecule, and exemplifies the use of a spectrophotometric titration in the determination of an ionization constant.

  19. Aspartic acid

    MedlinePlus

    ... Hormone production and release Normal nervous system function Plant sources of aspartic acid include: Legumes such as soybeans, garbanzo beans, and lentils Peanuts, almonds, walnuts, and flaxseeds Animal ...

  20. Activated recombinant adenovirus proteinases

    SciTech Connect

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  1. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  2. Multifunctional Environmental Smart Fertilizer Based on l-Aspartic Acid for Sustained Nutrient Release.

    PubMed

    Lü, Shaoyu; Feng, Chen; Gao, Chunmei; Wang, Xinggang; Xu, Xiubin; Bai, Xiao; Gao, Nannan; Liu, Mingzhu

    2016-06-22

    Fertilizer is one of the most important elements of modern agriculture. However, conventional fertilizer, when applied to crops, is vulnerable to losses through volatilization, leaching, nitrification, or other means. Such a loss limits crop yields and pollutes the environment. In an effort to enhance nutrient use efficiency and reduce environmental pollution, an environmental smart fertilizer was reported in the current study. Poly(aspartic acid) and a degradable macro-cross-linker based on l-aspartic acid were synthesized and introduced into the fertilizer as a superabsorbent to improve the fertilizer degradability and soil moisture-retention capacity. Sustained release behavior of the fertilizer was achieved in soil. Cumulative release of nitrogen and phosphorus was 79.8% and 64.4% after 30 days, respectively. The water-holding and water-retention capacities of soil with the superabsorbent are obviously higher than those of the control soil without superabsorbent. For the sample of 200 g of soil with 1.5 g of superabsorbent, the water-holding capacity is 81.8%, and the water-retention capacity remains 22.6% after 23 days. All of the current results in this study indicated that the as-prepared fertilizer has a promising application in sustainable modern agriculture. PMID:27244106

  3. Structure-based design of inhibitors of the aspartic protease endothiapepsin by exploiting dynamic combinatorial chemistry.

    PubMed

    Mondal, Milon; Radeva, Nedyalka; Köster, Helene; Park, Ahyoung; Potamitis, Constantinos; Zervou, Maria; Klebe, Gerhard; Hirsch, Anna K H

    2014-03-17

    Structure-based design (SBD) can be used for the design and/or optimization of new inhibitors for a biological target. Whereas de novo SBD is rarely used, most reports on SBD are dealing with the optimization of an initial hit. Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify bioactive ligands given that it enables the target to direct the synthesis of its strongest binder. We have designed a library of potential inhibitors (acylhydrazones) generated from five aldehydes and five hydrazides and used DCC to identify the best binder(s). After addition of the aspartic protease endothiapepsin, we characterized the protein-bound library member(s) by saturation-transfer difference NMR spectroscopy. Cocrystallization experiments validated the predicted binding mode of the two most potent inhibitors, thus demonstrating that the combination of de novo SBD and DCC constitutes an efficient starting point for hit identification and optimization.

  4. Antifouling gold surfaces grafted with aspartic acid and glutamic acid based zwitterionic polymer brushes.

    PubMed

    Li, Wenchen; Liu, Qingsheng; Liu, Lingyun

    2014-10-28

    We report two new amino acid based antifouling zwitterionic polymers, poly(N(4)-(2-methacrylamidoethyl)asparagine) (pAspAA) and poly(N(5)-(2-methacrylamidoethyl)glutamine) (pGluAA). The vinyl monomers were developed from aspartic acid and glutamic acid. Surface-initiated photoiniferter-mediated polymerization was employed to graft polymer brushes from gold surfaces. Different thickness of polymer brushes was controlled by varying UV irradiation time. The nonspecific adsorption from undiluted human blood serum and plasma was studied by surface plasmon resonance (SPR). With the polymer film as thin as 11-12 nm, the adsorption on pAspAA from serum and plasma was as low as 0.75 and 5.18 ng/cm(2), respectively, and 1.88 and 10.15 ng/cm(2), respectively, for pGluAA. The adsorption amount is comparable to or even better than other amino acid based zwitterionic polymers such as poly(serine methacrylate), poly(lysine methacrylamide), and poly(ornithine methacrylamide) and other widely used antifouling polymers such as poly(sulfobetaine methacrylate), even under thinner polymer film thickness. The pAspAA and pGluAA grafted surfaces also showed strong resistance to endothelial cell attachment. The possession of both zwitterionic structure and hydrophilic amide groups, biomimetic property, and multifunctionality make pAspAA and pGluAA promising candidates for biocompatible antifouling functionalizable materials. PMID:25262768

  5. Antifouling gold surfaces grafted with aspartic acid and glutamic acid based zwitterionic polymer brushes.

    PubMed

    Li, Wenchen; Liu, Qingsheng; Liu, Lingyun

    2014-10-28

    We report two new amino acid based antifouling zwitterionic polymers, poly(N(4)-(2-methacrylamidoethyl)asparagine) (pAspAA) and poly(N(5)-(2-methacrylamidoethyl)glutamine) (pGluAA). The vinyl monomers were developed from aspartic acid and glutamic acid. Surface-initiated photoiniferter-mediated polymerization was employed to graft polymer brushes from gold surfaces. Different thickness of polymer brushes was controlled by varying UV irradiation time. The nonspecific adsorption from undiluted human blood serum and plasma was studied by surface plasmon resonance (SPR). With the polymer film as thin as 11-12 nm, the adsorption on pAspAA from serum and plasma was as low as 0.75 and 5.18 ng/cm(2), respectively, and 1.88 and 10.15 ng/cm(2), respectively, for pGluAA. The adsorption amount is comparable to or even better than other amino acid based zwitterionic polymers such as poly(serine methacrylate), poly(lysine methacrylamide), and poly(ornithine methacrylamide) and other widely used antifouling polymers such as poly(sulfobetaine methacrylate), even under thinner polymer film thickness. The pAspAA and pGluAA grafted surfaces also showed strong resistance to endothelial cell attachment. The possession of both zwitterionic structure and hydrophilic amide groups, biomimetic property, and multifunctionality make pAspAA and pGluAA promising candidates for biocompatible antifouling functionalizable materials.

  6. Proteinases and their inhibitors in cells and tissues.

    PubMed

    Rappay, G

    1989-01-01

    A large body of evidence has been assembled to indicate the substantial importance of proteolytic processes in various physiological functions. It has recently become clear too that endo-acting peptide bond hydrolases provisionally characterized and classified at present as serine, cysteine, aspartic and metallo together with unknown catalytic mechanism proteinases sometimes act in cascades. They are controlled by natural proteinase inhibitors present in cells and body fluids. In the first part of the present monograph the author was concerned to present an overview on the morphological and physiological approach to localization, surveying reaction principles and methods suitable for visualization of proteolytic enzymes and their natural and synthetic inhibitors. In the second part the roles played by proteinases have been summarized from the point of view of cell biology. The selection of earlier and recent data reviewed on the involvement of proteolysis in the behavior of individual cells reveals that enzymes, whether they be exogeneous or intrinsic, can be effective and sensitive modulators of cellular growth and morphology. There exists a close correlation between malignant growth and degradation of cells. It appears likely that as yet unknown or at least so far inadequately characterized factors that influence the survival or the death of cells may turn out to be proteinases. The causal role of extracellular proteolysis in cancer cell metastases, in stopping cancer cell growth and in cytolysis remains for further investigated. Ovulation, fertilization and implantation are basic biological functions in which proteolytic enzymes play a key role. The emergence of new approaches in reproductive biology and a growing factual basis will inevitably necessitate a reevaluation of present knowledge of proteolytic processes involved. The molecular aspects of intracellular protein catabolism have been discussed in terms of the inhibition of lysosomal and/or non

  7. Multiple forms of calcium-dependent proteinase in crustacean muscle

    SciTech Connect

    Mykles, D.L.; Skinner, D.M.

    1986-01-01

    Four calcium-dependent proteinase (CDP) activities in lobster muscles have been resolved by high performance liquid chromatography. These activities differ in molecular weight and net charge. Though optimum activity occurred at high (5 and 10 mM) calcium at pH 6.8, the enzymes differ in activation at lower calcium concentrations. Only one of the CDPs is active at 100 ..mu..M calcium; none are active at 10 ..mu..M and below. Although all four CDPs are inhibited by the cysteine proteinase inhibitors leupeptin, E-64, and iodoacetamide, they show a differential response to the aspartic proteinase inhibitor pepstatin and the serine proteinase inhibitor PMSF. In contrast to CDPs from vertebrate tissues, crustacean muscles contain multiple forms that require calcium at millimolar levels. 17 refs., 6 figs.

  8. Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species.

    PubMed

    Arnórsdottir, Jóhanna; Smáradóttir, Rúna B; Magnússon, Olafur Th; Thorbjarnardóttir, Sigrídur H; Eggertsson, Gudmundur; Kristjánsson, Magnús M

    2002-11-01

    The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli. The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa. The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence. Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity. The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases. With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same. Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio-proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues. The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index. These factors may contribute to the adaptation of these proteinases to different temperature conditions.

  9. Molecular dynamic and docking interaction study of Heterodera glycines serine proteinase with Vigna mungo proteinase inhibitor.

    PubMed

    Prasad, C V S Siva; Gupta, Saurabh; Gaponenko, Alex; Tiwari, Murlidhar

    2013-08-01

    Many plants do produce various defense proteins like proteinase inhibitors (PIs) to protect them against various pests. PIs function as pseudosubstrates of digestive proteinase, which inhibits proteolysis in pests and leads to amino acid deficiency-based mortality. This work reports the structural interaction studies of serine proteinase of Heterodera glycines (SPHG) with Vigna mungo proteinase inhibitor (VMPI). 3D protein structure modeling, validation of SPHG and VMPI, and their putative protein-protein binding sites were predicted. Protein-protein docking followed by molecular dynamic simulation was performed to find the reliable confirmation of SPHG-VMPI complex. Trajectory analysis of each successive conformation concludes better interaction of first loop in comparison with second loop. Lysine residues of first loop were actively participating in complex formation. Overall, this study discloses the structural aspects and interaction mechanisms of VMPI with SPHG, and it would be helpful in the development of pest-resistant genetically modified crops.

  10. Proteinase expression during differentiation of human osteoclasts in vitro.

    PubMed

    Blair, H C; Sidonio, R F; Friedberg, R C; Khan, N N; Dong, S S

    2000-06-12

    Osteoclasts are macrophage-derived polykaryons that degrade bone in an acidic extracellular space. This differentiation includes expression of proteinases and acid transport proteins, cell fusion, and bone attachment, but the sequence of events is unclear. We studied two proteins expressed at high levels only in the osteoclast, cathepsin K, a thiol proteinase, and tartrate-resistant acid phosphatase (TRAP), and compared this expression with acid transport and bone degradation. Osteoclastic differentiation was studied using human apheresis macrophages cocultured with MG63 osteosarcoma cells, which produce cytokines including RANKL and CSF-1 that mediate efficient osteoclast formation. Immunoreactive cathepsin K appeared at 3-5 days. Cathepsin K activity was seen on bone substrate but not within cells, and cathepsin K increased severalfold during further differentiation and multinucleation from 7 to 14 days. TRAP also appeared at 3-5 d, independently of cell fusion or bone attachment, and TRAP activity reached much higher levels in osteoclasts attached to bone fragments. Two proteinases that occur in the precursor macrophages, cathepsin B, a thiol proteinase related to cathepsin K, and an unrelated lysosomal aspartate proteinase, cathepsin D, were also studied to determine the specificity of the differentiation events. Cathepsin B occurred at all times, but increased two- to threefold in parallel with cathepsin K. Cathepsin D activity did not change with differentiation, and secreted activity was not significant. In situ acid transport measurements showed increased acid accumulation after 7 days either in cells on osteosarcoma matrix or attached to bone, but bone pit activity and maximal acid uptake required 10-14 days. We conclude that TRAP and thiol proteinase expression begin at essentially the same time, and precede cell fusion and bone attachment. However, major increases in acid secretion and proteinases expression continue during cell fusion and bone

  11. The possible involvement of D-amino acids or their metabolites in Arabidopsis cysteine proteinase/cystatin N-dependent proteolytic pathway.

    PubMed

    Gholizadeh, A

    2015-01-01

    Cysteine proteinases and their inhibitors 'cystatins' play essential roles in plant growth and development. They are involved in various signaling pathways and in the response to wide ranges of biotic and abiotic environmental stresses. To investigate their possible influence from D-amino acids or their metabolism in vivo, Arabidopsis seedlings were allowed to grow under four physicochemically different D-amino acids including D-aspartate, D-serine, D-alanine and D-phenylalanine containing media. The reverse transcription polymerase chain reaction (R T-PCR) analysis of cysteine proteinase and cystatin gene expressions showed that the addition of D-amino acid to the plant growth media considerably induce the expression of proteinase transcript while decrease the expression level of inhibitor gene in the leaf and root tissues of the test plant in overall. Based on the obtained results the potential impact of D-amino acids or their metabolism on the activity of cysteine proteinase/cystatin-dependent proteolytic apparatus as well as their possible cooperation were predicted and discussed in the plant system.

  12. Electron density topography based model to explore N-methyl-D-aspartate receptor channel blockers

    NASA Astrophysics Data System (ADS)

    Ingle, Snehal V.; Joshi, Kaustubh A.

    2016-03-01

    The dwell time of a molecule in a voltage dependent NMDA receptor channel is an important factor in defining its activity as channel blocker. A model has been designed, based on quantum chemical descriptors like geometrical parameters, charge distribution, electron density topography and global reactivity descriptors, to shed lights on the dwell time of a channel blocker. Structure and charge distribution studies indicate polarization of molecules with the electron density located at the core of the molecule. Electron density topography reveals ring critical point (ρrcp), emerging as a signature parameter to understand the dwell time of a channel blocker molecule.

  13. Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin

    SciTech Connect

    Otto, H.; Marti, T.; Holz, M.; Mogi, T.; Lindau, M.; Khorana, H.G.; Heyn, M.P. )

    1989-12-01

    Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, (H+). In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition. The decay rate of N is proportional to (H+). By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time (log (tau)) linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.

  14. Magnesium-aspartate-based crystallization switch inspired from shell molt of crustacean

    PubMed Central

    Tao, Jinhui; Zhou, Dongming; Zhang, Zhisen; Xu, Xurong; Tang, Ruikang

    2009-01-01

    Many animals such as crustacean periodically undergo cyclic molt of the exoskeleton. During this process, amorphous calcium mineral phases are biologically stabilized by magnesium and are reserved for the subsequent rapid formation of new shell tissue. However, it is a mystery how living organisms can regulate the transition of the precursor phases precisely. We reveal that the shell mineralization from the magnesium stabilized precursors is associated with the presence of Asp-rich proteins. It is suggested that a cooperative effect of magnesium and Asp-rich compound can result into a crystallization switch in biomineralization. Our in vitro experiments confirm that magnesium increases the lifetime of amorphous calcium carbonate and calcium phosphate in solution so that the crystallization can be temporarily switched off. Although Asp monomer alone inhibits the crystallization of pure amorphous calcium minerals, it actually reduces the stability of the magnesium-stabilized precursors to switch on the transformation from the amorphous to crystallized phases. These modification effects on crystallization kinetics can be understood by an Asp-enhanced magnesium desolvation model. The interesting magnesium-Asp-based switch is a biologically inspired lesson from nature, which can be developed into an advanced strategy to control material fabrications. PMID:20007788

  15. Compartmentalization of proteinases and amylases in Nauphoeta cinerea midgut.

    PubMed

    Elpidina, E N; Vinokurov, K S; Gromenko, V A; Rudenskaya, Y A; Dunaevsky, Y E; Zhuzhikov, D P

    2001-12-01

    Compartmentalization of proteinases, amylases, and pH in the midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was studied in order to understand the organization of protein and starch digestion. Total proteolytic activity measured with azocasein was maximal at pH 11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the bulk of activity (67%) was found in PM. Total AM and PM preparations were fractionated on a Sephadex G-50 column and further analysed by means of activity electrophoresis and specific inhibitors and activators. The major activity in PM was classified as an unusual SH-dependent proteinase with M(r) 24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was 43-fold activated in the presence of 1 mM DTT, insensitive to synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases, strongly inhibited by STI, and displayed four active bands on zymograms. In PM, activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine proteinases were observed. Aspartic and metalloproteinases were not detected. In AM, activity of unusual SH-dependent proteinase also dominated and activity of chymotrypsin-like proteinase was observed, but their levels were much lower than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0, was quite the opposite. The major part of it (67%) was located in AM. Treatment of amylase preparation with proteinases from AM and PM reduced amylase activity twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and 8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with the optimal pH of amylase, located in this compartment, but the activity of proteinases, including the ability to degrade amylase, in such an environment is low. Active proteolysis takes place in the second half of PM, where pH of the gut is close to the optimal pH of proteinases.

  16. Co-factor activated recombinant adenovirus proteinases

    SciTech Connect

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  17. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  18. Picornaviral 3C cysteine proteinases have a fold similar to the chymotrypsin-like serine proteinases

    SciTech Connect

    Allaire,M.; Chernaia, M.; Malcolm, B.; James, M.

    1994-01-01

    The picornavirus family includes several pathogens such as poliovirus, rhinovirus (the major cause of the common cold), hepatitis A virus and the foot-and-mouth disease virus. Picornaviral proteins are expressed by direct translation of the genomic RNA into a single, large polyprotein precursor. Proteolysis of the viral polyprotein into the mature proteins is assured by the viral 3C enzymes, which are cysteine proteinases. Here we report the X-ray crystal structure at 2.3 {angstrom} resolution of the 3C proteinase from hepatitis A virus (HAV-3C). The overall architecture of HAV-3C reveals a fold resembling that of the chymotrypsin family of serine proteinases, which is consistent with earlier predictions. Catalytic residues include Cys 172 as nucleophile and His 44 as general base. The 3C cleavage specificity for glutamine residues is defined primarily by His 191. The overall structure suggests that an inter-molecular (trans) cleavage releases 3C and that there is an active proteinase in the polyprotein.

  19. Crystallization and preliminary X-ray investigation of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus.

    PubMed

    Tanokura, M; Matsuzaki, H; Iwata, S; Nakagawa, A; Hamaya, T; Takizawa, T; Takahashi, K

    1992-01-01

    Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase distinctly different in various properties from the family of pepsin-type aspartic proteinases, and so far it remains unknown which residues participate in the catalysis of the enzyme and how the mechanism operates. The acid proteinase A was crystallized from an ammonium sulfate solution by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1) with unit cell dimensions of a = 54.7 A, b = 70.4 A and c = 38.0 A. On the assumption that there is one enzyme molecule in the asymmetric unit, the calculated ratio of volume to unit protein mass (Vm) was 1.64 A3 per dalton. Diffraction data were collected up to a resolution higher than 1.5 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation. The crystal of proteinase A is, therefore, suitable for the structural analysis with a high resolution.

  20. Aspartate carbamoyltransferase from rat liver

    PubMed Central

    Bresnick, E.; Mossé, Helena

    1966-01-01

    1. Aspartate-carbamoyltransferase activity was concentrated from rat-liver preparations. Only l-aspartate, β-benzyl-l-aspartate and β-erythro-hydroxy-dl-aspartate were carbamoylated enzymically. The Km for l-aspartate and carbamoyl phosphate have been determined by three methods: colorimetric procedure, radioactive assay with [14C]aspartate and an assay with [14C]carbamoyl phosphate. 2. The Km for aspartate has been determined as a function of the pH; the pK of the functional group at the active site of the enzyme, pKe, was at pH9·0. Enzymic activity was diminished in the presence of N-ethylmaleimide, p-hydroxymercuribenzoate and the heavy metals Ag+, Hg2+, or Zn2+. The inhibitions could be prevented by mercaptoethanol. These findings suggested the association of a thiol group with the enzymic activity. 3. Enzymic activity was also decreased by sodium lauryl sulphate, urea and dioxan. Competitive inhibition (with l-aspartate) was manifested by maleate, succinate, oxaloacetate, β-erythro-hydroxy-dl-aspartate and β-benzyl-l-aspartate. The Ki for most of these inhibitions has been determined. 4. The properties of the liver enzyme are compared with those of Escherichia coli aspartate carbamoyltransferase and the implications of the findings are discussed. PMID:5339547

  1. Adult Schistosoma mansoni express cathepsin L proteinase activity.

    PubMed

    Smith, A M; Dalton, J P; Clough, K A; Kilbane, C L; Harrop, S A; Hole, N; Brindley, P J

    1994-09-01

    This report presents the deduced amino acid sequence of a novel cathepsin L proteinase from Schistosoma mansoni, and describes cathepsin L-like activity in extracts of adult schistosomes. Using consensus primers specific for cysteine proteinases, gene fragments were amplified from adult S. mansoni cDNA by PCR and cloned. One of these fragments showed marked identity to Sm31, the cathepsin B cysteine proteinase of adult S. mansoni, whereas another differed from Sm31 and was employed as a probe to isolate two cDNAs from an adult S. mansoni gene library. Together these cDNAs encoded a novel preprocathepsin L of 319 amino acids; this zymogen is predicted to be processed in vivo into a mature, active cathepsin L proteinase of 215 amino acids. Closest homologies were with cathepsins L from rat, mouse, and chicken (46-47% identity). Southern hybridization analysis suggested that only one or a few copies of the gene was present per genome, demonstrated that its locus was distinct from that of Sm31, and that a homologous sequence was present in Schistosoma japonicum. Because these results indicated that schistosomes expressed a cathepsin L proteinase, extracts of adult S. mansoni were examined for acidic, cysteine proteinase activity. Based on rates of cleavage of peptidyl substrates employed to discriminate between classes of cysteine proteinases, namely cathepsin L (Z-phe-arg-AMC), cathepsin B (Z-arg-arg-AMC) and cathepsin H (Bz-arg-AMC), the extracts were found to contain vigorous cathepsin L-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. A Deficiency in Aspartate Biosynthesis in Lactococcus lactis subsp. lactis C2 Causes Slow Milk Coagulation†

    PubMed Central

    Wang, Hua; Yu, Weizhu; Coolbear, Tim; O’Sullivan, Dan; McKay, Larry L.

    1998-01-01

    A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc−) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis. PMID:9572935

  3. Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment-Based Drug Design Facilitated by Dynamic Combinatorial Chemistry.

    PubMed

    Mondal, Milon; Radeva, Nedyalka; Fanlo-Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard; Hirsch, Anna K H

    2016-08-01

    Fragment-based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit-identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X-ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis-acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240-fold improvement in potency compared to the parent hits. Subsequent X-ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit-identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit-to-lead optimization.

  4. Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment-Based Drug Design Facilitated by Dynamic Combinatorial Chemistry.

    PubMed

    Mondal, Milon; Radeva, Nedyalka; Fanlo-Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard; Hirsch, Anna K H

    2016-08-01

    Fragment-based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit-identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X-ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis-acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240-fold improvement in potency compared to the parent hits. Subsequent X-ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit-identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit-to-lead optimization. PMID:27400756

  5. Exploring details about structure requirements based on novel CGRP receptor antagonists urethanamide, aspartate, succinate and pyridine derivatives by in silico methods

    NASA Astrophysics Data System (ADS)

    Li, Yan; He, Haoran; Wang, Jinghui; Han, Chunxiao; Feng, Jiaqi; Zhang, Shuwei; Yang, Ling

    2014-09-01

    The migraine never fails to afflict individuals in the world that knows no lack of such cases. CGRP (calcitonin gene-related peptide) is found closely related to migraine and olcegepant (BIBN4096) is effective in alleviating the pain. In our work, the combination of ligand- and receptor-based three-dimensional quantitative structure-activity relationship (3D-QSAR) studies along with molecular docking was applied to provide us insights about how urethanamide, pyridine and aspartate and succinate derivatives (novel CGRP receptor antagonists) play a part in inhibiting the activity of CGRP receptor. The optimal CoMSIA model shows the Q2 of 0.505, R2ncv of 0.992 and its accurate predictive ability was confirmed by checking out an independent test set which gave R2pred value of 0.885. Besides, the 3D contour maps help us identify how different groups affect the antagonist activity while connecting to some key positions. In addition, the docking analysis shows the binding site emerging as the distorted “V” shape and including two binding pockets: one of them is hydrophobic, fixing the structural part 3 of compound 80, the other anchors the part 1 of compound 80. The docking analysis also shows the interaction mechanism between compound 80 and CGRP receptor, similar to the interaction between olcegepant and CGRP receptor. The findings derived from this work reveal the mechanism of related antagonists and facilitate the future rational design of novel antagonists with higher potency.

  6. Critical aspartic acid residues in pseudouridine synthases.

    PubMed

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  7. An aspartate and a water molecule mediate efficient acid-base catalysis in a tailored antibody pocket

    SciTech Connect

    Debler, Erik W.; Müller, Roger; Hilvert, Donald; Wilson, Ian A.

    2009-12-01

    Design of catalysts featuring multiple functional groups is a desirable, yet formidable goal. Antibody 13G5, which accelerates the cleavage of unactivated benzisoxazoles, is one of few artificial enzymes that harness an acid and a base to achieve efficient proton transfer. X-ray structures of the Fab-hapten complexes of wild-type 13G5 and active-site variants now afford detailed insights into its mechanism. The parent antibody preorganizes Asp{sup H35} and Glu{sup L34} to abstract a proton from substrate and to orient a water molecule for leaving group stabilization, respectively. Remodeling the environment of the hydrogen bond donor with a compensatory network of ordered waters, as seen in the Glu{sup L34} to alanine mutant, leads to an impressive 10{sup 9}-fold rate acceleration over the nonenzymatic reaction with acetate, illustrating the utility of buried water molecules in bifunctional catalysis. Generalization of these design principles may aid in creation of catalysts for other important chemical transformations.

  8. An aspartate and a water molecule mediate efficient acid-base catalysis in a tailored antibody pocket.

    PubMed

    Debler, Erik W; Müller, Roger; Hilvert, Donald; Wilson, Ian A

    2009-11-01

    Design of catalysts featuring multiple functional groups is a desirable, yet formidable goal. Antibody 13G5, which accelerates the cleavage of unactivated benzisoxazoles, is one of few artificial enzymes that harness an acid and a base to achieve efficient proton transfer. X-ray structures of the Fab-hapten complexes of wild-type 13G5 and active-site variants now afford detailed insights into its mechanism. The parent antibody preorganizes Asp(H35) and Glu(L34) to abstract a proton from substrate and to orient a water molecule for leaving group stabilization, respectively. Remodeling the environment of the hydrogen bond donor with a compensatory network of ordered waters, as seen in the Glu(L34) to alanine mutant, leads to an impressive 10(9)-fold rate acceleration over the nonenzymatic reaction with acetate, illustrating the utility of buried water molecules in bifunctional catalysis. Generalization of these design principles may aid in creation of catalysts for other important chemical transformations.

  9. The cysteine proteinases of the pineapple plant.

    PubMed Central

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-01-01

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct. Images Fig. 4. Fig. 5. PMID:2327970

  10. The cysteine proteinases of the pineapple plant.

    PubMed

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-03-15

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

  11. Sensitive, hydrosoluble, macromolecular fluorogenic substrates for human immunodeficiency virus 1 proteinase.

    PubMed Central

    Anjuère, F; Monsigny, M; Lelièvre, Y; Mayer, R

    1993-01-01

    Hydrosoluble macromolecular fluorogenic substrates specific for the human immunodeficiency virus 1 (HIV-1) proteinase have been prepared. The fluoresceinyl peptide Ftc-epsilon-Ahx-Ser-Phe-Asn-Phe-Pro-Gln-Ile-Thr-(Gly)n, corresponding to the first cleavage site of HIV-1 gag-pol native precursor was linked to a water-soluble neutral (Lys)n derivative. The epsilon-aminohexanoyl residue (epsilon-Ahx) and the glycyl sequence were added in order to improve the stability of the substrate and the accessibility of the cleavage site to the HIV-1 proteinase respectively. This macro-molecular peptidic-substrate conjugate is significantly more water-soluble than the free peptide itself on a substrate molar concentration basis. The assay is based on the quantitative precipitation of the polymeric material by adding propan-2-ol whereas the fluorescent peptide moiety released upon proteolysis remains soluble in the supernatant. The proteinase activity is assessed by measuring the fluorescence of the supernatant. This assay allows the detection of a few fmol of HIV-1 proteinase, even in the presence of cell culture media, plasma or cell lysate and it gives accurate results within a large proteinase concentration range. The hydrosoluble macromolecular substrate is also suitable for determining the HIV-1 proteinase activity using 96-well microplates, allowing us to test accurately and rapidly numerous enzyme samples and/or the potency of new proteinase inhibitors. PMID:8489513

  12. Differential Inhibition of Helicoverpa armigera (Hubner) Gut Proteinases by Proteinase Inhibitors of Okra and It's Wild Relatives

    PubMed Central

    Udamale, Shilpa K.; Moharil, M. P.; Ugale, T. B.; Mankar, J. M.

    2013-01-01

    The seeds of ten genotypes and twenty-nine wild relatives of okra were analysed for the presence of trypsin, chymotrypsin, and Helicoverpa gut proteinases (HGPs) inhibitors (HGPIs), with the aim to identify potent inhibitors of H. armigera gut proteinases. Proteinase inhibitors (PIs) obtained from wild relatives of okra exhibited stronger inhibition of HGPs than the genotypes of okra. In in vitro inhibitory assay against HGPs, A. tuberculatus 90396 and 90515 showed high tryptic inhibitory (71.8% and 69.2%), chymotryptic inhibitory (68.5% and 66.2%), and Helicoverpa gut proteinase activity (70.2% and 68.2%). In electrophoretic profile showed the same variation in the number of trypsin inhibitors (TIs), chymotrypsin Inhibitors (CIs), and HGPIs isoforms with different intensities, whereas genotypes of okra mostly showed monomorphic profile. Maximum eight HGPIs isoforms were found in A. tuberculatus (90396 and 90515). In bioassay studies, significant reduction in weight of H. armigera larvae was found, when larvae fed on PIs obtained from A. tuberculatus (90396 and 90515). Thus, the result of the present investigation indicates that further exploration of PIs obtained from A. tuberculatus (90396 and 90515) will be helpful for developing PIs-based insect resistance management strategies. PMID:25937977

  13. Characterization and cloning of metallo-proteinase in the excretory/secretory products of the infective-stage larva of Trichinella spiralis.

    PubMed

    Lun, H M; Mak, C H; Ko, R C

    2003-05-01

    Inhibitor sensitivity assays using azocaesin and FTC-caesin as substrates showed that the excretory/secretory (E/S) products of the infective-stage larvae of Trichinella spiralis contained serine, metallo-, cysteine and aspartic proteinases. The activity of the metallo-proteinase was zinc ion dependent (within a range of ZnSO(4) concentrations). Gelatin-substrate gel electrophoresis revealed two bands of molecular mass 48 and 58 kDa which were sensitive to the metallo-proteinase inhibitor EDTA. The former peptide was probably a cleavage product of the latter. The authenticity of the 58 kDa metallo-proteinase as an E/S product was confirmed by immunoprecipitation. Using PCR and RACE reactions, a complete nucleotide sequence of the metallo-proteinase gene was obtained. It comprised 2,223 bp with an open reading frame encoding 604 amino acid residues. The 3' untranslated region consisted of 352 bp, including a polyadenylation signal AATAA. A consensus catalytic zinc-binding motif was present. The conserved domains suggest that the cloned metallo-proteinase belongs to the astacin family and occurs as a single copy gene with 11 introns and 10 exons. Cluster analysis showed that the sequence of the metallo-proteinase gene of T. spiralis resembles those of Caenorhabdites elegans and Strongyloides stercoralis. PMID:12743801

  14. Aspartic cathepsin D endopeptidase contributes to extracellular digestion in clawed lobsters Homarus americanus and Homarus gammarus.

    PubMed

    Rojo, Liliana; Muhlia-Almazan, Adriana; Saborowski, Reinhard; García-Carreño, Fernando

    2010-11-01

    Acid digestive proteinases were studied in the gastric fluids of two species of clawed lobster (Homarus americanus and Homarus gammarus). An active protein was identified in both species as aspartic proteinase by specific inhibition with pepstatin A. It was confirmed as cathepsin D by mass mapping, N-terminal, and full-length cDNA sequencing. Both lobster species transcribed two cathepsin D mRNAs: cathepsin D1 and cathepsin D2. Cathepsin D1 mRNA was detected only in the midgut gland, suggesting its function as a digestive enzyme. Cathepsin D2 mRNA was found in the midgut gland, gonads, and muscle. The deduced amino acid sequence of cathepsin D1 and cathepsin D2 possesses two catalytic DTG active-site motifs, the hallmark of aspartic proteinases. The putatively active cathepsin D1 has a molecular mass of 36.4 kDa and a calculated pI of 4.14 and possesses three potential glycosylation sites. The sequences showed highest similarities with cathepsin D from insects but also with another crustacean cathepsin D. Cathepsin D1 transcripts were quantified during a starvation period using real-time qPCR. In H. americanus, 15 days of starvation did not cause significant changes, but subsequent feeding caused a 2.5-fold increase. In H. gammarus, starvation caused a 40% reduction in cathepsin D1 mRNA, and no effect was observed with subsequent feeding. PMID:20169386

  15. Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.

    PubMed

    Hansen, Kasper B; Mullasseril, Praseeda; Dawit, Sara; Kurtkaya, Natalie L; Yuan, Hongjie; Vance, Katie M; Orr, Anna G; Kvist, Trine; Ogden, Kevin K; Le, Phuong; Vellano, Kimberly M; Lewis, Iestyn; Kurtkaya, Serdar; Du, Yuhong; Qui, Min; Murphy, T J; Snyder, James P; Bräuner-Osborne, Hans; Traynelis, Stephen F

    2010-06-01

    N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.

  16. Implementation of a Fluorescence-Based Screening Assay Identifies Histamine H3 Receptor Antagonists Clobenpropit and Iodophenpropit as Subunit-Selective N-Methyl-d-Aspartate Receptor Antagonists

    PubMed Central

    Hansen, Kasper B.; Mullasseril, Praseeda; Dawit, Sara; Kurtkaya, Natalie L.; Yuan, Hongjie; Vance, Katie M.; Orr, Anna G.; Kvist, Trine; Ogden, Kevin K.; Le, Phuong; Vellano, Kimberly M.; Lewis, Iestyn; Kurtkaya, Serdar; Du, Yuhong; Qui, Min; Murphy, T. J.; Snyder, James P.; Bräuner-Osborne, Hans

    2010-01-01

    N-Methyl-d-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca2+-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism. PMID:20197375

  17. [Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis].

    PubMed

    Dunaevskiĭ, Ia E; Gruban', T N; Beliakova, G A; Belozerskiĭ, M A

    2006-01-01

    The presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen). PMID:17205798

  18. [Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis].

    PubMed

    Dunaevskiĭ, Ia E; Gruban', T N; Beliakova, G A; Belozerskiĭ, M A

    2006-01-01

    The presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).

  19. NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5'-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly-L-lysine.

    PubMed

    Chan-Huot, Monique; Dos, Alexandra; Zander, Reinhard; Sharif, Shasad; Tolstoy, Peter M; Compton, Shara; Fogle, Emily; Toney, Michael D; Shenderovich, Ilya; Denisov, Gleb S; Limbach, Hans-Heinrich

    2013-12-01

    Using (15)N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5'-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-L-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using (15)N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed (13)C and (15)N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-L-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism. PMID:24147985

  20. Design and synthesis of procollagen C-proteinase inhibitors.

    PubMed

    Turtle, Eric; Chow, Nicholas; Yang, Charles; Sosa, Sergio; Bauer, Udo; Brenner, Mitch; Solow-Cordero, David; Ho, Wen-Bin

    2012-12-15

    Non-peptidic inhibitors of procollagen C-proteinase (PCP) were designed from substrate leads. Compounds were optimized for potency and selectivity, with N-substituted aryl sulfonamide hydroxamates having the best combination of these properties. Compounds 89 and 60 have IC(50) values of 10 and 80 nM, respectively, against PCP; excellent selectivity over MMP's 1, 2, and 9; and activity in cell-based collagen deposition assays.

  1. An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes

    PubMed Central

    Richardson, Graham; Ding, Haizhen; Rocheleau, Tom; Mayhew, George; Reddy, Erin; Han, Qian; Christensen, Bruce M.; Li, Jianyong

    2010-01-01

    A major pathway of beta-alanine synthesis in insects is through the alpha-decarboxylation of aspartate, but the enzyme involved in the decarboxylation of aspartate has not been clearly defined in mosquitoes and characterized in any insect species. In this study, we expressed two putative mosquito glutamate decarboxylase-like enzymes of mosquitoes and critically analyzed their substrate specificity and biochemical properties. Our results provide clear biochemical evidence establishing that one of them is an aspartate decarboxylase and the other is a glutamate decarboxylase. The mosquito aspartate decarboxylase functions exclusively on the production of beta-alanine with no activity with glutamate. Likewise the mosquito glutamate decarboxylase is highly specific to glutamate with essentially no activity with aspartate. Although insect aspartate decarboxylase shares high sequence identity with glutamate decarboxylase, we are able to closely predict aspartate decarboxylase from glutamate decarboxylase based on the difference of their active site residues. PMID:19842059

  2. The picornaviral 3C proteinases: cysteine nucleophiles in serine proteinase folds.

    PubMed Central

    Malcolm, B. A.

    1995-01-01

    The 3C proteinases are a novel group of cysteine proteinases with a serine proteinase-like fold that are responsible for the bulk of polyprotein processing in the Picornaviridae. Because members of this viral family are to blame for several ongoing global pandemic problems (rhinovirus, hepatitis A virus) as well as sporadic outbreaks of more serious pathologies (poliovirus), there has been continuing interest over the last two decades in the development of antiviral therapies. The recent determination of the structure of two of the 3C proteinases by X-ray crystallography opens the door for the application of the latest advances in computer-assisted identification and design of anti-proteinase therapeutic/chemoprophylactic agents. PMID:8520469

  3. Implantation serine proteinase 2 is a monomeric enzyme with mixed serine proteolytic activity and can silence signalling via proteinase activated receptors.

    PubMed

    Sharma, Navneet; Fahr, Jochen; Renaux, Bernard; Saifeddine, Mahmoud; Kumar, Rajeev; Nishikawa, Sandra; Mihara, Koichiro; Ramachandran, Rithwik; Hollenberg, Morley D; Rancourt, Derrick E

    2013-12-01

    Implantation serine proteinase 2 (ISP2), a S1 family serine proteinase, is known for its role in the critical processes of embryo hatching and implantation in the mouse uterus. Native implantation serine proteinases (ISPs) are co-expressed and co-exist as heterodimers in uterine and blastocyst tissues. The ISP1-ISP2 enzyme complex shows trypsin-like substrate specificity. In contrast, we found that ISP2, isolated as a 34 kDa monomer from a Pichia pastoris expression system, exhibited a mixed serine proteolytic substrate specificity, as determined by a phage display peptide cleavage approach and verified by the in vitro cleavage of synthetic peptides. Based upon the peptide sequence substrate selectivity, a database search identified many potential ISP2 targets of physiological relevance, including the proteinase activated receptor 2 (PAR2). The in vitro cleavage studies with PAR2-derived peptides confirmed the mixed substrate specificity of ISP2. Treatment of cell lines expressing proteinase-activated receptors (PARs) 1, 2, and 4 with ISP2 prevented receptor activation by either thrombin (PARs 1 and 4) or trypsin (PAR2). The disarming and silencing of PARs by ISP2 may play a role in successful embryo implantation.

  4. Proteinase K improves quantitative acylation studies.

    PubMed

    Fränzel, Benjamin; Fischer, Frank; Steegborn, Clemens; Wolters, Dirk Andreas

    2015-01-01

    Acetylation is a common PTM of proteins but is still challenging to analyze. Only few acetylome studies have been performed to tackle this issue. Yet, the detection of acetylated proteins in complex cell lysates remains to be improved. Here, we present a proteomic approach with proteinase K as a suitable protease to identify acetylated peptides quantitatively. We first optimized the digestion conditions using an artificial system of purified bovine histones to find the optimal protease. Subsequently, the capability of proteinase K was demonstrated in complex HEK293 cell lysates. Finally, SILAC in combination with MudPIT was used to show that quantification with proteinase K is possible. In this study, we identified a sheer number of 557 unique acetylated peptides originating from 633 acetylation sites.

  5. Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis.

    PubMed

    Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha; Edgerton, Mira

    2015-07-01

    Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

  6. An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis.

    PubMed

    Birsoy, Kıvanç; Wang, Tim; Chen, Walter W; Freinkman, Elizaveta; Abu-Remaileh, Monther; Sabatini, David M

    2015-07-30

    The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation. PMID:26232224

  7. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis

    PubMed Central

    Birsoy, Kıvanç; Wang, Tim; Chen, Walter; Freinkman, Elizaveta; Abu-Remaileh, Monther; Sabatini, David M.

    2015-01-01

    Summary The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation. PMID:26232224

  8. Hydrolytic activity of Virgibacillus sp. SK37, a starter culture of fish sauce fermentation, and its cell-bound proteinases.

    PubMed

    Sinsuwan, Sornchai; Rodtong, Sureelak; Yongsawatdigul, Jirawat

    2012-08-01

    Fish sauce production relies on a natural fermentation process requiring 12-18 months for process completion. Virgibacillus sp. SK37 has been shown to be a potential strain for fish sauce acceleration. However, hydrolytic activity of proteinases bound at cell surface of this strain has not been well elucidated. Addition of 0.2 % CaCl(2) (w/w) in conjunction with starter cultures of Virgibacillus sp. SK 37 increased protein hydrolysis as measured by α-amino group content throughout fermentation (P < 0.05). Cell-bound proteinases from Virgibacillus sp. SK 37 were extracted into a free form by incubating the washed cells in Ca(2+)-free buffer at 37 °C for 2 h. Cell-bound proteinases revealed molecular mass of 19, 20, 22, 32, 34, and 44 kDa based on a synthetic peptide zymogram. The proteinases showed subtilisin-like serine characteristics with the highest activity at 50 °C and pH 8 and 11. Activity of the extracted proteinases increased ~4 times at ≥100 mM CaCl(2). In addition, CaCl(2) enhanced thermal stability of the extracted proteinases. Enzymes showed proteolytic activity in either the absence or presence of 10 and 25 % NaCl toward fish muscle, soy protein isolate, and casein substrates. Cell-bound proteinases were likely to play an important role in protein hydrolysis during fish sauce fermentation. PMID:22806191

  9. Aspartate protects Lactobacillus casei against acid stress.

    PubMed

    Wu, Chongde; Zhang, Juan; Du, Guocheng; Chen, Jian

    2013-05-01

    The aim of this study was to investigate the effect of aspartate on the acid tolerance of L. casei. Acid stress induced the accumulation of intracellular aspartate in L. casei, and the acid-resistant mutant exhibited 32.5 % higher amount of aspartate than that of the parental strain at pH 4.3. Exogenous aspartate improved the growth performance and acid tolerance of Lactobacillus casei during acid stress. When cultivated in the presence of 50 mM aspartate, the biomass of cells increased 65.8 % compared with the control (without aspartate addition). In addition, cells grown at pH 4.3 with aspartate addition were challenged at pH 3.3 for 3 h, and the survival rate increased 42.26-fold. Analysis of the physiological data showed that the aspartate-supplemented cells exhibited higher intracellular pH (pHi), intracellular NH4 (+) content, H(+)-ATPase activity, and intracellular ATP pool. In addition, higher contents of intermediates involved in glycolysis and tricarboxylic acid cycle were observed in cells in the presence of aspartate. The increased contents of many amino acids including aspartate, arginine, leucine, isoleucine, and valine in aspartate-added cells may contribute to the regulation of pHi. Transcriptional analysis showed that the expression of argG and argH increased during acid stress, and the addition of aspartate induced 1.46- and 3.06-fold higher expressions of argG and argH, respectively, compared with the control. Results presented in this manuscript suggested that aspartate may protect L. casei against acid stress, and it may be used as a potential protectant during the production of probiotics. PMID:23292549

  10. Cold-adapted digestive aspartic protease of the clawed lobsters Homarus americanus and Homarus gammarus: biochemical characterization.

    PubMed

    Rojo, Liliana; García-Carreño, Fernando; de Los Angeles Navarrete del Toro, Maria

    2013-02-01

    Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K(M) value at 4°C, 10°C, and 25°C, and had 20-fold higher k(cat)/K(M) values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature. PMID:22648335

  11. Novel proteinase inhibitor promotes resistance to insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel Beta vulgaris serine proteinase inhibitor gene (BvSTI) and its protein are identified in response to insect feeding on B. vulgaris seedlings. BvSTI is cloned into an expression vector with constitutive promoter and transformed into Nicotiana benthamiana plants to assess BvSTI’s ability to ...

  12. Aspartate-Based CXCR4 Chemokine Receptor Binding of Cross-Bridged Tetraazamacrocyclic Copper(II) and Zinc(II) Complexes.

    PubMed

    Maples, Randall D; Cain, Amy N; Burke, Benjamin P; Silversides, Jon D; Mewis, Ryan E; D'huys, Thomas; Schols, Dominique; Linder, Douglas P; Archibald, Stephen J; Hubin, Timothy J

    2016-08-26

    The CXCR4 chemokine receptor is implicated in a number of diseases including HIV infection and cancer development and metastasis. Previous studies have demonstrated that configurationally restricted bis-tetraazamacrocyclic metal complexes are high-affinity CXCR4 antagonists. Here, we present the synthesis of Cu(2+) and Zn(2+) acetate complexes of six cross-bridged tetraazamacrocycles to mimic their coordination interaction with the aspartate side chains known to bind them to CXCR4. X-ray crystal structures for three new Cu(2+) acetate complexes and two new Zn(2+) acetate complexes demonstrate metal-ion-dependent differences in the mode of binding the acetate ligand concomitantly with the requisite cis-V-configured cross-bridged tetraazamacrocyle. Concurrent density functional theory molecular modelling studies produced an energetic rationale for the unexpected [Zn(OAc)(H2 O)](+) coordination motif present in all of the Zn(2+) cross-bridged tetraazamacrocycle crystal structures, which differs from the chelating acetate [Zn(OAc)](+) structures of known unbridged and side-bridged tetraazamacrocyclic Zn(2+) -containing CXCR4 antagonists. PMID:27458983

  13. [Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum].

    PubMed

    Ievleva, E V; Revina, T A; Kudriavtseva, N N; Sof'in, A V; Valueva, T A

    2006-01-01

    The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0-10.0). When studied by SDS-PAGE in the presence of beta-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30-60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.

  14. Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment.

    PubMed

    Shan, Zhifu; Yamasaki, Takeshi; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

    2016-07-01

    Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP(Sc)). PrP(Sc) is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP(C)) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP(Sc) (PrP(Sc)-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP(Sc) (PrP(Sc)-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP(Sc) can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119-127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP(Sc)-sen and PrP(Sc)-res even if all PrP(Sc) molecules were not detected. The analytical dynamic range for PrP(Sc) detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%-11% and 2.5-3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP(Sc) detection did not affect the following PrP(Sc) detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP(Sc) detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds. PMID:27565564

  15. Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment.

    PubMed

    Shan, Zhifu; Yamasaki, Takeshi; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

    2016-07-01

    Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP(Sc)). PrP(Sc) is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP(C)) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP(Sc) (PrP(Sc)-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP(Sc) (PrP(Sc)-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP(Sc) can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119-127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP(Sc)-sen and PrP(Sc)-res even if all PrP(Sc) molecules were not detected. The analytical dynamic range for PrP(Sc) detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%-11% and 2.5-3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP(Sc) detection did not affect the following PrP(Sc) detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP(Sc) detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.

  16. Silk gland-specific proteinase inhibitor serpin16 from the Bombyx mori shows cysteine proteinase inhibitory activity.

    PubMed

    Guo, Peng-Chao; Dong, Zhaoming; Xiao, Li; Li, Tao; Zhang, Yan; He, Huawei; Xia, Qingyou; Zhao, Ping

    2015-01-30

    Serpins (serine proteinase inhibitors) are widely distributed in different species and are well known for their inhibitory activities towards serine proteinases. Here, we report the functional characterization of Bombyx mori serpin16. Expression analysis showed that serpin16 was specifically expressed at high levels in the silk gland at both the transcriptional and translational levels. Moreover, homology modeling and multi-sequence alignment suggested that serpin16 had a canonical serpin fold, but it contained a unique reactive center loop, which was obviously shorter than that of typical serpins. Inhibitory activity analyses revealed that the target proteinase of serpin18 is a cysteine proteinase, rather than a serine proteinase. Furthermore, a Michaelis complex model of serpin16 with its target proteinase was constructed to explain the structural basis of how serpin16 recognizes the cysteine proteinase and its target specificity.

  17. N-methyl-D-aspartate receptor antibody-mediated neurological disease: results of a UK-based surveillance study in children

    PubMed Central

    Wright, Sukhvir; Hacohen, Yael; Jacobson, Leslie; Agrawal, Shakti; Gupta, Rajat; Philip, Sunny; Smith, Martin; Lim, Ming; Wassmer, Evangeline; Vincent, Angela

    2015-01-01

    Objective N-methyl-D-aspartate receptor antibody (NMDAR-Ab) encephalitis is a well-recognised clinico-immunological syndrome that presents with neuropsychiatric symptoms cognitive decline, movement disorder and seizures. This study reports the clinical features, management and neurological outcomes of paediatric NMDAR-Ab-mediated neurological disease in the UK. Design A prospective surveillance study. Children with NMDAR-Ab-mediated neurological diseases were voluntarily reported to the British Neurological Surveillance Unit (BPNSU) from November 2010 to December 2011. Initial and follow-up questionnaires were sent out to physicians. Results Thirty-one children fulfilled the criteria for the study. Eight presented during the study period giving an incidence of 0.85 per million children per year (95% CI 0.64 to 1.06); 23 cases were historical. Behavioural change and neuropsychiatric features were present in 90% of patients, and seizures and movement disorders both in 67%. Typical NMDAR-Ab encephalitis was reported in 24 children and partial phenotype without encephalopathy in seven, including predominantly psychiatric (four) and movement disorder (three). All patients received steroids, 22 (71%) received intravenous immunoglobulin, 9 (29%) received plasma exchange,and 10 (32%) received second-line immunotherapy. Of the 23 patients who were diagnosed early, 18 (78%) made a full recovery compared with only 1 of 8 (13%) of the late diagnosed patients (p=0.002, Fisher's exact test). Seven patients relapsed, with four needing additional second-line immunotherapy. Conclusions Paediatric NMDAR-Ab-mediated neurological disease appears to be similar to adult NMDAR-Ab encephalitis, but some presented with a partial phenotype. Early treatment was associated with a quick and often full recovery. PMID:25637141

  18. Diversity of digestive proteinases in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae.

    PubMed

    Vinokurov, K S; Elpidina, E N; Oppert, B; Prabhakar, S; Zhuzhikov, D P; Dunaevsky, Y E; Belozersky, M A

    2006-10-01

    The spectrum of Tenebrio molitor larval digestive proteinases was studied in the context of the spatial organization of protein digestion in the midgut. The pH of midgut contents increased from 5.2-5.6 to 7.8-8.2 from the anterior to the posterior. This pH gradient was reflected in the pH optima of the total proteolytic activity, 5.2 in the anterior and 9.0 in the posterior midgut. When measured at the pH and reducing conditions characteristic of each midgut section, 64% of the total proteolytic activity was in the anterior and 36% in the posterior midgut. In the anterior midgut, two-thirds of the total activity was due to cysteine proteinases, whereas the rest was from serine proteinases. In contrast, most (76%) of the proteolytic activity in the posterior midgut was from serine proteinases. Cysteine proteinases from the anterior were represented by a group of anionic fractions with similar electrophoretic mobility. Trypsin-like activity was predominant in the posterior midgut and was due to one cationic and three anionic proteinases. Chymotrypsin-like proteinases also were prominent in the posterior midgut and consisted of one cationic and four anionic proteinases, four with an extended binding site. Latent proteinase activity was detected in each midgut section. These data support a complex system of protein digestion, and the correlation of proteinase activity and pH indicates a physiological mechanism of enzyme regulation in the gut.

  19. Keratinolytic proteinase from Bacillus thuringiensis AD-12.

    PubMed

    Gegeckas, Audrius; Gudiukaitė, Renata; Citavicius, Donaldas

    2014-08-01

    A new isolated strain noted to produce a novel detergent-stable serine keratinolytic proteinase and identified as Bacillus thuringiensis AD-12. Native keratinolytic proteinase from B. thuringiensis (BtKER) was purified and characterized. The purified BtKER enzyme is a monomer with a molecular mass of 39kDa. Biochemical characterization assays revealed that the BtKER attained optimal activity at pH 7 and 30°C. Residual activity after 1h incubation at 50°C was higher than 80%. The enzyme was activated and stabilized by Mn(2+) and Li(+) metal ions but inactivated by organic solvents. Purified BtKER showed the highest substrate specificity toward keratin from wool>sodium caseinate>collagen>BSA>gelatin in descending order. BtKER is the first reported keratinolytic proteinase from B. thuringiensis and obtained results suggested that new characterized enzyme can be a powerful biocatalyst in peptide production associated to hydrolysis of keratinous and/or keratin-like waste.

  20. Characterization of the aspartate transcarbamoylase from Methanococcus jannaschii.

    PubMed

    Hack, E S; Vorobyova, T; Sakash, J B; West, J M; Macol, C P; Hervé, G; Williams, M K; Kantrowitz, E R

    2000-05-26

    The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli. Only the M. jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity. A purification protocol was devised for the Mj-PyrB and M. jannaschii PyrI (Mj-PyrI) gene products. Molecular weight measurements of the Mj-PyrB and Mj-PyrI gene products revealed that the Mj-PyrB gene product is a trimer and the Mj-PyrI gene product is a dimer. Preliminary characterization of the aspartate transcarbamoylase from M. jannaschii cell-free extract revealed that the enzyme has a similar molecular weight to that of the E. coli holoenzyme. Kinetic analysis of the M. jannaschii aspartate transcarbamoylase from the cell-free extract indicates that the enzyme exhibited limited homotropic cooperativity and little if any regulatory properties. The purified Mj-catalytic trimer exhibited hyperbolic kinetics, with an activation energy similar to that observed for the E. coli catalytic trimer. Homology models of the Mj-PyrB and Mj-PyrI gene products were constructed based on the three-dimensional structures of the homologous E. coli proteins. The residues known to be critical for catalysis, regulation, and formation of the quaternary structure from the well characterized E. coli aspartate transcarbamoylase were compared.

  1. Radiochemical microassay for aspartate aminotransferase activity in the nervous system

    SciTech Connect

    Garrison, D.; Beattie, J.; Namboodiri, M.A.

    1988-07-01

    A radiochemical procedure for measuring aspartate aminotransferase activity in the nervous system is described. The method is based on the exchange of tritium atoms at positions 2 and 3 of L-2,3-(/sup 3/H)aspartate with water when this amino acid is transaminated in the presence of alpha-ketoglutarate to form oxaloacetate. The tritiated water is separated from the radiolabeled aspartate by passing the reaction mixture over a cation exchange column. Confirmation that the radioactivity in the product is associated with water was obtained by separating it by anion exchange HPLC and by evaporation. The product formation is linear with time up to 120 min and with tissue in the 0.05- to 10-micrograms range. The apparent Km for aspartate in the rat brain homogenate is found to be 0.83 mM and that for alpha-ketoglutarate to be 0.12 mM. Methods that further improve the sensitivity of the assay are also discussed.

  2. Use of Recombinant Entamoeba histolytica Cysteine Proteinase 1 To Identify a Potent Inhibitor of Amebic Invasion in a Human Colonic Model▿

    PubMed Central

    Meléndez-López, Samuel G.; Herdman, Scott; Hirata, Ken; Choi, Min-Ho; Choe, Youngchool; Craik, Charles; Caffrey, Conor R.; Hansell, Elisabeth; Chávez-Munguía, Bibiana; Chen, Yen Ting; Roush, William R.; McKerrow, James; Eckmann, Lars; Guo, Jianhua; Stanley, Samuel L.; Reed, Sharon L.

    2007-01-01

    Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis. PMID:17513563

  3. Molt cycle-associated changes in calcium-dependent proteinase activity that degrades actin and myosin in crustacean muscle

    SciTech Connect

    Mykles, D.L.; Skinner, D.M.

    1982-01-01

    The role of calcium-dependent proteinase (CDP) in the proecdysial atrophy of crustacean claw muscle has been investigated. During atrophy the molar ratio of actin to myosin heavy chain decreased 31%, confirming earlier ultrastructural observations that the ratio of thin:thick myofilaments declined from 9:1 to 6:1 (D.L. Mykles and D.M. Skinner, 1981, J. Ultrastruct. Res. 75, 314 to 325). The release of TCA-soluble material in muscle homogenates at neutral pH was stimulated by Ca/sup 2 +/ and completely inhibited by EGTA. The specific degradation of the major myofibrillar proteins (actin, myosin heavy and light chains, paramyosin, tropomyosin, troponin-T, and troponin-I) was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic activity was more than twofold greater in proecdysial muscle homogenates. Degradation of myofibrillar proteins was inhibited by EGTA, and the two inhibitors of crysteine proteinases, leupeptin, and antipain, but not pepstatin, an inhibitor of aspartic proteinases. Unlike CDPs from vertebrate muscle, the CDP(s) in crab claw muscle degrades actin and myosin in addition to other myofibrillar proteins.

  4. Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

    PubMed Central

    2010-01-01

    Background Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. Conclusions BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora. PMID:20663211

  5. Properties of a subtilisin-like proteinase from a psychrotrophic Vibrio species comparison with proteinase K and aqualysin I.

    PubMed

    Kristjánsson, M M; Magnússon, O T; Gudmundsson, H M; Alfredsson, G A; Matsuzawa, H

    1999-03-01

    An extracellular serine proteinase purified from cultures of a psychrotrophic Vibrio species (strain PA-44) belongs to the proteinase K family of the superfamily of subtilisin-like proteinases. The enzyme is secreted as a 47-kDa protein, but under mild heat treatment (30 min at 40 degrees C) undergoes autoproteolytic cleavage on the carboxyl-side of the molecule to give a proteinase with a molecular mass of about 36 kDa that apparently shares most of the enzymatic characteristics and the stability of the 47-kDa protein. In this study, selected enzymatic properties of the Vibrio proteinase were compared with those of the related proteinases, proteinase K and aqualysin I, as representative mesophilic and thermophilic enzymes, respectively. The catalytic efficiency (kcat/Km) for the amidase activity of the cold-adapted enzyme against succinyl-AAPF-p-nitroanilide was significantly higher than that of its mesophilic and thermophilic counterparts, especially when compared with aqualysin I. The stability of the Vibrio proteinase, both towards heat and denaturants, was found to be significantly lower than of either proteinase K or aqualysin I. One or more disulfide bonds in the psychrotrophic proteinase are important for the integrity of the active enzyme structure, as disulfide cleavage, either by reduction with dithiothreitol or by sulfitolysis, led to a loss in its activity. Under the same conditions, aqualysin I was also partially inactivated by dithiothreitol, but the activity of proteinase K was unaffected. The disulfides of either proteinase K or aqualysin I were not reactive towards sulfitolysis, except under denaturing conditions, while all disulfides of the Vibrio proteinase reacted in absence of a denaturant. The reactivity of the disulfides of the proteins as a function of denaturant concentration followed the order: Vibrio proteinase > proteinase K > aqualysin I. The same order of reactivity was also observed for the inactivation of the enzymes by H2O2

  6. Inhibition of proteolytic activity of poliovirus and rhinovirus 2A proteinases by elastase-specific inhibitors.

    PubMed Central

    Molla, A; Hellen, C U; Wimmer, E

    1993-01-01

    A polyprotein cleavage assay has been developed to assay the proteolytic activities in vitro of the 2A proteinases encoded by poliovirus and human rhinovirus 14, which are representative members of the Enterovirus and Rhinovirus genera of picornaviruses, respectively. The elastase-specific substrate-based inhibitors elastatinal and methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK) inhibited both 2A proteinases in vitro. The electrophoretic mobilities of both 2A proteinases were reduced upon incubation with elastatinal, whereas the mobility of a Cys-109-->Ala poliovirus 2Apro mutant was unchanged, an observation suggesting that this inhibitor may have formed a covalent bond with the active-site Cys-109 nucleophile. Iodoacetamide, calpain inhibitor 1, and antipain inhibited poliovirus 2Apro. MPCMK caused a reduction in the yields of the enteroviruses poliovirus type 1 and coxsackievirus A21 and of human rhinovirus 2 in infected HeLa cells but did not affect the growth of encephalomyocarditis virus, a picornavirus of the Cardiovirus genus. MPCMK abrogated the shutoff of host cell protein synthesis that is induced by enterovirus and rhinovirus infection and reduced the synthesis of virus-encoded polypeptides in infected cells. These results indicate that the determinants of substrate recognition by 2A proteinases resemble those of pancreatic and leukocyte elastases. These results may be relevant to the development of broad-range chemotherapeutic agents against entero- and rhinoviruses. Images PMID:8392608

  7. Substrate specificities of pepstatin-insensitive carboxyl proteinases from gram-negative bacteria.

    PubMed

    Ito, M; Dunn, B M; Oda, K

    1996-10-01

    Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, and Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, are the first and second examples of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. The substrate specificities of PCP and XCP were studied using a series of synthetic chromogenic peptide substrates with the general structure, P5-P4-P3-P2-Phe-Nph-P2'-P3' (P5, P4, P3, P2, P2', P3': a variety of amino acids, Nph is p-nitro-L-phenylalanine, and the Phe-Nph bond is cleaved). PCP and XCP were shown to hydrolyze a synthetic substrate, Lys-Pro-Ala-Leu-Phe-Nph-Arg-Leu, most effectively among 28 substrates. The kinetic parameters of this peptide for PCP were Km = 6.3 microM, Kcat = 51.4 s-1, and kcat/Km = 8.16 microM-1.s-1. The kinetic parameters for XCP were Km = 3.6 microM, kcat = 52.2 s-1, and kcat/Km = 14.5 microM-1.s-1. PCP showed a stricter substrate specificity than XCP. That is, the specificity constant (kcat/Km) of each substrate for PCP was in general < 0.5 microM-1.s-1, but was drastically improved by the replacement of Lys by Leu at the P2 position. On the other hand, XCP showed a less stringent substrate specificity, with most of the peptides exhibiting reasonable kcat/Km values (> 1.0 microM-1.s-1). Thus it was found that the substrate specificities of PCP and XCP differ considerably, in spite of the high similarity in their primary structures. In addition, tyrostatin was found to be a competitive inhibitor for XCP, with a Ki value of 2.1 nM, as well as for PCP (Ki = 2.6 nM).

  8. A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

    PubMed Central

    Beveridge, A. J.

    1996-01-01

    The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis. PMID:8819168

  9. Secreted fungal aspartic proteases: A review.

    PubMed

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application. PMID:27137097

  10. Secreted fungal aspartic proteases: A review.

    PubMed

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application.

  11. Dataset of cocoa aspartic protease cleavage sites.

    PubMed

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-09-01

    The data provide information in support of the research article, "The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors" (Janek et al., 2016) [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. PMID:27508221

  12. Modulating the pH-activity profile of cellulase by substitution: replacing the general base catalyst aspartate with cysteinesulfinate in cellulase A from Cellulomonas fimi.

    PubMed

    Cockburn, Darrell W; Vandenende, Chris; Clarke, Anthony J

    2010-03-01

    Cellulase A (CenA) from Cellulomonas fimi is an inverting glycoside hydrolase and a member of family 6 of the CAZy database classification system. We replaced its putative catalytic base aspartyl residues, Aps392 and Asp216, with cysteinesulfinate using a combination of site-directed mutagenesis and chemical modification to investigate the applicability of this approach for the modulation of enzymatic properties. The substituted cysteinyl residues were oxidized to cysteinesulfinic acid with hydrogen peroxide, and the resulting protein products were demonstrated to retain their native structure. Oxidation of the Asp392Cys mutant enzyme restored 52% of wild-type activity when assessed at pH 7.5, whereas Asp216Cys CenA remained inactive. This suggests that Asp216 is not the catalytic base and provides further support for Asp392 performing this role. Similar substitution of the catalytic acid residue Asp252 or the catalytic nucleophile of the retaining enzyme Cel5A from Thermobifida fusca failed to produce active enzymes. This indicates a potential utility of this approach for uniquely identifying catalytic base residues. The replacement of Asp392 with cysteinesulfinate induced an acidic shift in the pH profile of the enzyme such that this enzyme derivative was more active than wild-type CenA below pH 5.5. These data demonstrate the potential of combining site-directed mutagenesis with chemical modification as a viable approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH.

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

    PubMed

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

    1992-01-01

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

  14. Sequential processing of lysosomal acid phosphatase by a cytoplasmic thiol proteinase and a lysosomal aspartyl proteinase.

    PubMed Central

    Gottschalk, S; Waheed, A; Schmidt, B; Laidler, P; von Figura, K

    1989-01-01

    BHK cells expressing human lysosomal acid phosphatase (LAP) transport LAP to lysosomes as an integral membrane protein. In lysosomes LAP is released from the membrane by proteolytic processing, which involves at least two cleavages at the C terminus of LAP. The first cleavage is catalysed by a thiol proteinase at the outside of the lysosomal membrane and removes the bulk of the cytoplasmic tail of LAP. The second cleavage is catalysed by an aspartyl proteinase inside the lysosomes and releases the luminal part of LAP from the membrane-spanning domain. The first cleavage at the cytoplasmic side of the lysosomal membrane depends on acidification of lysosomes and the second cleavage inside the lysosomes depends on prior processing of the cytoplasmic tail. These results suggest that the cytoplasmic tail controls the conformation of the luminal portion of LAP and vice versa. Images PMID:2684640

  15. [Characterization of thermal denaturation process of proteinase K by spectrometry].

    PubMed

    Zhang, Qi-Bing; Na, Xin-Zhu; Yin, Zong-Ning

    2013-07-01

    The effect of different temperatures on the activity and conformational changes of proteinase K was studied. Methods Proteinase K was treated with different temperatures, then denatured natural substrate casein was used to assay enzyme activity, steady-state and time-resolved fluorescence spectroscopy was used to study tertiary structure, and circular dichroism was used to study secondary structure. Results show with the temperature rising from 25 to 65 degrees C, the enzyme activity and half-life of proteinase K dropped, maximum emission wavelength red shifted from 335 to 354 nm with fluorescence intensity decreasing. Synchronous fluorescence intensity of tryptophan residues decreased and that of tyrosine residues increased. Fluorescence lifetime of tryptophan residues reduced from 4. 427 1 to 4. 032 4 ns and the fraction of alpha-helix dropped. It was concluded that it is simple and accurate to use steady-state/time-resolved fluorescence spectroscopy and circular dichroism to investigate thermal stability of proteinase K. Thermal denaturation of proteinase K followed a three-state process. Fluorescence intensity of proteinase K was affected by fluorescence resonance energy transfer from tyrosine to tryptophan residues. The alpha-helix was the main structure to maintain conformational stability of enzyme active site of proteinase K.

  16. Coevolution between pathogen-derived proteinases and proteinase inhibitors of host insects.

    PubMed

    Vilcinskas, Andreas

    2010-01-01

    Virulence is thought to coevolve as a result of reciprocal selection between pathogens and their hosts. This paper focuses on coevolution between microbial proteinases operating as virulence factors and host defense molecules of insects. Owing to shorter generation times and smaller genomes, microbes exhibit a high evolutionary adaptability in comparison with their hosts. Indeed, the latter can only compete with pathogens if they evolve mechanisms providing a comparable genetic plasticity. Gene or domain duplication and shuffling by recombination is the driving force behind the countermeasures in host defense effectors. Recent literature provides evidence for both diversifications of fungal proteinases involved in pathogenesis and expansion host proteinase inhibitors subsets contributing to insect innate immunity. For example, the pathogen-associated spectrum of proteolytic enzymes encompasses thermolysin-like metalloproteinases that putatively promoted the evolution of corresponding host inhibitors of these virulence factors which complement the insect repertoire of antimicrobial defense molecules. Beyond mutual diversification of effector molecules coevolution resulted also in sophisticated molecular adaptations of host insects such as sensing and feedback-loop regulation of microbial metalloproteinases and corresponding countermeasures of pathogens providing evasion of host immunity induced by these virulence factors.

  17. The amino acid sequence of the aspartate aminotransferase from baker's yeast (Saccharomyces cerevisiae).

    PubMed Central

    Cronin, V B; Maras, B; Barra, D; Doonan, S

    1991-01-01

    1. The single (cytosolic) aspartate aminotransferase was purified in high yield from baker's yeast (Saccharomyces cerevisiae). 2. Amino-acid-sequence analysis was carried out by digestion of the protein with trypsin and with CNBr; some of the peptides produced were further subdigested with Staphylococcus aureus V8 proteinase or with pepsin. Peptides were sequenced by the dansyl-Edman method and/or by automated gas-phase methods. The amino acid sequence obtained was complete except for a probable gap of two residues as indicated by comparison with the structures of counterpart proteins in other species. 3. The N-terminus of the enzyme is blocked. Fast-atom-bombardment m.s. was used to identify the blocking group as an acetyl one. 4. Alignment of the sequence of the enzyme with those of vertebrate cytosolic and mitochondrial aspartate aminotransferases and with the enzyme from Escherichia coli showed that about 25% of residues are conserved between these distantly related forms. 5. Experimental details and confirmatory data for the results presented here are given in a Supplementary Publication (SUP 50164, 25 pages) that has been deposited at the British Library Document Supply Centre, Boston Spa. Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5. PMID:1859361

  18. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  19. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  20. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  1. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  2. 21 CFR 582.5017 - Aspartic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  3. Purification, crystallization and preliminary crystallographic studies of a Kunitz-type proteinase inhibitor from tamarind (Tamarindus indica) seeds.

    PubMed

    Patil, Dipak N; Chaudhry, Anshul; Sharma, Ashwani K; Tomar, Shailly; Kumar, Pravindra

    2009-07-01

    A Kunitz-type proteinase inhibitor has been purified from tamarind (Tamarindus indica) seeds. SDS-PAGE analysis of a purified sample showed a homogeneous band corresponding to a molecular weight of 21 kDa. The protein was identified as a Kunitz-type proteinase inhibitor based on N-terminal amino-acid sequence analysis. It was crystallized by the vapour-diffusion method using PEG 6000. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 37.2, b = 77.1, c = 129.1 A. Diffraction data were collected to a resolution of 2.7 A. Preliminary crystallographic analysis indicated the presence of one proteinase inhibitor molecule in the asymmetric unit, with a solvent content of 44%.

  4. A novel cell penetrating aspartic protease inhibitor blocks processing and presentation of tetanus toxoid more efficiently than pepstatin A.

    PubMed

    Zaidi, Nousheen; Burster, Timo; Sommandas, Vinod; Herrmann, Timo; Boehm, Bernhard O; Driessen, Christoph; Voelter, Wolfgang; Kalbacher, Hubert

    2007-12-14

    Selective inhibition of enzymes involved in antigen processing such as cathepsin E and cathepsin D is a valuable tool for investigating the roles of these enzymes in the processing pathway. However, the aspartic protease inhibitors, including the highly potent pepstatin A (PepA), are inefficiently transported across the cell membrane and thus have limited access to antigen processing compartments. Previously described mannose-pepstatin conjugates were efficiently taken up by the cells via receptor mediated uptake. However, cells without mannose receptors are unable to take up these conjugates efficiently. The aim of the present study was to synthesize new cell-permeable aspartic protease inhibitors by conjugating pepstatin A with well-known cell penetrating peptides (CPPs). To achieve this, the most commonly used CPPs namely pAntp(43-58) (penetratin), Tat(49-60), and 9-mer of l-arginine (R9), were synthesized and coupled to pepstatin. The enzyme inhibitory properties of these bioconjugates and their cellular uptake into MCF7 (human breast cancer cell line), Boleths (EBV-transformed B-cell line) and dendritic cells (DC) were the focus of our study. We found that the bioconjugate PepA-penetratin (PepA-P) was the most efficient cell-permeable aspartic protease inhibitor tested, and was more efficient than unconjugated PepA. Additionally, we found that PepA-P efficiently inhibited the tetanus toxoid C-fragment processing in peripheral blood mononuclear cells (PBMC), primary DC and in primary B cells. Therefore, PepA-P can be used in studying the role of intracellular aspartic proteases in the MHC class II antigen processing pathway. Moreover, inhibition of tetanus toxoid C-fragment processing by PepA-P clearly implicates the role of aspartic proteinases in antigen processing.

  5. High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains.

    PubMed

    Yang, Hu; Zhai, Chao; Yu, Xianhong; Li, Zhezhe; Tang, Wei; Liu, Yunyun; Ma, Xiaojian; Zhong, Xing; Li, Guolong; Wu, Di; Ma, Lixin

    2016-06-01

    Proteinase K is widely used in scientific research and industries. This report was aimed to achieve high-level expression of proteinase K using Pichia pastoris GS115 as the host strain. The coding sequence of a variant of proteinase K that has higher activity than the wild type protein was chosen and optimized based on the codon usage preference of P. pastoris. The novel open reading frame was synthesized and a series of multi-copy expression vectors were constructed based on the pHBM905BDM plasmid, allowing for the tandem integration of multiple copies of the target gene into the genome of P. pastoris with a single recombination. These strains were used to study the correlation between the gene copy number and the expression level of proteinase K. The results of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the host genome stably. Meanwhile, the results of qPCR and enzyme activity assays indicated that the mRNA and protein expression levels of the target gene increased as the gene copy number increased. Moreover, the effect of gene dosage on the expression level of the recombinant protein was more obvious using high-density fermentation. The maximum expression level and enzyme activity of proteinase K, which were obtained from the recombinant yeast strain bearing 5 copies of the target gene after an 84-h induction, were approximately 8.069 mg/mL and 108,295 U/mL, respectively. The recombinant proteinase was purified and characterized. The optimum pH and temperature for the activity of this protease were approximately pH 11 and 55 °C, respectively. PMID:26892536

  6. High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains.

    PubMed

    Yang, Hu; Zhai, Chao; Yu, Xianhong; Li, Zhezhe; Tang, Wei; Liu, Yunyun; Ma, Xiaojian; Zhong, Xing; Li, Guolong; Wu, Di; Ma, Lixin

    2016-06-01

    Proteinase K is widely used in scientific research and industries. This report was aimed to achieve high-level expression of proteinase K using Pichia pastoris GS115 as the host strain. The coding sequence of a variant of proteinase K that has higher activity than the wild type protein was chosen and optimized based on the codon usage preference of P. pastoris. The novel open reading frame was synthesized and a series of multi-copy expression vectors were constructed based on the pHBM905BDM plasmid, allowing for the tandem integration of multiple copies of the target gene into the genome of P. pastoris with a single recombination. These strains were used to study the correlation between the gene copy number and the expression level of proteinase K. The results of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the host genome stably. Meanwhile, the results of qPCR and enzyme activity assays indicated that the mRNA and protein expression levels of the target gene increased as the gene copy number increased. Moreover, the effect of gene dosage on the expression level of the recombinant protein was more obvious using high-density fermentation. The maximum expression level and enzyme activity of proteinase K, which were obtained from the recombinant yeast strain bearing 5 copies of the target gene after an 84-h induction, were approximately 8.069 mg/mL and 108,295 U/mL, respectively. The recombinant proteinase was purified and characterized. The optimum pH and temperature for the activity of this protease were approximately pH 11 and 55 °C, respectively.

  7. [Inactivation of T4 phage in water environment using proteinase].

    PubMed

    Lü, Wen-zhou; Yang, Qing-xiang; Zhang, Yu; Yang, Min; Zhu, Chun-fang

    2004-09-01

    The inactivation effectiveness of proteinase to viruses was investigated by using T4 phage as a model virus. The results showed that the inactivation effectiveness of proteinase to T4 phage was obvious. In the optimum conditions and 67.5 u/mL concentration, the inactivation rate of proteinase K to T4 phage in sterilized water and in sewage achieved 99.4% and 49.4% respectively in an hour, and achieved >99.9% and 81.1% in three hours. The inactivation rate of the industrial proteinase 1398 to T4 phage in sterilized water achieved 74.4% in an hour. The effects of pH and temperature on the inactivation effectiveness was not evident.

  8. Purification of human leucocyte DNA: proteinase K is not necessary.

    PubMed

    Douglas, A M; Georgalis, A M; Benton, L R; Canavan, K L; Atchison, B A

    1992-03-01

    A rapid nontoxic method for the purification of DNA from human leucocytes is described. Preliminary experiments which tested different methods of DNA purification indicated that digestion of proteins with proteinase K was unnecessary. This led to the development of a simple procedure involving lysis of the cells in SDS followed by extraction with 6 M NaCl. The method described overcomes the requirement for lengthy incubations in the presence of expensive proteinase K and subsequent extraction with toxic chemicals.

  9. A low molecular weight proteinase inhibitor produced by T lymphocytes.

    PubMed Central

    Ganea, D; Teodorescu, M; Dray, S

    1986-01-01

    A low molecular weight (MW) proteinase inhibitor, between 6500 and 21,500 MW, appeared in the supernatant of rabbit spleen cells cultured at high density for 24 hr. The inhibitor inhibited the enzymatic activity of trypsin for both a high MW natural substrate, fibrinogen, and for a low MW artificial substrate, Chromozym TRY. The low MW proteinase inhibitor is protein in nature and is different, in terms of specificity for enzymes, MW and sensitivity to different physical or chemical treatments, from aprotinin, a low MW proteinase inhibitor (6500 MW) of bovine origin, and from the soybean trypsin inhibitor, a relatively high MW proteinase inhibitor (21,500 MW). The inhibitor was found in the supernatant of purified T cells but not B cells, and its production was increased in the presence of an optimal concentration of Con A. The possibility that this proteinase inhibitor has a role in the regulation of trypsin-like proteinases involved to the immune response remains to be investigated. Images Figure 4 PMID:2417942

  10. The induction of proteinases in corn and soybean by anoxia

    SciTech Connect

    VanToai, T.; Hwang, Shihying )

    1989-04-01

    This study characterized the anaerobic changes in proteinase activities in corn and soybean roots and to investigate the possibility that these changes might contribute to the differential anaerobiosis tolerance of the two species. After 24 h of anoxia, crude protein extracts from H60 corn and Keller soybean root tips (10cm) were assayed for proteinase activities at pH range from 4.5 to 9.5. Turnover of aberrant proteins was studied in seedlings labelled with {sup 3}H-leucine for 12 h under: (a) puromycin (0.64 mM) in air, (b) ethanol (1%) in air, (c) nitrogen and (d) air. After the treatment, the labelled proteins remaining in roots were determined every 2 h for 6 h. In both corn and soybean, activities of alkali proteinases increased, and activities of acid proteinases declined under anoxia. Neutral proteinases increase in anoxic corn roots, but decline in anoxic soybean roots. The protein turnover rate in corn treated with puromycin, ethanol and nitrogen was much higher than in control roots. The protein turnover rate in soybean roots treated with puromycin, ethanol was similar to the rate of the control. The results indicated that: (a) anoxic corn can degrade aberrant proteins, but anoxic soybean cannot, (b) the degradation of aberrant proteins in anoxic corn is accomplished by neutral proteinases, and (c) the accumulation of aberrant proteins in soybean might contribute to the susceptibility of this species to anoxia.

  11. The effect of calciums on molecular motions of proteinase K.

    PubMed

    Liu, Shu-Qun; Tao, Yan; Meng, Zhao-Hui; Fu, Yun-Xin; Zhang, Ke-Qin

    2011-02-01

    The native serine protease proteinase K binds two calcium cations. It has been reported that Ca(2+) removal decreased the enzyme's thermal stability and to some extent the substrate affinity, but has discrepant effects on catalytic activity of the enzyme. Molecular dynamics simulations were performed on the Ca(2+)-bound and Ca(2+)-free proteases to investigate the mechanism by which the calciums affect the structural stability, molecular motions, and catalytic activity of proteinase K. Very similar structural properties were observed between these two forms of proteinase K during simulations; and several long-lived hydrogen bonds and salt bridges common to both forms of proteinase K were found to be crucial in maintaining the local conformations around these two Ca(2+) sites. Although Ca(2+) removal enhanced the overall flexibility of proteinase K, the flexibility in a limited number of segments surrounding the substrate-binding pockets decreased. The largest differences in the equilibrium structures of the two simulations indicate that, upon the removal of Ca(2+), the large concerted motion originating from the Ca1 site can transmit to the substrate-binding regions but not to the catalytic triad residues. In conjunction with the large overlap of the essential subspaces between the two simulations, these results not only provide insight into the dynamics of the underlying molecular mechanism responsible for the unchanged enzymatic activity as well as the decreased thermal stability and substrate affinity of proteinase K upon Ca(2+) removal, but also complement the experimentally determined structural and biochemical data.

  12. An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels.

    PubMed

    Visal-Shah, S; Vrain, T C; Yelle, T C; Nguyen-Quoc, B; Michaud, D

    2001-08-01

    A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)-PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology.

  13. Purification and Characterization of a Keratinolytic Serine Proteinase from Streptomyces albidoflavus

    PubMed Central

    Bressollier, Philippe; Letourneau, François; Urdaci, Maria; Verneuil, Bernard

    1999-01-01

    Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70°C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH2-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K. PMID:10347045

  14. The two-component NS2B-NS3 proteinase represses DNA unwinding activity of the West Nile virus NS3 helicase.

    PubMed

    Chernov, Andrei V; Shiryaev, Sergey A; Aleshin, Alexander E; Ratnikov, Boris I; Smith, Jeffrey W; Liddington, Robert C; Strongin, Alex Y

    2008-06-20

    Similar to many flavivirus types including Dengue and yellow fever viruses, the nonstructural NS3 multifunctional protein of West Nile virus (WNV) with an N-terminal serine proteinase domain and an RNA triphosphatase, an NTPase domain, and an RNA helicase in the C-terminal domain is implicated in both polyprotein processing and RNA replication and is therefore a promising drug target. To exhibit its proteolytic activity, NS3 proteinase requires the presence of the cofactor encoded by the upstream NS2B sequence. During our detailed investigation of the biology of the WNV helicase, we characterized the ATPase and RNA/DNA unwinding activities of the full-length NS2B-NS3 proteinase-helicase protein as well as the individual NS3 helicase domain lacking both the NS2B cofactor and the NS3 proteinase sequence and the individual NS3 proteinase-helicase lacking only the NS2B cofactor. We determined that both the NS3 helicase and NS3 proteinase-helicase constructs are capable of unwinding both the DNA and the RNA templates. In contrast, the full-length NS2B-NS3 proteinase-helicase unwinds only the RNA templates, whereas its DNA unwinding activity is severely repressed. Our data suggest that the productive, catalytically competent fold of the NS2B-NS3 proteinase moiety represents an essential component of the RNA-DNA substrate selectivity mechanism in WNV and, possibly, in other flaviviruses. Based on our data, we hypothesize that the mechanism we have identified plays a role yet to be determined in WNV replication occurring both within the virus-induced membrane-bound replication complexes in the host cytoplasm and in the nuclei of infected cells.

  15. Developing novel anthelmintics from plant cysteine proteinases

    PubMed Central

    Behnke, Jerzy M; Buttle, David J; Stepek, Gillian; Lowe, Ann; Duce, Ian R

    2008-01-01

    Intestinal helminth infections of livestock and humans are predominantly controlled by treatment with three classes of synthetic drugs, but some livestock nematodes have now developed resistance to all three classes and there are signs that human hookworms are becoming less responsive to the two classes (benzimidazoles and the nicotinic acetylcholine agonists) that are licensed for treatment of humans. New anthelmintics are urgently needed, and whilst development of new synthetic drugs is ongoing, it is slow and there are no signs yet that novel compounds operating through different modes of action, will be available on the market in the current decade. The development of naturally-occurring compounds as medicines for human use and for treatment of animals is fraught with problems. In this paper we review the current status of cysteine proteinases from fruits and protective plant latices as novel anthelmintics, we consider some of the problems inherent in taking laboratory findings and those derived from folk-medicine to the market and we suggest that there is a wealth of new compounds still to be discovered that could be harvested to benefit humans and livestock. PMID:18761736

  16. The primary structure and characterization of carbohydrate chains of the extracellular glycoprotein proteinase inhibitor from latex of Carica papaya.

    PubMed

    Odani, S; Yokokawa, Y; Takeda, H; Abe, S; Odani, S

    1996-10-01

    A secretory proteinase inhibitor was isolated from the latex of green fruits of papaya (Carica papaya). The protein exhibited stoichiometric inhibition of bovine trypsin and alpha-chymotrypsin by the same site or overlapping binding sites. The complete covalent structure consisting of 184 amino acids and two disulfide bonds was determined by protein analysis. During the structural analysis, a procedure was established to separate very hydrophilic peptides by reverse-phase HPLC. The result revealed that the latex protein belongs to an extensively diverse plant protein family that includes inhibitors of serine, cysteine and aspartic proteases, a taste-modifying protein, wound responsive proteins, storage proteins, amylase inhibitors and even an oxidoreductase. In this superfamily, the latex proteinase inhibitor is most similar to the curious protein, miraculin, which makes sour food taste sweet. Two carbohydrate chains, each probably composed of (mannose)5, (xylose)1, (fucose)0-2, and (N-acetylglucosamine)2 residues, were attached to asparagine 84 and 90. Mass-spectrometric and compositional analysis suggested that they may represent a new class of plant xylose-containing carbohydrate chains with five mannose residues.

  17. The primary structure and characterization of carbohydrate chains of the extracellular glycoprotein proteinase inhibitor from latex of Carica papaya.

    PubMed

    Odani, S; Yokokawa, Y; Takeda, H; Abe, S; Odani, S

    1996-10-01

    A secretory proteinase inhibitor was isolated from the latex of green fruits of papaya (Carica papaya). The protein exhibited stoichiometric inhibition of bovine trypsin and alpha-chymotrypsin by the same site or overlapping binding sites. The complete covalent structure consisting of 184 amino acids and two disulfide bonds was determined by protein analysis. During the structural analysis, a procedure was established to separate very hydrophilic peptides by reverse-phase HPLC. The result revealed that the latex protein belongs to an extensively diverse plant protein family that includes inhibitors of serine, cysteine and aspartic proteases, a taste-modifying protein, wound responsive proteins, storage proteins, amylase inhibitors and even an oxidoreductase. In this superfamily, the latex proteinase inhibitor is most similar to the curious protein, miraculin, which makes sour food taste sweet. Two carbohydrate chains, each probably composed of (mannose)5, (xylose)1, (fucose)0-2, and (N-acetylglucosamine)2 residues, were attached to asparagine 84 and 90. Mass-spectrometric and compositional analysis suggested that they may represent a new class of plant xylose-containing carbohydrate chains with five mannose residues. PMID:8898891

  18. Identification of neutrophil elastase as the proteinase in burn wound fluid responsible for degradation of fibronectin.

    PubMed

    Grinnell, F; Zhu, M

    1994-08-01

    To identify proteinases responsible for fibronectin degradation in the wound environment we studied wound fluid obtained from burn patients. Immunoblotting experiments showed that extensive degradation of fibronectin had occurred in some burn wound fluid samples, in which case intact fibronectin molecules were undetectable, and the largest fibronectin fragment was 116 kDa. The 116-kDa fragment as well as a smaller 90-kDa fragment contained the fibronectin cell binding domain. These burn-fluid samples degraded freshly added fibronectin. Activity of the fibronectin-degrading enzyme was blocked by a broad-spectrum serine proteinase inhibitor or by specific neutrophil elastase inhibitors but not by metalloproteinase inhibitors or inhibitors of trypsin-like or chymotrypsin-like serine proteinases. Enzyme activity also was neutralized by antibodies against human neutrophil elastase. Incubation of fibronectin with burn wound fluid or purified human neutrophil elastase generated similar fibronectin-degradation products. Finally, direct assay of burn-wound-fluid samples with a synthetic elastase substrate showed a correlation between fluid-phase elastase activity and fibronectin degradation. Based on these findings, we conclude that burn-wound-fluid elastase is responsible for extensive fibronectin degradation. Acute elevation of elastase did not appear to hinder normal wound repair.

  19. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells

    PubMed Central

    Sun, Di; Chen, Shun; Cheng, Anchun; Wang, Mingshu

    2016-01-01

    The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3Cpros) of picornaviruses share similar spatial structures and it is becoming apparent that 3Cpro plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3Cpro are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3Cpro can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3Cpro and these essential factors, 3Cpro is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3Cpro are ongoing and a better understanding of the roles played by 3Cpro may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3Cpro is summarized. PMID:26999188

  20. Different cysteine proteinases involved in bone resorption and osteoclast formation.

    PubMed

    Brage, M; Abrahamson, M; Lindström, V; Grubb, A; Lerner, U H

    2005-06-01

    Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106 did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on cathepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase.

  1. Phospholipase, proteinase and haemolytic activities of Candida albicans isolated from oral cavities of patients with type 2 diabetes mellitus.

    PubMed

    Tsang, C S P; Chu, F C S; Leung, W K; Jin, L J; Samaranayake, L P; Siu, S C

    2007-10-01

    The aim of this study was to biotype and characterize phospholipase, proteinase and haemolytic activities of oral Candida albicans isolates from 210 Chinese patients with type 2 diabetes mellitus (DM) and 210 age- and sex-matched healthy controls. Seventy-six and 50 C. albicans isolates were obtained from type 2 DM patients and controls, respectively, using the oral rinse technique. The isolates were characterized with a biotyping system based on enzyme profiles, carbohydrate assimilation patterns and boric acid resistance of the yeasts, and the isolates were further tested for in vitro phospholipase, proteinase and haemolytic activities. The major biotypes of C. albicans isolates from the type 2 DM and control groups were A1R (42.1 %) and J1R (36.0 %), respectively. Significantly higher proteinase and haemolytic activities were found in the isolates from the type 2 DM group (P<0.05). Proteinase activity was higher in isolates from patients with > or =10 years of DM history than those with <10 years (P<0.05). Haemolytic activity was significantly higher in isolates from female DM patients than in those from male counterparts (P<0.05). These data provide evidence of increased extracellular enzyme activity in Candida isolates taken from DM patients.

  2. Neutrophil Elastase and Proteinase-3 Trigger G Protein-biased Signaling through Proteinase-activated Receptor-1 (PAR1)*

    PubMed Central

    Mihara, Koichiro; Ramachandran, Rithwik; Renaux, Bernard; Saifeddine, Mahmoud; Hollenberg, Morley D.

    2013-01-01

    Neutrophil proteinases released at sites of inflammation can affect tissue function by either activating or disarming signal transduction mediated by proteinase-activated receptors (PARs). Because PAR1 is expressed at sites where abundant neutrophil infiltration occurs, we hypothesized that neutrophil-derived enzymes might also regulate PAR1 signaling. We report here that both neutrophil elastase and proteinase-3 cleave the human PAR1 N terminus at sites distinct from the thrombin cleavage site. This cleavage results in a disarming of thrombin-activated calcium signaling through PAR1. However, the distinct non-canonical tethered ligands unmasked by neutrophil elastase and proteinase-3, as well as synthetic peptides with sequences derived from these novel exposed tethered ligands, selectively stimulated PAR1-mediated mitogen-activated protein kinase activation. This signaling was blocked by pertussis toxin, implicating a Gαi-triggered signal pathway. We conclude that neutrophil proteinases trigger biased PAR1 signaling and we describe a novel set of tethered ligands that are distinct from the classical tethered ligand revealed by thrombin. We further demonstrate the function of this biased signaling in regulating endothelial cell barrier integrity. PMID:24052258

  3. Effects of leupeptin on proteinase and germination of castor beans

    SciTech Connect

    Alpi, A.; Beevers, H.

    1981-10-01

    Leupeptin, tripeptide inhibitor of some proteinases, was shown previously to maintain the stability of several enzymes (isocitrate lyase, fumarase, and catalase) in crude extracts of castor bean endosperm. This reagent is now shown to inhibit the breakdown of water-soluble and crystalloid-storage proteins of the protein bodies isolated from castor beans by the SH-proteinase and it also inhibits the endopeptidase from mung beans. When suitably introduced into the endosperm of dry castor beans it strongly inhibits germination and seedling development. Application of leupeptin to endosperm halves removed from the seed prevents the normal development of enzymes concerned with gluconeogenesis from fat and drastically curtails sugar production. The results suggest that the SH-proteinase is intimately involved in the mobilization of storage proteins.

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

    PubMed

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

    1989-11-28

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

  5. Venom gland EST analysis of the saw-scaled viper, Echis ocellatus, reveals novel alpha9beta1 integrin-binding motifs in venom metalloproteinases and a new group of putative toxins, renin-like aspartic proteases.

    PubMed

    Wagstaff, Simon C; Harrison, Robert A

    2006-08-01

    Echis ocellatus is the most medically important snake in West Africa. However, the composition of its venom and the differential contribution of these venom components to the severe haemorrhagic and coagulopathic pathology of envenoming are poorly understood. To address this situation we assembled a toxin transcriptome based upon 1000 expressed sequence tags (EST) from a cDNA library constructed from pooled venom glands of 10 individual E. ocellatus. We used a variety of bioinformatic tools to construct a fully annotated venom-toxin transcriptome that was interrogated with a combination of BLAST annotation, gene ontology cataloguing and disintegrin-motif searching. The results of these analyses revealed an unusually abundant and diverse expression of snake venom metalloproteinases (SVMP) and a broad toxin-expression profile including several distinct isoforms of bradykinin-potentiating peptides, phospholipase A(2), C-type lectins, serine proteinases and l-amino oxidases. Most significantly, we identified for the first time a conserved alpha(9)beta(1) integrin-binding motif in several SVMPs, and a new group of putative venom toxins, renin-like aspartic proteases. PMID:16713134

  6. Differential inhibition of Helicoverpa armigera gut proteinases by proteinase inhibitors of pigeonpea (Cajanus cajan) and its wild relatives.

    PubMed

    Chougule, Nanasaheb P; Hivrale, Vandana K; Chhabda, Pavanjeet J; Giri, Ashok P; Kachole, Manvendra S

    2003-10-01

    The seeds of 36 pigeonpea [Cajanus cajan (L) Millsp.] cultivars, resistant and susceptible to pests and pathogens and 17 of its wild relatives were analysed for inhibitors of trypsin, chymotrypsin, and insect gut proteinases to identify potential inhibitors of insect (Helicoverpa armigera) gut enzymes. Proteinase inhibitors (PIs) of pigeonpea cultivars showed total inhibition of trypsin and chymotrypsin, and moderate inhibition potential towards H. armigera proteinases (HGP). PIs of wild relatives exhibited stronger inhibition of HGP, which was up to 87% by Rhynchosia PIs. Electrophoretic detection of HGPI proteins and inhibition of HGP isoforms by few pigeonpea wild relative PIs supported our enzyme inhibitor assay results. Present results indicate that PIs exhibit wide range of genetic diversity in the wild relatives of pigeonpea whereas pigeonpea cultivars (resistant as well as susceptible to pests and pathogens) are homogeneous. The potent HGPIs identified in this study need further exploration for their use in strengthening pigeonpea defence against H. armigera.

  7. A Green Polymerization of Aspartic Acid for the Undergraduate Organic Laboratory

    ERIC Educational Resources Information Center

    Bennett, George D.

    2005-01-01

    The green polymerization of aspartic acid carried out during an organic-inorganic synthesis laboratory course for undergraduate students is described. The procedure is based on work by Donlar Corporation, a Peru, Illinois-based company that won a Green Chemistry Challenge Award in 1996 in the Small Business category for preparing thermal…

  8. Proton transfer pathways in an aspartate-water cluster sampled by a network of discrete states

    NASA Astrophysics Data System (ADS)

    Reidelbach, Marco; Betz, Fridtjof; Mäusle, Raquel Maya; Imhof, Petra

    2016-08-01

    Proton transfer reactions are complex transitions due to the size and flexibility of the hydrogen-bonded networks along which the protons may "hop". The combination of molecular dynamics based sampling of water positions and orientations with direct sampling of proton positions is an efficient way to capture the interplay of these degrees of freedom in a transition network. The energetically most favourable pathway in the proton transfer network computed for an aspartate-water cluster shows the pre-orientation of water molecules and aspartate side chains to be a pre-requisite for the subsequent concerted proton transfer to the product state.

  9. Serine proteinases from barley malt may degrade beta-amylase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley seed proteinases are critically important to seed germination and malting in that they generate amino acids from seed N reserves, supporting embryo growth during germination and yeast fermentation during brewing. However, relatively little is known regarding the endogenous protein substrate ...

  10. Purification and characterization of a new serine proteinase from Bacillus subtilis with specificity for amino acids at P1 and P2 positions.

    PubMed

    Yamagata, A; Yoshida, N; Noda, K; Ito, A

    1995-12-01

    A proteinase was purified 230-fold to apparent homogeneity from culture filtrates of Bacillus subtilis by a series of column chromatographies on DE52, DEAE-Toyopearl, Cellulofine GC200M, and Mono-Q, using Boc-Ala-Ala-Pro-Ser-pNA as a substrate. The molecular weight of the proteinase was estimated to be 42,000 by SDS-PAGE in the presence of 2-mercaptoethanol. Studies on the substrate specificity with peptide p-nitroanilides and natural peptides revealed that this proteinase preferentially hydrolyzed the peptide bond on the carboxyl-terminal side of either serine or alanine residues at the P1 position and hydrophobic bulky amino acids at P2. It was most active at pH 9.5 for the hydrolysis of Boc-Ala-Ala-Pro-Ser-pNA. The enzyme was inactivated by diisopropyl fluorophosphate (DFP), but not by tosyl-L-phenylalanine chloromethylketone (TPCK) or by EDTA. Based on the reactivity toward substrates and inhibitors, this enzyme differs from elastase- or subtilisin-like proteinase, hence it is a new type of proteinase with specificity for amino acids at P1 and P2 positions. PMID:8519806

  11. Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: purification and characterization of a trypsin-like proteinase.

    PubMed

    Tsybina, T A; Dunaevsky, Y E; Belozersky, M A; Zhuzhikov, D P; Oppert, B; Elpidina, E N

    2005-03-01

    A new trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI 7.4. The enzyme was also characterized by temperature optimum at 55 degrees C, pH optimum at 8.5, and K(m) value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a trypsin-like serine proteinase stable within the pH range of 5.0-9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N-terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50-72% identity with other insect trypsin-like proteinases, and 44-50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.

  12. Medial temporal N-acetyl aspartate in pediatric major depression

    PubMed Central

    MacMaster, Frank P.; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S. Preeya; Buhagiar, Christian; Rosenberg, David R.

    2008-01-01

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD-case control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  13. Medial temporal N-acetyl-aspartate in pediatric major depression.

    PubMed

    MacMaster, Frank P; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S Preeya; Buhagiar, Christian; Rosenberg, David R

    2008-10-30

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD case-control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in the left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  14. A yeast arginine specific tRNA is a remnant aspartate acceptor

    PubMed Central

    Fender, Aurélie; Geslain, Renaud; Eriani, Gilbert; Giegé, Richard; Sissler, Marie; Florentz, Catherine

    2004-01-01

    High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNAAsp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA4Arg. This tRNA has a sequence strikingly similar to that of tRNAAsp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA4Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA4Arg into an aspartate acceptor, as efficient as tRNAAsp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA4Arg. The latent aspartate acceptor capacity in a contemporary tRNAArg leads to the proposal of an evolutionary link between tRNA4Arg and tRNAAsp genes. PMID:15452274

  15. Aspartic acid racemization in tooth enamel from living humans.

    PubMed Central

    Helfman, P M; Bada, J L

    1975-01-01

    The aspartic acid in human tooth enamel shows increasing racemization with age. This increase is not seen in the metabolically active protein hemoglobin. The rate constant for the racemization reaction of aspartic acid in human tooth enamel was found to be 8.29 X 10(-4) yr-1. This rate constant suggests that in any protein with a long in vivo lifetime, D-aspartic acid will accumulate with age (about 8% of total aspartic acid in enamel will be the D-enantiomer after 60 years). Thus, racemization may play some role in the aging process affecting metabolically stable tissues in long-lived homeotherms. Aspartic acid racemization in toogh enamel also provides a biochronological tool for assessing the age of living mammals. PMID:1059082

  16. Aspartate inhibits Staphylococcus aureus biofilm formation.

    PubMed

    Yang, Hang; Wang, Mengyue; Yu, Junping; Wei, Hongping

    2015-04-01

    Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp.

  17. Aspartate inhibits Staphylococcus aureus biofilm formation.

    PubMed

    Yang, Hang; Wang, Mengyue; Yu, Junping; Wei, Hongping

    2015-04-01

    Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp. PMID:25687923

  18. Ozone inactivation of human alpha 1-proteinase inhibitor

    SciTech Connect

    Johnson, D.A.

    1980-06-01

    Ozone decreased the trypsin, chymotrypsin, and elastase inhibitory activities of human alpha 1-proteinase inhibitor both in plasma and in solutions of the pure inhibitor. The total loss of porcine elastase inhibitory activity required 18 mol of ozone/mol of pure alpha 1-PI and approximately 850 mol of ozone/mol of alpha 1-PI in plasma. A corresponding loss of the ability to inhibit human leukocyte elastase was observed. Inactivated alpha 1-PI contains four residues of methionine sulfoxide, in addition to oxidized tryosine and tryptophan. Electrophoretic analysis demonstrated that the ozone-inactivated alpha 1-PI did not form normal complexes with serine proteinases. These findings suggest that the inhalation of ozone could inactivate alpha 1-PI on the airspace side of the lung to create a localized alpha 1-PI deficiency, which might contribute to the development of emphysema.

  19. Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

    SciTech Connect

    Onstott, T. C.; Aubrey, A.D.; Kieft, T L; Silver, B J; Phelps, Tommy Joe; Van Heerden, E.; Opperman, D. J.; Bada, J L.

    2014-01-01

    Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 C and 1 2 years for 3 km depth and 54 C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

  20. Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

    PubMed

    Onstott, T C; Magnabosco, C; Aubrey, A D; Burton, A S; Dworkin, J P; Elsila, J E; Grunsfeld, S; Cao, B H; Hein, J E; Glavin, D P; Kieft, T L; Silver, B J; Phelps, T J; van Heerden, E; Opperman, D J; Bada, J L

    2014-01-01

    Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 °C and 1-2 years for 3 km depth and 54 °C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 °C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

  1. Does Aspartic Acid Racemization Constrain the Depth Limit of the Subsurface Biosphere?

    NASA Technical Reports Server (NTRS)

    Onstott, T C.; Magnabosco, C.; Aubrey, A. D.; Burton, A. S.; Dworkin, J. P.; Elsila, J. E.; Grunsfeld, S.; Cao, B. H.; Hein, J. E.; Glavin, D. P.; Kieft, T. L.; Silver, B. J.; Phelps, T. J.; Heerden, E. Van; Opperman, D. J.; Bada, J. L.

    2013-01-01

    Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of approximately 89 years for 1 km depth and 27 C and 1-2 years for 3 km depth and 54 C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

  2. CP30, a cysteine proteinase involved in Trichomonas vaginalis cytoadherence.

    PubMed

    Mendoza-López, M R; Becerril-Garcia, C; Fattel-Facenda, L V; Avila-Gonzalez, L; Ruíz-Tachiquín, M E; Ortega-Lopez, J; Arroyo, R

    2000-09-01

    We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells. PMID:10948104

  3. CP30, a cysteine proteinase involved in Trichomonas vaginalis cytoadherence.

    PubMed

    Mendoza-López, M R; Becerril-Garcia, C; Fattel-Facenda, L V; Avila-Gonzalez, L; Ruíz-Tachiquín, M E; Ortega-Lopez, J; Arroyo, R

    2000-09-01

    We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells.

  4. CP30, a Cysteine Proteinase Involved in Trichomonas vaginalis Cytoadherence

    PubMed Central

    Mendoza-López, M. Remedios; Becerril-Garcia, Cecilia; Fattel-Facenda, Loriz V.; Avila-Gonzalez, Leticia; Ruíz-Tachiquín, Martha E.; Ortega-Lopez, Jaime; Arroyo, Rossana

    2000-01-01

    We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells. PMID:10948104

  5. Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

    PubMed

    Odei-Addo, Frank; Frost, Carminita; Smith, Nanette; Ogawa, Tomohisa; Muramoto, Koji; Oliva, Maria Luiza Vilela; Gráf, László; Naude, Ryno

    2014-10-01

    One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor. PMID:24090421

  6. Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates

    PubMed Central

    Bartlett, John D.

    2013-01-01

    This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These proteins and their respective null mice and human mutations are also evaluated to shed light on the mechanisms that cause nonsyndromic enamel malformations termed amelogenesis imperfecta. Pertinent controversies are addressed. For example, do any of these proteins have a critical function in addition to their role in enamel development? Does amelogenin initiate crystallite growth, does it inhibit crystallite growth in width and thickness, or does it do neither? Detailed examination of the null mouse literature provides unmistakable clues and/or answers to these questions, and this data is thoroughly analyzed. Striking conclusions from this analysis reveal that widely held paradigms of enamel formation are inadequate. The final section of this review weaves the recent data into a plausible new mechanism by which these enamel matrix proteins support and promote enamel development. PMID:24159389

  7. Aspartate Aminotransferase in Alfalfa Root Nodules 1

    PubMed Central

    Farnham, Mark W.; Griffith, Stephen M.; Miller, Susan S.; Vance, Carroll P.

    1990-01-01

    Aspartate aminotransferase (AAT) plays an important role in nitrogen metabolism in all plants and is particularly important in the assimilation of fixed N derived from the legume-Rhizoblum symbiosis. Two isozymes of AAT (AAT-1 and AAT-2) occur in alfalfa (Medicago sativa L.). Antibodies against alfalfa nodule AAT-2 do not recognize AAT-1, and these antibodies were used to study AAT-2 expression in different tissues and genotypes of alfalfa and also in other legume and nonlegume species. Rocket immunoelectrophoresis indicated that nodules of 38-day-old alfalfa plants contained about eight times more AAT-2 than did nodules of 7-day-old plants, confirming the nodule-enhanced nature of this isozyme. AAT-2 was estimated to make up 16, 15, 5, and 8 milligrams per gram of total soluble protein in mature nodules, roots, stems, and leaves, respectively, of effective N2-fixing alfalfa. The concentration of AAT-2 in nodules of ineffective non-N2-fixing alafalfa genotypes was about 70% less than that of effective nodules. Western blots of soluble protein from nodules of nine legume species indicated that a 40-kilodalton polypeptide that reacts strongly with AAT-2 antibodies is conserved in legumes. Nodule AAT-2 immunoprecipitation data suggested that amide- and ureide-type legumes may differ in expression and regulation of the enzyme. In addition, Western blotting and immunoprecipitations of AAT activity demonstrated that antibodies against alfalfa AAT-2 are highly cross-reactive with AAT enzyme protein in leaves of soybean (Glycine max L.), wheat (Triticum aestivum L.), and maize (Zea mays L.) and in roots of maize, but not with AAT in soybean and wheat roots. Results from this study indicate that AAT-2 is structurally conserved and localized in similar tissues among diverse species. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:16667896

  8. Neutrophil-derived Oxidants and Proteinases as Immunomodulatory Mediators in Inflammation

    PubMed Central

    Witko-Sarsat, V.

    1994-01-01

    Neutrophils generate potent microbicidal molecules via the oxygen-dependent pathway, leading to the generation of reactive oxygen intermediates (ROI), and via the non-oxygen dependent pathway, consisting in the release of serine proteinases and metalloproteinases stored in granules. Over the past years, the concept has emerged that both ROI and proteinases can be viewed as mediators able to modulate neutrophil responses as well as the whole inflammatory process. This is well illustrated by the oxidative regulation of proteinase activity showing that oxidants and proteinases acts is concert to optimize the microbicidal activity and to damage host tissues. ROI and proteinases can modify the activity of several proteins involved in the control of inflammatory process. Among them, tumour necrosis factor-α and interleukin-8, are elective targets for such a modulation. Moreover, ROI and proteinases are also able to modulate the adhesion process of neutrophils to endothelial cells, which is a critical step in the inflammatory process. PMID:18472951

  9. Dispersal of Bap-mediated Staphylococcus aureus biofilm by proteinase K.

    PubMed

    Kumar Shukla, Sudhir; Rao, Toleti Subba

    2013-02-01

    The dominant role of biofilm-associated protein (Bap) in Staphylococcus aureus biofilm development prompted us to investigate Bap as a potential target for proteinase-mediated biofilm dispersion. Biofilm assay in microtitre plates showed that proteinase K hampered the early adhesion of cells as well as biofilm development. Proteinase K treatment of 24- and 48-h-old biofilms showed enhanced dispersion of bap-positive S. aureus biofilm; however, proteinase K did not affect the bap-negative S. aureus biofilm. When antibiotics were used in combination with proteinase K, significant enhancement in antibiotic action was noticed against bap-positive S. aureus biofilm. This study establishes that antibiotics in combination with proteinase K can be used for controlling S. aureus biofilms in whose development Bap surface protein has a major role. We propose that Bap protein could be a potential target for therapeutic control of S. aureus infections (for example, bovine mastitis).

  10. Studies on proteinases from Calotropis gigantea latex. I. Purification and some properties of two proteinases containing carbohydrate.

    PubMed

    Abraham, K I; Joshi, P N

    1979-05-10

    Two proteinase containing carbohydrate, called calotropain-FI and calotropain-FII, were purified from Calotropis gigantea latex by CM-Sephadex C-50 chromatography. Both calotropain-FI and FII were found to be homogeneous by rechromatography on CM-Sephadex C-50, gel filtration on Sephadex G-100, electrophoresis on polyacrylamide gel and by N-terminal amino acid analysis. Some properties of these enzymes are reported.

  11. Proteinase inhibitor gene families: strategies for transformation to improve plant defenses against herbivores.

    PubMed

    Ryan, C A

    1989-01-01

    Recent evidence indicates that the presence of serine proteinase inhibitors in plant leaves can reduce predation by insects. Plants can now be transformed with proteinase inhibitor genes with strong promoters to express the inhibitor proteins in relatively high levels at specific times. Inhibitors having variable specificities against digestive proteinases of insects and pathogens can now be assessed for their possible role(s) in natural plant defense and for their potential usefulness in protecting crop plants against herbivores.

  12. A new crystal form of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus.

    PubMed

    Tanokura, M; Sasaki, H; Muramatsu, T; Iwata, S; Hamaya, T; Takizawa, T; Takahashi, K

    1993-10-01

    Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase, whose catalytic residues and mechanism remain to be elucidated. A new form of proteinase A crystals more suitable for crystallography than that obtained previously was prepared from an ammonium sulfate solution at pH 3.5 by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1), with unit cell dimensions of a = 69.75 +/- 0.06 A, b = 87.55 +/- 0.05 A, and c = 60.83 +/- 0.04 A. On the assumption of two enzyme molecules per asymmetric unit, the calculated volume to unit protein mass ratio (Vm) was 2.08 A3/Da. By assuming the specific volume to be 0.74 cm3/g, the solvent content (Vso1) was estimated to be 41%, i.e., much larger than that of the crystal form obtained previously at pH 2.0 (Vso1 = 26%). Diffraction data were collected up to a resolution higher than 1.6 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation.

  13. Cloning of a serine proteinase inhibitor from bovine brain: expression in the brain and characterization of its target proteinases.

    PubMed

    Nakaya, N; Nishibori, M; Kawabata, M; Saeki, K

    1996-12-01

    A cDNA encoding of the serine proteinase inhibitor (serpin), B-43, was cloned from the cDNA library of the bovine brain. It encoded 378 amino acids, and the MW of the protein was estimated to be 42.6 kDa, which is consistent with that of the native B-43 purified from the bovine brain. The homology search revealed that B-43 belongs to the ovalbumin branch of the serpin superfamily. Among them, B-43 was most homologous to human placental thrombin inhibitor (PI-6) and its murine counterpart, with the amino acid identity of 76% and 71%, respectively. Northern blot analysis showed that the size of the transcript was 1.4 kb, and that the expression of B-43 in the bovine brain varied depending on the brain regions, i.e. a lower level of expression was observed in the cerebral cortex and the hippocampus compared to the level of expression that was observed in the medulla oblongata. [35S]-labeled B-43 protein was synthesized in vitro by using a rabbit reticulocyte lysate system, which formed complexes with proteinases such as thrombin, trypsin, alpha-chymotrypsin, and 7S nerve growth factor (NGF), but not with urokinase or plasmin. These results, together with the immunohistochemical localization of B-43 in astrocytes and in some neurons which was observed in the previous study suggest that B-43 may be involved in the regulation of serine proteinases present in the brain or extravasated from the blood.

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

    PubMed

    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.

  15. Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion.

    PubMed

    Vinokurov, K S; Elpidina, E N; Oppert, B; Prabhakar, S; Zhuzhikov, D P; Dunaevsky, Y E; Belozersky, M A

    2006-10-01

    Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of "heavy" trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.

  16. Non-enzymic beta-decarboxylation of aspartic acid.

    NASA Technical Reports Server (NTRS)

    Doctor, V. M.; Oro, J.

    1972-01-01

    Study of the mechanism of nonenzymic beta-decarboxylation of aspartic acid in the presence of metal ions and pyridoxal. The results suggest that aspartic acid is first converted to oxalacetic acid by transamination with pyridoxal which in turn is converted to pyridoxamine. This is followed by decarboxylation of oxalacetic acid to form pyruvic acid which transaminates with pyridoxamine to form alanine. The possible significance of these results to prebiotic molecular evolution is briefly discussed.

  17. Functional properties of a cysteine proteinase from pineapple fruit with improved resistance to fungal pathogens in Arabidopsis thaliana.

    PubMed

    Wang, Wei; Zhang, Lu; Guo, Ning; Zhang, Xiumei; Zhang, Chen; Sun, Guangming; Xie, Jianghui

    2014-02-21

    In plant cells, many cysteine proteinases (CPs) are synthesized as precursors in the endoplasmic reticulum, and then are subject to post-translational modifications to form the active mature proteinases. They participate in various cellular and physiological functions. Here, AcCP2, a CP from pineapple fruit (Ananas comosus L.) belonging to the C1A subfamily is analyzed based on the molecular modeling and homology alignment. Transcripts of AcCP2 can be detected in the different parts of fruits (particularly outer sarcocarps), and gradually increased during fruit development until maturity. To analyze the substrate specificity of AcCP2, the recombinant protein was overexpressed and purified from Pichia pastoris. The precursor of purified AcCP2 can be processed to a 25 kDa active form after acid treatment (pH 4.3). Its optimum proteolytic activity to Bz-Phe-Val-Arg-NH-Mec is at neutral pH. In addition, the overexpression of AcCP2 gene in Arabidopsis thaliana can improve the resistance to fungal pathogen of Botrytis cinerea. These data indicate that AcCP2 is a multifunctional proteinase, and its expression could cause fruit developmental characteristics of pineapple and resistance responses in transgenic Arabidopsis plants.

  18. Developing a rapid throughput screen for detection of nematicidal activity of plant cysteine proteinases: the role of Caenorhabditis elegans cystatins.

    PubMed

    Phiri, A M; De Pomerai, D; Buttle, D J; Behnke, J M B

    2014-02-01

    Plant cysteine proteinases (CPs) from papaya (Carica papaya) are capable of killing parasitic nematode worms in vitro and have been shown to possess anthelmintic effects in vivo. The acute damage reported in gastrointestinal parasites has not been found in free-living nematodes such as Caenorhabditis elegans nor among the free-living stages of parasitic nematodes. This apparent difference in susceptibility might be the result of active production of cysteine proteinase inhibitors (such as cystatins) by the free-living stages or species. To test this possibility, a supernatant extract of refined papaya latex (PLS) with known active enzyme content was used. The effect on wild-type (Bristol N2) and cystatin null mutant (cpi-1(-/-) and cpi-2(-/-)) C. elegans was concentration-, temperature- and time-dependent. Cysteine proteinases digested the worm cuticle leading to release of internal structures and consequent death. Both cystatin null mutant strains were highly susceptible to PLS attack irrespective of the temperature and concentration of exposure, whereas wild-type N2 worms were generally resistant but far more susceptible to attack at low temperatures. PLS was able to induce elevated cpi-1 and cpi-2 cystatin expression. We conclude that wild-type C. elegans deploy cystatins CPI-1 and CPI-2 to resist CP attack. The results suggest that the cpi-1 or cpi-2 null mutants (or a double mutant combination of the two) could provide a cheap and effective rapid throughput C. elegans-based assay for screening plant CP extracts for anthelmintic activity.

  19. Impact of Mercury(II) on proteinase K catalytic center: investigations via classical and Born-Oppenheimer molecular dynamics.

    PubMed

    Panek, Jarosław J; Mazzarello, Riccardo; Novič, Marjana; Jezierska-Mazzarello, Aneta

    2011-02-01

    Mercury(II) has a strong affinity for the thiol groups in proteins often resulting in the disruption of their biological functions. In this study we present classical and first-principles, DFT-based molecular dynamics (MD) simulations of a complex of Hg(II) and proteinase K, a well-known serine protease with a very broad and diverse enzymatic activity. It contains a catalytic triad formed by Asp39, His69, and Ser224, which is responsible for its biological activity. It was found previously by X-ray diffraction experiments that the presence of Hg(II) inhibits the enzymatic action of proteinase K by affecting the stereochemistry of the triad. Our simulations predict that (i) the overall structure as well as the protein backbone dynamics are only slightly affected by the mercury cation, (ii) depending on the occupied mercury site, the hydrogen bonds of the catalytic triad are either severely disrupted (both bonds for mercury at site 1, and the His69-Ser224 contact for mercury at site 2) or slightly strengthened (the Asp39-His69 bond when mercury is at site 2), (iii) the network of hydrogen bonds of the catalytic triad is not static but undergoes constant fluctuations, which are significantly modified by the presence of the Hg(II) cation, influencing in turn the triad's ability to carry out the enzymatic function--these facts explain the experimental findings on the inhibition of proteinase K by Hg(II).

  20. An in-built proteinase inhibitor system for the protection of recombinant proteins recovered from transgenic plants.

    PubMed

    Rivard, Daniel; Anguenot, Raphaël; Brunelle, France; Le, Van Quy; Vézina, Louis-Philippe; Trépanier, Sonia; Michaud, Dominique

    2006-05-01

    Proteolytic degradation represents a significant barrier to the efficient production of several recombinant proteins in plants, both in vivo during their expression and in vitro during their recovery from source tissues. Here, we describe a strategy to protect recombinant proteins during the recovery process, based on the coexpression of a heterologous proteinase inhibitor acting as a 'mouse trap' against the host proteases during extraction. After confirming the importance of trypsin- and chymotrypsin-like activities in crude protein extracts of potato (Solanum tuberosum L.) leaves, transgenic lines of potato expressing either tomato cathepsin D inhibitor (CDI) or bovine aprotinin, both active against trypsin and chymotrypsin, were generated by Agrobacterium tumefaciens-mediated genetic transformation. Leaf crude protein extracts from CDI-expressing lines, showing decreased levels of cathepsin D-like and ribulose 1,5-bisphosphate carboxylase/oxygenase hydrolysing activities in vitro, conducted decreased turnover rates of the selection marker protein neomycin phosphotransferase II (NPTII) relative to the turnover rates measured for transgenic lines expressing only the marker protein. A similar stabilizing effect on NPTII was observed in leaf protein extracts from plant lines coexpressing bovine aprotinin, confirming the ability of ectopically expressed broad-spectrum serine proteinase inhibitors to reproduce the protein-stabilizing effect of low-molecular-weight proteinase inhibitors generally added to protein extraction media.

  1. Midgut proteinases of Sitotroga cerealella (Oliver) (Lepidoptera:Gelechiidae): Characterization and relationship to resistance in cereals

    SciTech Connect

    Wu, Lan.

    1989-01-01

    Midgut proteinases are vital to the insects which digest ingested food in the midgut. Insect midgut proteinases, therefore, have been considered as possible targets for the control of insect pests. Proteinaceous proteinase inhibitors are very attractive for their potential use in developing insect resistant plant varieties via genetic engineering. Sitotroga cerealella is one of the major storage pests of cereals, and no antibiotic resistance in wheat against this insect has been identified to date. A series of diagnostic inhibitors, thiol-reducing agents and a metal-ion chelator were used in the identification of proteinases in crude extracts from S. cerealella larval midguts with both protein and ester substrates. The partial inhibition of proteolytic activity in crude midgut extract toward ({sup 3}H)-methemoglobin by pepstatin A suggested the presence of another proteinase which was sensitive to pepstatin A. The optimum pH range for the proteolytic activity, however, indicated that the major midgut proteinases were not carboxyl proteinases. Two proteinases were successfully purified by a combination of fractionation with ammonium sulfate, gel permeation and anion exchange chromatography. Characterization of the enzymes with the purified enzyme preparations confirmed that the two major proteinases were serine endoproteinases with trypsin-like and chymotrypsin-like specificities respectively. Bioassays were conducted using the artificial seeds to test naturally occurring proteinaceous proteinase inhibitors of potential value. Soybean trypsin inhibitor and the Bowman-Birk proteinase inhibitor had adverse effects on the development of the insect. A predictive model was constructed to evaluate effects of seed resistance in conjunction with other control methods on S. cerealella population dynamics.

  2. [Aspartate aminotransferase--key enzyme in the human systemic metabolism].

    PubMed

    Otto-Ślusarczyk, Dagmara; Graboń, Wojciech; Mielczarek-Puta, Magdalena

    2016-01-01

    Aspartate aminotransferase is an organ-nonspecific enzyme located in many tissues of the human body where it catalyzes reversible reaction of transamination. There are two aspartate aminotransferase isoforms--cytoplasmic (AST1) and mitochondrial (AST2), that usually occur together and interact with each other metabolically. Both isoforms are homodimers containing highly conservative regions responsible for catalytic properties of enzyme. The common feature of all aspartate aminotransfeses is Lys - 259 residue covalent binding with prosthetic group - pyridoxal phosphate. The differences in the primary structure of AST isoforms determine their physico-chemical, kinetic and immunological properties. Because of the low concentration of L-aspartate (L-Asp) in the blood, AST is the only enzyme, which supply of this amino acid as a substrate for many metabolic processes, such as urea cycle or purine and pyrimidine nucleotides in the liver, synthesis of L-arginine in the kidney and purine nucleotide cycle in the brain and the skeletal muscle. AST is also involved in D-aspartate production that regulates the metabolic activity at the auto-, para- and endocrine level. Aspartate aminotransferase is a part of the malate-aspartate shuttle in the myocardium, is involved in gluconeogenesis in the liver and kidney, glyceroneogenesis in the adipose tissue, and synthesis of neurotransmitters and neuro-glial pathway in the brain. Recently, the significant role of AST in glutaminolysis - normal metabolic pathway in tumor cells, was demonstrated. The article is devoted the role of AST, known primarily as a diagnostic liver enzyme, in metabolism of various human tissues and organs. PMID:27117097

  3. An easy method for diagnosing macro-aspartate aminotransferase: a case series.

    PubMed

    Beşer, Omer Faruk; Laçinel, Sibel; Gülcü, Didem; Kutlu, Tufan; Cullu Çokuğraş, Fügen; Erkan, Tülay

    2014-10-01

    Macro-aspartate transaminase (macro-AST) must be considered when the aspartate transaminase (AST) level is chronically high without any liver, cardiac, or muscle disease. Many specialized laboratory techniques have been recommended for diagnosing macro-AST, including the polyethylene glycol immune precipitate technique, which is simple. This study presents a considerably easier method based on the studies of Davidson and Watson and Castiella et al. Our method is based on the decrease in the plasma AST level after storage of the macroenzyme at 2-8 °C for 5 days, and has the advantages of low cost, reliability, and practicality at any health center. In our eight cases of macro-AST, the AST activity at day 6 had decreased by more than 50% from day 1. This method is practical for primary healthcare facilities because of its easy application and accurate results, and obviated the need for unnecessary tests after diagnosis.

  4. Controlled intracellular proteolysis during postpartal involution of the uterus: characterization and regulation of an alkaline proteinase.

    PubMed

    Roth, M; Hoechst, M; Afting, E G

    1981-01-01

    The postpartal involution of the uterus is predominantly due to cellular hypotrophy. This implies an intracellular proteolytic system which must be carefully controlled pre and post partum. We have characterized and partially purified a proteinase with an alkaline pH-optimum of activity and a proteinase inhibitor protein which inhibits this proteinase very strongly. The alkaline proteinase copurifies with the actomyosin complex of the uterine myometrium and degrades the actomyosin complex with a concomitant loss of its myosin-ATPase activity. The alkaline proteinase is a very labile enzyme, markedly sensitive to SH-group modifying agents and has very high molecular weight at the present state of purification. This proteolytic enzyme could specifically be separated from the main components of the actomyosin complex by extraction with low ionic strength phosphate buffers. The proteinase inhibitor protein may control the activity of this alkaline proteinase during pregnancy and involution. The inhibitor protein raises 15-fold during pregnancy, possibly blocks important steps of intracellular proteolysis and permits organ growth. The dramatic fall of the inhibitor protein activity after parturition, which precedes the loss of weight, could release the proteolytic system, including the alkaline proteinase, and permits controlled intracellular degradation.

  5. Gelatinases and serine proteinase inhibitors of seminal plasma and the reproductive tract of turkey (Meleagris gallopavo).

    PubMed

    Kotłowska, M; Kowalski, R; Glogowski, J; Jankowski, J; Ciereszko, A

    2005-04-01

    This study examined proteolytic enzymes and serine proteinase inhibitors in turkey seminal plasma with relation to their distribution within the reproductive tract and to yellow semen syndrome (YSS). Proteases of blood plasma, extracts from the reproductive tract, and seminal plasma were analyzed by gelatin zymography. We found a clear regional distribution of proteolytic enzymes in the turkey reproductive tract. Each part was characterized by a unique profile of serine proteolytic enzymes of molecular weights ranging from 29 to 88 kDa. The ductus deferens was found to be a site of very intense proteolytic activity. Two metalloproteases of 58 and 66 kDa were detected in all parts of the reproductive tract and seminal plasma. Using electrophoretic methods for detection of anti-trypsin activity, we found three serine proteinase inhibitors in turkey seminal plasma. Two inhibitors were found in the testis and epididymis and a third in the ductus deferens and seminal plasma. Blood plasma was characterized by the presence of two metalloproteinases and one serine proteinase inhibitor (of low migration rate) that were also detected in the reproductive tract. Amidase and anti-trypsin activities (expressed per gram of protein) differed for yellow and white seminal plasma. We concluded that turkey seminal plasma contains metalloproteases, serine proteinases, and serine proteinase inhibitors. The metalloproteases and one proteinase inhibitor are related to blood proteinases but the other two inhibitors and serine proteinases seem to be unique for the reproductive tract.

  6. The isolation and properties of a non-pepsin proteinase from human gastric mucosa.

    PubMed Central

    Roberts, N B; Taylor, W H

    1978-01-01

    1. A non-pepsin proteinase, proteinase 2, was successfully isolated free from pepsinogen (by repetitive chromatography on DEAE- and CM-celluloses) from the gastric mucosa of a patient with a duodenal ulcer and the uninvaded mucosa of a patient with a gastric adenocarcinoma. 2. Proteinases 1a and 1b, found in gastric adenocarcinoma, were not found in the gastic mucosa of these patients. 3. Proteinase 2 was shown to have an asymmetrical broad pH-activity curve with a maximum over the pH range 3.0-3.7. 4. Proteolytic activity of proteinase 2 was inhibited by pepstatin; the concentration of pepstatin giving 50% inhibition is of the order of 3nm. 5. Inhibition of proteolytic activity by carbenoxolone and related triterpenoids indicated that at pH 4.0 proteinase 2 possesses structural characteristics relating it to the pepsins and at pH 7.4 to the pepsinogens. 6. The sites of cleavage of the B-chain of oxidized insulin for proteinase 2 at pH 1.7 and pH 3.5 were shown to be similar to those previously established for human pepsin 3 and for the cathepsin E of rabbit bone marrow. 7. The non-pepsin proteinase 2 (cathepsin) of human gastric mucosa has properties more similar to cathepsin E than to the cathepsins D. Images Fig. 2. Fig. 4. PMID:25649

  7. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

    PubMed Central

    Dougherty, W G; Semler, B L

    1993-01-01

    Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

  8. Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction.

    PubMed

    Genelhu, M S; Zanini, M S; Veloso, I F; Carneiro, A M; Lopes, M T; Salas, C E

    1998-09-01

    We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 microgram = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25 degrees C) for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.

  9. Activation of Proteinase 3 Contributes to Nonalcoholic Fatty Liver Disease and Insulin Resistance

    PubMed Central

    Toonen, Erik JM; Mirea, Andreea-Manuela; Tack, Cees J; Stienstra, Rinke; Ballak, Dov B; van Diepen, Janna A; Hijmans, Anneke; Chavakis, Triantafyllos; Dokter, Wim H; Pham, Christine TN; Netea, Mihai G; Dinarello, Charles A; Joosten, Leo AB

    2016-01-01

    Activation of inflammatory pathways is known to accompany development of obesity-induced nonalcoholic fatty liver disease (NAFLD), insulin resistance and type 2 diabetes. In addition to caspase-1, the neutrophil serine proteases proteinase 3, neutrophil elastase and cathepsin G are able to process the inactive proinflammatory mediators interleukin (IL)-1β and IL-18 to their bioactive forms, thereby regulating inflammatory responses. In this study, we investigated whether proteinase 3 is involved in obesity-induced development of insulin resistance and NAFLD. We investigated the development of NAFLD and insulin resistance in mice deficient for neutrophil elastase/proteinase 3 and neutrophil elastase/cathepsin G and in wild-type mice treated with the neutrophil serine proteinase inhibitor human α-1 antitrypsin. Expression profiling of metabolically relevant tissues obtained from insulin-resistant mice showed that expression of proteinase 3 was specifically upregulated in the liver, whereas neutrophil elastase, cathepsin G and caspase-1 were not. Neutrophil elastase/proteinase 3-deficient mice showed strongly reduced levels of lipids in the liver after being fed a high-fat diet. Moreover, these mice were resistant to high–fat–diet-induced weight gain, inflammation and insulin resistance. Injection of proteinase 3 exacerbated insulin resistance in caspase-1–/– mice, indicating that proteinase 3 acts independently of caspase-1. Treatment with α-1 antitrypsin during the last 10 d of a 16-wk high-fat diet reduced hepatic lipid content and decreased fasting glucose levels. We conclude that proteinase 3 is involved in NAFLD and insulin resistance and that inhibition of proteinase 3 may have therapeutic potential. PMID:27261776

  10. Cationic inhibitors of serine proteinases from buckwheat seeds.

    PubMed

    Tsybina, T A; Dunaevsky, Y E; Musolyamov, A K; Egorov, T A; Belozersky, M A

    2001-09-01

    Preparations of low molecular weight protein inhibitors of serine proteinases have been obtained from buckwheat (Fagopyrum esculentum) seeds by chromatography of seed extract on trypsin-Sepharose 4B, Mono-Q, and Mono-S ion exchangers (FPLC regime). Their molecular masses, determined by mass spectrometry, were 5203 (BWI-1c), 5347 (BWI-2c), 7760 (BWI-3c), and 6031 daltons (BWI-4c). All of the inhibitors possess high pH- and thermal stability in the pH range 2-12. In addition to trypsin, BWI-3c and BWI-4c inhibited chymotrypsin and subtilisin-like bacterial proteases. The N-terminal sequences of all of the inhibitors were determined: BWI-1c (23 residues), BWI-2c (33 residues), BWI-3c (18 residues), and BWI-4c (20 residues). In their physicochemical properties and N-terminal amino acid sequences, the buckwheat seed trypsin inhibitors BWI-3c and BWI-4c appear to belong to potato proteinase inhibitor I family. PMID:11703172

  11. Hormonal control of proteinase activity in squash cotyledons.

    PubMed

    Penner, D; Ashton, F M

    1967-06-01

    A crude proteolytic enzyme preparation which hydrolyzes casein was isolated from the cotyledons of squash seedlings. The presence of ethylene diamine tetraacetate or cysteine did not appreciably affect the activity of the preparation. During the course of germination, the level of proteolytic activity increased in the cotyledons of intact embryos through the third day and then decreased. The presence of the embryonic axis during the first 32 hours of germination was a prerequisite for the development of maximum proteolytic activity.The presence of a cytokinin, such as benzyladenine, kinetin, BTP [6-(benzylamino)-9-(2-tetrahydropyranyl)-9H-purine], or phenyladenine, in the culture solution could reproduce the effect of the embryonic axis. Other growth regulators did not produce this stimulation. High concentrations (1 mm) of all growth regulators examined were inhibitory. Various combinations of growth regulators failed to produce any synergistic effects. 6-Methylpurine inhibited the development of proteinase activity, and this inhibition was only partially restored by benzyladenine.A proteinase was partially purified from the cotyledons of 2-day-old squash seedlings and its synthesis was shown to be under the control of the embryonic axis.

  12. Purification and properties of an alkaline proteinase of Fusarium culmorum.

    PubMed

    Pekkarinen, Anja I; Jones, Berne L; Niku-Paavola, Marja-Leena

    2002-02-01

    The disease Fusarium head blight (scab) causes severe problems for farmers and for the industries that use cereals. It is likely that the fungi that cause scab (Fusarium spp.) use various enzymes when they invade grains. We are studying enzymes that the fungi may use to hydrolyze grain proteins. To do this, Fusarium culmorum was grown in a gluten-containing medium from which an alkaline serine proteinase with a molecular mass of 28.7 kDa was purified by size-exclusion and cation exchange chromatographies. The enzyme was maximally active at pH 8.3-9.6 and 50 degrees C, but was unstable under these conditions. It hydrolyzed the synthetic substrates N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and, to a lesser extent, N-succinyl-Ala-Ala-Pro-Leu p-nitroanilide. It was inhibited by phenylmethanesulfonyl fluoride and chymostatin, but not by soybean trypsin or Bowman-Birk inhibitors. Parts of the amino-acid sequence were up to 82% homologous with those of several fungal subtilisins. One of the active site amino acids was detected and it occupied the same relative position as in the other subtilisins. Therefore, on the basis of these characteristics, the proteinase is subtilisin-like. Purification of the enzyme was complicated by the fact that, when purified, it apparently underwent autolysis. The presence of extraneous protein stabilized the activity.

  13. Diisopropyl fluorophosphate labeling of sperm-associated proteinases

    SciTech Connect

    Odem, R.R.; Willand, J.L.; Polakoski, K.L. )

    1990-02-01

    Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.

  14. [Assay of urine cysteine proteinase in diagnosing gynecological malignant tumors].

    PubMed

    Peng, Z L

    1992-09-01

    Cysteine proteinases (CP) belong to the subclass of endopeptidase, and have been considered to play an important role in spreading cancer cells. Cysteine proteinases in urine (UCP) were determined in 71 healthy women, 76 patients with gynecological benign tumors and 125 cases (173 samples) with gynecological malignant tumors. Enzyme levels were assayed using the artificial substrate CSZ-Ala-Arg-AFC by detecting the release of free AFC with the aid of a fluorometer. The value ranged from upper 80% to 99% of UCP in 71 normal women and was calculated with the percentile method. The results showed that ROC curve displayed a highly sensitive character. The sensitivity and specificity for gynecological malignant tumor were 91.8%, and 71.7% respectively. The sensitivities of UCP for ovarian cancer, cervical cancer, carcinoma of endometrium and cancer of vulva were 96%, 91%, 85.7% and 72.7% respectively. Due to its high sensitivity. It was suggested that UCP assay can be a good screening test to distinguish gynecological malignancy from benign tumors. The accuracy of diagnosing gynecological malignancy may be improved if UCP assay is combined with other tests with higher specificity.

  15. Proteinases of Pseudomonas aeruginosa evoke mucin release by tracheal epithelium.

    PubMed Central

    Klinger, J D; Tandler, B; Liedtke, C M; Boat, T F

    1984-01-01

    We have determined the potential of exoproducts from pathogenic bacteria to stimulate the release of high molecular weight mucins from goblet cells of airway epithelium in a rabbit tracheal explant system. Culture supernatants from proteolytic strains of Pseudomonas aeruginosa and Serratia marcescens, but not supernatants from a number of non-proteolytic strains, released mucins from goblet cells. Highly purified elastase and alkaline proteinase from P. aeruginosa stimulated goblet cell mucin release in a dose-dependent fashion. Lipopolysaccharide, exotoxin A, and alginate of P. aeruginosa did not possess mucin release properties. Proteolytic activity was required for mucin release by P. aeruginosa elastase, but such release in goblet cells was not mediated by cyclic AMP. Morphologic studies suggested rapid release of mucins from goblet cells was response to elastase by a process resembling apocrine secretion. Several nonbacterial proteinases mimicked the effect of Pseudomonas proteases. These studies provide support for the hypothesis that bacterial and other play a role in the pathogenesis of mucus hypersecretion in acute and chronic lung infections. Images PMID:6568227

  16. Radioimmunoassay of aspartate aminotransferase isoenzymes in human serum

    SciTech Connect

    Leung, F.Y.; Niblock, A.E.; Henderson, A.R.

    1984-08-01

    A description is given of the development of a sensitive, specific radioimmunoassay for the cytoplasmic and mitochondrial isoenzymes of human aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase; EC 2.6.1.1). Isoenzymes from human heart tissue were purified to homogeneity and used to raise high-titer antisera in rabbits. The antisera were partly purified by selective column chromatography. The Bolton-Hunter reagent was used to radioiodinate the isoenzymes. The assay requires 100 microL of serum, includes a solid-phase second-antibody separation, and can be completed in less than 3 h. There was no cross reactivity between the two isoenzymes. As little as 5 micrograms (50 pmol) of each aspartate aminotransferase can be measured per liter of serum.

  17. Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites.

    PubMed

    Polonais, Valerie; Shea, Michael; Soldati-Favre, Dominique

    2011-08-01

    Aspartic proteases are important virulence factors for pathogens and are recognized as attractive drug targets. Seven aspartic proteases (ASPs) have been identified in Toxoplasma gondii genome. Bioinformatics and phylogenetic analyses regroup them into five monophyletic groups. Among them, TgASP1, a coccidian specific aspartic protease related to the food vacuole plasmepsins, is associated with the secretory pathway in non-dividing cells and relocalizes in close proximity to the nascent inner membrane complex (IMC) of daughter cells during replication. Despite a potential role for TgASP1 in IMC formation, the generation of a conventional knockout of the TgASP1 gene revealed that this protease is not required for T. gondii tachyzoite survival or for proper IMC biogenesis.

  18. Synthesis and In Vitro Evaluation of Aspartate Transcarbamoylase Inhibitors

    PubMed Central

    Coudray, Laëtitia; Pennebaker, Anne F.; Montchamp, Jean-Luc

    2009-01-01

    The design, synthesis, and evaluation of a series of novel inhibitors of aspartate transcarbamoylase (ATCase) are reported. Several submicromolar phosphorus-containing inhibitors are described, but all-carboxylate compounds are inactive. Compounds were synthesized to probe the postulated cyclic transition-state of the enzyme-catalyzed reaction. In addition, the associated role of the protonation state at the phosphorus acid moiety was evaluated using phosphinic and carboxylic acids. Although none of the synthesized inhibitors is more potent than N-phosphonacetyl-L-aspartate (PALA), the compounds provide useful mechanistic information, as well as the basis for the design of future inhibitors and/or prodrugs. PMID:19828320

  19. Structural Insights into a Novel Class of Aspartate Aminotransferase from Corynebacterium glutamicum

    PubMed Central

    Son, Hyeoncheol Francis; Kim, Kyung-Jin

    2016-01-01

    Aspartate aminotransferase from Corynebacterium glutamicum (CgAspAT) is a PLP-dependent enzyme that catalyzes the production of L-aspartate and α-ketoglutarate from L-glutamate and oxaloacetate in L-lysine biosynthesis. In order to understand the molecular mechanism of CgAspAT and compare it with those of other aspartate aminotransferases (AspATs) from the aminotransferase class I, we determined the crystal structure of CgAspAT. CgAspAT functions as a dimer, and the CgAspAT monomer consists of two domains, the core domain and the auxiliary domain. The PLP cofactor is found to be bound to CgAspAT and stabilized through unique residues. In our current structure, a citrate molecule is bound at the active site of one molecule and mimics binding of the glutamate substrate. The residues involved in binding of the PLP cofactor and the glutamate substrate were confirmed by site-directed mutagenesis. Interestingly, compared with other AspATs from aminotransferase subgroup Ia and Ib, CgAspAT exhibited unique binding sites for both cofactor and substrate; moreover, it was found to have unusual structural features in the auxiliary domain. Based on these structural differences, we propose that CgAspAT does not belong to either subgroup Ia or Ib, and can be categorized into a subgroup Ic. The phylogenetic tree and RMSD analysis also indicates that CgAspAT is located in an independent AspAT subgroup. PMID:27355211

  20. Limited proteolysis by macrophage elastase inactivities human. cap alpha. /sub 1/-proteinase inhibitor

    SciTech Connect

    Banda, M.J.; Clark, E.J.; Werb, Z.

    1980-12-01

    Ever since the initial description of ..cap alpha../sub 1/-proteinase inhibitor (..cap alpha../sub 1/PI), the role of this plasma glycoprotein and its allelic polymorphism in disease and in healthy physiology has been the subject of much investigation, ..cap alpha../sub 1/PI inactivates a number of serine proteinases, including granulocyte elastase, and thus affords protection from the connective tissue degradation mediated by this class of proteinases. Because an imbalance in the ratio between ..cap alpha../sub 1/PI and proteinase may contribute to the development of destructive lung diseases, proteinases have been implicated in the pathogenesis of pulmonary emphysema. Both macrophages and polymorphonuclear leukocytes have been implicated in disruption of the ..cap alpha../sub 1/PI-proteinase balance. In this report, a new mechanism for alteration of the ..cap alpha../sub 1/PI-proteinase balance is demonstrated. It was found that the purified form of macrophage elastase catalytically degrades and inactivates ..cap alpha../sub 1/PI so that it no longer inhibits the elastinolytic activity of granulocyte elastase.

  1. Aspartate analysis in formulations using a new enzyme sensor.

    PubMed

    Campanella, L; Aturki, Z; Sammartino, M P; Tomassetti, M

    1995-04-01

    A biosensor has been developed for the purpose of directly analysing aspartate in pharmaceutical formulations and aspartame in sweeteners. This biosensor consists of an ammonia-sensitive gas-diffusion electrode and the enzyme L-aspartase immobilized by means of polyazetidine on a dialysis membrane.

  2. [Phospholipase, proteinase and hemolytic activities of Candida albicans isolates obtained from clinical specimens].

    PubMed

    Yenişehirli, Gülgün; Bulut, Yunus; Tunçoglu, Ebru

    2010-01-01

    This study was aimed to determine the phospholipase, proteinase and hemolytic activities of Candida albicans strains isolated from clinical specimens. A total of 147 C. albicans strains isolated from blood (n = 29), respiratory specimens (n = 44), urine (n = 52), pus (n = 17) and stool (n = 5) were included in the study. Proteinase and phospholipase activities were determined in 81% and 76% of C. albicans isolates, respectively. All C. albicans isolates revealed beta-hemolytic activity on Sabouraud dextrose agar supplemented with 7% fresh sheep blood and 3% glucose. Phospholipase and proteinase positivity were highest among the respiratory isolates. Proteinase activity of respiratory (93%) and blood (83%) isolates were statistically significantly higher than that of urine (77%; p = 0.032), pus (65%; p = 0.007) and stool isolates (60%; p = 0.026). While phospholipase activity showed statistically significant difference between respiratory (84%) and pus (53%) isolates (p = 0.014), no statistically significant difference was determined for blood (79%), urine (75%) and stool (80%) isolates (p > 0.05). Two blood isolates with 4+ proteinase activity and 3 urine isolates with 3+ proteinase activity were phospholipase negative. One urine isolate with 4+ phospholipase activity and 4 with 3+ phospholipase activity were proteinase negative. Phospholipase and proteinase negative 1 isolate from stool and 1 isolate from pus were found to have 4+ hemolytic activity. In conclusion, besides proteinase and phospholipase enzyme activities, hemolytic activity may play an important role for the C.albicans infections. The pathogenetic role of these virulence factors should be evaluated by further clinical studies.

  3. Regulation of N-methyl-D-aspartate receptor expression and N-methyl-D-aspartate-induced cellular response during chronic hypoxia in differentiated rat PC12 cells.

    PubMed

    Kobayashi, S; Millhorn, D E

    2000-01-01

    The purpose of the present study was to examine the effect of chronic hypoxia on N-methyl-D-aspartate-mediated cellular responses in differentiated PC12 cells. PC12 cells were differentiated by treatment with nerve growth factor. Patch-clamp analysis in differentiated PC12 cells showed that extracellularly applied N-methyl-D-aspartate induced an inward current that was abolished by the presence of the N-methyl-D-aspartate receptor antagonist MK-801. Results from Ca(2+) imaging experiments showed that N-methyl-D-aspartate induced an elevation in intracellular free Ca(2+) which was also abolished by MK-801. We also examined the effect of hypoxia on the N-methyl-D-aspartate-induced current in nerve growth factor-treated cells. We found that the N-methyl-D-aspartate-induced inward current and the N-methyl-D-aspartate-induced elevation in intracellular free Ca(2+) were markedly attenuated by chronic hypoxia. We next examined the possibility that the reduced N-methyl-D-aspartate responsiveness was due to down-regulation of N-methyl-D-aspartate receptor levels. Northern blot and immunoblot analyses showed that both messenger RNA and protein levels for N-methyl-D-aspartate receptor subunit 1 were markedly decreased during hypoxia. However, the messenger RNA for N-methyl-D-aspartate receptor subunit 2C was increased, whereas the protein level for subunit 2C did not change. Our results indicate that differentiated PC12 cells express functional N-methyl-D-aspartate receptors and that chronic exposure to hypoxia attenuates the N-methyl-D-aspartate-induced Ca(2+) accumulation in these cells via down-regulation of N-methyl-D-aspartate receptor subunit 1. This mechanism may play an important role in protecting PC12 cells against hypoxic stress. PMID:11113364

  4. Synthesis and structure-activity relationships of a series of penicillin-derived HIV proteinase inhibitors containing a stereochemically unique peptide isostere.

    PubMed

    Holmes, D S; Bethell, R C; Cammack, N; Clemens, I R; Kitchin, J; McMeekin, P; Mo, C L; Orr, D C; Patel, B; Paternoster, I L

    1993-10-15

    A series of HIV-1 proteinase inhibitors was synthesized based upon a single penicillin derived thiazolidine moiety. Reaction of the C-4 carboxyl group with (R)-phenylalaninol gave amide 10 which was a moderately potent inhibitor of HIV-1 proteinase (IC50 = 0.15 microM). Further modifications based on molecular modeling studies led to compound 48 which contained a stereochemically unique statine-based isostere. This was a potent competitive inhibitor (Ki = 0.25 nM) with antiviral activity against HIV-1 in vitro (5 microM). Neither modification to the benzyl group in an attempt to improve interaction with the S2' pocket, nor introduction of a hydrogen bond donating group to interact with residue Gly48' resulted in improved inhibitory or antiviral activity. PMID:8230099

  5. Mammalian tolloid proteinases: role in growth factor signalling.

    PubMed

    Troilo, Helen; Bayley, Christopher P; Barrett, Anne L; Lockhart-Cairns, Michael P; Jowitt, Thomas A; Baldock, Clair

    2016-08-01

    Tolloid proteinases are essential for tissue patterning and extracellular matrix assembly. The members of the family differ in their substrate specificity and activity, despite sharing similar domain organization. The mechanisms underlying substrate specificity and activity are complex, with variation between family members, and depend on both multimerization and substrate interaction. In addition, enhancers, such as Twisted gastrulation (Tsg), promote cleavage of tolloid substrate, chordin, to regulate growth factor signalling. Although Tsg and mammalian tolloid (mTLD) are involved in chordin cleavage, no interaction has been detected between them, suggesting Tsg induces a change in chordin to increase susceptibility to cleavage. All members of the tolloid family bind the N terminus of latent TGFβ-binding protein-1, providing support for their role in TGFβ signalling. PMID:27391803

  6. Triacontanol negatively modulates the jasmonic acid-stimulated proteinase inhibitors in tomato (Lycopersicon esculentum).

    PubMed

    Ramanarayan, Krishnamurthy; Swamy, Gangadharamurthy Sivakumar

    2004-04-01

    Triacontanol (TRIA), a long chain aliphatic alcohol (C30H61OH) reverses the effect of jasmonic acid (JA) in inducing proteinase inhibitors (PIs) in tomato leaves. Porcine pancreas trypsin and Spodoptera litura gut proteinases were inhibited in the presence of leaf proteins treated with JA, and TRIA partially reverses this effect. Spodoptera litura larvae fed with tomato leaves treated with JA were reduced in body weight and TRIA is able to partially reverse this JA-induced effect. These results reflect the partial reversal effect of TRIA in down regulating the JA-induced production of proteinase inhibitors.

  7. Ozone effects on inhibitors of human neutrophil proteinases

    SciTech Connect

    Smith, C.E.; Stack, M.S.; Johnson, D.A.

    1987-02-15

    The effects of ozone on human alpha 1-proteinase inhibitor (A-1-PI), alpha 1-antichymotrypsin (A-1-Achy), bronchial leukocyte proteinase inhibitor (BLPI), and Eglin C were studied using in vitro exposures in phosphate-buffered solutions. Following ozone exposure, inhibitory activities against human neutrophil elastase (HNE) and/or cathepsin G (Cat G) were measured. Exposure of A-1-PI to 50 mol O3/mol protein resulted in a complete loss of HNE inhibitory activity, whereas A-1-Achy lost only 50% of its Cat G inhibitory activity and remained half active even after exposure to 250 mol of O3. At 40 mol O3/mol protein, BLPI lost 79% of its activity against HNE and 87% of its Cat G inhibitory activity. Eglin C, a leech-derived inhibitor, lost 81% of its HNE inhibitory activity and 92% of its ability to inhibit Cat G when exposed to 40 mol O3/mol. Amino acid analyses of ozone-exposed inhibitors showed destruction of Trp, Met, Tyr, and His with as little as 10 mol O3/mol protein, and higher levels of O3 resulted in more extensive oxidation of susceptible residues. The variable ozone susceptibility of the different amino acid residues in the four proteins indicated that oxidation was a function of protein structure, as well as the inherent susceptibility of particular amino acids. Exposure of A-1-PI and BLPI in the presence of the antioxidants, Trolox C (water soluble vitamin E) and ascorbic acid (vitamin C), showed that antioxidant vitamins may protect proteins from oxidative inactivation by ozone. Methionine-specific modification of BLPI reduced its HNE and Cat G inhibitory activities. Two moles of N-chlorosuccinimide per mole of BLPI methionine caused an 80% reduction in activity against Cat G, but only a 40% reduction in HNE inhibitory activity.

  8. Proinflammatory genes expression in granulocytes activated by native proteinase-binding fragments of anti-proteinase 3 IgG.

    PubMed

    Surmiak, M; Kaczor, M; Sanak, M

    2015-08-01

    The classical pathway of neutrophils activation due to cytoplasmic antineutrophil cytoplasmic antibodies (c-ANCA) involves specific antigen binding to proteinase-3 and activation of the immunoglobulin G receptors by the constant fragment of the antibody. A requirement for this double signaling was suggested also because proteinase-3 is presented within a complex of NB-1 glycoprotein lacking transmembrane domain. An integrin Mac-1 receptor was postulated to cooperate in neutrophil stimulation by anti-proteinase 3 (anti-PR3). A characteristic profile of transcriptional activation of neutrophils by c-ANCA was described by us previously. We ascertained mRNA expression of neutrophils following stimulation with antigen-binding fragments of native anti-PR3 IgG. Expression of targeted transcripts was compared with our previous results, in which intact anti-PR3 IgG was used. Human neutrophils were isolated from healthy volunteers negative for ANCAs. Antigen-binding fragments of human anti-PR3 were prepared from sera of patients with granulomatosis with polyangiitis. We analyzed reactive oxygen species production and abundance of mRNA of 151 genes by quantitative real time-PCR in neutrophils stimulated with anti-PR3 IgG F(ab)(2). We observed a consistent upregulation of 17 genes (CYSLTR1, HPGD, IL1R1, IL1RL1, MAPK1, MAPK8, NR3C1, PLA2G7, PTGDR, CD302, DNAJB1, F2R, F2RL1, IER3, RAC1, RPL41, PTGER3), whereas other 9 genes were up-regulated only in some donors. No reactive oxygen species production was observed in neutrophils stimulated with anti-PR3 F(ab)(2). Stimulation of neutrophils with F(ab)(2) of anti-PR3 autoantibodies activated cells to a lesser extent than intact IgG. However, several cellular pathways were up-regulated, involving calcium and phosphatidylinositol 3-kinase AKT, nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK) signaling. Interestingly, binding of F(ab)(2) to the PR-3 present on the surface of neutrophil is sufficient for lipid

  9. Homotropic effects in aspartate transcarbamoylase. What happens when the enzyme binds a single molecule of the bisubstrate analog N-phosphonacetyl-L-aspartate?

    PubMed

    Foote, J; Schachman, H K

    1985-11-01

    The active sites of aspartate transcarbamoylase from Escherichia coli were titrated by measuring the decrease in the enzyme-catalyzed arsenolysis of N-carbamoyl-L-aspartate caused by the addition of the tight-binding inhibitor, N-phosphonacetyl-L-aspartate. Because the enzyme is a poor catalyst for this non-physiological reaction, high concentrations are required for the assays (more than 1000-fold the dissociation constant of the reversibly bound inhibitor) and, therefore, virtually all of the bisubstrate analog is bound. From the endpoint of the titration, 5.7 active sites were calculated, in excellent agreement with the number, six, based on the structure of the enzyme. Simple inhibition was observed only when the molar ratio of inhibitor to enzyme exceeded five; under these conditions, as shown in earlier physical chemical studies, the R-conformational state of the enzyme is the sole or predominant species. At low ratios of inhibitor to enzyme, the addition of inhibitor caused an increase in activity which is attributable to the conversion of the enzyme from the low-activity T-state to the much more active R-state. Comparison of the linear increase in activity as a function of inhibitor concentration at the low molar ratio (0.01, i.e. 1 inhibitor/600 active sites) with the activity lost at the high ratio provided a direct value for the mean number of active sites converted from the T-state to the R-state as a result of the binding of one bisubstrate analog to an enzyme molecule. This number was four with Mg X ATP or carbamoyl phosphate present and 4.7 for the enzyme in the presence of Mg X PPi, values approaching or identical to the theoretical maximum, 4.7, for a concerted transition with all of the active sites of the molecule changing from the T- to R-state upon the formation of a binary complex of hexameric enzyme with a single inhibitor. With the enzyme in the absence of effectors or with Mg X CTP present, the titrations showed that an average of two and

  10. Successive Use of Non-Host Plant Proteinase Inhibitors Required for Effective Inhibition of Helicoverpa armigera Gut Proteinases and Larval Growth1

    PubMed Central

    Harsulkar, Abhay M.; Giri, Ashok P.; Patankar, Aparna G.; Gupta, Vidya S.; Sainani, Mohini N.; Ranjekar, Prabhakar K.; Deshpande, Vasanti V.

    1999-01-01

    We report on the efficacy of proteinase inhibitors (PIs) from three host plants (chickpea [Cicer arietinum], pigeonpea [Cajanus cajan], and cotton [Gossypium arboreum]) and three non-host (groundnut [Arachis hypogea], winged bean [Psophocarpus tetragonolobus], and potato [Solanum tuberosum]) in retarding the growth of Helicoverpa armigera larvae, a devastating pest of important crop plants. Enzyme assays and electrophoretic analysis of interaction of H. armigera gut proteinases (HGPs) with PIs revealed that non-host PIs inhibited HGP activity efficiently whereas host PIs were ineffective. In the electrophoretic assay, trypsin inhibitor activity bands were detected in all of the host and non-host plants, but HGP inhibitor activity bands were present only in non-host plants (except cotton in the host plant group). H. armigera larvae reared on a diet containing non-host PIs showed growth retardation, a reduction in total and trypsin-like proteinase activity, and the production of inhibitor-insensitive proteinases. Electrophoretic analysis of PI-induced HGP showed differential regulation of proteinase isoforms. Interestingly, HGP activity induced in response to dietary potato PI-II was inhibited by winged bean PIs. The optimized combination of potato PI-II and winged bean PIs identified in the present study and their proposed successive use has potential in developing H. armigera-resistant transgenic plants. PMID:10517841

  11. Synthesis of β-alanine from L-aspartate using L-aspartate-α-decarboxylase from Corynebacterium glutamicum.

    PubMed

    Shen, Yan; Zhao, Lianzhen; Li, Youran; Zhang, Liang; Shi, Guiyang

    2014-08-01

    β-Alanine is mainly produced by chemical methods in current industrial processes. Here, panD from Corynebacterium glutamicum encoding L-aspartate-α-decarboxylase (ADC) was cloned and expressed in Escherichia coli BL21(DE3). ADC C.g catalyzes the α-decarboxylation of L-aspartate to β-alanine. The purified ADC C.g was optimal at 55 °C and pH 6 with excellent stability at 16-37 °C and pH 4-7. A pH-stat directed, fed-batch feeding strategy was developed for enzymatic synthesis of β-alanine to keep the pH value within 6-7.2 and thus attenuate substrate inhibition. A maximum conversion of 97.2 % was obtained with an initial 5 g L-aspartate/l and another three feedings of 0.5 % (w/v) L-aspartate at 8 h intervals. The final β-alanine concentration was 12.85 g/l after 36 h. This is the first study concerning the enzymatic production of β-alanine by using ADC.

  12. Kinetic analysis of a general model of activation of aspartic proteinase zymogens involving a reversible inhibitor. II. Contribution of the uni- and bimolecular activation routes.

    PubMed

    Muñoz-López, A; Sotos-Lomas, A; Arribas, E; Escribano, J; Masia-Perez, J; Muñoz-Muñoz, J L; Varon, R

    2007-04-01

    From the kinetic study carried out in part I of this series (preceding article) an analysis quantifying the relative contribution to the global process of the uni- and bimolecular routes has been carried out. This analysis suggests a way to predict the time course of the relative contribution as well as the effect on this relative weight of the initial zymogen, inhibitor and activating enzyme concentrations.

  13. Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice.

    PubMed

    Quilis, Jordi; López-García, Belén; Meynard, Donaldo; Guiderdoni, Emmanuel; San Segundo, Blanca

    2014-04-01

    Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants.

  14. Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris.

    PubMed

    Zani, Marie-Louise; Nobar, Shila M; Lacour, Sandrine A; Lemoine, Soazig; Boudier, Christian; Bieth, Joseph G; Moreau, Thierry

    2004-06-01

    Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast alpha-factor cDNA and expressed in the Pichia pastoris yeast expression system. Full-length elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fast-acting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k(ass) values of 2-4 x 10(6) m(-1).s(-1), while dissociation rate constants k(diss) were found to be in the 10(-4) s(-1) range, indicating low reversibility of the complexes. The equilibrium dissociation constant K(i) for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from k(ass) and k(diss) values for neutrophil elastase and proteinase 3. K(i) values were found to be in the 10(-10) molar range and virtually identical for both inhibitors. Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases.

  15. Structures of aspartate aminotransferases from Trypanosoma brucei, Leishmania major and Giardia lamblia

    PubMed Central

    Abendroth, Jan; Choi, Ryan; Wall, Abigail; Clifton, Matthew C.; Lukacs, Christine M.; Staker, Bart L.; Van Voorhis, Wesley; Myler, Peter; Lorimer, Don D.; Edwards, Thomas E.

    2015-01-01

    The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm. PMID:25945710

  16. Structures of aspartate aminotransferases from Trypanosoma brucei, Leishmania major and Giardia lamblia.

    PubMed

    Abendroth, Jan; Choi, Ryan; Wall, Abigail; Clifton, Matthew C; Lukacs, Christine M; Staker, Bart L; Van Voorhis, Wesley; Myler, Peter; Lorimer, Don D; Edwards, Thomas E

    2015-05-01

    The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm. PMID:25945710

  17. Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination

    PubMed Central

    2012-01-01

    Background Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Results Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. Conclusions Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role

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

    PubMed

    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

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

    PubMed

    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

  20. A novel cysteine proteinase (CP65) of Trichomonas vaginalis involved in cytotoxicity.

    PubMed

    Alvarez-Sánchez, M E; Avila-González, L; Becerril-García, C; Fattel-Facenda, L V; Ortega-López, J; Arroyo, R

    2000-04-01

    The goal of this study was to demonstrate the participation in cellular damage of a Trichomonas vaginalis proteinase with a molecular mass of 65 kDa (CP65). By two dimensional gelatin-gel electrophoresis of trichomonad proteins we detected four spots with proteolytic activity on the 65 kDa region, but only one, pI 7.2, binds to the HeLa cell surface. By indirect immunofluorescence, rabbit antibodies against this proteinase localized the CP65 on the plasma membrane and in the cytoplasm of T. vaginalis. Pretreatment of parasites with the specific anti-CP65 antibody reduced trichomonal cytotoxicity to HeLa cell monolayers. The specific cysteine proteinase inhibitor, L-3-carboxy-2, 3-trans-epoxypropionyl-leucylamido (4-guanidino) butane (E64) abrogated the proteinase activity and reduced cytotoxicity levels of T. vaginalis in cell culture monolayers, indicating that the trichomonad CP65 is a cysteine proteinase. Activity of the CP65 proteinase was optimal at pH 5.5 and 37 degrees C, conditions similar to those of patients with trichomonosis. Also, this proteinase degraded some of the proteins found in the vagina, i.e. collagen IV and fibronectin, but not laminin-1 or haemoglobin. Finally, immunoprecipitation assays showed that sera and vaginal washes from trichomonosis patient possess anti-CP65 antibodies. In conclusion, results presented in this work demonstrate that the CP65 is a surface cysteine proteinase involved in T. vaginalis cytotoxicity to HeLa cell monolayers, as a virulence factor. It is immunogenic during human infection and degrades some extracellular matrix proteins, i.e. collagen IV and fibronectin. PMID:10764610

  1. A novel cysteine proteinase (CP65) of Trichomonas vaginalis involved in cytotoxicity.

    PubMed

    Alvarez-Sánchez, M E; Avila-González, L; Becerril-García, C; Fattel-Facenda, L V; Ortega-López, J; Arroyo, R

    2000-04-01

    The goal of this study was to demonstrate the participation in cellular damage of a Trichomonas vaginalis proteinase with a molecular mass of 65 kDa (CP65). By two dimensional gelatin-gel electrophoresis of trichomonad proteins we detected four spots with proteolytic activity on the 65 kDa region, but only one, pI 7.2, binds to the HeLa cell surface. By indirect immunofluorescence, rabbit antibodies against this proteinase localized the CP65 on the plasma membrane and in the cytoplasm of T. vaginalis. Pretreatment of parasites with the specific anti-CP65 antibody reduced trichomonal cytotoxicity to HeLa cell monolayers. The specific cysteine proteinase inhibitor, L-3-carboxy-2, 3-trans-epoxypropionyl-leucylamido (4-guanidino) butane (E64) abrogated the proteinase activity and reduced cytotoxicity levels of T. vaginalis in cell culture monolayers, indicating that the trichomonad CP65 is a cysteine proteinase. Activity of the CP65 proteinase was optimal at pH 5.5 and 37 degrees C, conditions similar to those of patients with trichomonosis. Also, this proteinase degraded some of the proteins found in the vagina, i.e. collagen IV and fibronectin, but not laminin-1 or haemoglobin. Finally, immunoprecipitation assays showed that sera and vaginal washes from trichomonosis patient possess anti-CP65 antibodies. In conclusion, results presented in this work demonstrate that the CP65 is a surface cysteine proteinase involved in T. vaginalis cytotoxicity to HeLa cell monolayers, as a virulence factor. It is immunogenic during human infection and degrades some extracellular matrix proteins, i.e. collagen IV and fibronectin.

  2. Behavior of aspartic acid as a corrosion inhibitor for steel

    SciTech Connect

    Kalota, D.J.; Silverman, D.C. )

    1994-02-01

    Corrosion inhibition of steel by aspartic acid (C[sub 4]H[sub 7]NO[sub 4]), an amino acid of low molecular weight, was found to depend strongly on pH. At a pH less than the ionization constant at [approximately]9.5 to 10 (measured at 25 C), C[sub 4]H[sub 7]NO[sub 4] appeared to accelerate corrosion. Above the pH, it acted as a corrosion inhibitor for steel. A specially constructed potential-pH diagram for iron (Fe) that incorporated C[sub 4]H[sub 7]NO[sub 4] showed the change in behavior was accompanied by the most stable thermodynamic state changing from an iron aspartate complex to iron oxide. Polymerized C[sub 4]H[sub 7]NO[sub 4] (polyaspartic acid) behaved in a similar manner. Some other amino acids of low molecular weight behaved similarly.

  3. Cloning, expression, and characterization of a milk-clotting aspartic protease gene (Po-Asp) from Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Chen, Liguo; Tan, Qi; Shang, Xiaodong; Ma, Aimin

    2014-02-01

    An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3' portion of cDNA in our previous work. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. The mature polypeptide with 327 amino acid residues had a calculated molecular mass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68-80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity.

  4. Enhancement of insecticides against codling moth (Lepidoptera: Tortricidae) with L-aspartate in laboratory and field experiments.

    PubMed

    Pszczolkowski, Maciej A; Brown, John J

    2014-06-01

    The idea of enhancing insecticide efficacy against phytophagous insects with feeding stimulators was proposed as early as the 1960s, and a number of insect feeding stimulators based on sugars, molasses, and cottonseed extracts, biologically active at relatively high (5% and higher) concentrations, have been advocated. Here, we show that an acidic amino acid, L-aspartate, stimulates feeding in codling moth neonates at much lower concentrations and acts as an effective tank-mixed additive for increasing efficacy of insecticides, reducing fruit damage, and increasing yield of the fruit. In laboratory experiments, 1 mg/ml L-aspartate increased foliage consumption by 40-60% and, when added to Assail 30 SG, Baythroid XL, Delegate WG, or Carbaryl 80S, maintained its feeding stimulatory properties and reduced LD50(s) by approximately 10 times. In a 3-yr field trial, addition of L-aspartate to the aforementioned insecticides at 395 g/ha reduced fruit damage from approximately 6%, on average to < 1% for first-generation codling moth, and from approximately 20 to approximately 5% for the second generation. Interestingly, addition of L-aspartate also increased the average weight of apples by 11-27%, as measured at the time of harvest.

  5. Vesicular uptake and exocytosis of l-aspartate is independent of sialin

    PubMed Central

    Morland, Cecilie; Nordengen, Kaja; Larsson, Max; Prolo, Laura M.; Farzampour, Zoya; Reimer, Richard J.; Gundersen, Vidar

    2013-01-01

    The mechanism of release and the role of l-aspartate as a central neurotransmitter are controversial. A vesicular release mechanism for l-aspartate has been difficult to prove, as no vesicular l-aspartate transporter was identified until it was found that sialin could transport l-aspartate and l-glutamate when reconstituted into liposomes. We sought to clarify the release mechanism of l-aspartate and the role of sialin in this process by combining l-aspartate uptake studies in isolated synaptic vesicles with immunocyotchemical investigations of hippocampal slices. We found that radiolabeled l-aspartate was taken up into synaptic vesicles. The vesicular l-aspartate uptake, relative to the l-glutamate uptake, was twice as high in the hippocampus as in the whole brain, the striatum, and the entorhinal and frontal cortices and was not inhibited by l-glutamate. We further show that sialin is not essential for exocytosis of l-aspartate, as there was no difference in ATP-dependent l-aspartate uptake in synaptic vesicles from sialin-knockout and wild-type mice. In addition, expression of sialin in PC12 cells did not result in significant vesicle uptake of l-aspartate, and depolarization-induced depletion of l-aspartate from hippocampal nerve terminals was similar in hippocampal slices from sialin-knockout and wild-type mice. Further, there was no evidence for nonvesicular release of l-aspartate via volume-regulated anion channels or plasma membrane excitatory amino acid transporters. This suggests that l-aspartate is exocytotically released from nerve terminals after vesicular accumulation by a transporter other than sialin.—Morland, C., Nordengen, K., Larsson, M., Prolo, L. M., Farzampour, Z., Reimer, R. J., Gundersen, V. Vesicular uptake and exocytosis of l-aspartate is independent of sialin. PMID:23221336

  6. Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers?

    PubMed

    Sugawara, Hiroaki; Shibuya, Kenichi; Yoshioka, Toshihito; Hashiba, Teruyoshi; Satoh, Shigeru

    2002-03-01

    Senescence of carnation petals is accompanied by autocatalytic ethylene production and wilting of the petals; the former is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes and the latter is related to the expression of a cysteine proteinase (CPase) gene. CPase is probably responsible for the degradation of proteins, leading to the decomposition of cell components and resultant cell death during the senescence of petals. The carnation plant also has a gene for the CPase inhibitor (DC-CPIn) that is expressed abundantly in petals at the full opening stage of flowers. In the present study, DC-CPIn cDNA was cloned and expressed in E. coli. The recombinant DC-CPIn protein completely inhibited the activities of a proteinase (CPase) extracted from carnation petals and papain. Northern blot analysis showed that the mRNA for CPase (DC-CP1) accumulated in large amounts, whereas that for DC-CPIn disappeared, corresponding to the onset of petal wilting in flowers undergoing natural senescence and exogenous ethylene-induced senescence. Based on these findings, a role of DC-CPIn in the regulation of petal wilting is suggested; DC-CPIn acts as a suppressor of petal wilting, which probably functions to fine-tune petal wilting in contrast to coarse tuning, the up-regulation of CPase activity by gene expression.

  7. Production and purification of antioxidant peptides from a mungbean meal hydrolysate by Virgibacillus sp. SK37 proteinase.

    PubMed

    Lapsongphon, Nawaporn; Yongsawatdigul, Jirawat

    2013-11-15

    Antioxidant peptides of mungbean meal hydrolysed by Virgibacillus sp. SK37 proteinases (VH), Alcalase (AH) and Neutrase (NH) were investigated. The antioxidant activities based on 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) radical-scavenging, ferric-reducing antioxidant power (FRAP) and metal chelation of VH were comparable to those of NH. VH was purified using ultrafiltration, ion exchange and gel filtration chromatography. The purified peptides (F37) from VH, which had the highest specific antioxidant activity, consisted of four peptides containing an arginine residue at their C-termini. In addition, the ABTS radical-scavenging activity of the purified peptides (F42) at 0.148mg/ml was comparable to that of 1mM of butylated hydroxytoluene (BHT). These two fractions were stable over a wide pH (4-10) and temperature (25-121°C) range. Virgibacillus sp. SK37 proteinase is a potential processing-aid for the production of a mungbean meal hydrolyzate with antioxidant properties. PMID:23790878

  8. Interference of Wegener's granulomatosis autoantibodies with neutrophil Proteinase 3 activity.

    PubMed

    van de Wiel, B A; Dolman, K M; van der Meer-Gerritsen, C H; Hack, C E; von dem Borne, A E; Goldschmeding, R

    1992-12-01

    Classic anti-neutrophil cytoplasmic autoantibodies (C-ANCA) are disease-specific markers of Wegener's granulomatosis (WG). The possible pathogenetic role of these autoantibodies, which are directed against Proteinase 3 (PR3), is not yet clear. We studied the effect of C-ANCA on PR3 proteolytic activity and on the complexation of PR3 with alpha 1-antitrypsin (alpha 1AT). C-ANCA IgG from eight patients with active WG significantly inhibited PR3 proteolytic activity, particularly towards elastin (median 84.2% inhibition). C-ANCA IgG significantly inhibited the complexation of PR3 with alpha 1AT (median 58.8% inhibition). Moreover, addition of purified PR3 to C-ANCA-positive sera from WG patients yielded less complexes with alpha 1AT (median 44.8%) compared with sera containing perinuclear anti-neutrophil cytoplasmic autoantibodies (P-ANCA) or ANCA-negative sera. These findings indicate the existence of a hitherto unknown property of C-ANCA, which may be of importance in the pathogenesis of WG.

  9. Proteinase 3-ANCA Vasculitis versus Myeloperoxidase-ANCA Vasculitis

    PubMed Central

    Hilhorst, Marc; van Paassen, Pieter

    2015-01-01

    In patients with GN or vasculitis, ANCAs are directed against proteinase 3 (PR3) or myeloperoxidase (MPO). The differences between PR3-ANCA-associated vasculitis (AAV) and MPO-AAV described in the past have been supplemented during the last decade. In this review, we discuss the differences between these two small-vessel vasculitides, focusing especially on possible etiologic and pathophysiologic differences. PR3-AAV is more common in northern parts of the world, whereas MPO-AAV is more common in southern regions of Europe, Asia, and the Pacific, with the exception of New Zealand and Australia. A genetic contribution has been extensively studied, and there is a high prevalence of the HLA-DPB1*04:01 allele in patients with PR3-AAV as opposed to patients with MPO-AAV and/or healthy controls. Histologically, MPO-AAV and PR3-AAV are similar but show qualitative differences when analyzed carefully. Clinically, both serotypes are difficult to distinguish, but quantitative differences are present. More organs are affected in PR3-AAV, whereas renal limited vasculitis occurs more often in patients with MPO-AAV. For future clinical trials, we advocate classifying patients by ANCA serotype as opposed to the traditional disease type classification. PMID:25956510

  10. The kinetics of proteinase K digestion of linear prion polymers.

    PubMed

    Masel, J; Jansen, V A

    1999-09-22

    Transmissible spongiform encephalopathies such as scrapie are caused by a protein-only infectious agent, known as a prion. It is not clear how a protein can be capable of replicating itself, and the mechanism remains controversial. One influential model hypothesizes that prions are nucleated, macroscopically linear polymers. We investigated the theoretical kinetics of this model and derived predictions which could be used to test the model. In the model, the polymerization and depolymerization rates are independent polymer size. This leads to an exponential size distribution at equilibrium. In agreement with a prediction stemming from this size distribution, the average size of PrP-res polymers was proportional to the square root of the concentration of PrP-res in a published study of in vitro conversion. Prion digestion by proteinase K (PK) is predicted to be biphasic. The second phase of digestion should be virtually independent of the PK concentration and should depend on the initial size distribution of prion polymers. For initially equilibrated polymers with an exponential size distribution, phase two digestion is exponential at a predicted rate. This rate varies in a defined way with the concentration used for equilibration and with other parameters which affect the average polymer size.

  11. Sweet potato SPAP1 is a typical aspartic protease and participates in ethephon-mediated leaf senescence.

    PubMed

    Chen, Hsien-Jung; Huang, Yu-Hsuan; Huang, Guan-Jhong; Huang, Shyh-Shyun; Chow, Te-Jin; Lin, Yaw-Huei

    2015-05-15

    Plant aspartic proteases are generally divided into three categories: typical, nucellin-like, and atypical aspartic proteases based on their gene and protein structures. In this report, a full-length cDNA SPAP1 was cloned from sweet potato leaves, which contained 1515 nucleotides (504 amino acids) and exhibited high amino acid sequence identity (ca. 51-72%) with plant typical aspartic proteases, including tomato LeAspP, potato StAsp, and wheat WAP2. SPAP1 also contained conserved DTG and DSG amino acid residues within its catalytic domain and plant specific insert (PSI) at the C-terminus. The cDNA corresponding to the mature protein (starting from the 66th to 311th amino acid residues) without PSI domain was constructed with pET30a expression vector for fusion protein and antibody production. RT-PCR and protein blot hybridization showed that SPAP1 expression level was the highest in L3 mature leaves, then gradually declined until L5 completely yellow leaves. Ethephon, an ethylene-releasing compound, also enhanced SPAP1 expression at the time much earlier than the onset of leaf senescence. Exogenous application of SPAP1 fusion protein promoted ethephon-induced leaf senescence, which could be abolished by pre-treatment of SPAP1 fusion protein with (a) 95 °C for 5 min, (b) aspartic protease inhibitor pepstatin A, and (c) anti-SPAP1 antibody, respectively. Exogenous SPAP1 fusion protein, whereas, did not significantly affect leaf senescence under dark. These data conclude that sweet potato SPAP1 is a functional typical aspartic protease and participates in ethephon-mediated leaf senescence. The SPAP1-promoted leaf senescence and its activity are likely not associated with the PSI domain. Interaction of ethephon-inducible components for effective SPAP1 promotion on leaf senescence is also suggested.

  12. Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase.

    PubMed

    Panchenko, M V; Stetler-Stevenson, W G; Trubetskoy, O V; Gacheru, S N; Kagan, H M

    1996-03-22

    Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.

  13. Disruption of prosomes by some bivalent metal ions results in the loss of their multicatalytic proteinase activity and cancels the nuclease resistance of prosomal RNA.

    PubMed Central

    Nothwang, H G; Coux, O; Bey, F; Scherrer, K

    1992-01-01

    Prosomes are ribonucleoprotein particles constituted by a variable set of about 20 proteins found associated with untranslated mRNA. In addition, they contain a small RNA, the presence of which has been an issue of controversy for a long time. The intact particles have a multicatalytic proteinase (MCP) activity and are very stable; we have never observed autodigestion of the particle by its intrinsic proteinase activity. Surprisingly it was found that Zn2+ and Cu2+ ions at concentrations of 0.1-1 mM disrupt the prosome particles isolated from HeLa cells and duck erythroblasts and abolish instantaneously its MCP activity, without altering the two-dimensional electrophoretic pattern of the constituent proteins. Fe2+, however, seems to induce autodegradation rather than dissociation of the prosome constituents. Most interestingly, protein or oligopeptide substrates protect the particle and its proteinase activity from disruption by Zn2+ or Cu2+. Nuclease-digestion assays reveal that the prosomal RNA, which is largely resistant in the intact particle, becomes digestible after dissociation of prosomes by Zn2+. These data give, for the first time, unambiguous proof of the presence of an RNA in the particle. Furthermore, they demonstrate a structure-function relationship between the complex and its enzyme activity, which seems to be based on the particle as an entity and not on the single constituent proteins. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:1445237

  14. Trichomonas vaginalis surface proteinase activity is necessary for parasite adherence to epithelial cells.

    PubMed Central

    Arroyo, R; Alderete, J F

    1989-01-01

    The role of cysteine proteinases in adherence of Trichomonas vaginalis NYH 286 to HeLa and human vaginal epithelial cells was evaluated. Only pretreatment of trichomonads, but not epithelial cells, with N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), an inhibitor of trichomonad cysteine proteinases, greatly diminished the ability of T. vaginalis to recognize and bind to epithelial cells. Leupeptin and L-1-tosylamide-2-phenylethyl chloromethyl ketone, other cysteine proteinase inhibitors, also decreased T. vaginalis cytadherence. Parasites incubated with TLCK and washed extensively still did not adhere to cells at levels equal to those seen for control trichomonads treated with phosphate-buffered saline or culture medium alone. Exposure of TLCK-treated organisms with other cysteine proteinases restored cytadherence levels, indicating that proteinase action on the parasite surface is prerequisite for host cell attachment. Concentrations of TLCK which inhibited cytadherence did not alter the metabolism of T. vaginalis, as determined by metabolic labeling of trichomonad proteins; the protein patterns of T. vaginalis in the presence and absence of TLCK were identical. Kinetics of TLCK-mediated inhibition of cytadherence of other T. vaginalis isolates with different levels of epithelial-cell parasitism were similar to the concentration-dependent inhibition seen for isolate NYH 286. Incubation of TLCK-treated, washed organisms in growth medium resulted in regeneration of adherence. Finally, treatment of T. vaginalis organisms with proteinase inhibitors for abrogation of cytadherence effectively rendered the trichomonads unable to kill host cells, which is consistent with the contact-dependent nature of host cytotoxicity. These data show for the first time the involvement of T. vaginalis cysteine proteinases in parasite attachment to human epithelial cells. These results have implications for future pharmacologic intervention at a key step in infection. PMID:2789190

  15. Distribution and evolution of the serine/aspartate racemase family in invertebrates.

    PubMed

    Uda, Kouji; Abe, Keita; Dehara, Yoko; Mizobata, Kiriko; Sogawa, Natsumi; Akagi, Yuki; Saigan, Mai; Radkov, Atanas D; Moe, Luke A

    2016-02-01

    Free D-amino acids have been found in various invertebrate phyla, while amino acid racemase genes have been identified in few species. The purpose of this study is to elucidate the distribution, function, and evolution of amino acid racemases in invertebrate animals. We searched the GenBank databases, and found 11 homologous serine racemase genes from eight species in eight different invertebrate phyla. The cloned genes were identified based on their maximum activity as Acropora millepora (Cnidaria) serine racemase (SerR) and aspartate racemase (AspR), Caenorhabditis elegans (Nematoda) SerR, Capitella teleta (Annelida) SerR, Crassostrea gigas (Mollusca) SerR and AspR, Dugesia japonica (Platyhelminthes) SerR, Milnesium tardigradum (Tardigrada) SerR, Penaeus monodon (Arthropoda) SerR and AspR and Strongylocentrotus purpuratus (Echinodermata) AspR. We found that Acropora, Aplysia, Capitella, Crassostrea and Penaeus had two amino acid racemase paralogous genes and these paralogous genes have evolved independently by gene duplication at their recent ancestral species. The transcriptome analyses using available SRA data and enzyme kinetic data suggested that these paralogous genes are expressed in different tissues and have different functions in vivo. Phylogenetic analyses clearly indicated that animal SerR and AspR are not separated by their particular racemase functions and form a serine/aspartate racemase family cluster. Our results revealed that SerR and AspR are more widely distributed among invertebrates than previously known. Moreover, we propose that the triple serine loop motif at amino acid positions 150-152 may be responsible for the large aspartate racemase activity and the AspR evolution from SerR. PMID:26352274

  16. Stable and Simple Immobilization of Proteinase K Inside Glass Tubes and Microfluidic Channels.

    PubMed

    Küchler, Andreas; Bleich, Julian N; Sebastian, Bernhard; Dittrich, Petra S; Walde, Peter

    2015-11-25

    Engyodontium album proteinase K (proK) is widely used for degrading proteinaceous impurities during the isolation of nucleic acids from biological samples, or in proteomics and prion research. Toward applications of proK in flow reactors, a simple method for the stable immobilization of proK inside glass micropipette tubes was developed. The immobilization of the enzyme was achieved by adsorption of a dendronized polymer-enzyme conjugate from aqueous solution. This conjugate was first synthesized from a polycationic dendronized polymer (denpol) and proK and consisted, on average, of 2000 denpol repeating units and 140 proK molecules, which were attached along the denpol chain via stable bis-aryl hydrazone bonds. Although the immobilization of proK inside the tube was based on nonspecific, noncovalent interactions only, the immobilized proK did not leak from the tube and remained active during prolonged storage at 4 °C and during continuous operation at 25 °C and pH = 7.0. The procedure developed was successfully applied for the immobilization of proK on a glass/PDMS (polydimethylsiloxane) microchip, which is a requirement for applications in the field of proK-based protein analysis with such type of microfluidic devices. PMID:26536248

  17. Stable and Simple Immobilization of Proteinase K Inside Glass Tubes and Microfluidic Channels.

    PubMed

    Küchler, Andreas; Bleich, Julian N; Sebastian, Bernhard; Dittrich, Petra S; Walde, Peter

    2015-11-25

    Engyodontium album proteinase K (proK) is widely used for degrading proteinaceous impurities during the isolation of nucleic acids from biological samples, or in proteomics and prion research. Toward applications of proK in flow reactors, a simple method for the stable immobilization of proK inside glass micropipette tubes was developed. The immobilization of the enzyme was achieved by adsorption of a dendronized polymer-enzyme conjugate from aqueous solution. This conjugate was first synthesized from a polycationic dendronized polymer (denpol) and proK and consisted, on average, of 2000 denpol repeating units and 140 proK molecules, which were attached along the denpol chain via stable bis-aryl hydrazone bonds. Although the immobilization of proK inside the tube was based on nonspecific, noncovalent interactions only, the immobilized proK did not leak from the tube and remained active during prolonged storage at 4 °C and during continuous operation at 25 °C and pH = 7.0. The procedure developed was successfully applied for the immobilization of proK on a glass/PDMS (polydimethylsiloxane) microchip, which is a requirement for applications in the field of proK-based protein analysis with such type of microfluidic devices.

  18. Two novel asparaginyl endopeptidase-like cysteine proteinases from the protist Trichomonas vaginalis: their evolutionary relationship within the clan CD cysteine proteinases.

    PubMed

    León-Félix, Josefina; Ortega-López, Jaime; Orozco-Solís, Ricardo; Arroyo, Rossana

    2004-06-23

    Cysteine proteinases (CPs) are important virulence factors of the protozoan parasite Trichomonas vaginalis. A total of six genes coding for cathepsin L-like CPs belonging to clan CA have been identified in T. vaginalis. At least 23 distinct spots with proteolytic activity have been detected by two-dimensional (2-D) substrate gel electrophoresis from in vitro grown parasites; however, only few of them have been characterized. In this work, we detected six spots with proteolytic activity and molecular weights between 25 and 35 kDa. The six proteinases correspond to two distinct CP families: the papain-like family, represented by four spots with pIs between 4.5 and 5.5; and the legumain-like family represented by two spots with pI 6.3 and 6.5. Next, we obtained two cDNAs encoding for legumain-like CPs from T. vaginalis, which were named Tvlegu-1 and Tvlegu-2. The size of these cDNA clones were 1225 and 1364 bp, which encoded for 388 and 415 amino acids, respectively. Their putative translation products have molecular masses of 42.8 and 47.2 kDa, corresponding to inactive legumain-like CP precursors. The two sequences share approximately 40% identity at the amino acid level. These protein products can be classified within a branch of the legumain-like family in clan CD cysteine proteinases due to their sensitivity to specific proteinases inhibitors, their DNA sequences, and phylogenetic reconstruction. However, they do not correspond either to the typical asparaginyl endopeptidase or the glycosylphosphatidylinositol (GPI): protein transamidase subfamilies. These results suggest that the TVLEGU-1 and TVLEGU-2 peptidases are likely to be part of a new subfamily within the legumain-like family of clan CD cysteine proteinases. Furthermore, they could be one of the missing links between prokaryotic and eukaryotic CPs in clan CD enzymes.

  19. Modeling N-methyl-D-aspartate-induced bursting in dopamine neurons.

    PubMed

    Li, Y X; Bertram, R; Rinzel, J

    1996-03-01

    Burst firing of dopaminergic neurons of the substantia nigra pars compacta can be induced in vitro by the glutamate agonist N-methyl-D-aspartate. It has been suggested that the interburst hyperpolarization is due to Na+ extrusion by a ouabain-sensitive pump [Johnson et al. (1992) Science 258, 665-667]. We formulate and explore a theoretical model, with a minimal number of currents, for this novel mechanism of burst generation. This minimal model is further developed into a more elaborate model based on observations of additional currents and hypotheses about their spatial distribution in dopaminergic neurons [Hounsgaard (1992) Neuroscience 50, 513-518; Llinás et al. (1984) Brain Res. 294, 127-132]. Using the minimal model, we confirm that interaction between the regenerative, inward N-methyl-D-aspartate-mediated current and the outward Na(+)-pump current is sufficient to generate the slow oscillation (approximately 0.5 Hz) underlying the burst. The negative-slope region of the N-methyl-D-aspartate channel's current-voltage relation is indispensable for this slow rhythm generation. The time-scale of Na(+)-handling determines the burst's slow frequency. Moreover, we show that, given the constraints of sodium handling, such bursting is best explained mechanistically by using at least two spatial, cable-like compartments: a soma where action potentials are produced and a dendritic compartment where the slow rhythm is generated. Our result is consistent with recent experimental evidence that burst generation originates in distal dendrites [Seutin et al. (1994) Neuroscience 58, 201-206]. Responses of the model to a number of electrophysiological and pharmacological stimuli are consistent with known responses observed under similar conditions. These include the persistence of the slow rhythm when the tetrodotoxin-sensitive Na+ channel is blocked and when the soma is voltage-clamped at -60 mV. Using our more elaborate model, we account for details of the observed

  20. Chiral Three-Dimensional Microporous Nickel Aspartate with Extended Ni-O-Ni Bonding

    SciTech Connect

    Anokhina,E.; Go, Y.; Lee, Y.; Vogt, T.; Jacobson, A.

    2006-01-01

    In the course of our investigation aimed at the preparation of homochiral coordination polymers using readily available in optically pure form ligands and building blocks of condensed metal polyhedra, we recently reported a one-dimensional nickel aspartate compound [Ni{sub 2}O(L-Asp)(H{sub 2}O){sub 2}]{center_dot}4H{sub 2}O (1) based on helical chains with extended Ni-O-Ni bonding. Here we report a new nickel aspartate [Ni{sub 2.5}(OH)(L-Asp){sub 2}]{center_dot}6.55H{sub 2}O (2) with a three-dimensional Ni-O-Ni connectivity that forms at a higher pH and is based on the same helices as in 1 which are connected by additional nickel octahedra to generate a chiral open framework with one-dimensional channels with minimum van der Waals dimensions of 8 x 5 Angstroms. The crystal structure of 2 was determined by synchrotron single-crystal X-ray diffraction on a 10 x 10 x 240 {micro}m crystal.

  1. Pediatric anti-N methyl D aspartate receptor encephalitis.

    PubMed

    Suri, Vinit; Sharma, Sushma; Gupta, Rohan; Sogani, S K; Mediratta, Sunit; Jadhao, Nilesh

    2013-05-01

    Anti-N Methyl D Aspartate Receptor encephalitis (anti-NMDARE) is a recently defined disease, which is probably more under-recognized than rare. We report a case of anti-NMDARE in a 13-years-old girl, who presented with intractable seizures. To the best of our knowledge, this is the second case of pediatric anti-NMDARE being reported from India. The need for a greater awareness of this disease and the subtle differences in clinical presentation between pediatric and adult patients are highlighted. PMID:24082929

  2. Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.

    PubMed

    Kummer, J A; Kamp, A M; Citarella, F; Horrevoets, A J; Hack, C E

    1996-04-19

    Human granzyme A is one of the serine proteinases present in the granules of cytotoxic T lymphocytes and natural killer cells. Granzymes are synthesized as inactive proenzymes with an amino-terminal prodipeptide, which is processed during transport of granzymes to the cytotoxic granules, where they are stored as active proteinases. In this study, we explored the possibility of producing recombinant granzymes. Recombinant human granzyme A zymogen was expressed in several eukaryotic cell lines (HepG2, Jurkat, and COS-1) after infection with a recombinant vaccinia virus containing full-length granzyme A cDNA. Immunoblot analysis of cell lysates showed that all infected cells produced a disulfide-linked homodimer of identical molecular weight as natural granzyme A. Infected HepG2 cells produced the largest amount of this protease (approximately 160 times more than lymphokine activated killer (LAK) cells). The recombinant protein only had high mannose type oligosaccharides as did the natural protein. Although infected HepG2 and COS cells contained high granzyme A antigen levels, lysates from these cells did not show any granzyme A proteolytic activity. However, the inactive proenzyme could be converted into active granzyme A by incubation with the thiol proteinase cathepsin C (dipeptidyl peptidase I). This study is the first to demonstrate expression of an active recombinant human cytotoxic lymphocyte proteinase and conversion of inactive progranzyme A into an active enzyme by cathepsin C. We suggest that a similar approach can be used for the production of other granzymes and related proteinases.

  3. Fluorometric determination of acid proteinase activity in Candida albicans strains from diabetic patients with vulvovaginal candidiasis.

    PubMed

    Yildirim, Zuhal; Kilic, Nedret; Kalkanci, Ayse

    2011-09-01

    Vulvovaginal candidiasis is one of the most frequent disorders in obstetrics and gynaecology. Approximately three-quarters of all adult women experience at least one episode of vulvovaginal candidiasis during their life span. Diabetes mellitus (DM) increases the rate of vaginal colonisation and infection with Candida species. The secreted acid proteinase might be especially relevant in the pathogenesis of vulvovaginal candidiasis. The aim of this study was to determine the acid proteinase activity in the samples of Candida albicans from diabetic patients with vulvovaginal candidiasis by a fluorometric method. Vaginal swabs were taken from 33 women (aged between 22 and 57 years) having symptoms of vaginitis. Patients were divided into three groups: control group, controlled diabetic group and uncontrolled diabetic group. The proteinase activity in the culture supernatants was determined by a modified fluorometric method. Acid proteinase activities were significantly increased in the uncontrolled diabetic group in comparison with both the control group and the controlled diabetic group (P < 0.05). Acid proteinase may play an important role in C. albicans pathogenesis in diabetic patients. Improving glucose control may reduce the risk of Candida colonisation and potentially symptomatic infection, among women with diabetes and hence may be useful even for weaker enzyme activity measurements.

  4. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

    PubMed Central

    Sigle, Leah Theresa; Ramalho-Ortigão, Marcelo

    2013-01-01

    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. PMID:24037187

  5. Molecular insights into mechanisms of lepidopteran serine proteinase resistance to natural plant defenses.

    PubMed

    Tamaki, Fábio K; Terra, Walter R

    2015-11-27

    Plants have a wide range of chemical defenses against predation, including substances that target digestive serine proteinases of herbivorous. Previous works demonstrated that lepidopteran insects have digestive serine proteinases resistant to plant proteinase inhibitors (PPIs) and ketone modifications, while coleopteran ones are sensitive to those plant defenses. This paper focuses on molecular aspects that lead lepidopteran serine proteinases to PPI and ketone modification resistance. Using biochemical experiments and computer 3D modeling we demonstrated that lepidopteran trypsins are more hydrophobic than coleopteran ones, a feature associated to trypsin oligomerization and decreased inhibition by PPI. Moreover, the determination of pKa values of chymotrypsin catalytic residues obtained by TPCK modification indicates that the environment around the active site of ketone-resistant and -sensitive chymotrypsins are different. Structural analysis using resistant and sensitive chymotrypsins data allowed us to point 2 hotspot regions around the active site that could explain the observed differences. Our set of results highlights features of serine proteinases important for understanding the resistance of insects to plant chemical defenses.

  6. NaCl-activated extracellular proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation.

    PubMed

    Sinsuwan, S; Rodtong, S; Yongsawatdigul, J

    2007-06-01

    Virgibacillus sp. SK37 exhibited high extracellular proteolytic activity in skim milk broth containing 10% NaCl. Optimum conditions of the crude proteinase were at pH 8.0 and 65 degrees C. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, suggesting the serine proteinase with a subtilisin-like characteristic. Proteolytic activity increased with NaCl concentration up to 20%. Ca(2+) activated the enzyme activity but reduced enzyme stability at 65 degrees C. Several proteinases with dominant molecular mass (MW) of 81, 67, 63, 50, 38, and 18 kDa were detected on native-polyacrylamide gel electrophoresis (native-PAGE) activity staining in the absence and presence of 25% NaCl. These results demonstrated that Virgibacillus sp. SK37 produced salt-activated extracellular proteinases. Virgibacillus sp. SK37 could be a promising strain for starter culture development used in fish sauce fermentation. PMID:17995713

  7. A low-molecular-weight inhibitor of the neutral proteinase from rat intestinal smooth muscle.

    PubMed Central

    Carney, I T; Curtis, C G; Kay, J K; Birket, N

    1980-01-01

    1. Rat intestinal smooth muscle was shown to contain endogenous inhibitory activity towards the neutral trypsin-like muscle proteinase described previously [Beynon & Kay (1978) Biochem. J. 173, 291--298]. 2. Comtamination of the muscle tissue by mucosal, blood and pancreatic inhibitors was shown to be unlikely. 3. The inhibitory activity was resolved into high- and low-molecular-weight components. 4. The low-molecular-weight component was purified to homogeneity. It has a molecular weight of approx. 9000 and was stable over the pH range 3--11. 5. It inhibited the muscle proteinase competitively (Ki congruent to t microM), but had no effect on any of the other proteinases tested. 6. Leupeptin also inhibited the muscle proteinase competitively (Ki congruent to 0.3 microM), whereas the low-molecular weight proteins gastrin, glucagon and insulin B-chain had very little effect. 7. A role for a weakly binding inhibitor in modulating the influence of the neutral proteinase on intracellular protein degradation is considered. Images Fig. 4. PMID:7396824

  8. Characterization of a secretory proteinase of Candida parapsilosis and evidence for the absence of the enzyme during infection in vitro.

    PubMed Central

    Rüchel, R; Böning, B; Borg, M

    1986-01-01

    The opportunistic yeastlike fungi of the genus Candida comprise three species which are proteolytic in vitro. Among them, C. albicans and C. tropicalis are of foremost medical importance. However, a strict correlation between extracellular proteolytic activity and virulence is opposed by the low virulence of the third proteolytic species, C. parapsilosis. We purified the secretory acid proteinase of C. parapsilosis (clinical isolate 265). The enzyme is a carboxyl proteinase (EC 3.4.23) like all other secretory Candida proteinases handled so far. Proteinase 265 is distinguished by a lower molecular weight (approximately 33,000); it has increased hydrophobicity, which accounts for inhibition of the enzyme by hemin, and required the presence of nonionic detergent in the initial steps of purification. The enzyme already undergoes alkaline denaturation at neutrality. Its activity is thus confined to the acid microenvironment of the fungal cell wall. Within this range, the enzyme may degrade immunoglobulins like immunoglobulin A1 (IgA1), IgA2, and secretory IgA. No indication was found for glycosylation of proteinase 265 and the related enzyme of C. albicans CBS 2730. However, the comparable proteinase of C. tropicalis 293 was identified as a manno protein. Antiserum against proteinase 265 cross-reacted strongly with corresponding enzymes from other Candida species. Antisera against proteinases of C. albicans and C. tropicalis reacted only weakly with proteinase 265. Thus, secretory Candida proteinases are likely to possess common and species-specific antigenic sites. In contrast to C. albicans, infection of phagocytes by C. parapsilosis 265 was not accompanied by secretion of fungal proteinase. This lack of induction of the enzyme under conditions of infection may account for the low virulence of most isolates of C. parapsilosis. Images PMID:3525413

  9. Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor.

    PubMed Central

    Boudier, C; Bieth, J G

    1994-01-01

    N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-1.s-1. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P'1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant kass. = 2.6 x 10(5) M-1.s-1, dissociation rate constant kdiss. = 2.9 x 10(-3) s-1 and equilibrium dissociation constant Ki = 1.1 x 10(-8) M. Comparison with the native inhibitor indicates that oxidation decreases kass. by a factor of 18.8 and increases kdiss. by a factor of 6.4, and therefore leads to a 120-fold increase in Ki. Yet, the oxidized inhibitor may still act as a potent elastase inhibitor in the upper respiratory tract where its concentration is 500-fold higher than Ki, i.e. where the elastase inhibition is pseudo-irreversible. Experiments in vitro with fibrous human lung elastin, the most important natural substrate of elastase, support this view: 1.35 microM elastase is fully inhibited by 5-6 microM oxidized inhibitor whether the enzyme-inhibitor complex is formed in the presence or absence of elastin and whether elastase is pre-adsorbed on elastin or not. PMID:7945266

  10. Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells

    SciTech Connect

    Kaellquist, Linda; Rosen, Hanna; Nordenfelt, Pontus; Calafat, Jero; Janssen, Hans; Persson, Ann-Maj; Hansson, Markus; Olsson, Inge

    2010-11-15

    Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 with clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating 'pro{sub C}'-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.

  11. Cofactoring and Dimerization of Proteinase-Activated Receptors

    PubMed Central

    Lin, Huilan; Liu, Allen P.; Smith, Thomas H.

    2013-01-01

    Proteinase-activated receptors (PARs) are G protein–coupled receptors that transmit cellular responses to extracellular proteases and have important functions in vascular physiology, development, inflammation, and cancer progression. The established paradigm for PAR activation involves proteolytic cleavage of the extracellular N terminus, which reveals a new N terminus that functions as a tethered ligand by binding intramolecularly to the receptor to trigger transmembrane signaling. Most cells express more than one PAR, which can influence the mode of PAR activation and signaling. Clear examples include murine PAR3 cofactoring of PAR4 and transactivation of PAR2 by PAR1. Thrombin binds to and cleaves murine PAR3, which facilitates PAR4 cleavage and activation. This process is essential for thrombin signaling and platelet activation, since murine PAR3 cannot signal alone. Although PAR1 and PAR4 are both competent to signal, PAR1 is able to act as a cofactor for PAR4, facilitating more rapid cleavage and activation by thrombin. PAR1 can also facilitate PAR2 activation through a different mechanism. Cleavage of the PAR1 N terminus by thrombin generates a tethered ligand domain that can bind intermolecularly to PAR2 to activate signaling. Thus, PARs can regulate each other’s activity by localizing thrombin when in complex with PAR3 and PAR4 or by cleaved PAR1, providing its tethered ligand domain for PAR2 activation. The ability of PARs to cofactor or transactivate other PARs would necessitate that the two receptors be in close proximity, likely in the form of a heterodimer. Here, we discuss the cofactoring and dimerization of PARs and the functional consequences on signaling. PMID:24064459

  12. Proteinase-activated receptors (PARs) as targets for antiplatelet therapy.

    PubMed

    Cunningham, Margaret; McIntosh, Kathryn; Bushell, Trevor; Sloan, Graeme; Plevin, Robin

    2016-04-15

    Since the identification of the proteinase-activated receptor (PAR) family as mediators of serine protease activity in the 1990s, there has been tremendous progress in the elucidation of their pathophysiological roles. The development of drugs that target PARs has been the focus of many laboratories for the potential treatment of thrombosis, cancer and other inflammatory diseases. Understanding the mechanisms of PAR activation and G protein signalling pathways evoked in response to the growing list of endogenous proteases has yielded great insight into receptor regulation at the molecular level. This has led to the development of new selective modulators of PAR activity, particularly PAR1. The mixed success of targeting PARs has been best exemplified in the context of inhibiting PAR1 as a new antiplatelet therapy. The development of the competitive PAR1 antagonist, vorapaxar (Zontivity), has clearly shown the value in targeting PAR1 in acute coronary syndrome (ACS); however the severity of associated bleeding with this drug has limited its use in the clinic. Due to the efficacy of thrombin acting via PAR1, strategies to selectively inhibit specific PAR1-mediated G protein signalling pathways or to target the second thrombin platelet receptor, PAR4, are being devised. The rationale behind these alternative approaches is to bias downstream thrombin activity via PARs to allow for inhibition of pro-thrombotic pathways but maintain other pathways that may preserve haemostatic balance and improve bleeding profiles for widespread clinical use. This review summarizes the structural determinants that regulate PARs and the modulators of PAR activity developed to date.

  13. Structural and functional characterization of aspartate racemase from the acidothermophilic archaeon Picrophilus torridus.

    PubMed

    Aihara, Takayuki; Ito, Toshiya; Yamanaka, Yasuaki; Noguchi, Keiichi; Odaka, Masafumi; Sekine, Masae; Homma, Hiroshi; Yohda, Masafumi

    2016-07-01

    Functional and structural characterizations of pyridoxal 5'-phosphate-independent aspartate racemase of the acidothermophilic archaeon Picrophilus torridus were performed. Picrophilus aspartate racemase exhibited high substrate specificity to aspartic acid. The optimal reaction temperature was 60 °C, which is almost the same as the optimal growth temperature. Reflecting the low pH in the cytosol, the optimal reaction pH of Picrophilus aspartate racemase was approximately 5.5. However, the activity at the putative cytosolic pH of 4.6 was approximately 6 times lower than that at the optimal pH of 5.5. The crystal structure of Picrophilus aspartate racemase was almost the same as that of other pyridoxal 5'-phosphate -independent aspartate racemases. In two molecules of the dimer, one molecule contained a tartaric acid molecule in the catalytic site; the structure of the other molecule was relatively flexible. Finally, we examined the intracellular existence of D-amino acids. Unexpectedly, the proportion of D-aspartate to total aspartate was not very high. In contrast, both D-proline and D-alanine were observed. Because Picrophilus aspartate racemase is highly specific to aspartate, other amino acid racemases might exist in Picrophilus torridus. PMID:27094682

  14. Role of aspartate 143 in Escherichia coli tRNA-guanine transglycosylase: alteration of heterocyclic substrate specificity.

    PubMed

    Todorov, Katherine Abold; Garcia, George A

    2006-01-17

    tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the post-transcriptional modification of certain tRNAs in their anticodon wobble positions with queuine. To maintain the correct Watson-Crick base pairing properties of the wobble base (and hence proper translation of the genetic code), TGT must recognize its heterocyclic substrate with high specificity. The X-ray crystal structure of a eubacterial TGT bound to preQ1 [Romier, C., et al. (1996) EMBO J. 15, 2850-2857] suggested that aspartate 143 (Escherichia coli TGT numbering) was involved in heterocyclic substrate recognition. Subsequent mutagenic and computational modeling studies from our lab [Todorov, K. A., et al. (2005) Biophys. J. 89 (3), 1965-1977] provided experimental evidence supporting this hypothesis. Herein, we report further studies probing the differential heterocyclic substrate recognition properties of the aspartate 143 mutant TGTs. Our results are consistent with one of the mutants exhibiting an inversion of substrate recognition preference (xanthine vs guanine) relative to that of the wild type, as evidenced by Km values. This confirms the key role of aspartate 143 in maintaining the anticodon identities of the queuine-containing tRNAs and suggests that TGT mutants could be developed that would alter the tRNA wobble base base pairing properties.

  15. Crystal structure of Saccharomyces cerevisiae cytosolic aspartate aminotransferase.

    PubMed Central

    Jeffery, C. J.; Barry, T.; Doonan, S.; Petsko, G. A.; Ringe, D.

    1998-01-01

    The crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 A resolution in the presence of the cofactor pyridoxal-5'-phosphate and the competitive inhibitor maleate. The structure was solved by the method of molecular replacement. The final value of the crystallographic R-factor after refinement was 23.1% with good geometry of the final model. The yeast cytoplasmic enzyme is a homodimer with two identical active sites containing residues from each subunit. It is found in the "closed" conformation with a bound maleate inhibitor in each active site. It shares the same three-dimensional fold and active site residues as the aspartate aminotransferases from Escherichia coli, chicken cytoplasm, and chicken mitochondria, although it shares less than 50% sequence identity with any of them. The availability of four similar enzyme structures from distant regions of the evolutionary tree provides a measure of tolerated changes that can arise during millions of years of evolution. PMID:9655342

  16. Wheat-germ aspartate transcarbamoylase. Purification and cold-lability.

    PubMed Central

    Grayson, J E; Yon, R J; Butterworth, P J

    1979-01-01

    1. Aspartate transcarbamoylase was purified approx. 3000-fold from wheat (Triticum vulgare) germ in 15-20% yield. The product has a specific activity of 14 mumol/min per mg of protein and is approx. 90% pure. The purification scheme includes the use of biospecific "imphilyte" chromatography as described by Yon [Biochem.J.(1977) 161, 233-237]. The enzyme was passed successively through columns of CPAD [N-(3-carboxypropionyl)aminodecyl]-Sepharose in the absence and presence respectively of the ligands UMP and L-aspartate. In the second passage the enzyme was specifically displaced away from impurities with which it co-migrated in the first passage. These two steps contributed a factor of 80 to the overall purification. 2. The enzyme is slowly inactivated on dilution at 0 degrees C and pH 7.0, the inactivation being partially reversible. A detailed investigation of the temperature- and pH-dependence of the cold-inactivation suggested that it was initiated by the perturbation of the pKa values of groups with a moderately high and positive heat of ionization, which were tentatively identified as histidine residues. These findings support a new concept of cold-lability proposed by Bock, Gilbert & Frieden [Biochem. Biophys. Res. Commun. (1975) 66, 564-569]. PMID:43131

  17. A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing.

    PubMed

    Xie, Jian; Owen, Timothy; Xia, Ke; Callahan, Brian; Wang, Chunyu

    2016-08-31

    Hedgehog (Hh) signaling is driven by the cholesterol-modified Hh ligand, generated by autoprocessing of Hh precursor protein. Two steps in Hh autoprocessing, N-S acyl shift and transesterification, must be coupled for efficient Hh cholesteroylation and downstream signal transduction. In the present study, we show that a conserved aspartate residue, D46 of the Hh autoprocessing domain, coordinates these two catalytic steps. Mutagenesis demonstrated that D46 suppresses non-native Hh precursor autoprocessing and is indispensable for transesterification with cholesterol. NMR measurements indicated that D46 has a pKa of 5.6, ∼2 units above the expected pKa of aspartate, due to a hydrogen-bond between protonated D46 and a catalytic cysteine residue. However, the deprotonated form of D46 side chain is also essential, because a D46N mutation cannot mediate cholesteroylation. On the basis of these data, we propose that the proton shuttling of D46 side chain mechanistically couples the two steps of Hh cholesteroylation. PMID:27529645

  18. Overview of Clinical Trial Program and Applicability of Insulin Degludec/Insulin Aspart in Diabetes Management.

    PubMed

    Bantwal, Ganapathi; Wangnoo, Subhash K; Shunmugavelu, M; Nallaperumal, S; Harsha, K P; Bhattacharyya, Arpandev

    2015-05-01

    Insulin degludec/insulin aspart (IDegAsp) is the first soluble coformulation combining a long-acting insulin degludec (IDeg) and rapid-acting insulin aspart (IAsp). In patients with uncontrolled type 2 diabetes (T2DM) previously treated with insulins, IDegAsp twice daily effectively improves glycated haemoglobin (HbA1c) and fasting plasma glucose (FPG) levels with fewer hypoglycaemic episodes versus premix insulins. Further, insulin initiation with IDegAsp once daily provides superior long-term glycaemic control compared to insulin glargine with similar FPG and insulin doses, and numerically lower rates of overall and nocturnal hypoglycaemia. In patients with type 1 diabetes mellitus (T1DM), IDegAsp once daily and IAsp at remaining meals provides more convenient three injection regimen per day over conventional 4-5 injections based basal-bolus therapy. IDegAsp is an appropriate and reasonable option for intensifying insulin therapy in patients with T2DM and a relatively less complex treatment option for the management of T1DM. PMID:26548031

  19. Elaboration of a fragment library hit produces potent and selective aspartate semialdehyde dehydrogenase inhibitors.

    PubMed

    Thangavelu, Bharani; Bhansali, Pravin; Viola, Ronald E

    2015-10-15

    Aspartate-β-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action. The ASADH enzyme family shares the same substrate binding and active site catalytic groups; however, the enzymes from representative bacterial and fungal species show different inhibition patterns when previously screened against low molecular weight inhibitors identified from fragment library screening. In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor docking, kinetic, and structure-activity relationship (SAR) studies have been used to guide ASADH inhibitor development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromolar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development of inhibitors against this validated drug-target enzyme.

  20. Poly(aspartic acid) (PAA) hydrolases and PAA biodegradation: current knowledge and impact on applications.

    PubMed

    Hiraishi, Tomohiro

    2016-02-01

    Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of β-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between β-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in β-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and β-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2. PMID:26695157

  1. Elaboration of a fragment library hit produces potent and selective aspartate semialdehyde dehydrogenase inhibitors.

    PubMed

    Thangavelu, Bharani; Bhansali, Pravin; Viola, Ronald E

    2015-10-15

    Aspartate-β-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action. The ASADH enzyme family shares the same substrate binding and active site catalytic groups; however, the enzymes from representative bacterial and fungal species show different inhibition patterns when previously screened against low molecular weight inhibitors identified from fragment library screening. In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor docking, kinetic, and structure-activity relationship (SAR) studies have been used to guide ASADH inhibitor development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromolar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development of inhibitors against this validated drug-target enzyme. PMID:26404410

  2. Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis

    PubMed Central

    Hernández, Hilda M.; Marcet, Ricardo; Sarracent, Jorge

    2014-01-01

    Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis. PMID:25348828

  3. Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis.

    PubMed

    Hernández, Hilda M; Marcet, Ricardo; Sarracent, Jorge

    2014-01-01

    Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis. PMID:25348828

  4. Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis.

    PubMed

    Hernández, Hilda M; Marcet, Ricardo; Sarracent, Jorge

    2014-01-01

    Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis.

  5. The anthelmintic efficacy of natural plant cysteine proteinases against Hymenolepis microstoma in vivo.

    PubMed

    Mansur, F; Luoga, W; Buttle, D J; Duce, I R; Lowe, A; Behnke, J M

    2015-09-01

    Little is known about the efficacy of cysteine proteinases (CP) as anthelmintics for cestode infections in vivo. Hymenolepis microstoma is a natural parasite of house mice, and provides a convenient model system for the assessment of novel drugs for anthelmintic activity against cestodes. The experiments described in this paper indicate that treatment of H. microstoma infections in mice with the supernatant of papaya latex (PLS), containing active cysteine proteinases, is only minimally efficacious. The statistically significant effects seen on worm burden and biomass showed little evidence of dose dependency, were temporary and the role of cysteine proteinases as the active principles in PLS was not confirmed by specific inhibition with E-64. Worm fecundity was not affected by treatment at the doses used. We conclude also that this in vivo host-parasite system is not sensitive enough to be used reliably for the detection of cestocidal activity of compounds being screened as potential, novel anthelmintics.

  6. Modification of standard proteinase K/phenol method for extraction of DNA from small tumour biopsies.

    PubMed

    Pitera, R; Pitera, J E; Mufti, G J; Salisbury, J R

    1993-09-01

    The standard proteinase K/phenol DNA isolation method was found to produce unsatisfactory yields of DNA from small tissue biopsies (less than 50 mg). The influences of the volume of cell lysis buffer and the amount of proteinase K on the final DNA yield and quality were studied, and an improved method was devised and compared with both the standard procedure and a phenol-free protocol. The optimal volume of cell lysis buffer was found to be 200 microliters per mg of tissue while the optimal amount of proteinase K was 60 micrograms per mg of tissue. A mean yield of 12 mu/mg tissue of pure, high molecular weight DNA was achieved from 50 frozen samples prepared by crushing. Yields from 20 microns thick cryostat sections reached 30 micrograms/mg.

  7. Human placental extract mediated inhibition of proteinase K: implications of heparin and glycoproteins in wound physiology.

    PubMed

    Sharma, Kanika; Mukherjee, Chaitali; Roy, Siddhartha; De, Debashree; Bhattacharyya, Debasish

    2014-09-01

    Efficient debridement of the wound bed following the removal of microbial load prevents its progression into a chronic wound. Bacterial infection and excessive proteolysis characterize impaired healing and therefore, their inhibition might restore the disturbed equilibrium in the healing process. Human placental extract exhibits reversible, non-competitive inhibition towards Proteinase K, a microbial protease, by stabilizing it against auto-digestion. Scattering and fluorescence studies followed by biochemical analysis indicated the involvement of a glycan moiety. Surface plasmon resonance demonstrated specific interaction of heparin with Proteinase K having Kd in μM range. Further, Proteinase K contains sequence motifs similar to other heparin-binding proteins. Molecular docking revealed presence of clefts suitable for binding of heparin-derived oligosaccharides. Comprehensive analysis of this inhibitory property of placental extract partly explains its efficacy in curing wounds with common bacterial infections.

  8. Toll-like receptors recognize distinct proteinase-resistant glycoconjugates in Campylobacter jejuni and Escherichia coli.

    PubMed

    Phongsisay, Vongsavanh; Hara, Hiromitsu; Fujimoto, Shuji

    2015-03-01

    Campylobacter jejuni causes gastroenteritis and autoimmune neuropathy Guillain-Barré syndrome. The mechanism by which C. jejuni infection results in such the hyperimmunity is not completely understood. Host immunity plays an important role in the disease pathogenesis; however, little is known how immune system recognizes this human pathogen. In this study, we report that Toll-like receptors recognize distinct proteinase K-resistant glycoconjugates in C. jejuni and Escherichia coli. Lipopolysaccharide is solely proteinase-resistant glycoconjugate in E. coli. In contrast, C. jejuni possesses at least five different components that are resistant to proteinase digestion and are capable of inducing NF-κB activation through TLR2 and TLR4. Possession of multiple activators of Toll-like receptors may be the unique strategy of C. jejuni to trigger hyperimmunity.

  9. Serine proteinase inhibition by the active site titrant N alpha-(N, N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester. A comparative study.

    PubMed

    Ascenzi, P; Balliano, G; Gallina, C; Polticelli, F; Bolognesi, M

    2000-02-01

    Kinetics for the hydrolysis of the chromogenic active-site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester (Dmc-azaOrn-ONp) catalysed by bovine beta-trypsin, bovine alpha-thrombin, bovine Factor Xa, human alpha-thrombin, human Factor Xa, human Lys77-plasmin, human urinary kallikrein, Mr 33 000 and Mr 54 000 species of human urokinase, porcine pancreatic beta-kallikrein-A and -B and Ancrod (the coagulating serine proteinase from the Malayan pit viper Agkistrodon rhodostoma venom) have been obtained between pH 6.0 and 8.0, at 21.0 degrees C, and analysed in parallel with those for the enzymatic cleavage of N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). The enzyme kinetics are consistent with the minimum three-step catalytic mechanism of serine proteinases, the rate-limiting step being represented by the deacylation process. Bovine beta-trypsin kinetics are modulated by the acid-base equilibrium of the His57 catalytic residue (pKa approximately 6.9). Dmc-azaOrn-ONp and Dmc-azaLys-ONp bind stoichiometrically to the serine proteinase active site, and allow the reliable determination of the active enzyme concentration between 1.0 x 10-6 M and 3.0 x 10-4 M. The affinity and the reactivity for Dmc-azaOrn-ONp (expressed by Ks and k+2/Ks, respectively) of the serine proteinases considered are much lower than those for Dmc-azaLys-ONp. The very different affinity and reactivity properties for Dmc-azaOrn-ONp and Dmc-azaLys-ONp have been related to the different size of the ornithine/lysine side chains, and to the ensuing different positioning of the active-site titrants upon binding to the enzyme catalytic centre (i.e. to P1-S1 recognition). These data represent the first detailed comparative investigation on the catalytic properties of serine proteinases towards an ornithine derivative (i. e. Dmc-azaOrn-ONp).

  10. Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants.

    PubMed

    de la Torre, Fernando; Cañas, Rafael A; Pascual, M Belén; Avila, Concepción; Cánovas, Francisco M

    2014-10-01

    In the chloroplasts and in non-green plastids of plants, aspartate is the precursor for the biosynthesis of different amino acids and derived metabolites that play distinct and important roles in plant growth, reproduction, development or defence. Aspartate biosynthesis is mediated by the enzyme aspartate aminotransferase (EC 2.6.1.1), which catalyses the reversible transamination between glutamate and oxaloacetate to generate aspartate and 2-oxoglutarate. Plastids contain two aspartate aminotransferases: a eukaryotic-type and a prokaryotic-type bifunctional enzyme displaying aspartate and prephenate aminotransferase activities. A general overview of the biochemistry, regulation, functional significance, and phylogenetic origin of both enzymes is presented. The roles of these plastidic aminotransferases in the biosynthesis of essential amino acids are discussed.

  11. Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence.

    PubMed

    Jones, M L; Larsen, P B; Woodson, W R

    1995-06-01

    The senescence of carnation (Dianthus caryophyllus L.) flower petals is regulated by the phytohormone ethylene and is associated with considerable catabolic activity including the loss of protein. In this paper we present the molecular cloning of a cysteine proteinase and show that its expression is regulated by ethylene and associated with petal senescence. A 1600 bp cDNA was amplified by polymerase chain reaction using a 5'-specific primer and 3'-nonspecific primer designed to amplify a 1-aminocyclopropane-1-carboxylate synthase cDNA from reverse-transcribed stylar RNA. The nucleotide sequence of the cloned product (pDCCP1) was found to share significant homology to several cysteine proteinases rather than ACC synthase. A single open reading frame of 428 amino acids was shown to share significant homology with other plant cysteine proteinases including greater than 70% identity with a cysteine proteinase from Arabidopsis thaliana. Amino acids in the active site of cysteine proteinases were conserved in the pDCCP1 peptide. RNA gel blot analysis revealed that the expression of pDCCP1 increased substantially with the onset of ethylene production and senescence of petals. Increased pDCCP1 expression was also associated with ethylene production in other senescing floral organs including ovaries and styles. The pDCCP1 transcript accumulated in petals treated with exogenous ethylene within 3 h and treatment of flowers with 2,5-norbornadiene, an inhibitor of ethylene action, prevented the increase in pDCCP1 expression in petals. The temporal and spatial patterns of pDCCP1 expression suggests a role for cysteine proteinase in the loss of protein during floral senescence.

  12. Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis.

    PubMed Central

    Pike, R N; Potempa, J; McGraw, W; Coetzer, T H; Travis, J

    1996-01-01

    Adhesins from oral bacteria perform an important function in colonizing target tissues within the dentogingival cavity. In Porphyromonas gingivalis certain of these adhesion proteins exist as a complex with either of two major proteinases referred to as gingipain R (arginine-specific gingipain) and gingipain K (lysine-specific gingipain) (R. N. Pike, W. T. McGraw, J. Potempa, and J. Travis, J. Biol. Chem. 269:406-411, 1994). With specific proteinase inhibitors, it was shown that hemagglutination by either proteinase-adhesin complex could occur independently of proteinase activity. Significantly, low concentrations of fibrinogen, fibronectin, and laminin inhibited hemagglutination, indicating that adherence to these proteins and not the hemagglutination activity was a primary property of the adhesin activity component of complexes. Binding studies with gingipain K and gingipain R suggest that interaction with fibrinogen is a major function of the adhesin domain, with dissociation constants for binding to fibrinogen being 4 and 8.5 nM, respectively. Specific association with fibronectin and laminin was also found. All bound proteins were degraded by the functional proteinase domain, with gingipain R being more active on laminin and fibronectin and gingipain K being more effective in the digestion of fibrinogen. Cumulatively, these data suggest that gingipain R and gingipain K, acting as proteinase-adhesin complexes, progressively attach to, degrade, and detach from target proteins. Since such complexes appear to be present on the surfaces of both vesicles and membranes of P. gingivalis, they may play an important role in the attachment of this bacterium to host cell surfaces. PMID:8631676

  13. Excessive fluoride induces endoplasmic reticulum stress and interferes enamel proteinases secretion.

    PubMed

    Wei, Wei; Gao, Yanhui; Wang, Cheng; Zhao, Lijun; Sun, Dianjun

    2013-06-01

    Protein retention in the enamel layer during tooth formation is well known to be associated with dental fluorosis but the underlying mechanism is unclear. The functions of the endoplasmic reticulum (ER) correlate directly with secreted protein metabolism. We used an ameloblast-derived cell line to determine whether excessive amounts of fluoride cause ER stress, and whether this interferes with the secretion of enamel matrix proteinases. ER stress activates a signaling network called the unfolded protein response (UPR). Here, we used real-time RT-PCR and immunofluorescence to study the effect of fluoride on the expression, translation, and secretion of UPR transcription factors in ameloblast-like cells. Measurement of both the gene and protein expression of UPR transcription factors indicated that high-dose fluoride increases the expression of UPR transcription factors in a dose-dependent manner. We also used ELISA to detect and quantify the enamel proteinases secreted by ameloblasts. We found a corresponding decrease in extracellular secretion of the enamel proteinases matrix metalloproteinase-20 and kallikrein-4, after exposure to fluoride. Furthermore, correlation analysis indicated that the expression of UPR transcription factors showed a strong inverse correlation with that of enamel proteinases. The results suggest that high-dose fluoride initiates an ER stress response in ameloblasts and induces the UPR, which interferes with the synthesis and secretion of enamel proteinases. Taken together, these results suggest that excessive ingestion of fluoride during tooth formation can decrease the secretion of proteinases, thus causing protein retention in the enamel layer, indicating that the ER stress response may be responsible for dental fluorosis.

  14. Functions of Manduca sexta hemolymph proteinases HP6 and HP8 in two innate immune pathways.

    PubMed

    An, Chunju; Ishibashi, Jun; Ragan, Emily J; Jiang, Haobo; Kanost, Michael R

    2009-07-17

    Serine proteinases in insect plasma have been implicated in two types of immune responses; that is, activation of prophenoloxidase (proPO) and activation of cytokine-like proteins. We have identified more than 20 serine proteinases in hemolymph of the tobacco hornworm, Manduca sexta, but functions are known for only a few of them. We report here functions of two additional M. sexta proteinases, hemolymph proteinases 6 and 8 (HP6 and HP8). HP6 and HP8 are each composed of an amino-terminal clip domain and a carboxyl-terminal proteinase domain. HP6 is an apparent ortholog of Drosophila Persephone, whereas HP8 is most similar to Drosophila and Tenebrio spätzle-activating enzymes, all of which activate the Toll pathway. proHP6 and proHP8 are expressed constitutively in fat body and hemocytes and secreted into plasma, where they are activated by proteolytic cleavage in response to infection. To investigate activation and biological activity of HP6 and HP8, we purified recombinant proHP8, proHP6, and mutants of proHP6 in which the catalytic serine was replaced with alanine, and/or the activation site was changed to permit activation by bovine factor Xa. HP6 was found to activate proPO-activating proteinase (proPAP1) in vitro and induce proPO activation in plasma. HP6 was also determined to activate proHP8. Active HP6 or HP8 injected into larvae induced expression of antimicrobial peptides and proteins, including attacin, cecropin, gloverin, moricin, and lysozyme. Our results suggest that proHP6 becomes activated in response to microbial infection and participates in two immune pathways; activation of PAP1, which leads to proPO activation and melanin synthesis, and activation of HP8, which stimulates a Toll-like pathway. PMID:19487692

  15. Salicylic Acid Inhibits Synthesis of Proteinase Inhibitors in Tomato Leaves Induced by Systemin and Jasmonic Acid.

    PubMed Central

    Doares, S. H.; Narvaez-Vasquez, J.; Conconi, A.; Ryan, C. A.

    1995-01-01

    Salicylic acid (SA) and acetylsalicylic acid (ASA), previously shown to inhibit proteinase inhibitor synthesis induced by wounding, oligouronides (H.M. Doherty, R.R. Selvendran, D.J. Bowles [1988] Physiol Mol Plant Pathol 33: 377-384), and linolenic acid (H. Pena-Cortes, T. Albrecht, S. Prat, E.W. Weiler, L. Willmitzer [1993] Planta 191: 123-128), are shown here to be potent inhibitors of systemin-induced and jasmonic acid (JA)-induced synthesis of proteinase inhibitor mRNAs and proteins. The inhibition by SA and ASA of proteinase inhibitor synthesis induced by systemin and JA, as well as by wounding and oligosaccharide elicitors, provides further evidence that both oligosaccharide and polypeptide inducer molecules utilize the octadecanoid pathway to signal the activation of proteinase inhibitor genes. Tomato (Lycopersicon esculentum) leaves were pulse labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the inhibitory effects of SA are shown to be specific for the synthesis of a small number of JA-inducible proteins that includes the proteinase inhibitors. Previous results have shown that SA inhibits the conversion of 13S-hydroperoxy linolenic acid to 12-oxo-phytodienoic acid, thereby inhibiting the signaling pathway by blocking synthesis of JA. Here we report that the inhibition of synthesis of proteinase inhibitor proteins and mRNAs by SA in both light and darkness also occurs at a step in the signal transduction pathway, after JA synthesis but preceding transcription of the inhibitor genes. PMID:12228577

  16. Proteinase, phospholipase, hyaluronidase and chondroitin-sulphatase production by Malassezia pachydermatis.

    PubMed

    Coutinho, S D; Paula, C R

    2000-02-01

    The production of four functional enzyme categories was investigated in 30 strains of Malassezia pachydermatis isolated from dogs with otitis or dermatitis. The most appropriate reading intervals for these assays were determined with the aid of statistical comparisons. All strains produced proteinase and chondroitin-sulphatase; hyaluronidase and phospholipase were produced by all skin isolates (15/15) and 14 out of 15 ear canal isolates. Strains from ear canals did not differ significantly as a group from skin strains in quantitative production of any of the four enzymes; production of proteinase and chondroitin-sulphatase in particular was markedly uniform. PMID:10746230

  17. Determination of proteinase 3-alpha 1-antitrypsin complexes in inflammatory fluids.

    PubMed

    Dolman, K M; van de Wiel, B A; Kam, C M; Abbink, J J; Hack, C E; Sonnenberg, A; Powers, J C; von dem Borne, A E; Goldschmeding, R

    1992-12-14

    Physiological inhibitors were tested for their in vitro interaction with neutrophil proteinase 3 (PR3). The major plasma proteinase inhibitor of PR3 is alpha 1AT. We have developed a radioimmunoassay (RIA) for quantitative detection of PR3-alpha 1AT complexes formed in vivo in inflammatory exudates such as synovial fluid and plasma from patients with sepsis. Levels of PR3-alpha 1AT complexes correlated significantly with levels of human neutrophil elastase (HNE)-alpha 1AT complexes. Thus, in vivo alpha 1AT not only protects against excessive HNE activity, but also against excessive PR3 activity.

  18. Effects of E-64, a cysteine proteinase inhibitor, on cowpea weevil growth, development, and fecundity

    SciTech Connect

    Murdock, L.L.; Shade, R.E.; Pomeroy, M.A.

    1988-06-01

    E-64, a specific inhibitor of cysteine proteinases, was incorporated into artificial seeds at low levels (0.01-0.25% by weight). It prolonged developmental time and increased mortality of the larval cowpea weevil, Callosobruchus maculatus (F.), in direct proportion to its concentration in the artificial seeds. The fecundity of females emerging from the artificial seeds was significantly decreased by E-64 concentrations of 0.06% and higher. These observations are compatible with the hypothesis that the midgut cysteine proteinase in C. maculatus is essential for normal growth and development.

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

    PubMed

    Qasim, Mohammad A

    2014-01-01

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

  20. Understanding and targeting a novel plant viral proteinase/substrate interaction. Final report, July 1, 1989--June 30, 1995

    SciTech Connect

    Dougherty, W.

    1995-10-01

    The past 3 years of funding have focused our efforts on trying to understand the molecular basis of a unique substrate interaction displayed by a viral proteinase. We have made good progress and during this funding period we have made four contributions to the scientific literature and have developed the application of the proteinase in the expression and purification of recombinant fusion proteins. A comprehensive review of virus-encoded proteinases, written during the funding period, emphazing the tremendous similarity of viral proteinases with their cellular counterparts and at the same time detail the unique characteristics which permit them to function in a cellular environment. The focus of the research effort was the tobacco etch virus (TEV) 27kDa NIa proteinase.

  1. Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

    PubMed

    Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

    2016-08-01

    Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop.

  2. Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

    PubMed

    Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

    2016-08-01

    Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. PMID:27329566

  3. High-molecular-mass multicatalytic proteinase complexes produced by the nitrogen-fixing actinomycete Frankia strain BR.

    PubMed Central

    Benoist, P; Müller, A; Diem, H G; Schwencke, J

    1992-01-01

    A major-high-molecular mass proteinase and seven latent minor proteinases were found in cell extracts and in concentrates of culture medium from Frankia sp. strain BR after nondenaturing electrophoresis in mixed gelatin-polyacrylamide gels. All of these complexes showed multicatalytic properties. Their molecular masses and their sedimentation coefficients varied from 1,300 kDa (28S) to 270 kDa (12S). The electroeluted 1,300-kDa proteinase complex dissociated into 11 low-molecular-mass proteinases (40 to 19 kDa) after sodium dodecyl sulfate activation at 30 degrees C and electrophoresis under denaturing conditions. All of these electroeluted proteinases hydrolyzed N-carbobenzoxy-Pro-Ala-Gly-Pro-4-methoxy-beta- naphthylamide, D-Val-Leu-Arg-4-methoxy-beta-naphthylamide, and Boc-Val-Pro-Arg-4-methyl-7-coumarylamide, whereas Suc-Leu-Leu-Val-Tyr-4-methyl-7-coumarylamide was cleaved only by the six lower-molecular-mass proteinases (27.5 to 19 kDa). Examination by electron microscopy of uranyl acetate-stained, electroeluted 1,300- and 650-kDa intracellular and extracellular proteinase complexes showed ring-shaped and cylindrical particles (10 to 11 nm in diameter, 15 to 16 nm long) similar to those of eukaryotic prosomes and proteasomes. Polyclonal antibodies raised against rat skeletal muscle proteasomes cross-reacted with all of the high-molecular-mass proteinase complexes and, after denaturation of the electroeluted 1,300-kDa band, with polypeptides of 35 to 38, 65, and 90 kDa. Electrophoresis of the activated cell extracts under denaturing conditions revealed 11 to 17 gelatinases from 40 to 19 kDa, including the 11 proteinases of the 1,300-kDa proteinase complex. The inhibition pattern of these proteinases is complex. Thiol-reactive compounds and 1-10-phenanthroline strongly inhibited all of the proteinases, but inhibitors against serine-type proteinases were also effective for most of them. Images PMID:1537794

  4. Crystal structure of Clostridium acetobutylicum Aspartate kinase (CaAK): An important allosteric enzyme for amino acids production.

    PubMed

    Manjasetty, Babu A; Chance, Mark R; Burley, Stephen K; Panjikar, Santosh; Almo, Steven C

    2014-09-01

    Aspartate kinase (AK) is an enzyme which is tightly regulated through feedback control and responsible for the synthesis of 4-phospho-L-aspartate from L-aspartate. This intermediate step is at an important branch point where one path leads to the synthesis of lysine and the other to threonine, methionine and isoleucine. Concerted feedback inhibition of AK is mediated by threonine and lysine and varies between the species. The crystal structure of biotechnologically important Clostridium acetobutylicum aspartate kinase (CaAK; E.C. 2.7.2.4; Mw=48,030Da; 437aa; SwissProt: Q97MC0) has been determined to 3Å resolution. CaAK acquires a protein fold similar to the other known structures of AKs despite the low sequence identity (<30%). It is composed of two domains: an N-terminal catalytic domain (kinase) domain and a C-terminal regulatory domain further comprised of two small domains belonging to the ACT domain family. Pairwise comparison of 12 molecules in the asymmetric unit helped to identify the bending regions which are in the vicinity of ATP binding site involved in domain movements between the catalytic and regulatory domains. All 12 CaAK molecules adopt fully open T-state conformation leading to the formation of three tetramers unique among other similar AK structures. On the basis of comparative structural analysis, we discuss tetramer formation based on the large conformational changes in the catalytic domain associated with the lysine binding at the regulatory domains. The structure described herein is homologous to a target in wide-spread pathogenic (toxin producing) bacteria such as Clostridium tetani (64% sequence identity) suggesting the potential of the structure solved here to be applied for modeling drug interactions. CaAK structure may serve as a guide to better understand and engineer lysine biosynthesis for the biotechnology industry.

  5. Monomeric 55-kDa guanidinobenzoatase switches to a serine proteinase activity upon tetramerization. Tetrameric proteinase SP 220 K appears as the native form.

    PubMed

    Poustis-Delpont, C; Thaon, S; Auberger, P; Gerardi-Laffin, C; Sudaka, P; Rossi, B

    1994-05-20

    Guanidinobenzoatases are cell surface enzymes present in cells capable of migration or remodeling. The guanidinobenzoatase purified to homogeneity from human renal carcinoma did not display gelatinase activity under the 55-kDa form (Poustis-Delpont, C., Descomps, R., Auberger, P., Delque-Bayer, P., Sudaka, P., and Rossi, B. (1992) Cancer Res. 52, 3622-3628). We bring new insights into the structure-activity relationships of this enzyme using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]diisopropyl fluorophosphate labeling, gelatin zymography, and immunodetection using a polyclonal antibody raised against the 55-kDa entity. Upon aggregation into a 220-kDa form, the enzyme exhibited [3H]diisopropyl fluorophosphate labeling and diisopropyl fluorophosphate-inhibitable gelatinase activity whereas its capability to cleave p-nitrophenyl p'-guanidinobenzoate as a substrate was abolished. Thus, the guanidinobenzoatase property appears as a feature of a 55-kDa inactive form of a serine proteinase subunit. After boiling in the presence of sodium dodecyl sulfate (3% w/v), the 220-kDa entity subjected to SDS-polyacrylamide gel electrophoresis could be dissociated into a 55-kDa protein as shown by silver staining. The resulting 55-kDa band remained [3H]diisopropyl fluorophosphate-labeled and reacted with anti-55-kDa guanidinobenzoatase antibodies, strongly suggesting that the 220-kDa proteinase was a noncovalently associated tetramer. Interestingly, Triton X-100 extracts of renal carcinoma plasma membranes exhibited a 220-kDa serine proteinase activity, as expressed in gelatin zymography, which was barely detectable in the non-tumoral counterpart. It is noteworthy that an anti-55-kDa guanidinobenzoatase reactive 220-kDa species was also observed in renal carcinoma plasma membranes extracts as assessed by Western blot, whereas it was hardly visible in the non-tumoral counterpart. No signal was immunodetected at M(r) 55,000 in renal carcinoma and kidney cortex

  6. Subunit structure of karatasin, the proteinase isolated from Bromelia plumieri (karatas).

    PubMed

    Montes, C; Amador, M; Cuevas, D; Cordoba, F

    1990-01-01

    Close to 15% of the karatasin proteinase activity in the fruit juice of Bromelia plumieri (karatas) is present outside dialysis Visking tubing in 7 days in 0.2 M acetate buffer (pH) 3.5 or 6.5) containing phenyl mercuric acetate. The small proteinase(s), distinct from the 85% activity in juice due to nondialysable karatasin with a reported Mr of 24,868, separates across Spectrapore (13 kDa) membranes but not across Spectrapore with 3.5 kDa average pore diameter. The dialyzed proteinase is named karatasin-D (K-D). Purified non-Dialysable karatasin can be dissociated to what seems to be K-D by incubation in a buffer solution, containing SDS and 2-mercaptoethanol with phenyl mercuric acetate, in dialysis experiments for 8 days at room temperature using Spectrapore 13 kDa tubing. Thus, native karatasin in B. plumieri fruit juice seem to be the result of association of 2 small molecular mass K-D subunits, linked together by disulfide bonds and electrostatic forces, in equilibrium with small amounts of free K-D molecules. The amino acid composition and partial sequence of karatasin up to the 14th position from the amino terminus have discrete analogies with papain and with stem bromelain.

  7. Production, purification and characterisation of recombinant Fahsin, a novel antistasin-type proteinase inhibitor.

    PubMed

    de Bruin, Eric C; Roem, Dorina; Bulder, Ingrid; Dieker, Miranda; Voerman, Gerard; Hack, C Erik

    2005-11-01

    Serine proteinases from inflammatory cells, including polymorphonuclear neutrophils, are involved in various inflammatory disorders, like pulmonary emphysema and rheumatoid arthritis. Inhibitors of these serine proteinases are potential drug candidates for the treatment of these disorders, since they prevent the unrestricted proteolysis. This study describes a novel specific antistasin-type inhibitor of neutrophil serine proteinases, we called Fahsin. This inhibitor was purified from the Nile leech Limnatis nilotica, sequenced and heterologously expressed using a synthetic gene in the methylotrophic yeast Pichia pastoris, yielding 0.5 g(-l) of the protein in the culture medium. Recombinant Fahsin was purified to homogeneity and characterised by N-terminal amino acid sequencing and mass spectrometry. Inhibition-kinetic analysis showed that recombinant Fahsin is a fast, tight-binding inhibitor of human neutrophil elastase with inhibition constant in the nanomolar range. Furthermore, recombinant Fahsin was, in contrast to various other neutrophil elastase inhibitors, insensitive to chemical oxidation and biological oxidation via myeloperoxidase-generated free oxygen radicals. Thus, Fahsin constitutes a novel member of a still expanding family of naturally occurring inhibitors of serine proteinases with potential therapeutic use for treatment of human diseases.

  8. Detergents modify proteinase K resistance of PrP Sc in different transmissible spongiform encephalopathies (TSEs).

    PubMed

    Breyer, Johanna; Wemheuer, Wiebke M; Wrede, Arne; Graham, Catherine; Benestad, Sylvie L; Brenig, Bertram; Richt, Jürgen A; Schulz-Schaeffer, Walter J

    2012-05-25

    Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrP(Sc)). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrP(Sc) against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorption assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrP(Sc) after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrP(Sc) can be very different. The results obtained here may be helpful during the development or improvement of a PrP(Sc) detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrP(Sc) that may stabilize the aggregates.

  9. Nutritional requirements and nitrogen-dependent regulation of proteinase activity of Lactobacillus helveticus CRL 1062.

    PubMed

    Hebert, E M; Raya, R R; De Giori, G S

    2000-12-01

    The nutritional requirements of Lactobacillus helveticus CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of L. helveticus CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM. The growth rate and the synthesis of their cell membrane-bound serine proteinases, but not of their intracellular leucyl-aminopeptidases, were influenced by the peptide content of the medium. The highest proteinase levels were found during cell growth in basal SCDM, while the synthesis of this enzyme was inhibited in SCDM supplemented with Casitone, Casamino Acids, or beta-casein. Low-molecular-mass peptides (<3,000 Da), extracted from Casitone, and the dipeptide leucylproline (final concentration, 5 mM) play important roles in the medium-dependent regulation of proteinase activity. The addition of the dipeptide leucylproline (5 mM) to SCDM reduced proteinase activity by 25%. PMID:11097908

  10. Proteinase K and the structure of PrPse: the good, the bad, and the ugly

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious proteins (prions) are, ironically, defined by their resistance to proteolytic digestion. A defining characteristic of the transmissible isoform of the prion protein (PrPSc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunod...

  11. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  12. Isolation and characterization of cancer procoagulant: a cysteine proteinase from malignant tissue.

    PubMed

    Falanga, A; Gordon, S G

    1985-09-24

    Cancer procoagulant, a proteolytic procoagulant enzyme, has been purified from rabbit V2 carcinoma extracts by two procedures. In the first, the protein was purified by benzamidine--Sepharose affinity chromatography, gel filtration chromatography, and phenyl-Sepharose hydrophobic chromatography. Antiserum was raised against the purified protein and was used to prepare an immunoadsorbent column. In the second, tumor extracts were purified by immunoaffinity chromatography followed by p-(chloromercuri)benzoate affinity chromatography. The second procedure was substantially quicker and easier. The final product of both procedures was homogeneous on the basis of analytical sodium dodecyl sulfate--polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weight was 68 000 and the isoelectric point 4.8. The proteinase activity of cancer procoagulant directly activated factor X, in the absence of factor VII, and was inhibited by 1 mM iodoacetamide and 0.1 mM mercury which are classic cysteine proteinase inhibitors. A carbohydrate analysis showed less than 1 mol of hexose or sialic acid/mol of protein. The amino acid analysis showed that serine (19.1%), glycine (18.77%), and glutamic acid (12.5%) were the prevalent amino acids. The amino acid composition of cancer procoagulant was substantially different than other known factor X activating proteinases or other cysteine proteinases including cathepsin B.

  13. Proteinases release /sup 35/S-labeled macromolecules from cultured airway epithelial cells

    SciTech Connect

    Varsano, S.; Borson, D.B.; Gold, M.; Forsberg, S.; Basbaum, C.B.; Nadel, J.A.

    1986-03-05

    To determine whether proteinases release radiolabeled macromolecules from airway cells devoid of secretory granules, they studied canine cultured tracheal epithelial cells grown to confluency. At this time the cells are bound by tight junctions, maintain anion transport, have a well developed glycocalyx, but contain no secretory granules. They labeled the cells with /sup 35/SO/sub 4/ (50..mu..ci/ml/24h) then changed the medium every 20 min and measured nondialyzable /sup 35/S released into the medium. Two h later, the rate of spontaneous release of /sup 35/S-labeled-macromolecules was 5700 +/- 1600 CPM/20 min (mean +/- SD). At this time trypsin, thermolysin, pseudomonas elastase and alkaline proteinase, each released /sup 35/S-labeled-macromolecules, whereas aspergillus acid proteinase did not. In more detailed studies, trypsin released /sup 35/S in a concentration dependent fashion, with a threshold below 10 units/ml and a response to 1000 units/ml of 1092 +/- 173% (mean +/- SD; n=5 cultures) above pre-trypsin baseline. Sepharose CL4B chromatography of the radiolabeled materials released by trypsin showed a void volume fraction (MW greater than or equal to 10/sup 6/), and a second, included fraction (MW 2-3 x 10/sup 5/). These results indicate that cultured airway epithelial cells synthesize macromolecules and release them into the medium, and that proteinases increase the rate of macromolecule release markedly.

  14. A triticale water-deficit-inducible phytocystatin inhibits endogenous cysteine proteinases in vitro.

    PubMed

    Chojnacka, Magdalena; Szewińska, Joanna; Mielecki, Marcin; Nykiel, Małgorzata; Imai, Ryozo; Bielawski, Wiesław; Orzechowski, Sławomir

    2015-02-01

    Water-deficit is accompanied by an increase in proteolysis. Phytocystatins are plant inhibitors of cysteine proteinases that belong to the papain and legumain family. A cDNA encoding the protein inhibitor TrcC-8 was identified in the vegetative organs of triticale. In response to water-deficit, increases in the mRNA levels of TrcC-8 were observed in leaf and root tissues. Immunoblot analysis indicated that accumulation of the TrcC-8 protein occurred after 72h of water-deficit in the seedlings. Using recombinant protein, inhibitory activity of TrcC-8 against cysteine proteases from triticale and wheat tissues was analyzed. Under water-deficit conditions, there are increases in cysteine proteinase activities in both plant tissues. The cysteine proteinase activities were inhibited by addition of the recombinant TrcC-8 protein. These results suggest a potential role for the triticale phytocystatin in modulating cysteine proteinase activities during water-deficit conditions. PMID:25462979

  15. Biased signalling and proteinase-activated receptors (PARs): targeting inflammatory disease.

    PubMed

    Hollenberg, M D; Mihara, K; Polley, D; Suen, J Y; Han, A; Fairlie, D P; Ramachandran, R

    2014-03-01

    Although it has been known since the 1960s that trypsin and chymotrypsin can mimic hormone action in tissues, it took until the 1990s to discover that serine proteinases can regulate cells by cleaving and activating a unique four-member family of GPCRs known as proteinase-activated receptors (PARs). PAR activation involves the proteolytic exposure of its N-terminal receptor sequence that folds back to function as a 'tethered' receptor-activating ligand (TL). A key N-terminal arginine in each of PARs 1 to 4 has been singled out as a target for cleavage by thrombin (PARs 1, 3 and 4), trypsin (PARs 2 and 4) or other proteases to unmask the TL that activates signalling via Gq , Gi or G12 /13 . Similarly, synthetic receptor-activating peptides, corresponding to the exposed 'TL sequences' (e.g. SFLLRN-, for PAR1 or SLIGRL- for PAR2) can, like proteinase activation, also drive signalling via Gq , Gi and G12 /13 , without requiring receptor cleavage. Recent data show, however, that distinct proteinase-revealed 'non-canonical' PAR tethered-ligand sequences and PAR-activating agonist and antagonist peptide analogues can induce 'biased' PAR signalling, for example, via G12 /13 -MAPKinase instead of Gq -calcium. This overview summarizes implications of this 'biased' signalling by PAR agonists and antagonists for the recognized roles the PARs play in inflammatory settings. PMID:24354792

  16. Isolation and characterization of a proteinase K sensitive PrPSc fraction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent studies have shown that a sizeable fraction of PrPSc present in prion-infected tissues is,contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in att...

  17. Proteinase from germinating bean cotyledons. Evidence for involvement of a thiol group in catalysis.

    PubMed

    Csoma, C; Polgár, L

    1984-09-15

    To degrade storage proteins germinating seeds synthesize proteinases de novo that can be inhibited by thiol-blocking reagents [Baumgartner & Chrispeels (1977) Eur. J. Biochem. 77, 223-233]. We have elaborated a procedure for isolation of such a proteinase from the cotyledons of Phaseolus vulgaris. The purification procedure involved fractionation of the cotyledon homogenate with acetone and with (NH4)2SO4 and successive chromatographies on DEAE-cellulose, activated thiol-Sepharose Sepharose and Sephacryl S-200. The purified enzyme has an Mr of 23,400, proved to be highly specific for the asparagine side chain and blocking of its thiol group resulted in loss of the catalytic activity. The chemical properties of the thiol group of the bean enzyme were investigated by acylation with t-butyloxycarbonyl-L-asparagine p-nitro-phenyl ester and by alkylations with iodoacetamide and iodoacetate. Deviations from normal pH-rate profile were observed, which indicated that the thiol group is not a simple functional group, but constitutes a part of an interactive system at the active site. The pKa value for acylation and the magnitude of the rate constant for alkylation with iodoacetate revealed that the bean proteinase possesses some properties not shared by papain and the other cysteine proteinases studied to date.

  18. A triticale water-deficit-inducible phytocystatin inhibits endogenous cysteine proteinases in vitro.

    PubMed

    Chojnacka, Magdalena; Szewińska, Joanna; Mielecki, Marcin; Nykiel, Małgorzata; Imai, Ryozo; Bielawski, Wiesław; Orzechowski, Sławomir

    2015-02-01

    Water-deficit is accompanied by an increase in proteolysis. Phytocystatins are plant inhibitors of cysteine proteinases that belong to the papain and legumain family. A cDNA encoding the protein inhibitor TrcC-8 was identified in the vegetative organs of triticale. In response to water-deficit, increases in the mRNA levels of TrcC-8 were observed in leaf and root tissues. Immunoblot analysis indicated that accumulation of the TrcC-8 protein occurred after 72h of water-deficit in the seedlings. Using recombinant protein, inhibitory activity of TrcC-8 against cysteine proteases from triticale and wheat tissues was analyzed. Under water-deficit conditions, there are increases in cysteine proteinase activities in both plant tissues. The cysteine proteinase activities were inhibited by addition of the recombinant TrcC-8 protein. These results suggest a potential role for the triticale phytocystatin in modulating cysteine proteinase activities during water-deficit conditions.

  19. A novel Entamoeba histolytica cysteine proteinase, EhCP4, is key for invasive amebiasis and a therapeutic target.

    PubMed

    He, Chen; Nora, George P; Schneider, Eric L; Kerr, Iain D; Hansell, Elizabeth; Hirata, Ken; Gonzalez, David; Sajid, Mohammed; Boyd, Sarah E; Hruz, Petr; Cobo, Eduardo R; Le, Christine; Liu, Wei-Ting; Eckmann, Lars; Dorrestein, Pieter C; Houpt, Eric R; Brinen, Linda S; Craik, Charles S; Roush, William R; McKerrow, James; Reed, Sharon L

    2010-06-11

    Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design.

  20. A Novel Entamoeba histolytica Cysteine Proteinase, EhCP4, Is Key for Invasive Amebiasis and a Therapeutic Target*

    PubMed Central

    He, Chen; Nora, George P.; Schneider, Eric L.; Kerr, Iain D.; Hansell, Elizabeth; Hirata, Ken; Gonzalez, David; Sajid, Mohammed; Boyd, Sarah E.; Hruz, Petr; Cobo, Eduardo R.; Le, Christine; Liu, Wei-ting; Eckmann, Lars; Dorrestein, Pieter C.; Houpt, Eric R.; Brinen, Linda S.; Craik, Charles S.; Roush, William R.; McKerrow, James; Reed, Sharon L.

    2010-01-01

    Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design. PMID:20378535

  1. Improved DNA content histograms from formalin-fixed, paraffin-embedded liver tissue by proteinase K digestion.

    PubMed

    Albro, J; Bauer, K D; Hitchcock, C L; Wittwer, C T

    1993-01-01

    An improved method for the enzymatic digestion of formalin-fixed, paraffin-embedded liver tissue for DNA content analysis by flow cytometry is presented. Forty samples of histologically normal liver were alternately digested by the traditional pepsin method or a new method utilizing proteinase K and heat. Sixteen (40%) of the pepsin-digested samples had apparent DNA aneuploid peaks by flow cytometry. False DNA aneuploid peaks were not present in any of the histograms obtained after proteinase K digestion. Microscopy showed that the pepsin-digested samples had residual cytoplasmic remnants which contained fluorescent material. Samples digested with proteinase K had few cytoplasmic remnants. The average G0/G1 coefficient of variation after proteinase K treatment was lower (41%) and the fluorescent intensity higher (128%) than the pepsin-treated samples. The apparent mean S-phase (a combination of S-phase cells and underlying debris) after proteinase K digestion was 35% of the pepsin-treated samples. Primary and secondary tumors of the liver that were DNA aneuploid after pepsin treatment were also DNA aneuploid after proteinase K treatment. A modified digestion protocol utilizing proteinase K and heat can provide superior results for DNA content analysis of formalin-fixed, paraffin-embedded liver tissue.

  2. Properties of Copolymers of Aspartic Acid and Aliphatic Dicarboxylic Acids Prepared by Reactive Extrusion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspartic acid may be prepared chemically or by the fermentation of carbohydrates. Currently, low molecular weight polyaspartic acids are prepared commercially by heating aspartic acid at high temperatures (greater than 220 degrees C) for several hours in the solid state. In an effort to develop a ...

  3. Structural Analysis of the Ligand-Binding Domain of the Aspartate Receptor Tar from Escherichia coli.

    PubMed

    Mise, Takeshi

    2016-07-01

    The Escherichia coli cell-surface aspartate receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni(2+). These signals are transmitted from the extracellular region of Tar to the cytoplasmic region via the transmembrane domain. The mechanism by which extracellular signals are transmitted into the cell through conformational changes in Tar is predicted to involve a piston displacement of one of the α4 helices of the homodimer. To understand the molecular mechanisms underlying the induction of Tar activity by an attractant, the three-dimensional structures of the E. coli Tar periplasmic domain with and without bound aspartate, Asp-Tar and apo-Tar, respectively, were determined. Of the two ligand-binding sites, only one site was occupied, and it clearly showed the electron density of an aspartate. The slight changes in conformation and the electrostatic surface potential around the aspartate-binding site were observed. In addition, the presence of an aspartate stabilized residues Phe-150' and Arg-73. A pistonlike displacement of helix α4b' was also induced by aspartate binding as predicted by the piston model. Taken together, these small changes might be related to the induction of Tar activity and might disturb binding of the second aspartate to the second binding site in E. coli. PMID:27292793

  4. Activities of amylase, proteinase, and lipase enzymes from Lactococcus chungangensis and its application in dairy products.

    PubMed

    Konkit, Maytiya; Kim, Wonyong

    2016-07-01

    Several enzymes are involved in the process of converting milk to lactic acid and coagulated milk to curd and, therefore, are important in dairy fermented products. Amylase, proteinase, and lipase are enzymes that play an important role in degrading milk into monomeric molecules such as oligosaccharides, amino acids, and fatty acids, which are the main molecules responsible for flavors in cheese. In the current study, we determined the amylase, proteinase, and lipase activities of Lactococcus chungangensis CAU 28(T), a bacterial strain of nondairy origin, and compared them with those of the reference strain, Lactococcus lactis ssp. lactis KCTC 3769(T), which is commonly used in the dairy industry. Lactococcus chungangensis CAU 28(T) and L. lactis ssp. lactis KCTC 3769(T) were both found to have amylase, proteinase, and lipase activities in broth culture, cream cheese, and yogurt. Notably, the proteinase and lipase activities of L. chungangensis CAU 28(T) were higher than those of L. lactis ssp. lactis KCTC 3769(T), with proteinase activity of 10.50 U/mL in tryptic soy broth and 8.64 U/mL in cream cheese, and lipase activity of 100 U/mL of tryptic soy broth, and 100 U/mL of cream cheese. In contrast, the amylase activity was low, with 5.28 U/mL in tryptic soy broth and 8.86 U/mL in cream cheese. These enzyme activities in L. chungangensis CAU 28(T) suggest that this strain has potential to be used for manufacturing dairy fermented products, even though the strain is of nondairy origin. PMID:27108177

  5. Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

    PubMed

    Smigocki, Ann C; Ivic-Haymes, Snezana; Li, Haiyan; Savić, Jelena

    2013-01-01

    Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI) was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

  6. A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae.

    PubMed

    Elpidina, E N; Tsybina, T A; Dunaevsky, Y E; Belozersky, M A; Zhuzhikov, D P; Oppert, B

    2005-08-01

    A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.

  7. Proteinases in Naegleria Fowleri (strain NF3), a pathogenic amoeba: a preliminary study.

    PubMed

    Mat Amin, Nakisah

    2004-12-01

    Naegleria fowleri is a free-living amoeba, known as a causative agent for a fatal disease of the central nervous system (CNS) in man such as Primary amoebic meningoencephalitis (PAM). Factors contributing to its pathogenicity and its distribution in the environment have been investigated by previous researchers. In case of its pathogenicity, several enzymes such as phospolipase A and sphingomyelinase, have been proposed to probably act as aggressors in promoting PAM but no study so far have been conducted to investigate the presence of proteinase enzyme in this amoeba although a 56kDa cystein proteinase enzyme has been identified in Entamoeba histolytica as an important contributing factor in the amoeba's virulence. In this preliminary study, a pathogenic amoeba, Naegleria fowleri (strain NF3) was examined for the presence of proteinases. Samples of enzymes in this amoeba were analysed by electrophoresis using SDS-PAGE-gelatin gels. The results showed that this amoeba possesses at least two high molecular weight proteinases on gelatin gels; their apparent molecular weights are approximately 128 kDa and approximately 170 kDa. Band of approximately 128 kDa enzyme is membrane-associated and its activity is higher at alkaline pH compared with lower pH; at lower pH, its activity is greatly stimulated by DTT. The approximately 170 kDa band enzyme appears to be inactivated at pH 8.0, at lower ph its activity is higher and DTT-dependance. The activity of this enzyme is partially inhibited by inhibitor E-64 but markedly inhibited to antipain suggesting it belongs to the cysteine proteinase group.

  8. Immunological cross-reactivity of the major allergen from perennial ryegrass (Lolium perenne), Lol p I, and the cysteine proteinase, bromelain.

    PubMed

    Pike, R N; Bagarozzi, D; Travis, J

    1997-04-01

    Antibodies prepared in rabbits against the major allergen from ryegrass (Lolium perenne), Lol p I, cross-reacted with the cysteine proteinase bromelain from pineapple and vice versa. Deglycosylation of the proteins showed that the cross-reaction was based on recognition of the carbohydrate moiety of the allergen, but for bromelain the cross-reaction was most likely due to a combination of factors. The results indicate that the carbohydrate residues from these allergens play an important role in cross-reactions found between them and possibly those from other species.

  9. 1-deoxynojirimycin impairs oligosaccharide processing of alpha 1-proteinase inhibitor and inhibits its secretion in primary cultures of rat hepatocytes.

    PubMed

    Gross, V; Andus, T; Tran-Thi, T A; Schwarz, R T; Decker, K; Heinrich, P C

    1983-10-25

    1-Deoxynojirimycin was found to inhibit oligosaccharide processing of rat alpha 1-proteinase inhibitor. In normal hepatocytes alpha 1-proteinase inhibitor was present in the cells as a 49,000 Mr high mannose type glycoprotein with oligosaccharide side chains having the composition Man9GlcNAc and Man8GlcNAc with the former in a higher proportion. Hepatocytes treated with 5 mM 1-deoxynojirimycin accumulated alpha 1-proteinase inhibitor as a 51,000 Mr glycoprotein with carbohydrate side chains of the high mannose type, containing glucose as measured by their sensitivity against alpha-glucosidase, the largest species being Glc3Man9GlcNAc. Conversion to complex oligosaccharides was inhibited by the drug. In addition, increasing concentrations of 1-deoxynojirimycin inhibited glycosylation resulting in the formation of some alpha 1-proteinase inhibitor with two instead of three oligosaccharide side chains. 5 mM 1-deoxynojirimycin inhibited the secretion of alpha 1-proteinase inhibitor by about 50%, whereas secretion of albumin was unaffected. The oligosaccharides of alpha 1-proteinase inhibitor secreted from 1-deoxynojirimycin-treated cells were characterized by their susceptibility to endoglucosaminidase H, incorporation of [3H]galactose, and [3H]fucose and concanavalin A-Sepharose chromatography. It was found that 1-deoxynojirimycin did not completely block oligosaccharide processing, resulting in the formation of alpha 1-proteinase inhibitor molecules carrying one or two complex type oligosaccharides. Only these alpha 1-proteinase inhibitor molecules processed to the complex type in one or two of their oligosaccharide chains were nearly exclusively secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of alpha 1-proteinase inhibitor. PMID:6226656

  10. The M358R variant of α(1)-proteinase inhibitor inhibits coagulation factor VIIa.

    PubMed

    Sheffield, William P; Bhakta, Varsha

    2016-02-12

    The naturally occurring M358R mutation of the plasma serpin α1-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg-Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg-Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10(2) M(-1)sec(-1). We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. PMID:26797521

  11. Elevated neutrophil membrane expression of proteinase 3 is dependent upon CD177 expression

    PubMed Central

    Abdgawad, M; Gunnarsson, L; Bengtsson, A A; Geborek, P; Nilsson, L; Segelmark, M; Hellmark, T

    2010-01-01

    Proteinase 3 (PR3) is a major autoantigen in anti-neutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double-positive or double-negative for mPR3 and CD177. The proportion of double-positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony-stimulating factor and granulocyte–macrophage colony-stimulating factor. Pro-PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double-positive cells. In cells sorted based on membrane expression, CD177–mRNA was several-fold higher in mPR3+ cells. When exogenous PR3 was added to CD177-transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene. PMID:20491791

  12. Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium

    PubMed Central

    Winton, Helen L; Wan, Hong; Cannell, Mark B; Thompson, Philip J; Garrod, David R; Stewart, Geoffrey A; Robinson, Clive

    1998-01-01

    House dust mite (HDM) allergens with cysteine and serine proteinase activity are risk factors for allergic sensitization and asthma. A simple method to fractionate proteinase activity from HDM faecal pellets into cysteine and serine class activity is described. Both proteinase fractions increased the permeability of epithelial cell monolayers. The effects of the serine proteinase fraction were inhibited by 4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The effects of the cysteine proteinase fraction could be inhibited by E-64. No reciprocity of action was found. Treatment of epithelial monolayers with either proteinase fraction caused breakdown of tight junctions (TJs). AEBSF inhibited TJ breakdown caused by the serine proteinase fraction, whereas E-64 inhibited the cysteine proteinase fraction. Agarose gel electrophoresis revealed that the proteinases induced DNA cleavage which was inhibited by the matrix metalloproteinase inhibitor BB-250. Compound E-64 inhibited DNA fragmentation caused by the cysteine proteinase fraction, but was without effect on the serine proteinase fraction. Staining of proteinase-treated cells with annexin V (AV) and propidium iodide (PI) revealed a diversity of cellular responses. Some cells stained only with AV indicating early apoptosis, whilst others were dead and stained with both AV and PI. HDM proteinases exert profound effects on epithelial cells which will promote allergic sensitization; namely disruption of intercellular adhesion, increased paracellular permeability and initiation of cell death. Attenuation of these actions by proteinase inhibitors leads to the conclusion that compounds designed to be selective for the HDM enzymes may represent a novel therapy for asthma. PMID:9720772

  13. A sycamore cell wall polysaccharide and a chemically related tomato leaf polysaccharide possess similar proteinase inhibitor-inducing activities.

    PubMed

    Ryan, C A; Bishop, P; Pearce, G

    1981-09-01

    A large pectic polysaccharide, called rhamnogalacturonan I, that is solubilized by a fungal endo-alpha-1,4-polygalacturonase from the purified walls of suspension-cultured sycamore cells possesses proteinase inhibitor-inducing activity similar to that of the proteinase inhibitor-inducing factor, a pectic-like oligosaccharide fraction isolated from tomato leaves. This suggests that the proteinase inhibitor-inducing activity resides in particular polysaccharide fragments which can be released when plant cell walls are exposed to appropriate enzyme degradation as a result of either wounding or pest attack.

  14. Induction of a heparin-stimulated serine proteinase in sex accessory gland tumors of the Lobund-Wistar rat.

    PubMed

    Wilson, Michael J; Lind, Jeremy; Sinha, Akhouri A

    2015-08-01

    Induction of new proteinase activities that may process growth factors, modify cell surface receptors, cleave extracellular matrix proteins, etc. is considered fundamental in carcinogenesis. The purpose of this study was to characterize a novel proteinase activity induced in sex accessory gland cancers (about 70% in seminal vesicles) of adult male Lobund-Wistar rats by a single injection of N-nitroso-N-methylurea (NMU; 25mg/kg) followed by implanted testosterone propionate (45mg in silastic tubing every 2months) treatment for 10-14months. A 28kDa proteinase activity was detected in tumor extracts using SDS-gelatin gel zymography with incubations done without CaCl2. Its activity was stimulated 15 fold by heparin (optimal activity 1.5-3.0μg/lane) added to the tissue extract-SDS sample buffer prior to electrophoresis. No 28kDa heparin-stimulated proteinase (H-SP) was found in the dorsal, lateral and anterior (coagulating gland) prostate lobes or seminal vesicles of untreated adult rats, but there was a 26-30kDa Ca(2+)-independent proteinase activity in the ventral prostate that showed limited heparin stimulation. The 28kDa H-SP was completely inhibited by 1.0mM 4-(2-aminoethyl)benzenesulfonylfluoride (AESBF) indicating that it was a serine-type proteinase. Other types of proteinase inhibitors were without effect, including serine proteinase inhibitors benzamidine, tranexamic acid and ε-aminocaproic acid. Proteinase activities of about 28kDa were found with casein, fibrinogen or carboxymethylated transferrin as substrate, however, these activities were not stimulated by heparin. Similar levels of activities of the 28kDa H-SP were found in primary tumors and their metastases, but little/no activity was detected in serum, even from rats with large tumor volume and metastases. These data demonstrate overexpression of a heparin-stimulated 28kDa serine proteinase in the primary tumors of sex accessory gland cancers and their metastases. This proteinase either does not

  15. Age-Related Changes in D-Aspartate Oxidase Promoter Methylation Control Extracellular D-Aspartate Levels and Prevent Precocious Cell Death during Brain Aging.

    PubMed

    Punzo, Daniela; Errico, Francesco; Cristino, Luigia; Sacchi, Silvia; Keller, Simona; Belardo, Carmela; Luongo, Livio; Nuzzo, Tommaso; Imperatore, Roberta; Florio, Ermanno; De Novellis, Vito; Affinito, Ornella; Migliarini, Sara; Maddaloni, Giacomo; Sisalli, Maria Josè; Pasqualetti, Massimo; Pollegioni, Loredano; Maione, Sabatino; Chiariotti, Lorenzo; Usiello, Alessandro

    2016-03-01

    The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that d-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of d-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate. PMID:26961959

  16. Proteolysis of the peanut allergen Ara h 1 by an endogenous aspartic protease.

    PubMed

    Wilson, Karl A; Tan-Wilson, Anna

    2015-11-01

    The 7S and 11S globulins of peanuts are subjected to proteolysis two days after seed imbibition, with Ara h 1 and the arachin acidic chains being among the first storage proteins to be mobilized. Proteolytic activity was greatest at pH 2.6-3 and is inhibited by pepstatin A, characteristic of an aspartic protease. This activity persists in seedling cotyledons up to at least 8 days after imbibition. In vitro proteolysis of Ara h 1 at pH 2.6 by extracts of cotyledons from seedlings harvested 24 h after seed imbibition generates newly appearing bands on SDS-PAGE. Partial sequences of Ara h 1 that were obtained through LC-MS/MS analysis of in-gel trypsin digests of those bands, combined with information on fragment size, suggest that proteolysis begins in the region that links the two cupin domains to produce two 33/34 kD fragments, each one encompassing an intact cupin domain. The later appearance of two 18 and 10/11 kD fragments can be explained by proteolysis within an exposed site in the cupin domains of each of the 33/34 kD fragments. The same or similar proteolytic activity was observed in developing seeds, but Ara h 1 remains intact through seed maturation. This is partly explained by the observation that acidification of the protein storage vacuoles, demonstrated by vacuolar accumulation of acridine orange that was dissipated by a membrane-permeable base, occurs only after germination. These findings suggest a method for use of the seed aspartic protease in reducing peanut allergy due to Ara h 1.

  17. N-methyl-D-aspartate promotes the survival of cerebellar granule cells in culture.

    PubMed

    Balázs, R; Jørgensen, O S; Hack, N

    1988-11-01

    Our previous studies on the survival-promoting influence of elevated concentrations of extracellular K+ ([K+]e) on cultured cerebellar granule cells led to the proposal that depolarization in vitro mimics the effect of the earliest afferent inputs received by the granule cells in vivo. This, in turn, might be mediated through the stimulation of excitatory amino acid receptors, in particular the N-methyl-D-aspartate-preferring subtype gating ion channels which are also permeable to Ca2+. Here we report that N-methyl-D-aspartate indeed has a dramatic effect on the survival in culture of cells derived from dissociated cerebella of 7-8-day-old rats and cultured in media containing 'low' [K+]e (5-15 mM). In addition to the visual inspection of the cultures, the effect of N-methyl-D-aspartate was quantitatively evaluated, using estimates related to the number of viable cells (determination of DNA and of reduction rate of a tetrazolium salt). Furthermore, proteins which are relatively enriched in either nerve cells (neuronal cell adhesion molecule, D3-protein and synaptin) or in glia (glutamine synthetase) were also measured. The findings showed that the rescue of cells by N-methyl-D-aspartate involved primarily nerve cells and that the survival requirement for N-methyl-D-aspartate, as for high K+, developed between 2 and 4 days in vitro. The effect depended on both the concentration of N-methyl-D-aspartate and the degree of depolarization of the cells: both the potency and the efficacy of N-methyl-D-aspartate were increased as [K+]e was raised from 5 to 15 mM, at which range K+ on its own has little if any influence on granule cell survival. These characteristics are consistent with the voltage-dependence of ion conductance through the N-methyl-D-aspartate receptor-linked channel. The most pronounced effect of N-methyl-D-aspartate was obtained in the presence of 15 mM K+, when cell survival approached that obtained in 'control' cultures (grown in 25 mM K

  18. New paradigm for allosteric regulation of Escherichia coli aspartate transcarbamoylase.

    PubMed

    Cockrell, Gregory M; Zheng, Yunan; Guo, Wenyue; Peterson, Alexis W; Truong, Jennifer K; Kantrowitz, Evan R

    2013-11-12

    For nearly 60 years, the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and five X-ray structures determined in the absence and presence of a Mg(2+) concentration within the physiological range. In the presence of 2 mM divalent cations (Mg(2+), Ca(2+), Zn(2+)), CTP does not significantly inhibit the enzyme, while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor of ATCase in vivo is the combination of CTP, UTP, and a divalent cation, and the actual allosteric activator is a divalent cation with ATP or ATP and GTP. The structural data reveals that two NTPs can bind to each allosteric site with a divalent cation acting as a bridge between the triphosphates. Thus, the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal that the catalytic chains undergo essentially no alternations; however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal region of the regulatory chains exists in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed.

  19. Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis.

    PubMed Central

    Abrahamson, M; Wikström, M; Potempa, J; Renvert, S; Hall, A

    1997-01-01

    AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans was studied by measuring inhibition of enzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-terminal sequence analysis of cystatin C products resulting from the interactions. RESULTS: Gingival crevicular fluid contained cystatin C at a concentration of approximately 15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, released cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal region of cystatin C at physiological pH values. The modified cystatin C resulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (Ki 5 nM) than that of full length cystatin C. A 50 kDa thiol stimulated proteinase, gingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitory activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10-bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. CONCLUSIONS: The physiological control of cathepsin B activity is impeded in

  20. Observing N-Acetyl Aspartate via Both Its N-Acetyl and Its Strongly Coupled Aspartate Groups in in VivoProton Magnetic Resonance Spectroscopy

    NASA Astrophysics Data System (ADS)

    Wilman, Alan H.; Allen, Peter S.

    1996-12-01

    The ∼2.6 ppm aspartate multiplet ofN-acetyl aspartate (NAA) is considered a potential source of additional information onN-acetyl aspartatein vivo.Because the aspartate multiplet is the AB part of a strongly coupled ABX system it gives rise, as is shown in the analysis presented, to a significant field-strength dependence in the echo-time-dependent modulations of the response to typical spatial-localization sequences. The echo-time dependence of this response is developed analytically, not only for the STEAM and the PRESS localization sequences, but also for a spin-echo sequence. It is then verified experimentally at 2.35 T. The field-strength dependence of the response is demonstrated by evaluating the changes in the echo-time-dependent responses to each of the three sequences at field strengths of 1.5, 2.35, and 4.0 T. By means of these results, the preferred sequence (PRESS) can be optimized for the NAA aspartate multiplet at each field strength, as is illustrated with the human brain spectra obtainedin vivoat 1.5 T. Thesein vivospectra compare the optimal, long TE timing (163 ms) with a suboptimal TE (70 ms), for the observation of the ∼2.6 ppm aspartate resonances of NAA.

  1. Clinical experience with insulin detemir, biphasic insulin aspart and insulin aspart in people with type 2 diabetes: Results from the Hyderabad cohort of the A1chieve study

    PubMed Central

    Santosh, R.; Mehrotra, Ravi; Sastry, N. G.

    2013-01-01

    Background: The A1chieve, a multicentric (28 countries), 24-week, non-interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with T2DM (n = 66,726) in routine clinical care across four continents. Materials and Methods: Data was collected at baseline, at 12 weeks and at 24 weeks. This short communication presents the results for patients enrolled from Hyderabad, India. Results: A total of 1249 patients were enrolled in the study. Four different insulin analogue regimens were used in the study. Patients had started on or were switched to biphasic insulin aspart (n = 893), insulin detemir (n = 158), insulin aspart (n = 124), basal insulin plus insulin aspart (n = 19) and other insulin combinations (n = 54). At baseline glycaemic control was poor for both insulin naïve (mean HbA1c: 9.0%) and insulin user (mean HbA1c: 9.5%) groups. After 24 weeks of treatment, both the groups showed improvement in HbA1c (insulin naïve: −0.9%, insulin users: −1.1%). SADRs including major hypoglycaemic events or episodes did not occur in any of the study patients. Conclusion: Starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia. PMID:24404501

  2. Identification and metabolic role of the mitochondrial aspartate-glutamate transporter in Saccharomyces cerevisiae.

    PubMed

    Cavero, S; Vozza, A; del Arco, A; Palmieri, L; Villa, A; Blanco, E; Runswick, M J; Walker, J E; Cerdán, S; Palmieri, F; Satrústegui, J

    2003-11-01

    The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspartate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identified Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomyces cerevisiae. On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast. PMID:14622413

  3. Effect of added proteinases and level of starter culture on the formation of biogenic amines in raw milk Manchego cheese.

    PubMed

    Fernández-García, E; Tomillo, J; Núñez, M

    1999-11-15

    The influence of two proteinases (Bacillus subtilis neutral proteinase and Micrococcus sp. cysteine proteinase) and two starter culture levels (0.1% and 1%) on biogenic amine formation has been studied in raw ewes' milk Manchego cheese. Amino acid decarboxylating micro-organisms were determined on tyrosine enriched selective media. Biogenic amines were analysed by capillary electrophoresis in citrate buffer at pH 3.6. Addition of proteinases and level of starter culture did not influence the population of micro-organisms with amino acid decarboxylating activity, which represented on average 1% of the bacterial population in 30-day-old cheeses. Tyramine and histamine were detected in all batches of cheese from day 30. Concentrations of tyramine and histamine were higher in cheeses made from milk with neutral proteinase (up to 356 and 284 mg kg(-1), respectively, after 90 days) than in cheeses made from milk with cysteine proteinase (up to 269 and 189 mg kg(-1), respectively) or with no proteinase added (up to 305 and 226 mg kg(-1), respectively). Formation of tyramine and histamine was also favoured in cheeses made with 1% starter culture with respect to cheeses made with only 0.1% starter culture, probably due to the higher pH values of the former cheeses. After 90 days of ripening, concentrations of 10-20 mg kg(-1) phenylethylamine were observed in 9 of the 12 batches, and levels < 10 mg kg(-1) tryptamine were only detected in 3 batches, with no significant relationship between the concentration of these amines and proteinase addition or level of starter culture. PMID:10733250

  4. [Properties of extracellular proteinase--an activator of protein C in blood plasma formed by Aspergillus ochraceus].

    PubMed

    Osmolovskiĭ, A A; Kreĭer, V G; Baranova, N A; Kurakov, A V; Egorov, N S

    2015-01-01

    The properties of an extracellular proteinase activating plasma protein C isolated from the culture supernatant of A. ochraceus VKM F-4104D have been studied. This enzyme demonstrated a substrate specificity absent of hydrolyzing activity toward chromogenic proteinase substrates. On the basis of inhibitory analysis, the protein C-activating proteinase from A. ochraceus VKM F-4104D appeared to be a serine proteinase, together with that isolated from the venom of Agkistrodon contortrix contortrix. The isolated enzyme was a nonglycosylated protein with a molecular weight of about 33 kDa, pI 6.0 with an observed optimal activity under a pH of 8.0-9.0 and 37°C. A comparison of the properties of the protein C-activating proteinase formed by A. ochraceus and the enzyme derived from the venom of Agkistrodon contortrix contortrix demonstrated a similarity in their properties; however, proteinase from the micromycete appeared to be in the nonglycosylated state and possessed the ability to hydrolyze the chromogenic plasmin substrate H-D-Val-Leu-Lys-pNA. PMID:25842908

  5. A trypsin-like proteinase in the midgut of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae): purification, characterization, and host plant inhibitors.

    PubMed

    Ranjbar, Mina; Zibaee, Arash; Sendi, Jalal Jalali

    2014-01-01

    A trypsin-like proteinase was purified and characterized in the midgut of Ectomyelois ceratoniae. A purification process that used Sepharyl G-100 and DEAE-cellulose fast flow chromatographies revealed a proteinase with specific activity of 66.7 μmol/min/mg protein, recovery of 27.04 and purification fold of 23.35. Molecular weight of the purified protein was found to be 35.8 kDa. Optimal pH and temperature were obtained 9 and 20°C for the purified trypsin proteinase, respectively. The purified enzyme was significantly inhibited by PMSF, TLCK, and SBTI as specific inhibitors of trypsins in which TLCK showed the highest inhibitory effect. Trypsin proteinase inhibitors were extracted from four varieties of pomegranate including Brait, Torsh-Sabz, May-Khosh, and Shirin by ion exchange chromatography. It was found that fractions 17-20 of Brait; fractions 18 and 21-26 of Torsh-Sabz; fractions 1-7, 11-17, and 19-21 of May-Khosh and fraction 8 for Shirin showed presence of trypsin inhibitor in these host. Comparison of their inhibitory effects on the purified trypsin proteinase of E. ceratoniae demonstrated that fractions from May-khosh variety had the highest effect on the enzyme among other extracted fractions. Characterization of serine proteinases of insects mainly trypsins is one of the promising methods to decrease population and damages via extracting their inhibitors and providing resistant varieties.

  6. The trichomonad cysteine proteinase TVCP4 transcript contains an iron-responsive element.

    PubMed

    Solano-González, Eduardo; Burrola-Barraza, Eduviges; León-Sicairos, Claudia; Avila-González, Leticia; Gutiérrez-Escolano, Lorena; Ortega-López, Jaime; Arroyo, Rossana

    2007-06-26

    The differential expression of the Trichomonas vaginalis cysteine proteinase TVCP4 by iron at the protein synthesis level and the prediction of an iron-responsive element (IRE)-like stem-loop structure at the 5'-region of the T. vaginalis cysteine proteinase 4 gene (tvcp4) mRNA suggest a post-transcriptional mechanism of iron regulation in trichomonads mediated by an IRE/IRP-like system. Gel-shifting, UV cross-linking and competition experiments demonstrated that this IRE-like structure specifically bound to human iron regulatory protein-1. IRP-like cytoplasmic proteins that bound human ferritin IRE sequence transcripts at low-iron conditions were also found in trichomonads. Thus, a post-transcriptional regulatory mechanism by iron for tvcp4 mediated by IRE/IRP-like interactions was found. PMID:17553495

  7. Luminal proteinases from Plodia interpunctella and the hydrolysis of Bacillus thuringiensis CryIA(c) protoxin.

    PubMed

    Oppert, B; Kramer, K J; Johnson, D; Upton, S J; Mcgaughey, W H

    1996-06-01

    The ability of proteinases in gut extracts of the Indianmeal moth, Plodia interpunctella, to hydrolyze Bacillus thuringiensis (Bt) protoxin, casein, and rho-nitroanilide substrates was investigated. A polyclonal antiserum to protoxin CryIA(c) was used in Western blots to demonstrate slower protoxin processing by gut enzymes from Bt subspecies entomocidus-resistant larvae than enzymes from susceptible or kurstaki-resistant strains. Enzymes from all three strains hydrolyzed N-alpha-benzoyl-L-arginine rho-nitroanilide, N-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, and N-succinyl-ala-ala-pro-leucine rho-nitroanilide. Zymograms and activity blots were used to estimate the apparent molecular masses, number of enzymes, and relative activities in each strain. Several serine proteinase inhibitors reduced gut enzyme activities, with two soybean trypsin inhibitors, two potato inhibitors, and chymostatin the most effective in preventing protoxin hydrolysis.

  8. Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis.

    PubMed Central

    Carlsson, J; Herrmann, B F; Höfling, J F; Sundqvist, G K

    1984-01-01

    Various strains of black-pigmented Bacteroides species were grown on horse blood agar and suspended in human serum. After various times of incubation the effect of the bacteria on the serum was evaluated by polyacrylamide gel electrophoresis and "rocket" immunoelectrophoresis. The formation of trichloroacetic acid-soluble material in the suspensions and the capacity of the treated sera to inhibit the activity of trypsin were also determined. The two tested strains of Bacteroides gingivalis (W83, H185) degraded most serum proteins, including the plasma proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin. They did not, however, degrade alpha-1-antichymotrypsin. Bacteroides intermedius NCTC 9336, Bacteroides asaccharolyticus NCTC 9337, and an asaccharolytic oral strain different from B. gingivalis (BN11a-f) did not degrade the plasma proteinase inhibitors. These strains were, however, able to inactivate the capacity of serum to inhibit the activity of trypsin. Images PMID:6198282

  9. The trichomonad cysteine proteinase TVCP4 transcript contains an iron-responsive element.

    PubMed

    Solano-González, Eduardo; Burrola-Barraza, Eduviges; León-Sicairos, Claudia; Avila-González, Leticia; Gutiérrez-Escolano, Lorena; Ortega-López, Jaime; Arroyo, Rossana

    2007-06-26

    The differential expression of the Trichomonas vaginalis cysteine proteinase TVCP4 by iron at the protein synthesis level and the prediction of an iron-responsive element (IRE)-like stem-loop structure at the 5'-region of the T. vaginalis cysteine proteinase 4 gene (tvcp4) mRNA suggest a post-transcriptional mechanism of iron regulation in trichomonads mediated by an IRE/IRP-like system. Gel-shifting, UV cross-linking and competition experiments demonstrated that this IRE-like structure specifically bound to human iron regulatory protein-1. IRP-like cytoplasmic proteins that bound human ferritin IRE sequence transcripts at low-iron conditions were also found in trichomonads. Thus, a post-transcriptional regulatory mechanism by iron for tvcp4 mediated by IRE/IRP-like interactions was found.

  10. Competitive inhibition of nitric oxide synthase by p-aminobenzamidine, a serine proteinase inhibitor.

    PubMed

    Venturini, G; Menegatti, E; Ascenzi, P

    1997-03-01

    p-Aminobenzamidine competitively inhibits bovine trypsin, human and bovine thrombin, and human plasmin, all of which act on substrates containing preferentially the L-arginyl side chain at their P1 position. Considering the structural and functional similarity between p-aminobenzamidine and the L-arginyl side chain in trypsin-like serine proteinases, we investigated the interaction of p-aminobenzamidine with mouse brain nitric oxide synthase (NOS), which uses L-arginine as the substrate for generating NO and L-citrulline. p-Aminobenzamidine is a competitive NOS inhibitor (Ki = 1.2 x 10(-4) M, at pH 7.5 and 37.0 degrees C), but not an NO precursor. Therefore, p-aminobenzamidine affects the NO production and the trypsin-like serine proteinase action. PMID:9125158

  11. Primary structure of a cysteine proteinase inhibitor from the fruit of avocado (Persea americana Mill).

    PubMed

    Kimura, M; Ikeda, T; Fukumoto, D; Yamasaki, N; Yonekura, M

    1995-12-01

    The complete amino acid sequence of a proteinaceous cysteine proteinase inhibitor from the fruit of avocado (avocado cystatin) is presented. The protein consists of 100 amino acid residues and has a molecular mass of 11,300 Da. Comparison of this sequence with sequences of plant cysteine proteinase inhibitors (phytocystatins), including oryzacystatins I and II from rice seeds, cowpea cystatin, and corn cystatin, showed that the avocado cystatin molecule has 60% and 54% residues identical with the two forms of the rice seed proteins, oryzacystatins I and II, respectively, and 64% and 63% with the cowpea and corn proteins, respectively. The totally conserved sequence, Gln-Val-Val-Ala-Gly, among several of the animal cystatins as well as phytocystatins, is at positions 47-51 in the avocado cystatin molecule.

  12. Modified TB rapid test by proteinase K for rapid diagnosis of pleural tuberculosis.

    PubMed

    Yari, Shamsi; Hadizadeh Tasbiti, Alireza; Ghanei, Mostafa; Shokrgozar, Mohammad Ali; Fateh, Abolfazl; Yari, Fatemeh; Bahrmand, Ahmadreza

    2016-03-01

    The diagnosis of pleural tuberculosis continues to be a challenge due to the low sensitivity of traditional diagnostic methods. Better and more rapid tests are needed for diagnosis of pleural TB. In this study, pleural fluids were tested with rapid test to determine Mycobacterium tuberculosis (MTB antigen). Affinity chromatography was used to purify specific polyclonal antibodies against MTB antigen. Pleural samples after decontamination were treated with proteinase K. Rapid test for pleural fluids was prepared by specific antibody. Rapid test was performed on 85 pleural fluid patients. The patients had a mean age of 46.55 ± 15.96 years and 38 were men. The performance of rapid test, using proteinase K, was found to be the most impressive: sensitivity 93%, specificity 94%, PPV 90%, and NPV 96% compared with adenosine deaminase test (ADA), PCR, smear, and culture. The present study did demonstrate that modified TB rapid test can substantially improve the diagnosis of extrapulmonary TB.

  13. The procollagen N-proteinases ADAMTS2, 3 and 14 in pathophysiology.

    PubMed

    Bekhouche, Mourad; Colige, Alain

    2015-01-01

    Collagen fibers are the main components of most of the extracellular matrices where they provide a structural support to cells, tissues and organs. Fibril-forming procollagens are synthetized as individual chains that associate to form homo- or hetero-trimers. They are characterized by the presence of a central triple helical domain flanked by amino and carboxy propeptides. Although there are some exceptions, these two propeptides have to be proteolytically removed to allow the almost spontaneous assembly of the trimers into collagen fibrils and fibers. While the carboxy-propeptide is mainly cleaved by proteinases from the tolloid family, the amino-propeptide is usually processed by procollagen N-proteinases: ADAMTS2, 3 and 14. This review summarizes the current knowledge concerning this subfamily of ADAMTS enzymes and discusses their potential involvement in physiopathological processes that are not directly linked to fibrillar procollagen processing. PMID:25863161

  14. Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus.

    PubMed

    Boonrod, Kajohn; Füllgrabe, Marc W; Krczal, Gabi; Wassenegger, Michael

    2011-10-01

    The multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, processes the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (ZYMV) HC-Pro. Several Escherichia coli-expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.

  15. Pharmacology of triheteromeric N-Methyl-D-Aspartate Receptors.

    PubMed

    Cheriyan, John; Balsara, Rashna D; Hansen, Kasper B; Castellino, Francis J

    2016-03-23

    The N-Methyl-D-Aspartate Receptors (NMDARs) are heteromeric cation channels involved in learning, memory, and synaptic plasticity, and their dysregulation leads to various neurodegenerative disorders. Recent evidence has shown that apart from the GluN1/GluN2A and GluN1/GluN2B diheteromeric ion channels, the NMDAR also exists as a GluN1/GluN2A/GluN2B triheteromeric channel that occupies the majority of the synaptic space. These GluN1/GluN2A/GluN2B triheteromers exhibit pharmacological and electrophysiological properties that are distinct from the GluN1/GluN2A and GluN1/GluN2B diheteromeric subtypes. However, these receptors have not been characterized with regards to their inhibition by conantokins, as well as their allosteric modulation by polyamines and extracellular protons. Here, we show that the GluN1/GluN2A/GluN2B triheteromeric channels showed less sensitivity to GluN2B-specific conantokin (con)-G and con-RlB, and subunit non-specific con-T, compared to the GluN2A-specific inhibitor TCN-201. Also, spermine modulation of GluN1/GluN2A/GluN2B triheteromers switched its nature from potentiation to inhibition in a pH dependent manner, and was 2.5-fold slower compared to the GluN1/GluN2B diheteromeric channels. Unraveling the distinctive functional attributes of the GluN1/GluN2A/GluN2B triheteromers is physiologically relevant since they form an integral part of the synapse, which will aid in understanding spermine/pH-dependent potentiation of these receptors in pathological settings. PMID:26917100

  16. Isolation and characterization of two forms of an acidic bromelain stem proteinase.

    PubMed

    Harrach, T; Eckert, K; Maurer, H R; Machleidt, I; Machleidt, W; Nuck, R

    1998-05-01

    Two forms of an acidic bromelain proteinase isolated from crude bromelain, an extract from pineapple stem, were found by a two-step FPLC purification procedure. The basic main components were removed by cation exchange chromatography and the breakthrough fraction was further resolved by anion exchange chromatography into 15 protein fractions, only two of which, called SBA/a and SBA/b, were proteolytically active. These components were characterized by electrospray mass spectroscopy (ESMS), isoelectric focusing, N-terminal amino acid sequence analysis, monosaccharide analysis, and enzymatic parameters. The molecular masses of SBA/a and SBA/b were determined by ESMS to be 23,550 and 23,560, respectively. The isoelectric points (pI) of the two bands of SBA/a were 4.8 and 4.9; SBA/b focused as a single band at pI = 4.8. Partial N-terminal amino acid sequences (11 residues) were identical to SBA/a and SBA/b and identical with those of stem bromelain, the basic main proteinase of the pineapple stem, and fruit bromelain, the acidic main proteinase of the pineapple fruit. Both components are highly glycosylated; hydrolysis of SBA/a yielded about twofold more monosaccharide per protein than SBA/b. The comparison of the catalytic properties of SBA/a with those of SBA/b revealed no relevant differences in the hydrolysis of three peptidyl-NH-Mec substrates and in the inhibition profiles using chicken cystatin and E-64, indicating that these components can be considered as two forms of a single enzyme. Both forms are scarcely inhibited by chicken cystatin and slowly inactivated by E-64, hence are nontypical cysteine proteinases of the papain superfamily.

  17. Identification and characterization of the immunogenic cytotoxic TvCP39 proteinase gene of Trichomonas vaginalis.

    PubMed

    Ramón-Luing, Lucero de Los Ángeles; Rendón-Gandarilla, Francisco Javier; Puente-Rivera, Jonathan; Ávila-González, Leticia; Arroyo, Rossana

    2011-10-01

    TvCP39 is a 39 kDa cysteine proteinase (CP) involved in Trichomonas vaginalis cytotoxicity that has been found in vaginal secretions and is immunogenic in patients with trichomonosis. The goal of this work was to identify, clone, express, and characterize the tvcp39 gene. The tvcp39 gene was identified using a proteomic approach, and the complete gene was amplified using PCR, cloned, and sequenced. TvCP39 is encoded by a 915-bp cathepsin L-like CP gene. A fragment corresponding to the mature region (TvCP39r) was expressed, purified, and used to produce rabbit polyclonal antibodies and in functional assays. In one- and two-dimensional western blot assays, the anti-TvCP39r antibody reacted with two protein bands of ~28 and 27 kDa and three spots of ~28, 27, and 24 kDa in trichomonad proteinase-rich extracts that could correspond to the mature and processed fragments of the TvCP39 peptidase. The anti-TvCP39r antibody reacted with the parasitic surface and the native TvCP39 present in vaginal washes from patients with trichomonosis. Moreover, the recombinant TvCP39 protein bound to the surface of HeLa cells and protected HeLa cell monolayers from trichomonal destruction in a concentration-dependent manner. In conclusion, our data support TvCP39 as one of the surface proteinases that is glycosylated and is involved in trichomonal cytotoxicity. Thus, TvCP39 is the first glycosylated cysteine proteinase detected in T. vaginalis. PMID:21777690

  18. Effect of acute ozone exposure on the proteinase-antiproteinase balance in the rat lung

    SciTech Connect

    Pickrell, J.A.; Gregory, R.E.; Cole, D.J.; Hahn, F.F.; Henderson, R.F.

    1987-04-01

    Lung disease may result from a persisting proteinase excess or a depletion of antiproteinase in pulmonary parenchyma. We investigated the in vivo effect of a 48-hr exposure to ozone at 0.5, 1.0, or 1.5 ppm on proteinase and antiproteinase activity of rat lungs. Elastase inhibitory capacities of serum, lung tissue, and airway washings were measured as indicators of antielastase activity. Trypsin inhibitory capacity was measured using an esterolytic procedure. Proteinase was measured as radioactive release from a /sup 14/C-globin substrate. The 48-hr exposures to O/sub 3/ at levels up to 1 ppm produced concentration-dependent decreases of 35-80% of antiproteinase activities in serum and in lung tissue. However, exposure to 1.5 ppm O/sub 3/ resulted in no decrease in antiproteinase activities. Acid proteinase activities (pH 4.2) were increased 65-120% by exposure to 1 or 1.5 ppm O/sub 3/, which correlated with inflammatory cells noted histologically. At 1.5 ppm O/sub 3/, pulmonary edema and hemorrhage were noted in histologic sections. These changes led to a flooding of the alveoli with up to 40 times normal protein levels and a greater than fivefold increase in airway antiproteinase. These data suggest that serum and soluble lung tissue antiproteinase activity decreased upon exposure to low levels of ozone. However, if O/sub 3/ exposure is high enough to produce pulmonary hemorrhage, antiproteinase may increase following serum exudation. These changes may be important in the development of ozone-induced lung diseases, especially emphysema.

  19. Benzyloxycarbonylphenylalanylcitrulline p-nitroanilide as a substrate for papain and other plant cysteine proteinases.

    PubMed Central

    Gray, C J; Boukouvalas, J; Szawelski, R J; Wharton, C W

    1984-01-01

    After preliminary assays, with papain, bromelain and ficin, on a range of citrulline p-nitroanilides, values of Km and kcat. for the papain-catalysed hydrolysis of three derivatives, N alpha- benzyloxycarbonylcitrulline p-nitroanilide, benzyloxycarbonylphenylalanylcitrulline p-nitroanilide and benzyloxycarbonylglycylphenylalanylcitrulline p-nitroanilide, were obtained. It is concluded that benzyloxycarbonylphenylalanylcitrulline p-nitroanilide is a highly selective substrate for the sensitive detection and assay of the plant cysteine proteinases. PMID:6721861

  20. Benzyloxycarbonylphenylalanylcitrulline p-nitroanilide as a substrate for papain and other plant cysteine proteinases.

    PubMed

    Gray, C J; Boukouvalas, J; Szawelski, R J; Wharton, C W

    1984-04-01

    After preliminary assays, with papain, bromelain and ficin, on a range of citrulline p-nitroanilides, values of Km and kcat. for the papain-catalysed hydrolysis of three derivatives, N alpha- benzyloxycarbonylcitrulline p-nitroanilide, benzyloxycarbonylphenylalanylcitrulline p-nitroanilide and benzyloxycarbonylglycylphenylalanylcitrulline p-nitroanilide, were obtained. It is concluded that benzyloxycarbonylphenylalanylcitrulline p-nitroanilide is a highly selective substrate for the sensitive detection and assay of the plant cysteine proteinases.

  1. Identification and characterization of the immunogenic cytotoxic TvCP39 proteinase gene of Trichomonas vaginalis.

    PubMed

    Ramón-Luing, Lucero de Los Ángeles; Rendón-Gandarilla, Francisco Javier; Puente-Rivera, Jonathan; Ávila-González, Leticia; Arroyo, Rossana

    2011-10-01

    TvCP39 is a 39 kDa cysteine proteinase (CP) involved in Trichomonas vaginalis cytotoxicity that has been found in vaginal secretions and is immunogenic in patients with trichomonosis. The goal of this work was to identify, clone, express, and characterize the tvcp39 gene. The tvcp39 gene was identified using a proteomic approach, and the complete gene was amplified using PCR, cloned, and sequenced. TvCP39 is encoded by a 915-bp cathepsin L-like CP gene. A fragment corresponding to the mature region (TvCP39r) was expressed, purified, and used to produce rabbit polyclonal antibodies and in functional assays. In one- and two-dimensional western blot assays, the anti-TvCP39r antibody reacted with two protein bands of ~28 and 27 kDa and three spots of ~28, 27, and 24 kDa in trichomonad proteinase-rich extracts that could correspond to the mature and processed fragments of the TvCP39 peptidase. The anti-TvCP39r antibody reacted with the parasitic surface and the native TvCP39 present in vaginal washes from patients with trichomonosis. Moreover, the recombinant TvCP39 protein bound to the surface of HeLa cells and protected HeLa cell monolayers from trichomonal destruction in a concentration-dependent manner. In conclusion, our data support TvCP39 as one of the surface proteinases that is glycosylated and is involved in trichomonal cytotoxicity. Thus, TvCP39 is the first glycosylated cysteine proteinase detected in T. vaginalis.

  2. A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

    PubMed

    Burlini, N; Magnani, P; Villa, A; Macchi, F; Tortora, P; Guerritore, A

    1992-08-21

    A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.

  3. Biased signalling and proteinase-activated receptors (PARs): targeting inflammatory disease

    PubMed Central

    Hollenberg, M D; Mihara, K; Polley, D; Suen, J Y; Han, A; Fairlie, D P; Ramachandran, R

    2014-01-01

    Although it has been known since the 1960s that trypsin and chymotrypsin can mimic hormone action in tissues, it took until the 1990s to discover that serine proteinases can regulate cells by cleaving and activating a unique four-member family of GPCRs known as proteinase-activated receptors (PARs). PAR activation involves the proteolytic exposure of its N-terminal receptor sequence that folds back to function as a ‘tethered’ receptor-activating ligand (TL). A key N-terminal arginine in each of PARs 1 to 4 has been singled out as a target for cleavage by thrombin (PARs 1, 3 and 4), trypsin (PARs 2 and 4) or other proteases to unmask the TL that activates signalling via Gq, Gi or G12/13. Similarly, synthetic receptor-activating peptides, corresponding to the exposed ‘TL sequences’ (e.g. SFLLRN—, for PAR1 or SLIGRL— for PAR2) can, like proteinase activation, also drive signalling via Gq, Gi and G12/13, without requiring receptor cleavage. Recent data show, however, that distinct proteinase-revealed ‘non-canonical’ PAR tethered-ligand sequences and PAR-activating agonist and antagonist peptide analogues can induce ‘biased’ PAR signalling, for example, via G12/13-MAPKinase instead of Gq-calcium. This overview summarizes implications of this ‘biased’ signalling by PAR agonists and antagonists for the recognized roles the PARs play in inflammatory settings. Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5 PMID:24354792

  4. SARS CoV main proteinase: The monomer-dimer equilibrium dissociation constant.

    PubMed

    Graziano, Vito; McGrath, William J; Yang, Lin; Mangel, Walter F

    2006-12-12

    The SARS coronavirus main proteinase (SARS CoV main proteinase) is required for the replication of the severe acute respiratory syndrome coronavirus (SARS CoV), the virus that causes SARS. One function of the enzyme is to process viral polyproteins. The active form of the SARS CoV main proteinase is a homodimer. In the literature, estimates of the monomer-dimer equilibrium dissociation constant, KD, have varied more than 65,0000-fold, from <1 nM to more than 200 microM. Because of these discrepancies and because compounds that interfere with activation of the enzyme by dimerization may be potential antiviral agents, we investigated the monomer-dimer equilibrium by three different techniques: small-angle X-ray scattering, chemical cross-linking, and enzyme kinetics. Analysis of small-angle X-ray scattering data from a series of measurements at different SARS CoV main proteinase concentrations yielded KD values of 5.8 +/- 0.8 microM (obtained from the entire scattering curve), 6.5 +/- 2.2 microM (obtained from the radii of gyration), and 6.8 +/- 1.5 microM (obtained from the forward scattering). The KD from chemical cross-linking was 12.7 +/- 1.1 microM, and from enzyme kinetics, it was 5.2 +/- 0.4 microM. While each of these three techniques can present different, potential limitations, they all yielded similar KD values.

  5. Identification of monomeric alpha-macroglobulin proteinase inhibitors in birds, reptiles, amphibians and mammals, and purification and characterization of a monomeric alpha-macroglobulin proteinase inhibitor from the American bullfrog Rana catesbeiana.

    PubMed Central

    Rubenstein, D S; Thøgersen, I B; Pizzo, S V; Enghild, J J

    1993-01-01

    The alpha-macroglobulins are classified as broad-spectrum inhibitors because of their ability to entrap proteinases of different specificities and catalytic class. Tetrameric and dimeric alpha-macroglobulins have been identified in a wide variety of organisms including those as primitive as the mollusc Octopus vulgaris; however, monomeric alpha-macroglobulin proteinase inhibitors have been previously identified only in rodents. The monomeric alpha-macroglobulin proteinase inhibitors are believed to be analogous to the evolutionary precursor of the multimeric members of this family exemplified by the tetrameric human alpha 2-macroglobulin. Until now, monomeric alpha-macroglobulin proteinase inhibitors have only been identified in rodents and have therefore been considered an evolutionary anomaly. However, in this report we have utilized several sensitive assays to screen various plasmas and sera for the presence of monomeric alpha-macroglobulins, and our results suggest that monomeric alpha-macroglobulin proteinase inhibitors are present in organisms belonging to the avian, reptilian, amphibian and mammalian classes of the chordate phylum. This indicates that these proteins are more widespread than previously recognized and that their presence in rodents is not an anomaly. To demonstrate further that the identified proteins were indeed monomeric alpha-macroglobulin proteinase inhibitors, we purified the monomeric alpha-macroglobulin from the American bullfrog Rana catesbeiana. We conclude that this protein is a monomer of 180 kDa on the basis of its behaviour on (i) pore-limit gel electrophoresis, (ii) non-reducing and reducing SDS/PAGE and (iii) gel-filtration chromatography. In addition, we demonstrate that this protein is an alpha-macroglobulin proteinase inhibitor by virtue of (i) its ability to inhibit proteinases of different catalytic class, (ii) the presence of a putative internal beta-cysteinyl-gamma-glutamyl thioester and (iii) an inhibitory mechanism

  6. Identification of monomeric alpha-macroglobulin proteinase inhibitors in birds, reptiles, amphibians and mammals, and purification and characterization of a monomeric alpha-macroglobulin proteinase inhibitor from the American bullfrog Rana catesbeiana.

    PubMed

    Rubenstein, D S; Thøgersen, I B; Pizzo, S V; Enghild, J J

    1993-02-15

    The alpha-macroglobulins are classified as broad-spectrum inhibitors because of their ability to entrap proteinases of different specificities and catalytic class. Tetrameric and dimeric alpha-macroglobulins have been identified in a wide variety of organisms including those as primitive as the mollusc Octopus vulgaris; however, monomeric alpha-macroglobulin proteinase inhibitors have been previously identified only in rodents. The monomeric alpha-macroglobulin proteinase inhibitors are believed to be analogous to the evolutionary precursor of the multimeric members of this family exemplified by the tetrameric human alpha 2-macroglobulin. Until now, monomeric alpha-macroglobulin proteinase inhibitors have only been identified in rodents and have therefore been considered an evolutionary anomaly. However, in this report we have utilized several sensitive assays to screen various plasmas and sera for the presence of monomeric alpha-macroglobulins, and our results suggest that monomeric alpha-macroglobulin proteinase inhibitors are present in organisms belonging to the avian, reptilian, amphibian and mammalian classes of the chordate phylum. This indicates that these proteins are more widespread than previously recognized and that their presence in rodents is not an anomaly. To demonstrate further that the identified proteins were indeed monomeric alpha-macroglobulin proteinase inhibitors, we purified the monomeric alpha-macroglobulin from the American bullfrog Rana catesbeiana. We conclude that this protein is a monomer of 180 kDa on the basis of its behaviour on (i) pore-limit gel electrophoresis, (ii) non-reducing and reducing SDS/PAGE and (iii) gel-filtration chromatography. In addition, we demonstrate that this protein is an alpha-macroglobulin proteinase inhibitor by virtue of (i) its ability to inhibit proteinases of different catalytic class, (ii) the presence of a putative internal beta-cysteinyl-gamma-glutamyl thioester and (iii) an inhibitory mechanism

  7. Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α.

    PubMed

    Steinberger, Jutta; Kontaxis, Georg; Rancan, Chiara; Skern, Tim

    2013-09-01

    The foot-and-mouth disease virus leader proteinase (Lb(pro)) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lb(pro) L200F provide structural evidence for intramolecular self-processing. (15)N-HSQC measurements of Lb(pro) L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLb(pro), lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lb(pro), stably binds its own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lb(pro) and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lb(pro).

  8. Clearance of human native, proteinase-complexed, and proteolytically inactivated C1-inhibitor in rats.

    PubMed

    de Smet, B J; de Boer, J P; Agterberg, J; Rigter, G; Bleeker, W K; Hack, C E

    1993-01-01

    C1-inhibitor is the only known inhibitor of the classical pathway of complement and the major inhibitor of the contact pathway of coagulation. Like other serine proteinase inhibitors, C1-inhibitor can exist in three conformations, ie, the native, the proteinase-complexed, and the proteolytically inactivated form. Here we studied the plasma elimination kinetics of these three forms of human C1-inhibitor in rats. The clearance of the complexed form of C1-inhibitor appeared to be the most rapid and depended in part on the proteinase involved (observed plasma t1/2 was 20 minutes for C1s-C1-inhibitor, 32 minutes for kallikrein-C1-inhibitor, and 47 minutes for beta XIIa-C1-inhibitor), whereas that of native C1-inhibitor was the slowest (observed plasma t1/2 4.5 hours). Inactivated C1-inhibitor was cleared with an apparent plasma t1/2 of 1.6 hours. Thus, the short plasma t1/2 of complexed relative to native C1-inhibitor explains why in patients only low concentrations of C1-inhibitor complexes may be observed despite activation of the contact and/or complement systems.

  9. Norovirus Proteinase-Polymerase and Polymerase Are Both Active Forms of RNA-Dependent RNA Polymerase

    PubMed Central

    Belliot, Gaël; Sosnovtsev, Stanislav V.; Chang, Kyeong-Ok; Babu, Vijay; Uche, Uzo; Arnold, Jamie J.; Cameron, Craig E.; Green, Kim Y.

    2005-01-01

    In vitro mapping studies of the MD145 norovirus (Caliciviridae) ORF1 polyprotein identified two stable cleavage products containing the viral RNA-dependent RNA polymerase (RdRp) domains: ProPol (a precursor comprised of both the proteinase and polymerase) and Pol (the mature polymerase). The goal of this study was to identify the active form (or forms) of the norovirus polymerase. The recombinant ProPol (expressed as Pro−Pol with an inactivated proteinase domain to prevent autocleavage) and recombinant Pol were purified after synthesis in bacteria and shown to be active RdRp enzymes. In addition, the mutant His-E1189A-ProPol protein (with active proteinase but with the natural ProPol cleavage site blocked) was active as an RdRp, confirming that the norovirus ProPol precursor could possess two enzymatic activities simultaneously. The effects of several UTP analogs on the RdRp activity of the norovirus and feline calicivirus Pro−Pol enzymes were compared and found to be similar. Our data suggest that the norovirus ProPol is a bifunctional enzyme during virus replication. The availability of this recombinant ProPol enzyme might prove useful in the development of antiviral drugs for control of the noroviruses associated with acute gastroenteritis. PMID:15681440

  10. Fibronectin-Degrading Activity of Trypanosoma cruzi Cysteine Proteinase Plays a Role in Host Cell Invasion

    PubMed Central

    Maeda, Fernando Yukio; Cortez, Cristian; Izidoro, Mario Augusto; Juliano, Luiz

    2014-01-01

    Trypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insect-stage metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, is not clear. Using T. cruzi CL strain metacyclic forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to metacyclic forms and was digested by cruzipain, the major T. cruzi cysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronectin expression was stimulated by treatment of epithelial HeLa cells with transforming growth factor beta, the parasite invasion was reduced. Treatment of HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas the treatment of parasites with cysteine proteinase inhibitor had the opposite effect. Metacyclic trypomastigote entry into HeLa cells was not affected by anti-β1 integrin antibody but was inhibited by anti-fibronectin antibody. Overall, our results have indicated that the cysteine proteinase of T. cruzi metacyclic forms, through its fibronectin-degrading activity, is implicated in host cell invasion. PMID:25267835

  11. Corticosteroid-binding globulin, a structural basis for steroid transport and proteinase-triggered release.

    PubMed

    Klieber, Michael A; Underhill, Caroline; Hammond, Geoffrey L; Muller, Yves A

    2007-10-01

    Corticosteroid-binding globulin (CBG) is a serine proteinase inhibitor (serpin) family member that transports glucocorticoids in blood and regulates their access to target cells. The 1.9A crystal structure of rat CBG shows that its steroid-binding site resembles the thyroxin-binding site in the related serpin, thyroxin-binding globulin, and mutagenesis studies have confirmed the contributions of key residues that constitute the steroid-binding pocket. Unlike thyroxin-bound thyroxin-binding globulin, the cortisol-bound CBG displays an "active" serpin conformation with the proteinase-sensitive, reactive center loop (RCL) fully expelled from the regulatory beta-sheet A. Moreover, the CBG structure allows us to predict that complete insertion of the proteolytically cleaved RCL into the serpin fold occurs in concert with a displacement and unwinding of helix D that would disrupt the steroid-binding site. This allosteric coupling between RCL positioning and occupancy of the CBG steroid-binding site, which resembles the ligand (glycosamino-glycan)-dependent activation of the thrombin inhibitory serpins heparin cofactor II and anti-thrombin RCLs, ensures both optimal recognition of CBG by target proteinases and efficient release of steroid to sites of action.

  12. Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation.

    PubMed Central

    Burnett, T J; Shankweiler, G W; Hageman, J H

    1986-01-01

    Cells of Bacillus subtilis 168 (trpC2) growing and sporulating in a single chemically defined medium carried out intracellular protein degradation and increased their levels of intracellular serine protease-1 in a manner very similar to what had previously been reported for cells sporulating in nutrient broth. The results were interpreted to mean that these processes are intrinsic to sporulation rather than medium dependent. To determine the cause of these increases in specific activity of proteinases, we purified the protease, prepared rabbit immunoglobulins directed against it, and monitored changes in protease antigen levels by performing rocket immunoelectrophoresis. In cells sporulating in nutrient broth, the protease antigen levels increased about 7-fold, whereas the specific activity increased about 150-fold, for an activation of about 20-fold. In cells sporulating in the single chemically defined sporulation medium, the protease antigen increased about 10-fold, whereas the specific activity increased at least 400-fold, for an activation of about 40-fold. These results were interpreted to mean that a posttranslational event activated the protease in vivo; a previously described endogenous proteinase inhibitor was confirmed to be present in the strain used. Chloramphenicol added to the cultures inhibited both the increases in antigen levels and in the specific activity of the proteinase. PMID:3079745

  13. Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions.

    PubMed

    Hernández-Gutiérrez, Rodolfo; Avila-González, Leticia; Ortega-López, Jaime; Cruz-Talonia, Fernando; Gómez-Gutierrez, Guillermo; Arroyo, Rossana

    2004-01-01

    Trichomonosis, a chronic sexually transmitted disease, remains a public health problem affecting yearly over 170 million people worldwide. This disease is caused by Trichomonas vaginalis, a protozoan flagellate rich in cysteine proteinases (CPs). Although CPs are involved in trichomonal cytopathogenicity, only few of them have been defined as virulence factors. In this study, we characterize a T. vaginalis 39-kDa proteinase (CP39) found in vaginal secretions from patients with trichomonosis. The CP39 proteinase bound to HeLa epithelial cells, vaginal epithelial cells (VECs), and human prostatic cancer cells (DU-145). CP39 did not bind to a human colon cancer (CaCo) cell line, suggesting tissue-specific binding. CP39 was found in six fresh trichomonad isolates tested. In two-dimensional gels, CP39 appeared as a single spot with a pI 4.5. CP39 is inhibited by E-64, stable at 50 degrees C, and active in a wide pH range (3.6-9.0), with an optimum pH at 7.0. In addition, CP39 degraded collagens I, III, IV, and V, human fibronectin, human hemoglobin, and human immunoglobulins A and G. Indirect immunofluorescence detected CP39 on the parasite surface with specific polyclonal antibody to purified CP39. Finally, CP39 was found to be immunogenic, as evidenced by detection on immunoblots with serum of patients with trichomonosis, but not control individuals. These data suggest that CP39 may play a role during trichomonal infection.

  14. Several murine metastasizing tumors possess a cysteine proteinase with cancer procoagulant characteristics.

    PubMed

    Falanga, A; Bolognese Dalessandro, A P; Casali, B; Roncaglioni, M C; Donati, M B

    1987-06-15

    Cancer Procoagulant (CP), a cysteine proteinase which triggers blood coagulation by directly activating Factor X (FX) in the absence of Factor VII (F VII), has recently been isolated from rabbit V2 carcinoma and biochemically characterized. We have studied the procoagulant activity of tissue extracts from 4 murine experimental tumors in order to define whether or not a F VII-independent activity with cysteine proteinase characteristics was present. The tumors studied were: Lewis lung carcinoma (3LL), B16 melanoma (B16), JW sarcoma (JWS) and the M4 variant of the mFS6 fibrosarcoma (M4). Extracts from 3LL, B16 and JWS tumor initiated coagulation in both the presence and absence of F VII, their procoagulant activity was sensitive to iodoacetamide (1 mM) and mercury chloride (0.1 mM). The procoagulant of M4 extract was dependent on the presence of F VII and was not significantly affected by the cysteine proteinase inhibitors. An Ouchterlony double immunodiffusion study showed immunological cross-reactivity of all but M4 extracts to a polyclonal antibody to purified CP. The present study suggests that the procoagulant(s) present in the murine tumors 3LL, B16 and JWS are enzymatically and immunologically indistinguishable from cancer procoagulant of the rabbit V2 carcinoma.

  15. [Characteristics of proteinase digestive function in invertebrates--inhabitants of cold seas].

    PubMed

    Mukhin, V A; Smirnova, E B; Novikov, V Iu

    2007-01-01

    Digestive proteinases of various taxa of invertebrates of the Northern seas have been studied: crustaceans Paralithodes camtchaticus, Pandalus borealis; molluscs Chlamys islandicus, Buccinum undatum, Serripes groenlandicus, and echinoderms Strongylocentrotus droebachiensis, Cucumaria frondosa, Asterias rubens, and Grossaster papposus. The presence of two proteolytic activity peaks in the acid (pH 2.5-3.5) and low alkaline zones (pH 7.5-8.5) and a similar proteinase spectrum have been revealed in digestive organs of the studied animals. The proteolytic activity in digestive organs of the Barents Sea invertebrates exceeds significantly that of terrestrial homoiothermal animals, which seems to be an extensive compensation for poor differentiation of the digestive system and for low substrate specificity of the enzymes as well as for cold conditions of the habitat. The principal qualitative difference between vertebrates and invertebrates consists in that the latter have no pepsin activity, but do have the cathepsin activity that is absent in vertebrate digestive organs. Contribution to the acid proteolysis is made by lysosomal cathepsins, rather than by pepsins. Activity in the alkaline and neutral pH zones is provided by serine proteinases. In digestive cavities of invertebrates, hydrolysis of proteins and mechanical processing of food occur only in the low alkaline zone, whereas acid proteolysis has intracellular lysosomal localization.

  16. A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests.

    PubMed

    Pernas, M; Sánchez-Monge, R; Gómez, L; Salcedo, G

    1998-12-01

    Cystatin CsC, a cysteine proteinase inhibitor from chestnut (Castanea sativa) seeds, has been purified and characterized. Its full-length cDNA clone was isolated from an immature chestnut cotyledon library. The inhibitor was expressed in Escherichia coli and purified from bacterial extracts. Identity of both seed and recombinant cystatin was confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis, two-dimensional electrophoresis and N-terminal sequencing. CsC has a molecular mass of 11,275 Da and pI of 6.9. Its amino acid sequence includes all three motifs that are thought to be essential for inhibitory activity, and shows significant identity to other phytocystatins, especially that of cowpea (70%). Recombinant CsC inhibited papain (Ki 29 nM), ficin (Ki 65 nM), chymopapain (Ki 366 nM), and cathepsin B (Ki 473 nM). By contrast with most cystatins, it was also effective towards trypsin (Ki 3489 nM). CsC is active against digestive proteinases from the insect Tribolium castaneum and the mite Dermatophagoides farinae, two important agricultural pests. Its effects on the cysteine proteinase activity of two closely related mite species revealed the high specificity of the chestnut cystatin.

  17. Ethylene regulates the expression of a cysteine proteinase gene during germination of chickpea (Cicer arietinum L.).

    PubMed

    Cervantes, E; Rodríguez, A; Nicolás, G

    1994-05-01

    Synthetic oligonucleotides corresponding to conserved regions of cysteine proteinases were used as primers in the RT-PCR amplification of a fragment of cDNA corresponding to a region of a cysteine proteinase gene expressed during germination of chickpea (cac for Cicer arietinum cysteine proteinase). The identity of the PCR-amplified fragment was confirmed by sequencing and the fragment used as a probe to investigate the pattern of cac gene expression during germination and its hormonal regulation. The corresponding transcript is undetected in the seed during embryogenesis and before imbibition, being detected 24 h after imbibition. Ablation of the embryonic axis before imbibition results in a dramatic decrease in the amount of transcript detected. Expression of the cac transcript in excised cotyledons is restored in the presence of aqueous extracts from embryonic axes and also by incubating the excised cotyledons in 1 mM ethephon. Experiments with various known inhibitors of ethylene action indicate that ethylene activates the expression of cac gene in the cotyledons of chickpea during normal germination.

  18. Cloning and characterization of an Eimeria acervulina sporozoite gene homologous to aspartyl proteinases.

    PubMed

    Laurent, F; Bourdieu, C; Kaga, M; Chilmonczyk, S; Zgrzebski, G; Yvoré, P; Péry, P

    1993-12-01

    A lambda ZapII cDNA library was constructed using mRNA from Eimeria acervulina sporulated oocysts and screened with monoclonal antibodies raised against Eimeria tenella sporulated oocytes. Monoclonal antibody N3C8B12 identified a clone (6S2) potentially encoding an aspartyl proteinase since significant homology with cathepsin D, pepsin and renin proteinases was revealed by sequence comparisons. The 1500-bp cDNA fragment containing the coccidial gene was subcloned into pGEX-FA expression vector, leading to the production of an 80-kDa fusion protein (FA6S2) which was used to immunize rabbits. The anti-FA6S2 rabbit sera revealed a single 43-kDa protein present in Eimeria acervulina, Eimeria tenella, Eimeria maxima and Eimeria falciformis sporulated oocyst antigens. Indirect immunofluorescence and electron microscopy with mAb N3C8B12 localized the putative aspartyl proteinase in the refractile bodies of Eimeria tenella sporozoites.

  19. N-Methyl-D-Aspartate Receptor Activation May Contribute to Glufosinate Neurotoxicity

    EPA Science Inventory

    N-Methyl-D-aspartate Receptor Activation May Contribute to Glufosinate Neurotoxicity Glufosinate (GLF) at high levels in mammals causes convulsions through a mechanism that is not completely understood. The structural similarity of GLF to glutamate (GLU) implicates the glutamate...

  20. Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells

    PubMed Central

    Sullivan, Lucas B.; Gui, Dan Y.; Hosios, Aaron M.; Bush, Lauren N.; Freinkman, Elizaveta; Vander Heiden, Matthew G.

    2015-01-01

    Summary Mitochondrial respiration is important for cell proliferation, however the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis. PMID:26232225

  1. Atomic resolution crystal structure of Sapp2p, a secreted aspartic protease from Candida parapsilosis.

    PubMed

    Dostál, Jiří; Pecina, Adam; Hrušková-Heidingsfeldová, Olga; Marečková, Lucie; Pichová, Iva; Řezáčová, Pavlina; Lepšík, Martin; Brynda, Jiří

    2015-12-01

    The virulence of the Candida pathogens is enhanced by the production of secreted aspartic proteases, which therefore represent possible targets for drug design. Here, the crystal structure of the secreted aspartic protease Sapp2p from Candida parapsilosis was determined. Sapp2p was isolated from its natural source and crystallized in complex with pepstatin A, a classical aspartic protease inhibitor. The atomic resolution of 0.83 Å allowed the protonation states of the active-site residues to be inferred. A detailed comparison of the structure of Sapp2p with the structure of Sapp1p, the most abundant C. parapsilosis secreted aspartic protease, was performed. The analysis, which included advanced quantum-chemical interaction-energy calculations, uncovered molecular details that allowed the experimentally observed equipotent inhibition of both isoenzymes by pepstatin A to be rationalized.

  2. Chiral Asymmetric Structures in Aspartic Acid and Valine Crystals Assessed by Atomic Force Microscopy.

    PubMed

    Teschke, Omar; Soares, David Mendez

    2016-03-29

    Structures of crystallized deposits formed by the molecular self-assembly of aspartic acid and valine on silicon substrates were imaged by atomic force microscopy. Images of d- and l-aspartic acid crystal surfaces showing extended molecularly flat sheets or regions separated by single molecule thick steps are presented. Distinct orientation surfaces were imaged, which, combined with the single molecule step size, defines the geometry of the crystal. However, single molecule step growth also reveals the crystal chirality, i.e., growth orientations. The imaged ordered lattice of aspartic acid (asp) and valine (val) mostly revealed periodicities corresponding to bulk terminations, but a previously unreported molecular hexagonal lattice configuration was observed for both l-asp and l-val but not for d-asp or d-val. Atomic force microscopy can then be used to identify the different chiral forms of aspartic acid and valine crystals.

  3. Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells.

    PubMed

    Sullivan, Lucas B; Gui, Dan Y; Hosios, Aaron M; Bush, Lauren N; Freinkman, Elizaveta; Vander Heiden, Matthew G

    2015-07-30

    Mitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis.

  4. Aspartic Peptidases of Human Pathogenic Trypanosomatids: Perspectives and Trends for Chemotherapy

    PubMed Central

    Santos, L.O.; Garcia-Gomes, A.S.; Catanho, M.; Sodré, C.L.; Santos, A.L.S.; Branquinha, M.H.; d’Avila-Levy, C.M.

    2013-01-01

    Aspartic peptidases are proteolytic enzymes present in many organisms like vertebrates, plants, fungi, protozoa and in some retroviruses such as human immunodeficiency virus (HIV). These enzymes are involved in important metabolic processes in microorganisms/virus and play major roles in infectious diseases. Although few studies have been performed in order to identify and characterize aspartic peptidase in trypanosomatids, which include the etiologic agents of leishmaniasis, Chagas’ disease and sleeping sickness, some beneficial properties of aspartic peptidase inhibitors have been described on fundamental biological events of these pathogenic agents. In this context, aspartic peptidase inhibitors (PIs) used in the current chemotherapy against HIV (e.g., amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) were able to inhibit the aspartic peptidase activity produced by different species of Leishmania. Moreover, the treatment of Leishmania promastigotes with HIV PIs induced several perturbations on the parasite homeostasis, including loss of the motility and arrest of proliferation/growth. The HIV PIs also induced an increase in the level of reactive oxygen species and the appearance of irreversible morphological alterations, triggering parasite death pathways such as programed cell death (apoptosis) and uncontrolled autophagy. The blockage of physiological parasite events as well as the induction of death pathways culminated in its incapacity to adhere, survive and escape of phagocytic cells. Collectively, these results support the data showing that parasites treated with HIV PIs have a significant reduction in the ability to cause in vivo infection. Similarly, the treatment of Trypanosoma cruzi cells with pepstatin A showed a significant inhibition on both aspartic peptidase activity and growth as well as promoted several and irreversible morphological changes. These studies indicate that aspartic peptidases can be promising targets in

  5. The standard enthalpies of formation of crystalline N-(carboxymethyl)aspartic acid and its aqueous solutions

    NASA Astrophysics Data System (ADS)

    Lytkin, A. I.; Chernyavskaya, N. V.; Volkov, A. V.; Nikol'Skii, V. M.

    2007-07-01

    The energy of combustion of N-(carboxymethyl)aspartic acid (CMAA) was determined by bomb calorimetry in oxygen. The standard enthalpies of combustion and formation of crystalline N-(carboxymethyl)aspartic acid were calculated. The heat effects of solution of crystalline CMAA in water and a solution of sodium hydroxide were measured at 298.15 K by direct calorimetry. The standard enthalpies of formation of CMAA and its dissociation products in aqueous solution were determined.

  6. Proteinase A, a storage-globulin-degrading endopeptidase of vetch (Vicia sativa L.) seeds, is not involved in early steps of storage-protein mobilization.

    PubMed

    Becker, C; Senyuk, V I; Shutov, A D; Nong, V H; Fischer, J; Horstmann, C; Müntz, K

    1997-09-01

    Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25,244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydryl-endopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna mungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination.

  7. L-aspartic acid transport by cat erythrocytes

    SciTech Connect

    Chen, C.W.; Preston, R.L.

    1986-03-01

    Cat and dog red cells are unusual in that they have no Na/K ATPase and contain low K and high Na intracellularly. They also show significant Na dependent L-aspartate (L-asp) transport. The authors have characterized this system in cat RBCs. The influx of /sup 3/H-L-asp (typically 2..mu..M) was measured in washed RBCs incubated for 60 s at 37/sup 0/C in medium containing 140 mM NaCl, 5 mM Kcl, 2 mM CaCl/sub 2/, 15 mM MOPS pH 7.4, 5 mM glucose, and /sup 14/C-PEG as a space marker. The cells were washed 3 times in the medium immediately before incubation which was terminated by centrifuging the RBCs through a layer of dibutylphthalate. Over an L-asp concentration range of 0.5-1000..mu..M, influx obeyed Michaelis-Menten kinetics with a small added linear diffusion component. The Kt and Jmax of the saturable component were 5.40 +/- 0.34 ..mu..M and 148.8 +/- 7.2 ..mu..mol 1. cell/sup -1/h/sup -1/ respectively. Replacement of Na with Li, K, Rb, Cs or choline reduce influx to diffusion. With the addition of asp analogues (4/sup +/M L-asp, 40/sup +/M inhibitor), the following sequence of inhibition was observed (range 80% to 40% inhib.): L-glutamate > L-cysteine sulfonate > D-asp > L-cysteic acid > D-glutamate. Other amino acids such as L-alanine, L-proline, L-lysine, L-cysteine, and taurine showed no inhibition (<5%). These data suggest that cat red cells contain a high-affinity Na dependent transport system for L-asp, glutamate, and closely related analogues which resembles that found in the RBCs of other carnivores and in neural tissues.

  8. The catalytic role of aspartic acid-92 in a human dual-specific protein-tyrosine-phosphatase.

    PubMed

    Denu, J M; Zhou, G; Guo, Y; Dixon, J E

    1995-03-14

    The mechanism of catalysis for the human dual-specific (vaccinia H1-related) protein-tyrosine-phosphatase was investigated. The pH dependence of the kcat value is bell-shaped when p-nitrophenyl phosphate was employed as a model substrate. The kcat/Km pH profile rises with a slope of 2 and decreases with a slope of -1, indicating that two groups must be unprotonated and one group must be protonated for activity. An amino acid residue with an apparent pKa value of 5.5 +/- 0.2 must be unprotonated and a residue with a pKa value of 5.7 must be unprotonated for activity. The pKa value of the catalytic cysteine-124 (C124) was 5.6 +/- 0.1. The aspartic acid-92-asparagine (D92N) mutant enzyme was 100-fold less active than the native enzyme and exhibited the loss of the basic limb in the pH profiles, suggesting that in the native enzyme D92 must be protonated for activity. The D92 residue is conserved throughout the entire family of dual-specific phosphatases. Mutants glutamic acid-6-glutamine, glutamic acid-32-glutamine, aspartic acid-14-asparagine, and aspartic acid-110-asparagine had less than a 2-fold effect on the kinetic parameters when compared to native enzyme. Based upon the lack of a "burst" in rapid reaction kinetics, formation of the intermediate is rate-limiting with both native and D92N mutant enzymes. In agreement with rate-limiting formation of the intermediate, the pKa value of 5.5 for the group which must be unprotonated for activity was assigned to C124.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Aspartate-90 and arginine-269 of hamster aspartate transcarbamylase affect the oligomeric state of a chimaeric protein with an Escherichia coli maltose-binding domain.

    PubMed Central

    Qiu, Y; Davidson, J N

    1998-01-01

    Residues Asp-90 and Arg-269 of Escherichia coli aspartate transcarbamylase seem to interact at the interface of adjacent catalytic subunits. Alanine substitutions at the analogous positions in the hamster aspartate transcarbamylase of a chimaeric protein carrying an E. coli maltose-binding domain lead to changes in both the kinetics of the enzyme and the quaternary structure of the protein. The Vmax for the Asp-90-->Ala and Arg-269-->Ala substitutions is decreased to 1/21 and 1/50 respectively, the [S]0.5 for aspartate is increased 540-fold and 826-fold respectively, and the [S]0.5 for carbamoyl phosphate is increased 60-fold for both. These substitutions decrease the oligomeric size of the protein. Whereas the native chimaeric protein behaves as a pentamer, the Asp-90 variant is a trimer and the Arg-269 variant is a dimer. The altered enzymes also exhibit marked decreases in thermal stability and are inactivated at much lower concentrations of urea than is the unaltered enzyme. Taken together, these results are consistent with the hypothesis that both Asp-90 and Arg-269 have a role in the enzymic function and structural integrity of hamster aspartate transcarbamylase. PMID:9425105

  10. A Trypanosoma cruzi-secreted 80 kDa proteinase with specificity for human collagen types I and IV.

    PubMed Central

    Santana, J M; Grellier, P; Schrével, J; Teixeira, A R

    1997-01-01

    Specific interactions between parasites and extracellular matrix components are an important mechanism in the dissemination of Chagas' disease. Binding of the extracellular matrix proteins to Trypanosoma cruzi receptors has been described as a significant step in this phenomenon. In this study, a specific proteinase activity was identified in cell-free extracts of amastigote, trypomastigote and epimastigote forms of T. cruzi using the collagenase fluorogenic substrate N-Suc-Gly-Pro-Leu-Gly-Pro-7-amido-4-methylcoumarin. Isolation of this activity was achieved by a four-step FPLC procedure. Optimal enzyme activity was found to occur at pH 8.0 and was associated with a single T. cruzi 80 kDa protein (Tc 80 proteinase) on SDS/PAGE under reducing conditions. An internal peptide sequence of Tc 80 proteinase was obtained (AGDNYTPPE), and no similarity was found to previously described proteinases of T. cruzi. This enzyme activity is strongly inhibited by HgCl2, tosyl-lysylchloromethane ('TLCK') p-chloromercuribenzoate and benzyloxycarbonyl-Phe-Ala-diazomethane. The purified enzyme was able to hydrolyse purified human [14C]collagen types I and IV at neutral pH, but not 14C-labelled BSA, rat laminin, rabbit IgG or small proteins such as insulin or cytochrome c. In addition, Tc 80 proteinase activity was found to be secreted by T. cruzi forms infective to mammalian cells. Furthermore we demonstrated that purified Tc 80 proteinase mediates native collagen type I hydrolysis in rat mesentery. This feature is compared with that of Clostridium histolyticum collagenase. These findings suggest that Tc 80 proteinase may facilitate T. cruzi host-cell infection by degrading the collagens of the extracellular matrix and could represent a good target for Chagas' disease chemotherapy. PMID:9224638

  11. Phospholipase and Aspartyl Proteinase Activities of Candida Species Causing Vulvovaginal Candidiasis in Patients with Type 2 Diabetes Mellitus.

    PubMed

    Bassyouni, Rasha H; Wegdan, Ahmed Ashraf; Abdelmoneim, Abdelsamie; Said, Wessam; AboElnaga, Fatma

    2015-10-01

    Few research had investigated the secretion of phospholipase and aspartyl proteinase from Candida spp. causing infection in females with type 2 diabetes mellitus. This research aimed to investigate the prevalence of vulvovaginal candidiasis (VVC) in diabetic versus non-diabetic women and compare the ability of identified Candida isolates to secrete phospholipases and aspartyl proteinases with characterization of their genetic profile. The study included 80 females with type 2 diabetes mellitus and 100 non-diabetic females within the child-bearing period. Candida strains were isolated and identified by conventional microbiological methods and by API Candida. The isolates were screened for their extracellular phospholipase and proteinase activities by culturing them on egg yolk and bovine serum albumin media, respectively. Detection of aspartyl proteinase genes (SAP1 to SAP8) and phospholipase genes (PLB1, PLB2) were performed by multiplex polymerase chain reaction. Our results indicated that vaginal candidiasis was significantly higher among the diabetic group versus nondiabetic group (50% versus 20%, respectively) (p = 0.004). C. albicans was the most prevalent species followed by C. glabrata in both groups. No significant association between diabetes mellitus and phospholipase activities was detected (p = 0.262), whereas high significant proteinase activities exhibited by Candida isolated from diabetic females were found (82.5%) (p = 0.000). Non-significant associations between any of the tested proteinase or phospholipase genes and diabetes mellitus were detected (p > 0.05). In conclusion, it is noticed that the incidence of C. glabrata causing VVC is increased. The higher prevalence of vaginal candidiasis among diabetics could be related to the increased aspartyl proteinase production in this group of patients.

  12. Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): molecular characterization and transcriptional response upon immune stimulation.

    PubMed

    Wickramaarachchi, W D Niroshana; De Zoysa, Mahanama; Whang, Ilson; Wan, Qiang; Lee, Jehee

    2013-09-01

    Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. Proteinase inhibitors play a key role in regulating the activity of the respective proteinases. Among serine proteinase inhibitors, kazal-type proteinase inhibitors (KPIs) are widely found in mammals, avians, and a variety of invertebrates. In this study, we describe the identification of a kazal-type serine proteinase inhibitor (Ab-KPI) from the disk abalone, Haliotis discus discus, which is presumably involved in innate immunity. The full-length cDNA of Ab-KPI includes 600 bp nucleotides with an open reading frame (ORF) encoding a polypeptide of 143 amino acids. The deduced amino acid sequence of Ab-KPI contains a putative 17-amino acid signal peptide and two tandem kazal domains with high similarity to other kazal-type SPIs. Each kazal domain consists of reactive site (P1) residue containing a leucine (L), and a threonine (T) located in the second amino acid position after the second conserved cysteine of each domain. Temporal expression of Ab-KPI was assessed by real time quantitative PCR in hemocytes and mantle tissue following bacterial and viral hemorrhagic septicemia virus (VHSV) challenge, and tissue injury. At 6 h post-bacterial and -VHSV challenge, Ab-KPI expression in hemocytes was increased 14-fold and 4-fold, respectively, compared to control samples. The highest up-regulations upon tissue injury were shown at 9 h and 12 h in hemocytes and mantle, respectively. The transcriptional modulation of Ab-KPI following bacterial and viral challenges and tissue injury indicates that it might be involved in immune defense as well as wound healing process in abalone.

  13. Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

    PubMed

    Salvati, L; Mattu, M; Polticelli, F; Tiberi, F; Gradoni, L; Venturini, G; Bolognesi, M; Ascenzi, P

    2001-06-01

    Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.

  14. Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α

    SciTech Connect

    Steinberger, Jutta; Rancan, Chiara; Skern, Tim

    2013-09-01

    The foot-and-mouth disease virus leader proteinase (Lb{sup pro}) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lb{sup pro} L200F provide structural evidence for intramolecular self-processing. {sup 15}N-HSQC measurements of Lb{sup pro} L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLb{sup pro}, lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lb{sup pro}, stably binds its own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lb{sup pro} and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lb{sup pro}. - Highlights: • We examine self-processing of the leader protease of foot-and-mouth disease virus. • NMR analysis strongly supports intramolecular self-processing. • Self-processing is a dynamic process with no stable complex. • Structural comparison with nsp1α of PRRSV which forms stable intramolecular complex. • Subdomain orientation explains differences in stability of intramolecular complexes.

  15. Protein digestion in cereal aphids (Sitobion avenae) as a target for plant defence by endogenous proteinase inhibitors.

    PubMed

    Pyati, Prashant; Bandani, Ali R; Fitches, Elaine; Gatehouse, John A

    2011-07-01

    Gut extracts from cereal aphids (Sitobion avenae) showed significant levels of proteolytic activity, which was inhibited by reagents specific for cysteine proteases and chymotrypsin-like proteases. Gut tissue contained cDNAs encoding cathepsin B-like cysteine proteinases, similar to those identified in the closely related pea aphid (Acyrthosiphon pisum). Analysis of honeydew (liquid excreta) from cereal aphids fed on diet containing ovalbumin showed that digestion of ingested proteins occurred in vivo. Protein could partially substitute for free amino acids in diet, although it could not support complete development. Recombinant wheat proteinase inhibitors (PIs) fed in diet were antimetabolic to cereal aphids, even when normal levels of free amino acids were present. PIs inhibited proteolysis by aphid gut extracts in vitro, and digestion of protein fed to aphids in vivo. Wheat subtilisin/chymotrypsin inhibitor, which was found to inhibit serine and cysteine proteinases, was more effective in both inhibitory and antimetabolic activity than wheat cystatin, which inhibited cysteine proteases only. Digestion of ingested protein is unlikely to contribute significantly to nutritional requirements when aphids are feeding on phloem, and the antimetabolic activity of dietary proteinase inhibitors is suggested to result from effects on proteinases involved in degradation of endogenous proteins.

  16. A Sulfhydryl Reagent Modulates Systemic Signaling for Wound-Induced and Systemin-Induced Proteinase Inhibitor Synthesis.

    PubMed Central

    Narvaez-Vasquez, J.; Orozco-Cardenas, M. L.; Ryan, C. A.

    1994-01-01

    The sulfhydryl group reagent p-chloromecuribenzene sulfonic acid (PCMBS), an established inhibitor of active apoplastic phloem loading of sucrose in several plant species, is shown to be a powerful inhibitor of wound-induced and systemin-induced activation of proteinase inhibitor synthesis and accumulation in leaves of tomato plants (Lycopersicon esculentum cv Castlemart). PCMBS, supplied to young tomato plants through their cut stems, blocks accumulation of proteinase inhibitors in leaves in response to wounding. The application of systemin directly to fresh wounds enhances systemic accumulation of proteinase inhibitors to levels higher than wounding alone. Placed on fresh wounds, PCMBS severely inhibits systemic induction of proteinase inhibitors, in both the presence and absence of exogenous systemin. PCMBS inhibition can be reversed by cysteine, dithiothreitol, and glutathione. Radiolabeled systemin placed on fresh wounds is readily transported from the wounded leaves to upper leaves. However, in the presence of PCMBS, radiolabeled systemin is not transported away from wound sites. Induction of proteinase inhibitor I synthesis by oligouronides (degree of polymerization [almost equal to] 20), linolenic acid, or methyl jasmonate was not inhibited by PCMBS. The cumulative data support a possible role for sulfhydryl groups in mediating the translocation of systemin from wound sites to distal receptor sites in tomato plants and further support a role for systemin as a systemic wound signal. PMID:12232239

  17. Insight derived from molecular dynamics simulation into substrate-induced changes in protein motions of proteinase K.

    PubMed

    Tao, Yan; Rao, Zi-He; Liu, Shu-Qun

    2010-10-01

    Because of the significant industrial, agricultural and biotechnological importance of serine protease proteinase K, it has been extensively investigated using experimental approaches such as X-ray crystallography, site-directed mutagenesis and kinetic measurement. However, detailed aspects of enzymatic mechanism such as substrate binding, release and relevant regulation remain unstudied. Molecular dynamics (MD) simulations of the proteinase K alone and in complex with the peptide substrate AAPA were performed to investigate the effect of substrate binding on the dynamics/molecular motions of proteinase K. The results indicate that during simulations the substrate-complexed proteinase K adopt a more compact and stable conformation than the substrate-free form. Further essential dynamics (ED) analysis reveals that the major internal motions are confined within a subspace of very small dimension. Upon substrate binding, the overall flexibility of the protease is reduced; and the noticeable displacements are observed not only in substrate-binding regions but also in regions opposite the substrate-binding groove/pockets. The dynamic pockets caused by the large concerted motions are proposed to be linked to the substrate recognition, binding, orientation and product release; and the significant displacements in regions opposite the binding groove/pockets are considered to play a role in modulating the dynamics of enzyme-substrate interaction. Our simulation results complement the biochemical and structural studies, highlighting the dynamic mechanism of the functional properties of proteinase K.

  18. Induction of release and up-regulated gene expression of interleukin (IL)-8 in A549 cells by serine proteinases

    PubMed Central

    Wang, Haiyan; Zheng, Yanshan; He, Shaoheng

    2006-01-01

    Background Hypersecretion of cytokines and serine proteinases has been observed in asthma. Since protease-activated receptors (PARs) are receptors of several serine proteinases and airway epithelial cells are a major source of cytokines, the influence of serine proteinases and PARs on interleukin (IL)-8 secretion and gene expression in cultured A549 cells was examined. Results A549 cells express all four PARs at both protein and mRNA levels as assessed by flow cytometry, immunofluorescence microscopy and reverse transcription polymerase chain reaction (PCR). Thrombin, tryptase, elastase and trypsin induce a up to 8, 4.3, 4.4 and 5.1 fold increase in IL-8 release from A549 cells, respectively following 16 h incubation period. The thrombin, elastase and trypsin induced secretion of IL-8 can be abolished by their specific inhibitors. Agonist peptides of PAR-1, PAR-2 and PAR-4 stimulate up to 15.6, 6.6 and 3.5 fold increase in IL-8 secretion, respectively. Real time PCR shows that IL-8 mRNA is up-regulated by the serine proteinases tested and by agonist peptides of PAR-1 and PAR-2. Conclusion The proteinases, possibly through activation of PARs can stimulate IL-8 release from A549 cells, suggesting that they are likely to contribute to IL-8 related airway inflammatory disorders in man. PMID:16696869

  19. Characterisation and mutational analysis of an ORF 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein.

    PubMed

    Liu, D X; Brown, T D

    1995-06-01

    Coronavirus gene expression involves proteolytic processing of the mRNA 1-encoded polyproteins by viral and cellular proteinases. Recently, we have demonstrated that an ORF 1b-encoded 100-kDa protein is proteolytically cleaved from the 1a/1b fusion polyprotein by a viral-specific proteinase of the picornavirus 3C proteinase group (3C-like proteinase). In this report, the 3C-like proteinase has been further analysed by internal deletion of a 2.3-kb fragment between the 3C-like proteinase-encoding region and ORF 1b and by substitution mutations of its catalytic centre as well as the two predicted cleavage sites flanking the 100-kDa protein. The results show that internal deletion of ORF 1a sequences from nucleotide 9911 to 12227 does not influence the catalytic activity of the proteinase in processing of the 1a/1b polyprotein to the 100-kDa protein species. Site-directed mutagenesis studies have confirmed that the predicted nucleophilic cysteine residue (Cys2922) and a histidine residue encoded by ORF 1a from nucleotide 8985 to 8987 (His2820) are essential for the catalytic activity of the proteinase, and that the QS(G) dipeptide bonds are its target cleavage sites. Substitution mutations of the third component of the putative catalytic triad, the glutamic acid 2843 (Glu2843) residue, however, do not affect the processing to the 100-kDa protein. In addition, cotransfection experiment shows that the 3C-like proteinase is capable of trans-cleavage of the 1a/1b polyprotein. These studies have confirmed the involvement of the 3C-like proteinase domain in processing of the 1a/1b polyprotein, the predicted catalytic centre of the proteinase, and its cleavage sites. PMID:7778277

  20. Aspartate-β-hydroxylase induces epitope-specific T cell responses in hepatocellular carcinoma.

    PubMed

    Tomimaru, Yoshito; Mishra, Sasmita; Safran, Howard; Charpentier, Kevin P; Martin, William; De Groot, Anne S; Gregory, Stephen H; Wands, Jack R

    2015-03-01

    Hepatocellular carcinoma (HCC) has a poor prognosis due to high recurrence rate. Aspartate-β-hydroxylase (ASPH) is a highly conserved transmembrane protein, which is over expressed in HCC and promotes a malignant phenotype. The capability of ASPH protein-derived HLA class I and II peptides to generate antigen specific CD4(+) and CD8(+) immune responses is unknown. Therefore, these studies aim to define the epitope specific components required for a peptide based candidate vaccine. Monocyte-derived dendritic cells (DCs) generated from the peripheral blood mononuclear cells (PBMCs) of HCC patients were loaded with ASPH protein. Helper CD4(+) T cells and CD8(+) cytotoxic T lymphocytes (CTLs) were co-incubated with the DCs; T cell activation was evaluated by flow cytometric analysis. Immunoinformatics tools were used to predict HLA class I- and class II-restricted ASPH sequences, and the corresponding peptides were synthesized. The immunogenicity of each peptide in cultures of human PBMCs was determined by IFN-γ ELISpot assay. ASPH protein-loaded DCs activated both CD4(+) and CD8(+) T cells contained within the PBMC population derived from HCC patients. Furthermore, the predicted HLA class I- and class II-restricted ASPH peptides were significantly immunogenic. Both HLA class I- and class II-restricted peptides derived from ASPH induce T cell activation in HCC. We observed that ASPH protein and related peptides were highly immunogenic in patients with HCC and produce the type of cellular immune responses required for generation of anti-tumor activity.

  1. Lactate oxidation at the mitochondria: a lactate-malate-aspartate shuttle at work

    PubMed Central

    Kane, Daniel A.

    2014-01-01

    Lactate, the conjugate base of lactic acid occurring in aqueous biological fluids, has been derided as a “dead-end” waste product of anaerobic metabolism. Catalyzed by the near-equilibrium enzyme lactate dehydrogenase (LDH), the reduction of pyruvate to lactate is thought to serve to regenerate the NAD+ necessary for continued glycolytic flux. Reaction kinetics for LDH imply that lactate oxidation is rarely favored in the tissues of its own production. However, a substantial body of research directly contradicts any notion that LDH invariably operates unidirectionally in vivo. In the current Perspective, a model is forwarded in which the continuous formation and oxidation of lactate serves as a mitochondrial electron shuttle, whereby lactate generated in the cytosol of the cell is oxidized at the mitochondria of the same cell. From this perspective, an intracellular lactate shuttle operates much like the malate-aspartate shuttle (MAS); it is also proposed that the two shuttles are necessarily interconnected in a lactate-MAS. Among the requisite features of such a model, significant compartmentalization of LDH, much like the creatine kinase of the phosphocreatine shuttle, would facilitate net cellular lactate oxidation in a variety of cell types. PMID:25505376

  2. The identification of gene duplication and the role of secreted aspartyl proteinase 1 in Candida parapsilosis virulence.

    PubMed

    Horváth, Péter; Nosanchuk, Joshua D; Hamari, Zsuzsanna; Vágvölgyi, Csaba; Gácser, Attila

    2012-03-15

    In this study, we analyzed the role of Candida parapsilosis-secreted aspartyl proteinase isoenzyme 1 (SAPP1) in virulence. The in silico analysis of SAPP1 sequence revealed a 2871 base pair-duplicated region (SAPP1a and SAPP1b) in the genome of C. parapsilosis. We generated homozygous ΔΔsapp1a, ΔΔsapp1b, and ΔΔsapp1a-ΔΔsapp1b mutants. Notably, Sapp1 production in an inducer medium was reduced by approximately 50% in the ΔΔsapp1a and ΔΔsapp1b mutants, but the other validated SAPP gene (SAPP2) was not affected. In contrast, Sapp2 production was increased in the ΔΔsapp1a-ΔΔsapp1b mutant relative to wild-type (WT) yeast. The ΔΔsapp1a-ΔΔsapp1b strain was hypersusceptible to human serum and was attenuated in its capacity to damage host-effector cells. The phagocytosis and killing of ΔΔsapp1a-ΔΔsapp1b yeasts by human peripheral blood mononuclear cells (PBMCs) and PBMC-derived macrophages (PBMC-DM) was significantly enhanced relative to WT. Phagolysosomal fusion in PBMC-DMs occurred more than twice as frequently with ingested ΔΔsapp1a-ΔΔsapp1b yeast cells compared with WT.

  3. D-Aspartate Modulates Nociceptive-Specific Neuron Activity and Pain Threshold in Inflammatory and Neuropathic Pain Condition in Mice

    PubMed Central

    Boccella, Serena; Vacca, Valentina; Errico, Francesco; Marinelli, Sara; Squillace, Marta; Di Maio, Anna; Vitucci, Daniela; Palazzo, Enza; De Novellis, Vito; Maione, Sabatino; Pavone, Flaminia; Usiello, Alessandro

    2015-01-01

    D-Aspartate (D-Asp) is a free D-amino acid found in the mammalian brain with a temporal-dependent concentration based on the postnatal expression of its metabolizing enzyme D-aspartate oxidase (DDO). D-Asp acts as an agonist on NMDA receptors (NMDARs). Accordingly, high levels of D-Asp in knockout mice for Ddo gene (Ddo−/−) or in mice treated with D-Asp increase NMDAR-dependent processes. We have here evaluated in Ddo−/− mice the effect of high levels of free D-Asp on the long-term plastic changes along the nociceptive pathway occurring in chronic and acute pain condition. We found that Ddo−/− mice show an increased evoked activity of the nociceptive specific (NS) neurons of the dorsal horn of the spinal cord (L4–L6) and a significant decrease of mechanical and thermal thresholds, as compared to control mice. Moreover, Ddo gene deletion exacerbated the nocifensive responses in the formalin test and slightly reduced pain thresholds in neuropathic mice up to 7 days after chronic constriction injury. These findings suggest that the NMDAR agonist, D-Asp, may play a role in the regulation of NS neuron electrophysiological activity and behavioral responses in physiological and pathological pain conditions. PMID:25629055

  4. Exposure to tobacco-derived materials induces overproduction of secreted proteinases in mast cells

    SciTech Connect

    Small-Howard, Andrea; Turner, Helen . E-mail: hturner@queens.org

    2005-04-15

    Mast cells reside at interfaces with the environment, including the mucosa of the respiratory and gastrointestinal tracts. This localization exposes mast cells to inhaled, or ingested, environmental challenges. In the airways of smokers, resident immune cells will be in contact with the condensed components of cigarette smoke. Mast cells are of particular interest due to their ability to promote airway remodeling and mucus hypersecretion. Clinical data show increased levels of mast cell-secreted tryptase and increased numbers of degranulated mast cells in the lavage and bronchial tissue of smokers. Since mast cell-secreted proteinases (MCPTs), including tryptases, contribute to pathological airway remodeling, we investigated the relationship between mast cell proteinases and smoke exposure. We exposed a mast cell line to cigarette smoke condensate (CSC). We show that CSC exposure increases MCPT levels in mast cells using an assay for tryptase-type MCPT activity. We hypothesized that this increase in MCPT activity reflects a CSC-induced increase in the cytosolic pool of proteinase molecules, via stimulation of MCPT transcription. Transcript array data suggested that mRNA changes in response to CSC were limited in number and peaked after 3 h of CSC exposure. However, we noted marked transcriptional regulation of several MCPT genes. CSC-induced changes in the mRNA levels for MCPTs were confirmed using quantitative RT-PCR. Taken together, our data suggest that chronic exposure to cigarette smoke up-regulates MCPT levels in mast cells at both the protein and the mRNA level. We suggest that the pathological airway remodeling that has been described in clinical studies of smoke inhalation may be attributable to MCPT overproduction in vivo.

  5. Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions.

    PubMed

    Hernández-Gutiérrez, Rodolfo; Avila-González, Leticia; Ortega-López, Jaime; Cruz-Talonia, Fernando; Gómez-Gutierrez, Guillermo; Arroyo, Rossana

    2004-01-01

    Trichomonosis, a chronic sexually transmitted disease, remains a public health problem affecting yearly over 170 million people worldwide. This disease is caused by Trichomonas vaginalis, a protozoan flagellate rich in cysteine proteinases (CPs). Although CPs are involved in trichomonal cytopathogenicity, only few of them have been defined as virulence factors. In this study, we characterize a T. vaginalis 39-kDa proteinase (CP39) found in vaginal secretions from patients with trichomonosis. The CP39 proteinase bound to HeLa epithelial cells, vaginal epithelial cells (VECs), and human prostatic cancer cells (DU-145). CP39 did not bind to a human colon cancer (CaCo) cell line, suggesting tissue-specific binding. CP39 was found in six fresh trichomonad isolates tested. In two-dimensional gels, CP39 appeared as a single spot with a pI 4.5. CP39 is inhibited by E-64, stable at 50 degrees C, and active in a wide pH range (3.6-9.0), with an optimum pH at 7.0. In addition, CP39 degraded collagens I, III, IV, and V, human fibronectin, human hemoglobin, and human immunoglobulins A and G. Indirect immunofluorescence detected CP39 on the parasite surface with specific polyclonal antibody to purified CP39. Finally, CP39 was found to be immunogenic, as evidenced by detection on immunoblots with serum of patients with trichomonosis, but not control individuals. These data suggest that CP39 may play a role during trichomonal infection. PMID:15363938

  6. Influence of immunoprotection on genetic variability of cysteine proteinases from Haemonchus contortus adult worms.

    PubMed

    Martín, S; Molina, J M; Hernández, Y I; Ferrer, O; Muñoz, Ma C; López, A; Ortega, L; Ruiz, A

    2015-11-01

    The limitations associated with the use of anthelmintic drugs in the control of gastrotintestinal nematodosis, such as the emergence of anthelmintic resistance, have stimulated the study of the immunological control of many parasites. In the case of Haemonchus contortus, several vaccination trials using native and recombinant antigens have been conducted. A group of antigens with demonstrated immunoprotective value are cathepsin B - like proteolytic enzymes of the cysteine proteinase type. These enzymes, which have been observed in both excretory-secretory products and somatic extracts of H. contortus, may vary among different geographic isolates and on strains isolated from different hosts, or even from the same host, as has been demonstrated in some comparative studies of genetic variability. In the present study, we evaluated the genetic variability of the worms that fully developed their endogenous cycle in immunised sheep and goat in order to identify the alleles of most immunoprotective value. To address these objectives, groups of sheep and goats were immunised with PBS soluble fractions enriched for cysteine proteinases from adult worms of H. contortus from either a strain of H. contortus isolated from goats of Gran Canaria Island (SP) or a strain isolated from sheep of North America (NA). The results confirmed the immunoprophylactic value of this type of enzyme against haemonchosis in both sheep and goats in association with increased levels of specific IgG. The genetic analysis demonstrated that the immunisation had a genetic selection on proteinase-encoding genes. In all the immunised animals, allelic frequencies were statistically different from those observed in non-immunised control animals in the four analysed genes. The reduction in the allelic frequencies suggests that parasites expressing these proteases are selectively targeted by the vaccine, and hence they should be considered in any subunit vaccine approach to control haemonchosis in small

  7. Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis.

    PubMed

    Carmona, A K; Puccia, R; Oliveira, M C; Rodrigues, E G; Juliano, L; Travassos, L R

    1995-07-01

    An exocellular proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine proteinases.

  8. Influence of immunoprotection on genetic variability of cysteine proteinases from Haemonchus contortus adult worms.

    PubMed

    Martín, S; Molina, J M; Hernández, Y I; Ferrer, O; Muñoz, Ma C; López, A; Ortega, L; Ruiz, A

    2015-11-01

    The limitations associated with the use of anthelmintic drugs in the control of gastrotintestinal nematodosis, such as the emergence of anthelmintic resistance, have stimulated the study of the immunological control of many parasites. In the case of Haemonchus contortus, several vaccination trials using native and recombinant antigens have been conducted. A group of antigens with demonstrated immunoprotective value are cathepsin B - like proteolytic enzymes of the cysteine proteinase type. These enzymes, which have been observed in both excretory-secretory products and somatic extracts of H. contortus, may vary among different geographic isolates and on strains isolated from different hosts, or even from the same host, as has been demonstrated in some comparative studies of genetic variability. In the present study, we evaluated the genetic variability of the worms that fully developed their endogenous cycle in immunised sheep and goat in order to identify the alleles of most immunoprotective value. To address these objectives, groups of sheep and goats were immunised with PBS soluble fractions enriched for cysteine proteinases from adult worms of H. contortus from either a strain of H. contortus isolated from goats of Gran Canaria Island (SP) or a strain isolated from sheep of North America (NA). The results confirmed the immunoprophylactic value of this type of enzyme against haemonchosis in both sheep and goats in association with increased levels of specific IgG. The genetic analysis demonstrated that the immunisation had a genetic selection on proteinase-encoding genes. In all the immunised animals, allelic frequencies were statistically different from those observed in non-immunised control animals in the four analysed genes. The reduction in the allelic frequencies suggests that parasites expressing these proteases are selectively targeted by the vaccine, and hence they should be considered in any subunit vaccine approach to control haemonchosis in small

  9. Effects of pH on the association between the inhibitor cystatin and the proteinase chymopapain.

    PubMed

    Reyes-Espinosa, Francisco; Arroyo-Reyna, Alfonso; Garcia-Gutierrez, Ponciano; Serratos, Iris N; Zubillaga, Rafael A

    2014-01-01

    Cysteine proteinases are involved in many aspects of physiological regulation. In humans, some cathepsins have shown another function in addition to their role as lysosomal proteases in intracellular protein degradation; they have been implicated in the pathogenesis of several heart and blood vessel diseases and in cancer development. In this work, we present a fluorometric and computational study of the binding of one representative plant cysteine proteinase, chymopapain, to one of the most studied inhibitors of these proteinases: chicken cystatin. The binding equilibrium constant, Kb, was determined in the pH range between 3.5 and 10.0, revealing a maximum in the affinity at pH 9.0. We constructed an atomic model for the chymopapain-cystatin dimer by docking the individual 3D protein structures; subsequently, the model was refined using a 100 ns NPT molecular dynamics simulation in explicit water. Upon scrutiny of this model, we identified 14 ionizing residues at the interface of the complex using a cutoff distance of 5.0 Å. Using the pKa values predicted with PROPKA and a modified proton-linkage model, we performed a regression analysis on our data to obtain the composite pKavalues for three isoacidic residues. We also calculated the electrostatic component of the binding energy (ΔGb,elec) at different pH values using an implicit solvent model and APBS software. The pH profile of this calculated energy compares well with the experimentally obtained binding energy, ΔGb. We propose that the residues that form an interchain ionic pair, Lys139A from chymopapain and Glu19B from cystatin, as well as Tyr61A and Tyr67A from chymopapain are the main residues responsible for the observed pH dependence in the chymopapain- cystatin affinity.

  10. Effects of pH on the association between the inhibitor cystatin and the proteinase chymopapain.

    PubMed

    Reyes-Espinosa, Francisco; Arroyo-Reyna, Alfonso; Garcia-Gutierrez, Ponciano; Serratos, Iris N; Zubillaga, Rafael A

    2014-01-01

    Cysteine proteinases are involved in many aspects of physiological regulation. In humans, some cathepsins have shown another function in addition to their role as lysosomal proteases in intracellular protein degradation; they have been implicated in the pathogenesis of several heart and blood vessel diseases and in cancer development. In this work, we present a fluorometric and computational study of the binding of one representative plant cysteine proteinase, chymopapain, to one of the most studied inhibitors of these proteinases: chicken cystatin. The binding equilibrium constant, Kb, was determined in the pH range between 3.5 and 10.0, revealing a maximum in the affinity at pH 9.0. We constructed an atomic model for the chymopapain-cystatin dimer by docking the individual 3D protein structures; subsequently, the model was refined using a 100 ns NPT molecular dynamics simulation in explicit water. Upon scrutiny of this model, we identified 14 ionizing residues at the interface of the complex using a cutoff distance of 5.0 Å. Using the pKa values predicted with PROPKA and a modified proton-linkage model, we performed a regression analysis on our data to obtain the composite pKavalues for three isoacidic residues. We also calculated the electrostatic component of the binding energy (ΔGb,elec) at different pH values using an implicit solvent model and APBS software. The pH profile of this calculated energy compares well with the experimentally obtained binding energy, ΔGb. We propose that the residues that form an interchain ionic pair, Lys139A from chymopapain and Glu19B from cystatin, as well as Tyr61A and Tyr67A from chymopapain are the main residues responsible for the observed pH dependence in the chymopapain- cystatin affinity. PMID:25426863

  11. Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals.

    PubMed

    El-Ahwany, Eman; Rabia, Ibrahim; Nagy, Faten; Zoheiry, Mona; Diab, Tarek; Zada, Suher

    2012-03-01

    Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships. PMID:22451733

  12. Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion.

    PubMed

    Abubakar, A; Saito, T; Kitazawa, H; Kawai, Y; Itoh, T

    1998-12-01

    Whey protein was digested with one of seven kinds of proteases at 37 degrees C (trypsin, proteinase K, actinase E, thermolysin, or papain) or at 25 degrees C (pepsin or chymotrypsin) for 24 h. The digested samples were assayed for the inhibitory activity of angiotensin-converting enzyme and for changes in the systolic blood pressure caused in spontaneously hypertensive rats after gastric intubation. The strongest depressive effect on the systolic blood pressure (-55 mm Hg) was observed at 6 h after gastric intubation of the whey protein that was digested by proteinase K. Finally, six peptides were chromatographically isolated from the proteinase K digest by a combination of hydrophobic reversed-phase HPLC and gel filtration. The amino acid sequences and their origins were clarified as follows: Val-Tyr-Pro-Phe-Pro-Gly [beta-casein (CN); f 59-64], Gly-Lys-Pro (beta 2-microglobulin; f 18-20), Ile-Pro-Ala (beta-lactoglobulin; f 78-80), Phe-Pro (serum albumin; f 221-222; beta-CN, f 62-63, f 157-158, and f 205-206), Val-Tyr-Pro (beta-CN; f 59-61), and Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro (beta-CN; f 80-90). Chemical synthesis of these six peptides confirmed that all peptides, except an undecapeptide, have antihypertensive activity in spontaneously hypertensive rats. The synthetic tripeptide Ile-Pro-Ala, originating from beta-lactoglobulin, showed the strongest antihypertensive activity (-31 mm Hg).

  13. Detection of D-aspartate in tau proteins associated with Alzheimer paired helical filaments.

    PubMed

    Kenessey, A; Yen, S H; Liu, W K; Yang, X R; Dunlop, D S

    1995-03-27

    Paired helical filaments (PHF) characteristic of Alzheimer neurofibrillary lesions are known to contain a modified form of microtubule associated protein tau. These proteins, PHF-tau, differ from normal tau in the extent and the site of phosphorylation. To determine whether PHF-tau, tau proteins from normal adult brains (N-tau), tau proteins from Alzheimer brains not associated with PHF (A-tau), and tau proteins from fetal brains (F-tau) differ in racemization, these proteins were compared for their D-aspartate content. The results demonstrated that PHF-tau contain more D-aspartate than N-tau, A-tau and F-tau. The average percentage D-aspartate for these proteins, after a correction for background, are 4.9%, 2.8%, 1.6%, and 1% for PHF-tau, N-tau, A-tau and F-tau, respectively. It remains to be determined if the increase in D-aspartate is a consequence of PHF formation. It is also unknown if the change in D-aspartate content in PHF-tau is associated with phosphorylation, which alters the susceptibility of tau to proteolysis.

  14. Uptake and metabolism of (14C)-aspartate by developing kernels of maize (Zea mays L. )

    SciTech Connect

    Muhitch, M.J. )

    1990-05-01

    Pulse-chase experiments were performed to determine the metabolic fate of (14C)-aspartate in the pedicel region and subsequent uptake into the endosperm. Kernels were removed from the cob, leaving the pedicel attached but removing glumes, palea, and lemma. The basal tips were incubated in (14C)-aspartate for 0.5 h, followed by a 2 h chase period with unlabeled aspartate. In contrast to a previous study in which 70% of the 14C from aspartate was recovered in the organic acid fraction (Lyznik, et al., Phytochemistry 24: 425, 1985), only 20 to 25% of the radioactivity found in the 2 h chase period. While a small amount of the 14C transiently appeared in alanine at the beginning of the chase period, the most heavily labeled non-fed amino acid was glutamine, which accounted for 21% of the radioactivity within the pedicel amino acid fraction by 0.5 h into the chase period. There was no evidence for asparagine synthesis within the pedicel region of the kernel. 14C recovered from the endosperm in the form of amino acids were aspartate (60%), glutamine (20%), glutamate (15%), and alanine (5%). These results suggest that some of the maternally supplied amino acids undergo metabolic conversion to other amino acids before being taken up by the endosperm.

  15. Interaction between L-aspartate and the brucite [Mg(OH)2]-water interface

    NASA Astrophysics Data System (ADS)

    Estrada, Charlene F.; Sverjensky, Dimitri A.; Pelletier, Manuel; Razafitianamaharavo, Angélina; Hazen, Robert M.

    2015-04-01

    The interaction of biomolecules at the mineral-water interface could have played a prominent role in the emergence of more complex organic species in life's origins. Serpentinite-hosted hydrothermal vents may have acted as a suitable environment for this process to occur, although little is known about biomolecule-mineral interactions in this system. We used batch adsorption experiments and surface complexation modeling to study the interaction of L-aspartate onto a thermodynamically stable product of serpentinization, brucite [Mg(OH)2], over a wide range of initial aspartate concentrations at four ionic strengths governed by [Mg2+] and [Ca2+]. We observed that up to 1.0 μmol of aspartate adsorbed per m2 of brucite at pH ∼ 10.2 and low Mg2+ concentrations (0.7 × 10-3 M), but surface adsorption decreased at high Mg2+ concentrations (5.8 × 10-3 M). At high Ca2+ concentrations (4.0 × 10-3 M), aspartate surface adsorption doubled (to 2.0 μmol m-2), with Ca2+ adsorption at 29.6 μmol m-2. We used the extended triple-layer model (ETLM) to construct a quantitative thermodynamic model of the adsorption data. We proposed three surface reactions involving the adsorption of aspartate (HAsp-) and/or Ca2+ onto brucite:

  16. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    SciTech Connect

    Lund, Leif R; Romer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J; Dano, Keld; Werb, Zena

    1996-01-01

    Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin

  17. Design, synthesis and inhibitory effect of pentapeptidyl chloromethyl ketones on proteinase K.

    PubMed

    Kore, Anilkumar R; Shanmugasundaram, Muthian

    2010-12-01

    The synthesis and proteolytic inhibitor function of new modified pentapeptide MeOSuc-AAAPF-CH(2)Cl 6 is described. The efficacy of 6 in inhibiting the proteolytic activity of proteinase K at a concentration of 0.10 mM allows a signal to be obtained for an exogenous target ('Xeno RNA') at 29 PCR cycles (i.e., Ct=29), whereas the control MeOSuc-AAAPV-CH₂Cl 1 requires a 7.5-fold higher concentration (0.75 mM) to produce the same Ct.

  18. Biochemical and biological characterization of two serine proteinases from Colombian Crotalus durissus cumanensis snake venom.

    PubMed

    Patiño, Arley Camilo; Pereañez, Jaime Andrés; Gutiérrez, José María; Rucavado, Alexandra

    2013-03-01

    Two clotting serine proteinases, named Cdc SI and Cdc SII, were isolated and characterized for the first time from Colombian Crotalus durissus cumanensis snake venom. The enzymes were purified using two chromatographic steps: molecular exclusion on Sephacryl S-200 and RP-HPLC on C8 Column. The molecular masses of the proteins, determined by MALDI-TOF mass spectrometry, were 28,561.4 and 28,799.2 Da for Cdc SI and Cdc SII, respectively. The aim of the present study was to evaluate enzymatic, coagulant and toxic properties of the two enzymes. The serine proteinases hydrolyzed specific chromogenic substrate (BaPNA) and exhibited a Michaelis-Menten behavior. Cdc SI had V(max) of 0.038 ± 0.003 nmol/min and K(M) of 0.034 ± 0.017 mM, while Cdc SII displayed values of V(max) of 0.267 ± 0.011 nmol/min and K(M) of 0.145 ± 0.023 mM. N-terminal sequences were VIGGDEXNIN and VIGGDICNINEHNFLVALYE for Cdc SI and Cdc SII, respectively. Molecular masses, N-terminal sequences, inhibition assays, and enzymatic profile suggest that Cdc SI and Cdc SII belong to the family of snake venom thrombin-like enzymes. These serine proteinases differed in their clotting activity on human plasma, showing a minimum coagulant dose of 25 μg and 0.571 μg for Cdc SI and Cdc SII, respectively. Enzymes also showed coagulant activity on bovine fibrinogen and degraded chain α of this protein. Toxins lack hemorrhagic and myotoxic activities, but are capable to induce defibrin(ogen)ation, moderate edema, and an increase in vascular permeability. These serine proteinases may contribute indirectly to the local hemorrhage induced by metalloproteinases, by causing blood clotting disturbances, and might also contribute to cardiovascular alterations characteristic of patients envenomed by C. d. cumanensis in Colombia.

  19. The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.

    PubMed Central

    Gobbetti, M; Smacchi, E; Corsetti, A

    1996-01-01

    A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively. PMID:8795211

  20. Racemization reaction of aspartic Acid and its use in dating fossil bones.

    PubMed

    Bada, J L; Protsch, R

    1973-05-01

    In the time interval datable by radiocarbon, and at the temperatures of most archeological sites, a substantial amount of racemization of aspartic acid takes place. By determination of the amount of racemization of aspartic acid in bones from a particular location which have been dated by the radiocarbon technique, it is possible to calculate the in situ first-order rate constant for interconversion of the L- and D enantiomers of aspartic acid. Once this "calibration" has been calculated, the reaction can be used to date other bones from the deposit that are either too old to be dated by radiocarbon or that are too small for radiocarbon dating. The only assumption required with this approach is that the average temperature experienced by the "calibration" sample is representative of the average temperature experienced by older samples. This "calibration" technique is used herein to date bones from the Olduvai Gorge area in Tanzania, Africa.

  1. Structure of RC1339/APRc from Rickettsia conorii, a retropepsin-like aspartic protease

    PubMed Central

    Li, Mi; Gustchina, Alla; Cruz, Rui; Simões, Marisa; Curto, Pedro; Martinez, Juan; Faro, Carlos; Simões, Isaura; Wlodawer, Alexander

    2015-01-01

    The crystal structures of two constructs of RC1339/APRc from Rickettsia conorii, consisting of either residues 105–231 or 110–231 followed by a His tag, have been determined in three different crystal forms. As predicted, the fold of a monomer of APRc resembles one-half of the mandatory homodimer of retroviral pepsin-like aspartic proteases (retropepsins), but the quaternary structure of the dimer of APRc differs from that of the canonical retropepsins. The observed dimer is most likely an artifact of the expression and/or crystallization conditions since it cannot support the previously reported enzymatic activity of this bacterial aspartic protease. However, the fold of the core of each monomer is very closely related to the fold of retropepsins from a variety of retroviruses and to a single domain of pepsin-like eukaryotic enzymes, and may represent a putative common ancestor of monomeric and dimeric aspartic proteases. PMID:26457434

  2. Distinguishing Aspartic and Isoaspartic Acids in Peptides by Several Mass Spectrometric Fragmentation Methods

    NASA Astrophysics Data System (ADS)

    DeGraan-Weber, Nick; Zhang, Jun; Reilly, James P.

    2016-09-01

    Six ion fragmentation techniques that can distinguish aspartic acid from its isomer, isoaspartic acid, were compared. MALDI post-source decay (PSD), MALDI 157 nm photodissociation, tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) charge tagging in PSD and photodissociation, ESI collision-induced dissociation (CID), electron transfer dissociation (ETD), and free-radical initiated peptide sequencing (FRIPS) with CID were applied to peptides containing either aspartic or isoaspartic acid. Diagnostic ions, such as the y-46 and b+H2O, are present in PSD, photodissociation, and charge tagging. c•+57 and z-57 ions are observed in ETD and FRIPS experiments. For some molecules, aspartic and isoaspartic acid yield ion fragments with significantly different intensities. ETD and charge tagging appear to be most effective at distinguishing these residues.

  3. A radiochemical assay for argininosuccinate synthetase with [U-14C]aspartate.

    PubMed

    Ratner, S

    1983-12-01

    A simple and sensitive radiochemical procedure to assay argininosuccinate synthetase activity in crude tissue homogenates and lysates of cultured cells is described. The new method depends on the location of 14C, uniformly, in the four carbons of aspartate. On incubation in the presence of excess of L-[U-14C]aspartate, L-citrulline, ATP, and an ATP-generating system, argininosuccinase and arginase, the [14C]fumarate formed is measured as the sum of malate and fumarate. After acidification the latter two acids are separated from [14C]aspartate on a small Dowex-50 column by elution with a few milliliters of water; the unutilized amino acid substrates remain on the column. With a specific radioactivity of 9 X 10(4) cpm, 1 to 2 nmol of product can be accurately measured under kinetically optimum conditions. PMID:6660522

  4. Attractant Signaling by an Aspartate Chemoreceptor Dimer with a Single Cytoplasmic Domain

    NASA Astrophysics Data System (ADS)

    Gardina, Paul J.; Manson, Michael D.

    1996-10-01

    Signal transduction across cell membranes often involves interactions among identical receptor subunits, but the contribution of individual subunits is not well understood. The chemoreceptors of enteric bacteria mediate attractant responses by interrupting a phosphotransfer circuit initiated at receptor complexes with the protein kinase CheA. The aspartate receptor (Tar) is a homodimer, and oligomerized cytoplasmic domains stimulate CheA activity much more than monomers do in vitro. Intragenic complementation was used to show in Escherichia coli that heterodimers containing one full-length and one truncated Tar subunit mediated responses to aspartate in the presence of full-length Tar homodimers that could not bind aspartate. Thus, a Tar dimer containing only one cytoplasmic domain can initiate an attractant (inhibitory) signal, although it may not be able to stimulate kinase activity of CheA.

  5. Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro.

    PubMed Central

    Marra, E; Doonan, S; Saccone, C; Quagliariello, E

    1977-01-01

    1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo. PMID:883959

  6. Racemization Reaction of Aspartic Acid and Its Use in Dating Fossil Bones

    PubMed Central

    Bada, Jeffrey L.; Protsch, Reiner

    1973-01-01

    In the time interval datable by radiocarbon, and at the temperatures of most archeological sites, a substantial amount of racemization of aspartic acid takes place. By determination of the amount of racemization of aspartic acid in bones from a particular location which have been dated by the radiocarbon technique, it is possible to calculate the in situ first-order rate constant for interconversion of the L- and D enantiomers of aspartic acid. Once this “calibration” has been calculated, the reaction can be used to date other bones from the deposit that are either too old to be dated by radiocarbon or that are too small for radiocarbon dating. The only assumption required with this approach is that the average temperature experienced by the “calibration” sample is representative of the average temperature experienced by older samples. This “calibration” technique is used herein to date bones from the Olduvai Gorge area in Tanzania, Africa. PMID:16592082

  7. Adsorption of L-aspartate to rutile (α-TiO 2): Experimental and theoretical surface complexation studies

    NASA Astrophysics Data System (ADS)

    Jonsson, Caroline M.; Jonsson, Christopher L.; Estrada, Charlene; Sverjensky, Dimitri A.; Cleaves, H. James, II; Hazen, Robert M.

    2010-04-01

    Interactions between aqueous amino acids and mineral surfaces influence many geochemical processes from biomineralization to the origin of life. However, the specific reactions involved and the attachment mechanisms are mostly unknown. We have studied the adsorption of L-aspartate on the surface of rutile (α-TiO 2, pH PPZC = 5.4) in NaCl(aq) over a wide range of pH, ligand-to-solid ratio and ionic strength, using potentiometric titrations and batch adsorption experiments. The adsorption is favored below pH 6 with a maximum of 1.2 μmol of adsorbed aspartate per m 2 of rutile at pH 4 in our experiments. The adsorption decreases at higher pH because the negatively charged aspartate molecule is repelled by the negatively charged rutile surface above pH PPZC. At pH values of 3-5, aspartate adsorption increases with decreasing ionic strength. The adsorption of aspartate on rutile is very similar to that previously published for glutamate ( Jonsson et al., 2009). An extended triple-layer model was used to provide a quantitative thermodynamic characterization of the aspartate adsorption data. Two reaction stoichiometries identical in reaction stoichiometry to those for glutamate were needed. At low surface coverages, aspartate, like glutamate, may form a bridging-bidentate surface species binding through both carboxyl groups, i.e. "lying down" on the rutile surface. At high surface coverages, the reaction stoichiometry for aspartate was interpreted differently compared to glutamate: it likely involves an outer-sphere or hydrogen bonded aspartate surface species, as opposed to a partly inner-sphere complex for glutamate. Both the proposed aspartate species are qualitatively consistent with previously published ATR-FTIR spectroscopic results for aspartate on amorphous titanium dioxide. The surface complexation model for aspartate was tested against experimental data for the potentiometric titration of aspartate in the presence of rutile. In addition, the model correctly

  8. [Characteristics of the Effect of Cestodes Parasitizing the Fish Intestine on the Activity of the Host Proteinases].

    PubMed

    Izvekova, G I; Solovyev, M M

    2016-01-01

    The activity and spectrum of proteinases in the intestines of host fishes change upon infestation with cestodes. Serine proteinases are found to make a greater contribution to the total proteolytic activity. The reduction of proteolytic activity is associated with adsorption of the enzymes of the host on the surface of cestodes, and the increase in the activity is caused by the injury of the intestinal mucosa by the attachment apparatuses of cestodes. The inhibition of proteainase activity indicates the possible participation of microbiota enzymes in protein hydrolyses.

  9. Aspartate embedding depth affects pHLIP's insertion pKa.

    PubMed

    Fendos, Justin; Barrera, Francisco N; Engelman, Donald M

    2013-07-01

    We have used the pHlow insertion peptide (pHLIP) family to study the role of aspartate embedding depth in pH-dependent transmembrane peptide insertion. pHLIP binds to the surface of a lipid bilayer as a largely unstructured monomer at neutral pH. When the pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pHLIP insertion is reversible when the pH is adjusted back to a neutral value. One of the critical events facilitating pHLIP insertion is the protonation of aspartates in the spanning domain of the peptide: the negative side chains of these residues convert to uncharged, polar forms, facilitating insertion by altering the hydrophobicity of the spanning domain. To examine this protonation mechanism further, we created pHLIP sequence variants in which the two spanning aspartates (D14 and D25) were moved up or down in the sequence. We hypothesized that the aspartate depth in the inserted state would directly affect the proton affinity of the acidic side chains, altering the pKa of pH-dependent insertion. To this end, we also mutated the arginine at position 11 to determine whether arginine snorkeling modulates the insertion pKa by affecting the aspartate depth. Our results indicate that both types of mutations change the insertion pKa, supporting the idea that the aspartate depth is a participating parameter in determining the pH dependence. We also show that pHLIP's resistance to aggregation can be altered with our mutations, identifying a new criterion for improving the performance of pHLIP in vivo when targeting acidic disease tissues such as cancer and inflammation. PMID:23721379

  10. Crystallization and preliminary X-ray diffraction analysis of the periplasmic domain of the Escherichia coli aspartate receptor Tar and its complex with aspartate

    SciTech Connect

    Mise, Takeshi; Matsunami, Hideyuki; Samatey, Fadel A.; Maruyama, Ichiro N.

    2014-08-27

    The periplasmic domain of the E. coli aspartate receptor Tar was cloned, expressed, purified and crystallized with and without bound ligand. The crystals obtained diffracted to resolutions of 1.58 and 1.95 Å, respectively. The cell-surface receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni{sup 2+}. To understand the molecular mechanisms underlying the induction of Tar activity by its ligands, the Escherichia coli Tar periplasmic domain with and without bound aspartate (Asp-Tar and apo-Tar, respectively) were each crystallized in two different forms. Using ammonium sulfate as a precipitant, crystals of apo-Tar1 and Asp-Tar1 were grown and diffracted to resolutions of 2.10 and 2.40 Å, respectively. Alternatively, using sodium chloride as a precipitant, crystals of apo-Tar2 and Asp-Tar2 were grown and diffracted to resolutions of 1.95 and 1.58 Å, respectively. Crystals of apo-Tar1 and Asp-Tar1 adopted space group P4{sub 1}2{sub 1}2, while those of apo-Tar2 and Asp-Tar2 adopted space groups P2{sub 1}2{sub 1}2{sub 1} and C2, respectively.

  11. Retrograde transport of (/sup 3/H)-D-aspartate label by cochlear and vestibular efferent neurons

    SciTech Connect

    Schwarz, D.W.; Schwarz, I.E.

    1988-01-01

    (/sup 3/H)-D-aspartic acid was injected into the inner ear of rats. After a six hour survival time, labeled cells were found at all locations known to contain efferent cochlear or vestibular neurons. Most labeled neurons were found in the ipsilateral lateral superior olivary nucleus (LSO), although both ventral nuclei of the trapezoid body (VTB), group E, and the caudal pontine reticular nucleus (CPR) just adjacent to the ascending limb of the facial nerve also contained labeled cells. Because not all efferent neurons in the rat could be previously shown to be cholinergic, aspartate and glutamate are efferent transmitter candidates.

  12. A serine proteinase homologue, SPH-3, plays a central role in insect immunity.

    PubMed

    Felföldi, Gabriella; Eleftherianos, Ioannis; Ffrench-Constant, Richard H; Venekei, István

    2011-04-15

    Numerous vertebrate and invertebrate genes encode serine proteinase homologues (SPHs) similar to members of the serine proteinase family, but lacking one or more residues of the catalytic triad. These SPH proteins are thought to play a role in immunity, but their precise functions are poorly understood. In this study, we show that SPH-3 (an insect non-clip domain-containing SPH) is of central importance in the immune response of a model lepidopteran, Manduca sexta. We examine M. sexta infection with a virulent, insect-specific, Gram-negative bacterium Photorhabdus luminescens. RNA interference suppression of bacteria-induced SPH-3 synthesis severely compromises the insect's ability to defend itself against infection by preventing the transcription of multiple antimicrobial effector genes, but, surprisingly, not the transcription of immune recognition genes. Upregulation of the gene encoding prophenoloxidase and the activity of the phenoloxidase enzyme are among the antimicrobial responses that are severely attenuated on SPH-3 knockdown. These findings suggest the existence of two largely independent signaling pathways controlling immune recognition by the fat body, one governing effector gene transcription, and the other regulating genes encoding pattern recognition proteins.

  13. Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K.

    PubMed

    Sang, Peng; Yang, Qiong; Du, Xing; Yang, Nan; Yang, Li-Quan; Ji, Xing-Lai; Fu, Yun-Xin; Meng, Zhao-Hui; Liu, Shu-Qun

    2016-01-01

    To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs) at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB) numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein. PMID:26907253

  14. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance.

    PubMed

    Lin, Chia-Wei; Su, Mei-Hsiu; Lin, Yu-Tsung; Chung, Chien-Hung; Ku, Hsin-Mei

    2015-07-01

    Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana. PMID:26184285

  15. Isolation, characterization and antifungal activity of proteinase inhibitors from Capsicum chinense Jacq. Seeds.

    PubMed

    Dias, Germana Bueno; Gomes, Valdirene Moreira; Pereira, Umberto Zottich; Ribeiro, Suzanna F Ferreira; Carvalho, André O; Rodrigues, Rosana; Machado, Olga L Tavares; Fernandes, Kátia Valevski Sales; Ferreira, André Teixeira S; Perales, Jonas; Da Cunha, Maura

    2013-01-01

    Capsicum species belong to the Solanaceae family and have great social, economic and agronomical significance. The present research presents data on the isolation and characterization of Capsicum chinense Jacq. peptides which were scrutinized in relation to their toxicity towards a diverse set of yeast species. The protein extract was separated with C18 reverse-phase chromatography in high performance liquid chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Tricine-SDS-PAGE of the PEF2 revealed peptides with molecular masses of approximately 5.0 and 8.5 kDa. These PEFs also exhibited strong antifungal activity against different yeasts. In the presence of the PEF2, Candida tropicalis exhibited morphological changes, including cellular agglomeration and formation of pseudohyphae. Determined N-terminal sequences of PEF2 and PEF3 were proven to be highly homologous to serine proteinase inhibitors, when analysed by comparative database sequence tools. For this reason were performed protease inhibitory activity assay. The PEFs displayed high inhibitory activity against trypsin and low inhibitory activity against chymotrypsin. PEF2 and PEF3 were considerably unsusceptible to a broad interval of pH and temperatures. Due to the myriad of application of Proteinase inhibitors (PIs) in fields ranging from plant protection against pathogens and pests to medicine such as in cancer and virus replication inhibition, the discovery of new PIs with new properties are of great interest.

  16. Diversity in proteinase specificity of thermophilic lactobacilli as revealed by hydrolysis of dairy and vegetable proteins.

    PubMed

    Pescuma, Micaela; Espeche Turbay, María Beatriz; Mozzi, Fernanda; Font de Valdez, Graciela; Savoy de Giori, Graciela; Hebert, Elvira María

    2013-09-01

    Ability of industrially relevant species of thermophilic lactobacilli strains to hydrolyze proteins from animal (caseins and β-lactoglobulin) and vegetable (soybean and wheat) sources, as well as influence of peptide content of growth medium on cell envelope-associated proteinase (CEP) activity, was evaluated. Lactobacillus delbrueckii subsp. lactis (CRL 581 and 654), L. delbrueckii subsp. bulgaricus (CRL 454 and 656), Lactobacillus acidophilus (CRL 636 and 1063), and Lactobacillus helveticus (CRL 1062 and 1177) were grown in a chemically defined medium supplemented or not with 1 % Casitone. All strains hydrolyzed mainly β-casein, while degradation of αs-caseins was strain dependent. Contrariwise, κ-Casein was poorly degraded by the studied lactobacilli. β-Lactoglobulin was mainly hydrolyzed by CRL 656, CRL 636, and CRL 1062 strains. The L. delbrueckii subsp. lactis strains, L. delbrueckii subsp. bulgaricus CRL 656, and L. helveticus CRL 1177 degraded gliadins in high extent, while the L. acidophilus and L. helveticus strains highly hydrolyzed soy proteins. Proteinase production was inhibited by Casitone, the most affected being the L. delbrueckii subsp. lactis species. This study highlights the importance of proteolytic diversity of lactobacilli for rational strain selection when formulating hydrolyzed dairy or vegetable food products.

  17. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance

    PubMed Central

    Lin, Chia-Wei; Su, Mei-Hsiu; Lin, Yu-Tsung; Chung, Chien-Hung; Ku, Hsin-Mei

    2015-01-01

    Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana. PMID:26184285

  18. Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR.

    PubMed

    Müller, A; Benoist, P; Diem, H G; Schwencke, J

    1991-12-01

    To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest.

  19. Characterization of a New Cell Envelope Proteinase PrtP from Lactobacillus rhamnosus CGMCC11055.

    PubMed

    Guo, Tingting; Ouyang, Xudong; Xin, Yongping; Wang, Yue; Zhang, Susu; Kong, Jian

    2016-09-21

    Cell envelope proteinases (CEPs) play essential roles in lactic acid bacteria growth in milk and health-promoting properties of fermented dairy products. The genome of Lactobacillus rhamnosus CGMCC11055 possesses two putative CEP genes prtP and prtR2, and the PrtP displays the distinctive domain organization from PrtR2 reported. The PrtP was purified and biochemically characterized. The results showed that the optimal activity occurred at 44 °C, pH 6.5. p-Amidinophenylmethylsulfonyl fluoride obviously inhibited enzymatic activity, suggesting PrtP was a member of serine proteinases. Under the optimal conditions, β-casein was a favorite substrate over αS1- and κ-casein, and 35 oligopeptides were identified in the β-casein hydrolysate, including the phosphoserine peptide and bioactive isoleucine-proline-proline. By analysis of the amino acid sequences of those oligopeptides, proline was the preferred residue at the breakdown site. Therefore, we speculated that PrtP was a new type of CEPs from Lb. rhamnosus. PMID:27585760

  20. Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K

    PubMed Central

    Sang, Peng; Yang, Qiong; Du, Xing; Yang, Nan; Yang, Li-Quan; Ji, Xing-Lai; Fu, Yun-Xin; Meng, Zhao-Hui; Liu, Shu-Qun

    2016-01-01

    To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs) at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB) numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein. PMID:26907253

  1. Proteinase-activated receptors 1 and 2 mediate contraction of human oesophageal muscularis mucosae.

    PubMed

    Chang, B-S; Chang, J-C; Huang, S-C

    2010-01-01

    Proteinase-activated receptors 1 and 2 mediate contraction of the human gallbladder. In the present study, we investigated effects mediated by proteinase-activated receptors (PARs) in the human oesophagus by measuring contraction of muscularis mucosae strips isolated from the human oesophagus. Both PAR(1) agonists (thrombin, SFLLRN-NH(2) and TFLLR-NH(2)) and PAR(2) agonists (trypsin, 2-furoyl-LIGRLO-NH(2) and SLIGKV-NH(2)) caused concentration-dependent contraction. In contrast, PAR(1) and PAR(2) control peptides did not cause contraction. The existence of PAR(1) and PAR(2) in the human oesophageal muscularis mucosae was confirmed by immunohistochemistry and reverse transcription-polymerase chain reaction. On the other hand, PAR(4) agonists, GYPGKF-NH(2), GYPGQV-NH(2) and AYPGKF-NH(2), did not cause contraction or relaxation in resting or carbachol-contracted muscularis mucosae strips, suggesting that PAR(4) is not involved in human oesophageal motility. The contractile responses to SFLLRN-NH(2) and trypsin in the human oesophagus were insensitive to atropine and tetrodotoxin, indicating that the contractile response was not neurally mediated. Taken together, these results demonstrate that PAR(1) and PAR(2) but not PAR(4) mediate contraction in human oesophageal muscularis mucosae. PAR(1) and PAR(2) may influence human oesophageal motility. PMID:19694963

  2. Morphological confocal microscopy in arthropods and the enhancement of autofluorescence after proteinase K extraction.

    PubMed

    Valdecasas, Antonio G; Abad, Angela

    2011-02-01

    Procedures to study the molecular and morphological characteristics of microscopic organisms are often incompatible with each other. Therein, the realization of alternatives that make the characterization of these features compatible and simultaneously permit the deposition of the original material as a voucher sample into a reference collection is one of the foremost goals of biodiversity studies. In this study, we show that genomic extraction does not necessarily compromise the detailed study of the external morphology of microscopic organisms, and to do so, we used a group of aquatic mites (Acari, Hydrachnidia) as a test group. Hydrachnidia morphology is difficult to study when specimens have been stored in pure ethanol; however, proteinase K extraction leaves them flexible and easy to dissect, while, at the same time, maintaining all of their diagnostic features intact. Furthermore, autofluorescence is significantly enhanced after proteinase extraction. Our study was conducted with aquatic mites that were stored in absolute ethanol in the field and processed for DNA extraction using a Qiagen QIAamp minikit. Before and after molecular extraction, a laser scanning confocal microscopy morphological examination was carried out.

  3. Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K.

    PubMed

    Sang, Peng; Yang, Qiong; Du, Xing; Yang, Nan; Yang, Li-Quan; Ji, Xing-Lai; Fu, Yun-Xin; Meng, Zhao-Hui; Liu, Shu-Qun

    2016-02-19

    To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs) at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB) numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein.

  4. Proteinase K-catalyzed synthesis of linear and star oligo(L-phenylalanine) conjugates.

    PubMed

    Ageitos, Jose M; Baker, Peter J; Sugahara, Michihiro; Numata, Keiji

    2013-10-14

    Chemoenzymatic synthesis of peptides is a green and clean chemical reaction that offers high yields without using organic synthesis and serves as an alternative to traditional peptide synthesis methods. This report describes the chemoenzymatic synthesis of oligo(L-phenylalanine) mediated by proteinase K from Tritirachium album, which is one of the most widely used proteases in molecular biological studies. The synthesized linear oligo-phenylalanine showed a unique self-assembly in aqueous solutions. To further functionalize linear oligo(L-phenylalanine) as a low-molecular-weight gelator, it was cosynthesized with tris(2-aminoethyl)amine to obtain star-oligo(L-phenylalanine), which was bioconjugated to demonstrate its self-assembly into fluorescent fibers. The self-assembled fibers of star-oligo(L-phenylalanine) formed fibrous networks with various branching ratios, which depended on the molecular weights and molecular aspect ratios of star-oligo(L-phenylalanine). This is the first study to demonstrate that proteinase K is a suitable enzyme for chemoenzymatic cosynthesis of oligopeptides and star-shaped heteropeptides.

  5. Proteinases involved in the degradation of trypsin inhibitor in germinating mung beans.

    PubMed

    Wilson, K A; Tan-Wilson, A L

    1983-01-01

    The mung bean (Vigna radiata (L.) Wilczek) trypsin inhibitor (MBTI) is rapidly modified by limited proteolysis during the early stages of seedling growth. Using an electrophoretic assay that separates the unmodified inhibitor (MBTI-F) and the first two modified species (MBTI-E and -C), a pH optimum of approximately 4 was found for the modification reaction. The inhibitor modifying activity is initially low in ungerminated seeds, with the reaction F leads to E being the primary reaction catalyzed. Activity catalyzing the production of MBTI-C appears on the first day of germination. This activity (F leads to E leads to C) increases up to 6 days after inhibition, at which time the cotyledons begin to abscise. The activity converting MBTI-F and -E to MBTI-C was strongly inhibited by phenylmethylsulfonyl fluoride (3.3 mM) but only weakly by iodoacetate (9 mM) and not at all by pepstatin A (9 microM), leupeptin (18 microM), or EDTA (5 mM). These results suggest the involvement of proteinases other than the major endopeptidase of the germinating seed, vicilin peptidohydrolase. This conclusion is further supported by gel filtration of the extracts of cotyledons on Sephacryl S-200. At least three proteinases are present in germinated cotyledons capable of modifying MBTI-F to MBTI-C and/or -E. All are distinguishable from vicilin peptidohydrolase on the basis of their molecular weight and inhibition by low molecular weight organic reagents.

  6. Proteinases, their receptors and inflammatory signalling: the Oxford South Parks Road connection*

    PubMed Central

    Hollenberg, M D

    2015-01-01

    In keeping with the aim of the Paton Memorial Lecture to ‘facilitate the historical study of pharmacology’, this overview, which is my distinct honour to write, represents a ‘Janus-like’ personal perspective looking both backwards and forwards at the birth and growth of ‘receptor molecular pharmacology’ with special relevance to inflammatory diseases. The overview begins in the Oxford Department of Pharmacology in the mid-1960s and then goes on to provide a current perspective of signalling by proteinases. Looking backwards, the synopsis describes the fruitful Oxford Pharmacology Department infrastructure that Bill Paton generated in keeping with the blueprint begun by his predecessor, J H Burn. Looking forwards, the overview illustrates the legacy of that environment in generating some of the first receptor ligand-binding data and providing the inspiration and vision for those like me who were training in the department at the same time. With apologies, I mention only in passing a number of individuals who benefitted from the ‘South Parks Road connection’ using myself as one of the ‘outcome study’ examples. It is also by looking forward that I can meet the complementary aim of summarizing the lecture presented at a ‘BPS 2014 Focused Meeting on Cell Signalling’ to provide an overview of the role of proteinases and their signalling mechanisms in the setting of inflammation. PMID:25521749

  7. The murine Spi-2 proteinase inhibitor locus: a multigene family with a hypervariable reactive site domain.

    PubMed Central

    Inglis, J D; Hill, R E

    1991-01-01

    We have isolated 10 closely linked members of a proteinase inhibitor multigene family from the inbred mouse strain 129. These sequences, termed the Serine Proteinase Inhibitor 2 (Spi-2) genes, appear to have been derived from a common ancestor represented in man by the single copy alpha 1-antichymotrypsin gene. The genes are clustered on two cloned genomic DNA segments spanning 220 kb, and have at least partially retained the intragenic structure of the ancestral Spi-2 gene. Sequence analysis from the final coding exon indicates that most of the mouse genes may be competent to encode functional proteins, some with a predictable inhibitory spectrum, and several representing novel inhibitor types. An oligonucleotide probe designed to one reactive centre sequence enabled the isolation of the cognate expressed transcript from a liver cDNA library. However, whether expressed or not, the reactive centre regions of all the sequences have diverged at a rapid rate relative to structurally defined flanking sequences. The divergence is also appreciably greater than that occurring in an adjacent non-coding sequence. This phenomenon has established novel potential inhibitory specificities, while maintaining a functional inhibitor structure. PMID:1991447

  8. Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE-ESI-MS/MS.

    PubMed

    Gahoual, Rabah; Beck, Alain; François, Yannis-Nicolas; Leize-Wagner, Emmanuelle

    2016-02-01

    Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post-translational modifications (PTMs) require comprehensive characterization because of their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually become an analytical tool of choice to characterize PTMs; however, some modifications are still challenging because of sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here, we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE-ESI-MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE-ESI-MS/MS method was applied for the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples, and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and, as a consequence, enables to distinguish the formation of l-aspartic acid or d-aspartic acid generated from deaN. Separation based on peptide modification allowed, as supported by the ESI

  9. Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE-ESI-MS/MS.

    PubMed

    Gahoual, Rabah; Beck, Alain; François, Yannis-Nicolas; Leize-Wagner, Emmanuelle

    2016-02-01

    Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post-translational modifications (PTMs) require comprehensive characterization because of their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually become an analytical tool of choice to characterize PTMs; however, some modifications are still challenging because of sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here, we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE-ESI-MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE-ESI-MS/MS method was applied for the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples, and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and, as a consequence, enables to distinguish the formation of l-aspartic acid or d-aspartic acid generated from deaN. Separation based on peptide modification allowed, as supported by the ESI

  10. Molecular characterization, expression and function analysis of a five-domain Kazal-type serine proteinase inhibitor from pearl oyster Pinctada fucata.

    PubMed

    Zhang, Dianchang; Ma, Jianjun; Jiang, Shigui

    2014-03-01

    Serine proteinase inhibitors represent an expanding superfamily of endogenous inhibitors that are regulate proteolytic events and involved in a variety of physiological and immunological processes. A five-domain Kazal-type serine proteinase inhibitor (poKSPI) was identified and characterized from pearl oyster Pinctada fucata based on expressed sequence tag (EST) analysis. The full-length cDNA was 737 bp with an open reading frame (ORF) 660 bp encoding a 219 amino acid protein a theoretical molecular weight (Mw) of 23.3 kDa and an isoelectric point (pI) of 8.40. A putative signal peptide of 19 amino acid residues and five tandem Kazal domains were identified. Four of the Kazal domains had the highly conserved motif sequences with six cysteine residues responsible for the formation of disulfide bridges. The deduced amino acid sequence of the poKSPI shared high homology with KSPIs from Hirudo medicinalis. The poKSPI mRNA could be detected in all examined tissues, the expression level of the poKSPI mRNA was the highest in mantle and gonad, while the lowest in haemocyte and intestine. After LPS challenge, the expression level of the poKSPI mRNA in digestive gland was significantly up-regulated at 4 h post-challenge and reached the peak at 12 h post-challenge, which was 4.23-fold higher than control group; the expression level of the poKSPI mRNA in gill was also significantly up-regulated at 8 and 12 h post-challenge, which were 4.48 and 2.26-fold higher than control group. After Vibrio alginolyticus challenge, the expression levels of the poKSPI mRNA in digestive gland were significantly up-regulated at 8, 12, 48 and 72 h post-challenge, which were 1.70, 1.79, 3.89 and 5.69-fold higher than control group, respectively; the expression level of the poKSPI mRNA in gill was significantly up-regulated at 24 h post-challenge, which was 5.30-fold higher than control group. The recombinant poKSPI protein could inhibit chymotrypsin and trypsin activities in dose

  11. Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.

    PubMed Central

    Burck, P J; Berg, D H; Luk, T P; Sassmannshausen, L M; Wakulchik, M; Smith, D P; Hsiung, H M; Becker, G W; Gibson, W; Villarreal, E C

    1994-01-01

    The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein

  12. N-methyl-d-aspartate antibody encephalitis: temporal progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes

    PubMed Central

    Irani, Sarosh R.; Bera, Katarzyna; Waters, Patrick; Zuliani, Luigi; Maxwell, Susan; Zandi, Michael S.; Friese, Manuel A.; Galea, Ian; Kullmann, Dimitri M.; Beeson, David; Lang, Bethan; Bien, Christian G.

    2010-01-01

    Antibodies to the N-methyl-d-aspartate subtype of glutamate receptor have been associated with a newly-described encephalopathy that has been mainly identified in young females with ovarian tumours. However, the full clinical spectrum and treatment responses are not yet clear. We established a sensitive cell-based assay for detection of N-methyl-d-aspartate receptor antibodies in serum or cerebrospinal fluid, and a quantitative fluorescent immunoprecipitation assay for serial studies. Although there was marked intrathecal synthesis of N-methyl-d-aspartate receptor antibodies, the absolute levels of N-methyl-d-aspartate receptor antibodies were higher in serum than in cerebrospinal fluid. N-methyl-d-aspartate receptor antibodies were of the immunoglobulin G1 subclass and were able to activate complement on N-methyl d-aspartate receptor-expressing human embryonic kidney cells. From questionnaires returned on 44 N-methyl-d-aspartate receptor antibody-positive patients, we identified a high proportion without a detected tumour (35/44, 80%: follow-up 3.6–121 months, median 16 months). Among the latter were 15 adult females (43%), 10 adult males (29%) and 10 children (29%), with four in the first decade of life. Overall, there was a high proportion (29%) of non-Caucasians. Good clinical outcomes, as defined by reductions in modified Rankin scores, correlated with decreased N-methyl-d-aspartate receptor antibody levels and were associated with early (<40 days) administration of immunotherapies in non-paraneoplastic patients (P < 0.0001) and earlier tumour removal in paraneoplastic patients (P = 0.02). Ten patients (23%) who were first diagnosed during relapses had no evidence of tumours but had received minimal or no immunotherapy during earlier episodes. Temporal analysis of the onset of the neurological features suggested progression through two main stages. The time of onset of the early features, characterized by neuropsychiatric symptoms and seizures preceded by a

  13. Identification of a small molecule [beta]-secretase inhibitor that binds without catalytic aspartate engagement

    SciTech Connect

    Steele, Thomas G.; Hills, Ivory D.; Nomland, Ashley A.; de León, Pablo; Allison, Timothy; McGaughey, Georgia; Colussi, Dennis; Tugusheva, Katherine; Haugabook, Sharie J.; Espeseth, Amy S.; Zuck, Paul; Graham, Samuel L.; Stachel, Shawn J.

    2010-09-02

    A small molecule inhibitor of beta-secretase with a unique binding mode has been developed. Crystallographic determination of the enzyme-inhibitor complex shows the catalytic aspartate residues in the active site are not engaged in inhibitor binding. This unprecedented binding mode in the field of aspartyl protease inhibition is described.

  14. The anomalous kinetics of coupled aspartate aminotransferase and malate dehydrogenase. Evidence for compartmentation of oxaloacetate.

    PubMed Central

    Bryce, C F; Williams, D C; John, R A; Fasella, P

    1976-01-01

    Cytoplasmic aspartate aminotransferase and malate dehydrogenase were purified from pig heart. Kinetic parameters were determined for the separate reaction catalysed by each enzyme and used to predict the course of the coupled reaction: (see article). Although a lag phase should have been easily seen, none was detected. The same coupled reaction was also carried out by using radioactive aspartate in the presence of unlabelled oxaloacetate. The reaction was quenched with HClO4 after 70 ms and the specific radioactivity of the malate produced in this system was found to be essentially the same as that of the original aspartate. These results show that oxaloacetate produced by the aspartate aminotransferase is converted into malate by malate dehydrogenase before it equilibrates with the pool of unlabelled oxaloacetate and are consistent with a proposal that the enzymes are associated in a complex. However, no physical evidence of the existence of a complex could be found. An alternative means of compartmentation of the intermediate as an unstable isomer is considered. Images Fig. 2. Fig. 3. PMID:942372

  15. Immobilized cells of recombinant Escherichia coli strain for continuous production of L-aspartic acid.

    PubMed

    Szymańska, Grazyna; Sobierajski, Bogusław; Chmiel, Aleksander

    2011-01-01

    For L-aspartic acid biosynthesis, high production cells of Escherichia coli mutant B-715 and P1 were immobilized in chitosan gel using a technique developed in our laboratory. The immobilization process reduced initial activity of the intact cells, however, the biocatalyst produced was very stabile for long-term use in multi-repeated batch or continuous processes. Temperature influence on the conversion of ammonium fumarate to L-aspartic acid was investigated. In long-term experiments, over 603 hours, the temperature 40 degrees C was found to be the best for both biocatalyst stability and high conversion rate. The optimum substrate concentration was 1.0 M. Continuous production of L-aspartic acid was investigated in three types of column bioreactors characterized by different volumes as well as different high to biocatalyst bed volume rations (Hz/Vz). The highest conversion rate, 99.8%, and the productivity 6 g/g/h (mass of L-aspartic acid per dry mass of cells in biocatalyst per time unit) was achieved in the bioreactor with the highest value Hz/Vz = 3.1, and liquid hour space velocity value of 5.2, defined as the volume of feeding substrate passed per volume of catalyst in bioreactor per one hour. PMID:21905626

  16. Aspartic acid in the hippocampus: a biomarker for postoperative cognitive dysfunction.

    PubMed

    Hu, Rong; Huang, Dong; Tong, Jianbin; Liao, Qin; Hu, Zhonghua; Ouyang, Wen

    2014-01-15

    This study established an aged rat model of cognitive dysfunction using anesthesia with 2% isoflurane and 80% oxygen for 2 hours. Twenty-four hours later, Y-maze test results showed that isoflurane significantly impaired cognitive function in aged rats. Gas chromatography-mass spectrometry results showed that isoflurane also significantly increased the levels of N,N-diethylacetamide, n-ethylacetamide, aspartic acid, malic acid and arabinonic acid in the hippocampus of isoflurane-treated rats. Moreover, aspartic acid, N,N-diethylacetamide, n-ethylacetamide and malic acid concentration was positively correlated with the degree of cognitive dysfunction in the isoflurane-treated rats. It is evident that hippocampal metabolite changes are involved in the formation of cognitive dysfunction after isoflurane anesthesia. To further verify these results, this study cultured hippocampal neurons in vitro, which were then treated with aspartic acid (100 μmol/L). Results suggested that aspartic acid concentration in the hippocampus may be a biomarker for predicting the occurrence and disease progress of cognitive dysfunction.

  17. Aspartic acid in the hippocampus: a biomarker for postoperative cognitive dysfunction

    PubMed Central

    Hu, Rong; Huang, Dong; Tong, Jianbin; Liao, Qin; Hu, Zhonghua; Ouyang, Wen

    2014-01-01

    This study established an aged rat model of cognitive dysfunction using anesthesia with 2% isoflurane and 80% oxygen for 2 hours. Twenty-four hours later, Y-maze test results showed that isoflurane significantly impaired cognitive function in aged rats. Gas chromatography-mass spectrometry results showed that isoflurane also significantly increased the levels of N,N-diethylacetamide, n-ethylacetamide, aspartic acid, malic acid and arabinonic acid in the hippocampus of isoflurane-treated rats. Moreover, aspartic acid, N,N-diethylacetamide, n-ethylacetamide and malic acid concentration was positively correlated with the degree of cognitive dysfunction in the isoflurane-treated rats. It is evident that hippocampal metabolite changes are involved in the formation of cognitive dysfunction after isoflurane anesthesia. To further verify these results, this study cultured hippocampal neurons in vitro, which were then treated with aspartic acid (100 μmol/L). Results suggested that aspartic acid concentration in the hippocampus may be a biomarker for predicting the occurrence and disease progress of cognitive dysfunction. PMID:25206795

  18. Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group.

    PubMed

    Bungard, Christopher J; Williams, Peter D; Ballard, Jeanine E; Bennett, David J; Beaulieu, Christian; Bahnck-Teets, Carolyn; Carroll, Steve S; Chang, Ronald K; Dubost, David C; Fay, John F; Diamond, Tracy L; Greshock, Thomas J; Hao, Li; Holloway, M Katharine; Felock, Peter J; Gesell, Jennifer J; Su, Hua-Poo; Manikowski, Jesse J; McKay, Daniel J; Miller, Mike; Min, Xu; Molinaro, Carmela; Moradei, Oscar M; Nantermet, Philippe G; Nadeau, Christian; Sanchez, Rosa I; Satyanarayana, Tummanapalli; Shipe, William D; Singh, Sanjay K; Truong, Vouy Linh; Vijayasaradhi, Sivalenka; Wiscount, Catherine M; Vacca, Joseph P; Crane, Sheldon N; McCauley, John A

    2016-07-14

    A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile. PMID:27437081

  19. Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group.

    PubMed

    Bungard, Christopher J; Williams, Peter D; Ballard, Jeanine E; Bennett, David J; Beaulieu, Christian; Bahnck-Teets, Carolyn; Carroll, Steve S; Chang, Ronald K; Dubost, David C; Fay, John F; Diamond, Tracy L; Greshock, Thomas J; Hao, Li; Holloway, M Katharine; Felock, Peter J; Gesell, Jennifer J; Su, Hua-Poo; Manikowski, Jesse J; McKay, Daniel J; Miller, Mike; Min, Xu; Molinaro, Carmela; Moradei, Oscar M; Nantermet, Philippe G; Nadeau, Christian; Sanchez, Rosa I; Satyanarayana, Tummanapalli; Shipe, William D; Singh, Sanjay K; Truong, Vouy Linh; Vijayasaradhi, Sivalenka; Wiscount, Catherine M; Vacca, Joseph P; Crane, Sheldon N; McCauley, John A

    2016-07-14

    A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

  20. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  1. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  2. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  3. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  4. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  5. A thermostable L-aspartate oxidase: a new tool for biotechnological applications.

    PubMed

    Bifulco, Davide; Pollegioni, Loredano; Tessaro, Davide; Servi, Stefano; Molla, Gianluca

    2013-08-01

    L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0-10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.

  6. Solution structure of PMP-C: a new fold in the group of small serine proteinase inhibitors.

    PubMed

    Mer, G; Hietter, H; Kellenberger, C; Renatus, M; Luu, B; Lefèvre, J F

    1996-04-26

    The solution structure and the disulfide pairings of a 36-residue proteinase inhibitor isolated from the insect Locusta migratoria have been determined using NMR spectroscopy and simulated annealing calculations. The peptide, termed PMP-C, was previously shown to inhibit bovine alpha-chymotrypsin as well as human leukocyte elastase, and was also found to block high-voltage-activated Ca2+ currents in rat sensory neurones. PMP-C has a prolate ellipsoid shape and adopts a tertiary fold hitherto unobserved in the large group of small "canonical" proteinase inhibitors. The over-all fold consists mainly of three strands arranged in a right-handed twisted, antiparallel, beta-sheet that demarcates a cavity, together with a linear amino-terminal segment oriented almost perpendicular to the three strands of the beta-sheet. Inside the cavity a phenyl ring constitutes the centre of a hydrophobic core. The proteinase binding loop is located in the carboxy-terminal part of the molecule, between two cysteine residues involved in disulfide bridges. Its conformation resembles that found in other small canonical proteinase inhibitors. A comparison of PMP-C structure with the recently published solution structure of the related peptide PMP-D2 shows that the most significant differences are complementary changes involved in the stabilization of similar folds. This comparison led us to review the structure of PMP-D2 and to identify two salt bridges in PMP-D2.

  7. Alpha-2-macroglobulin functions as an inhibitor of fibrinolytic, clotting, and neutrophilic proteinases in sepsis: studies using a baboon model.

    PubMed

    de Boer, J P; Creasey, A A; Chang, A; Abbink, J J; Roem, D; Eerenberg, A J; Hack, C E; Taylor, F B

    1993-12-01

    Alpha-2-macroglobulin (alpha 2M) may function as a proteinase inhibitor in vivo. Levels of this protein are decreased in sepsis, but the reason these levels are low is unknown. Therefore, we analyzed the behavior of alpha 2M in a baboon model for sepsis. Upon challenge with a lethal (4 baboons) or a sublethal (10 baboons) dose of Escherichia coli, levels of inactivated alpha 2M (i alpha 2M) steadily increased, the changes being more pronounced in the animals that received the lethal dose. The rise in i alpha 2M significantly correlated with the increase of thrombin-antithrombin III, plasmin-alpha 2-antiplasmin, and, to a lesser extent, with that of elastase-alpha 1-antitrypsin complexes, raising the question of involvement of fibrinolytic, clotting, and neutrophilic proteinases in the inactivation of alpha 2M. Experiments with chromogenic substrates confirmed that thrombin, plasmin, elastase, and cathepsin G indeed had formed complexes with alpha 2M. Changes in alpha 2M similar to those observed in the animals that received E. coli occurred in baboons challenged with Staphylococcus aureus, indicating that alpha 2M formed complexes with the proteinases just mentioned in gram-positive sepsis as well. We conclude that alpha 2M in this baboon model for sepsis is inactivated by formation of complexes with proteinases, derived from activated neutrophils and from fibrinolytic and coagulation cascades. We suggest that similar mechanisms may account for the decreased alpha 2M levels in clinical sepsis.

  8. DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms.

    PubMed

    Nguyen, Uyen T; Burrows, Lori L

    2014-09-18

    Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (<50% of control) to polystyrene. Treatment of established 72h biofilms with 100μg/ml of DNase for 24h induced incomplete biofilm dispersal, with <25% biofilm remaining compared to control. In contrast, addition of proteinase K completely inhibited biofilm formation, and 72h biofilms-including those grown under stimulatory conditions-were completely dispersed with 100μg/ml proteinase K. Generally-regarded-as-safe proteases bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25μg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms.

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

    PubMed

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

    2005-05-01

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

  10. Effects of cysteine proteinase inhibitors scN and E-64 on southern corn rootworm larval development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The southern corn rootworm (SCRW) can be a serious pest of peanut pods. A laboratory bioassay was developed to test feeding cysteine proteinase inhibitors soyacystatin N (scN) and E-64 against southern corn rootworm reared on artificial diet to determine the effects on larvae development and mortal...

  11. Enzymatic degradation of poly(L-lactide) film by proteinase K: quartz crystal microbalance and atomic force microscopy study.

    PubMed

    Yamashita, Koichi; Kikkawa, Yoshihiro; Kurokawa, Kenji; Doi, Yoshiharu

    2005-01-01

    Enzymatic degradation of the poly(L-lactide) (PLLA) amorphous film by proteinase K has been investigated by combination of the complementary techniques of quartz crystal microbalance and atomic force microscopy (AFM). The erosion rate increased with increasing enzyme concentrations and attained to be constant under the condition of [proteinase K] > 100 microg/mL. The amount of the enzyme molecules adsorbed to the film was quantitatively evaluated at various concentrations by AFM, and it revealed that the erosion rate is determined by the amount of adsorbed enzyme. Adsorption of proteinase K was irreversible despite lack of the binding domain, so that the enzyme molecules on the film surface could be observed directly by AFM. Transformation of the enzyme molecule caused by packing in high density on the surface was observed at higher enzyme concentrations. The "footprint" of the individual proteinase K molecule on the PLLA film after enzymatic degradation suggests that the enzyme moves on the surface to hydrolyze the film around it.

  12. Mapping of the Proteinase B Structural Gene PRB1, in SACCHAROMYCES CEREVISIAE and Identification of Nonsense Alleles within the Locus

    PubMed Central

    Zubenko, George S.; Mitchell, Aaron P.; Jones, Elizabeth W.

    1980-01-01

    We report the mapping of the structural gene for proteinase B, PRB1. It is located 1.1 cM proximal to CAN1 on the left arm of chromosome V of Saccharomyces cerevisiae. We have identified 34 amber and 12 ochre mutations among the 126 prb1 mutations in our collection. PMID:7009321

  13. Simultaneous isolation of the yeast cytosol and well-preserved mitochondria with negligible contamination by vacuolar proteinases.

    PubMed

    Schwencke, J; Canut, H; Flores, A

    1983-06-13

    Disruption of yeast spheroplasts by DEAE-dextran in isoosmotic conditions allows isolation of relatively undamaged subcellular fractions from yeast. The preservation of mitochondria and vacuoles permits the simultaneous isolation of the cytosol with negligible contamination by vacuolar proteinases and therefore, virtually eliminates proteolytic artefacts.

  14. Divalent metals stabilize cellular prion proteins and alter the rate of proteinase-K dependent limited proteolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The key biochemical event in the pathogenesis of prion diseases is the conversion of normal cellular prion proteins (PrP**c) to the proteinase K (PK) resistant, abnormal form (PrP**sc); however, the cellular mechanisms underlying the conversion remain enigmatic. Binding of divalent ca...

  15. Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1.

    PubMed

    Sakaguchi, Masayoshi; Osaku, Kanae; Maejima, Susumu; Ohno, Nao; Sugahara, Yasusato; Oyama, Fumitaka; Kawakita, Masao

    2014-01-01

    The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure.

  16. DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms.

    PubMed

    Nguyen, Uyen T; Burrows, Lori L

    2014-09-18

    Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (<50% of control) to polystyrene. Treatment of established 72h biofilms with 100μg/ml of DNase for 24h induced incomplete biofilm dispersal, with <25% biofilm remaining compared to control. In contrast, addition of proteinase K completely inhibited biofilm formation, and 72h biofilms-including those grown under stimulatory conditions-were completely dispersed with 100μg/ml proteinase K. Generally-regarded-as-safe proteases bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25μg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms. PMID:25043896

  17. Alpha-2-macroglobulin functions as an inhibitor of fibrinolytic, clotting, and neutrophilic proteinases in sepsis: studies using a baboon model.

    PubMed Central

    de Boer, J P; Creasey, A A; Chang, A; Abbink, J J; Roem, D; Eerenberg, A J; Hack, C E; Taylor, F B

    1993-01-01

    Alpha-2-macroglobulin (alpha 2M) may function as a proteinase inhibitor in vivo. Levels of this protein are decreased in sepsis, but the reason these levels are low is unknown. Therefore, we analyzed the behavior of alpha 2M in a baboon model for sepsis. Upon challenge with a lethal (4 baboons) or a sublethal (10 baboons) dose of Escherichia coli, levels of inactivated alpha 2M (i alpha 2M) steadily increased, the changes being more pronounced in the animals that received the lethal dose. The rise in i alpha 2M significantly correlated with the increase of thrombin-antithrombin III, plasmin-alpha 2-antiplasmin, and, to a lesser extent, with that of elastase-alpha 1-antitrypsin complexes, raising the question of involvement of fibrinolytic, clotting, and neutrophilic proteinases in the inactivation of alpha 2M. Experiments with chromogenic substrates confirmed that thrombin, plasmin, elastase, and cathepsin G indeed had formed complexes with alpha 2M. Changes in alpha 2M similar to those observed in the animals that received E. coli occurred in baboons challenged with Staphylococcus aureus, indicating that alpha 2M formed complexes with the proteinases just mentioned in gram-positive sepsis as well. We conclude that alpha 2M in this baboon model for sepsis is inactivated by formation of complexes with proteinases, derived from activated neutrophils and from fibrinolytic and coagulation cascades. We suggest that similar mechanisms may account for the decreased alpha 2M levels in clinical sepsis. PMID:7693593

  18. [Anti-N-methyl-D-aspartate receptor encephalitis associated with ovarian teratoma: Description of a case and anesthetic implications].

    PubMed

    Arteche Andrés, M A; Zugasti Echarte, O; de Carlos Errea, J; Pérez Rodríguez, M; Leyún Pérez de Zabalza, R; Azcona Calahorra, M A

    2015-10-01

    N-methyl-D-aspartate receptor encephalitis is an autoimmune encephalitis relationated or not with a neoplasm. Although its incidence is unknown, probably remains underdiagnosed. Epidemiological studies place it as the second cause of immune-mediated encephalitis and the first in patients aged less of 30 years. It shows neuropsychiatric symptoms and autonomic instability. After diagnosis, based on the detection of antibodies in serum or cerebrospinal fluid, an occult malignancy must be investigated. While increasing number of cases have been diagnosed and the important role of this receptor in general anesthesia mechanisms, the interaction of the disease with anesthetic agents and perioperative stress is unknown. We describe the case of a patient with encephalitis associated to ovarian teratoma that underwent gynaecological laparoscopy.

  19. Discovery of the first inhibitors of bacterial enzyme D-aspartate ligase from Enterococcus faecium (Aslfm).

    PubMed

    Škedelj, Veronika; Perdih, Andrej; Brvar, Matjaž; Kroflič, Ana; Dubbée, Vincent; Savage, Victoria; O'Neill, Alex J; Solmajer, Tom; Bešter-Rogač, Marija; Blanot, Didier; Hugonnet, Jean-Emmanuel; Magnet, Sophie; Arthur, Michel; Mainardi, Jean-Luc; Stojan, Jure; Zega, Anamarija

    2013-09-01

    The D-aspartate ligase of Enterococcus faecium (Aslfm) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrug-resistant E. faecium. Although there is currently little available information regarding the structural characteristics of Aslfm, we exploited the knowledge that this enzyme belongs to the ATP-grasp superfamily to target its ATP binding site. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes. A series of amino-oxazoles derived from bacterial biotin carboxylase inhibitors showed low micromolar activity. The most potent inhibitor compound 12, inhibits Aslfm with a Ki value of 2.9 μM. In the second design stage, a validated ligand-based pharmacophore modeling approach was used, taking the newly available inhibition data of an initial series of compounds into account. Experimental evaluation of the virtual screening hits identified two novel structural types of Aslfm inhibitors with 7-amino-9H-purine (18) and 7-amino-1H-pyrazolo[3,4-d]pyrimidine (30 and 34) scaffolds, and also with Ki values in the low micromolar range. Investigation the inhibitors modes of action confirmed that these compounds are competitive with respect to the ATP molecule. The binding of inhibitors to the target enzyme was also studied using isothermal titration calorimetry (ITC). Compounds 6, 12, 18, 30 and 34 represent the first inhibitors of Aslfm reported to date, and are an important step forward in combating infections due to E. faecium.

  20. Examining the Amine Functionalization in Dicarboxylates: Photoelectron Spectroscopy and Theoretical Studies of Aspartate and Glutamate

    SciTech Connect

    Deng, Shihu; Hou, Gao-Lei; Kong, Xiangyu; Valiev, Marat; Wang, Xue B.

    2014-06-30

    Aspartate (Asp2-) and Glutamate (Glu2-), two doubly charged conjugate bases of the corresponding amino acids were investigated using low temperature negative ion photoelectron spectroscopy (NIPES) and ab-initio calculations. The effect of amine functionalization was studied by a direct comparison to the parent dicarboxylate species (-CO2–(CH2)n–CO2-, DCn2-) -- succinate (DC22-) and propionate (DC32-). Experimentally the addition of amine group for n = 2 case (DC22-, Asp2-) significantly improves the stability of the resultant Asp2- dianionic species, albeit that NIPES shows only a small increase in adiabatic electron detachment energy (ADE) (+0.05eV). In contrast, for n = 3 (DC32-, Glu2-), much larger ADE increase is observed (+0.15eV). Similar results are obtained through ab-initio calculations. The latter indicates that increased stability of Asp2- can be attributed to the lowering of the energy of singlet dianion state due to hydrogen bonding effects. The effect of the amino group on the doublet monoanion state is more complicated, and results in the weakening of the binding of the adjacent carboxylate group due to electronic structure resonance effects. This conclusion is confirmed by the analysis of NIPES results that show enhanced production of near zero kinetic energy electrons observed experimentally for amine-functionalized species.

  1. Aspartate-β-hydroxylase Induces Epitope-specific T Cell Responses in Hepatocellular Carcinoma

    PubMed Central

    Tomimaru, Yoshito; Mishra, Sasmita; Safran, Howard; Charpentier, Kevin P.; Martin, William; De Groot, Anne S.; Gregory, Stephen H.; Wands, Jack R.

    2015-01-01

    Hepatocellular carcinoma (HCC) has a poor prognosis due to high recurrence rate. Aspartate-β-hydroxylase (ASPH) is a highly conserved transmembrane protein, which is over expressed in HCC and promotes a malignant phenotype. The capability of ASPH protein-derived HLA Class I and II peptides to generate antigen specific CD4+ and CD8+ immune responses is unknown. Therefore, these studies aim to define the epitope specific components required for a peptide based candidate vaccine. Monocyte-derived dendritic cells (DCs) generated from the peripheral blood mononuclear cells (PBMCs) of HCC patients were loaded with ASPH protein. Helper CD4+ T cells and CD8+ cytotoxic T lymphocytes (CTLs) were co-incubated with the DCs; T cell activation was evaluated by flow cytometric analysis. Immunoinformatics tools were used to predict HLA class I- and class II-restricted ASPH sequences, and the corresponding peptides were synthesized. The immunogenicity of each peptide in cultures of human PBMCs was determined by IFN-γ ELISpot assay. ASPH protein-loaded DCs activated both CD4+ and CD8+ T cells contained within the PBMC population derived from HCC patients. Furthermore, the predicted HLA class I- and class II-restricted ASPH peptides were significantly immunogenic. Both HLA class I- and class II-restricted peptides derived from ASPH induce T cell activation in HCC. We observed that ASPH protein and related peptides were highly immunogenic in patients with HCC and produce the type of cellular immune responses required for generation of anti-tumor activity. PMID:25629522

  2. Identification and characterization of a serine-like proteinase of the murine coronavirus MHV-A59.

    PubMed Central

    Lu, Y; Lu, X; Denison, M R

    1995-01-01

    Gene 1 of the murine coronavirus, MHV-A59, encodes approximately 800 kDa of protein products within two overlapping open reading frames (ORFs 1a and 1b). The gene is expressed as a polyprotein that is processed into individual proteins, presumably by virus-encoded proteinases. ORF 1a has been predicted to encode proteins with similarity to viral and cellular proteinases, such as papain, and to the 3C proteinases of the picornaviruses (A. E. Gorbalenya, A. P. Donchenko, V. M. Blinov, and E. V. Koonin, FEBS Lett. 243:103-114, 1989; A. E. Gorbalenya, E. V. Koonin, A. P. Donchenko, and V. M. Blinov, Nucleic Acids Res. 17:4847-4861, 1989). We have cloned into a T7 transcription vector a cDNA fragment containing the putative 3C-like proteinase domain of MHV-A59, along with portions of the flanking hydrophobic domains. The construct was used to express a polypeptide in a combined in vitro transcription-translation system. Major polypeptides with molecular masses of 38 and 33 kDa were detected at early times, whereas polypeptides with molecular masses of 32 and 27 kDa were predominant after 30 to 45 min and appeared to be products of specific proteolysis of larger precursors. Mutations at the putative catalytic histidine and cysteine residues abolished the processing of the 27-kDa protein. Translation products of the pGpro construct were able to cleave the 27-kDa protein in trans from polypeptides expressed from the noncleaving histidine or cysteine mutants. The amino-terminal cleavage of the 27-kDa protein occurred at a glutamine-serine dipeptide as previously predicted. This study provides experimental confirmation that the coronaviruses express an active proteinase within the 3C-like proteinase domain of gene 1 ORF 1a and that this proteinase utilizes at least one canonical QS dipeptide as a cleavage site in vitro. PMID:7745703

  3. Shrimp Serine Proteinase Homologues PmMasSPH-1 and -2 Play a Role in the Activation of the Prophenoloxidase System

    PubMed Central

    Jearaphunt, Miti; Amparyup, Piti; Sangsuriya, Pakkakul; Charoensapsri, Walaiporn; Senapin, Saengchan; Tassanakajon, Anchalee

    2015-01-01

    Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gram-negative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system. PMID:25803442

  4. Alterations in cysteine proteinase content of rat lung associated with development of Pneumocystis carinii infection.

    PubMed Central

    Hayes, D J; Stubberfield, C R; McBride, J D; Wilson, D L

    1991-01-01

    The rate of hydrolysis of three cysteine-type proteinase substrates, N-benzyloxycarbonyl-Arg-Arg-4-methyl-7-coumarylamide (AMC) (cathepsin B), Arg-AMC (cathepsin H), and N-benzyloxycarbonyl-Phe-Arg-AMC (cathepsin L), were determined in rat lung throughout the time course of the induction of Pneumocystis carinii infection by immunosuppression. Cathepsin B-like and cathepsin L-like activities fell below control values initially, but from week 8 of the immunosuppressive treatment significant increases above the control were noted. Cathepsin H-like activity was greater than control levels from week 3, and by week 12 it was 7,600% of the mean control value. When compared with the relative degree of infection, as assessed from the number of cysts present in lung impression smears, cathepsin B-like and cathepsin L-like activities were significantly increased only at heavy parasite burdens while cathepsin H-like activity displayed a close correlation with parasite number (r = 0.884; P less than 0.001). Activity was detected in lysates of purified P. carinii with all three substrates. Treatment of heavily infected animals with co-trimoxazole cleared the lungs of P. carinii, and this was accompanied by a marked reduction in proteinase activity, in particular, cathepsin H-like activity, which fell from 108- to 3-fold the mean control value following drug treatment. Analysis of cathepsin H isozyme patterns by fluorography following isoelectric focusing revealed differences between treated and control lung samples. In the immunosuppressed group, there was a time-dependent increase in the intensity of some of the bands observed in the controls and an appearance of several novel bands which corresponded to bands observed in lysates of P. carinii. It is likely, therefore, that the increased proteinase activity observed in the treated group is due, at least in part, to isozymes from P. carinii; consequently, cathepsin H-like activity might be of use diagnostically in the

  5. Serum concentrations of canine alpha(1)-proteinase inhibitor in cobalamin-deficient Yorkshire Terrier dogs.

    PubMed

    Grützner, Niels; Heilmann, Romy M; Bridges, Cory S; Suchodolski, Jan S; Steiner, Jörg M

    2013-05-01

    Fecal canine alpha1-proteinase inhibitor (cα1-PI) concentration has been reported to be increased in dogs with protein-losing enteropathy due to the loss of cα1-PI into the gastrointestinal tract. A chronic loss of cα1-PI may theoretically deplete serum cα1-PI, potentially altering the proteinase-to-proteinase inhibitor balance. Protein-losing enteropathy has been reported to occur frequently in certain dog breeds such as Yorkshire Terriers and to be associated with hypocobalaminemia. The objective was to compare serum cα1-PI concentrations in Yorkshire Terriers with and without cobalamin (COB) deficiency. Serum samples from 52 COB-deficient and 69 normocobalaminemic Yorkshire Terriers, which had been submitted to the Gastrointestinal Laboratory (2008-2011; College Station, TX), were included retrospectively. Serum cα1-PI concentrations were measured using an in-house radioimmunoassay and compared between Yorkshire Terriers with and without COB deficiency using a Mann-Whitney U test. A Fisher exact test was used to evaluate whether a decreased serum cα1-PI concentration is associated with COB deficiency in Yorkshire Terriers. Serum cα1-PI concentrations were significantly lower in COB-deficient Yorkshire Terriers (median: 1,016 mg/l, range: 315-3,945 mg/l) than in normocobalaminemic Yorkshire Terriers (median: 1,665 mg/l, range: 900-2,970 mg/l; P < 0.0001). One-fourth (n = 13) of the COB-deficient Yorkshire Terriers had a serum cα1-PI concentration below the lower limit of the reference interval (<732 mg/l), and COB deficiency was associated with decreased serum cα1-PI concentrations (P < 0.0001). In the current study, serum cα1-PI concentrations are significantly lower in COB-deficient Yorkshire Terriers when compared to normocobalaminemic Yorkshire Terriers. Further studies are needed to determine the functional and potential prognostic implications of serum cα1-PI concentrations in dogs with gastrointestinal disease.

  6. Structural Insights into the Activation and Inhibition of Histo-Aspartic Protease from Plasmodium falciparum

    SciTech Connect

    Bhaumik, Prasenjit; Xiao, Huogen; Hidaka, Koushi; Gustchina, Alla; Kiso, Yoshiaki; Yada, Rickey Y.; Wlodawer, Alexander

    2012-09-17

    Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 {angstrom} resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.

  7. Racemization of aspartic acid and phenylalanine in the sweetener aspartame at 100 degrees C.

    PubMed Central

    Boehm, M F; Bada, J L

    1984-01-01

    The racemization half-lives (i.e., the time required to reach a D/L = 0.33) at pH 6.8 for aspartic acid and phenylalanine in the sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester) were determined to be 13 and 23 hours, respectively, at 100 degrees C. Racemization at this pH does not occur in aspartame but rather in its diketopiperazine decomposition product. Our results indicate that the use of aspartame to sweeten neutral pH foods and beverages that are then heated at elevated temperature could generate D-aspartic acid and D-phenylalanine. The nutritive consequences of these D-amino acids in the human diet are not well established, and thus aspartame should probably not be used as a sweetener when the exposure of neutral pH foods and beverages to elevated temperatures is required. At pH 4, a typical pH of most foods and beverages that might be sweetened with aspartame, the half-lives are 47 hours for aspartic acid and 1200 hours for phenylalanine at 100 degrees C. Racemization at pH 4 takes place in aspartame itself. Although the racemization rates at pH 4 are slow and no appreciable racemization of aspartic acid and phenylalanine should occur during the normal use of aspartame, some food and beverage components could conceivably act as catalysts. Additional studies are required to evaluate whether the use of aspartame as a sugar substitute might not in turn result in an increased human consumption of D-aspartic acid and D-phenylalanine. PMID:6591191

  8. Racemization of aspartic acid and phenylalanine in the sweetener aspartame at 100 degrees C.

    PubMed

    Boehm, M F; Bada, J L

    1984-08-01

    The racemization half-lives (i.e., the time required to reach a D/L = 0.33) at pH 6.8 for aspartic acid and phenylalanine in the sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester) were determined to be 13 and 23 hours, respectively, at 100 degrees C. Racemization at this pH does not occur in aspartame but rather in its diketopiperazine decomposition product. Our results indicate that the use of aspartame to sweeten neutral pH foods and beverages that are then heated at elevated temperature could generate D-aspartic acid and D-phenylalanine. The nutritive consequences of these D-amino acids in the human diet are not well established, and thus aspartame should probably not be used as a sweetener when the exposure of neutral pH foods and beverages to elevated temperatures is required. At pH 4, a typical pH of most foods and beverages that might be sweetened with aspartame, the half-lives are 47 hours for aspartic acid and 1200 hours for phenylalanine at 100 degrees C. Racemization at pH 4 takes place in aspartame itself. Although the racemization rates at pH 4 are slow and no appreciable racemization of aspartic acid and phenylalanine should occur during the normal use of aspartame, some food and beverage components could conceivably act as catalysts. Additional studies are required to evaluate whether the use of aspartame as a sugar substitute might not in turn result in an increased human consumption of D-aspartic acid and D-phenylalanine.

  9. Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

    PubMed Central

    Kim, Won-Tae; Kong, Hyun-Hee; Ha, Young-Ran; Hong, Yeon-Chul; Jeong, Hae Jin; Yu, Hak Sun

    2006-01-01

    The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba. PMID:17170574

  10. Formation of biogenic amines in raw milk Hispánico cheese manufactured with proteinases and different levels of starter culture.

    PubMed

    Fernandez-García, E; Tomillo, J; Nuñez, M

    2000-11-01

    Two proteinases, a neutral proteinase from Bacillus subtilis and a cysteine proteinase from Micrococcus sp., were used to accelerate the ripening process of raw cow's milk Hispánico cheese, a semihard variety. Two levels (0.1% and 1%) of a commercial starter culture containing Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were added for cheese manufacture. The influence of both factors, proteinase addition and level of starter culture, on the growth of amino acid-decarboxylating microorganisms and on the formation of biogenic amines during cheese ripening was investigated in duplicate experiments. The population of tyrosine decarboxylase-positive bacteria, which represented less than 1% of the total bacterial population in most cheese samples, and tyrosine decarboxylase-positive lactobacilli was not influenced by proteinase addition or level of starter culture. Tyramine was detected in all batches of cheese from day 30. Its concentration was significantly (P < 0.05) influenced by proteinase addition but not by the level of starter culture and increased with cheese age. After 90 days of ripening, 103 to 191 mg/kg of tyramine was found in the different cheese batches. Histamine was not detected until day 60 in cheese with neutral proteinase and 1% starter culture and until day 90 in the rest of the cheeses. The concentration of this amine did not exceed 20 mg/kg in any of the batches investigated. Phenylethylamine and tryptamine were not found in any of the samples.

  11. Development of novel radiogallium-labeled bone imaging agents using oligo-aspartic acid peptides as carriers.

    PubMed

    Ogawa, Kazuma; Ishizaki, Atsushi; Takai, Kenichiro; Kitamura, Yoji; Kiwada, Tatsuto; Shiba, Kazuhiro; Odani, Akira

    2013-01-01

    (68)Ga (T 1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting (68)Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle (67)Ga, with the previously described (67)Ga-DOTA complex conjugated bisphosphonate, (67)Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with (67)Ga, resulting in (67)Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of (67)Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, (67)Ga-DOTA-(Asp)8, (67)Ga-DOTA-(Asp)11, and (67)Ga-DOTA-(Asp)14 showed high accumulation in bone (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of (67)Ga-DOTA-(Asp)n was lower than that of (67)Ga-DOTA-Bn-SCN-HBP, blood clearance of (67)Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of (67)Ga-DOTA-(Asp)11 and (67)Ga-DOTA-(Asp)14 were comparable with those of (67)Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of (68)Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases. PMID:24391942

  12. Insights into the behaviour of biomolecules on the early Earth: The concentration of aspartate by layered double hydroxide minerals

    NASA Astrophysics Data System (ADS)

    Grégoire, Brian; Erastova, Valentina; Geatches, Dawn L.; Clark, Stewart J.; Greenwell, H. Christopher; Fraser, Donald G.

    2016-03-01

    The role of mineral surfaces in concentrating and facilitating the polymerisation of simple protobiomolecules during the Hadean and Archean has been the subject of much research in order to constrain the conditions that may have led to the origin of life on early Earth. Here we examine the adsorption of the amino acid aspartate on layered double hydroxide minerals, and use a combined computer simulation - experimental spectroscopy approach to gain insight into the resulting structures of the host-aspartate material. We show that the uptake of aspartate occurs in alkaline solution by anion exchange of the dianion form of aspartate, rather than by surface adsorption. Anion exchange only occurs at values of pH where a significant population of aspartate has the amino group deprotonated, and is then highly efficient up to the mineral anion exchange capacity.

  13. Effect of aspartame and aspartate loading upon plasma and erythrocyte free amino acid levels in normal adult volunteers.

    PubMed

    Stegink, L D; Filer, L J; Baker, G L

    1977-10-01

    Aspartame is a dipeptide (L-aspartyl-L-phenylalanyl-methyl ester) with a sweeting potential 180 to 200 times that of sucrose. Questions have been raised about potential toxic effects of its constituent amino acids, aspartate and phenylalanine when the compound is ingested in large amounts. Plasma and erythrocyte amino acid levels were measured in 12 normal subjects after administration of either Aspartame (34 mg/kg) or equimolar quantities of aspartate (13 mg/kg) in a crossover design. No changes in either plasma or erythrocyte aspartate levels were noted at any time after either Aspartame or aspartate ingestion. Plasma phenylalanine levels decrease slightly after aspartate loading, and increased from fasting levels (4.9 +/- 1 mumoles/100 ml) to 10.7 +/- 1.9 mumoles/100 ml about 45 to 60 minutes after Aspartame loading. Phenylalanine levels returned to baseline by 4 hours. Erythrocyte phenylalanine levels showed similar changes.

  14. The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases.

    PubMed Central

    Kay, G; Bailie, J R; Halliday, I M; Nelson, J; Walker, B

    1992-01-01

    The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase. Images Fig. 2. Fig. 3. Fig. 4. PMID:1575691

  15. Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle

    PubMed Central

    Azanza, Jean-Louis; Raymond, Jacques; Robin, Jean-Michel; Cottin, Patrick; Ducastaing, André

    1979-01-01

    Ca2+-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial–Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or α1-casein were hydrolysed with a maximum rate at 30°C, pH7.5, and with 5mm-CaCl2, but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, α-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry 15, 2150–2158]. The Ca2+-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca2+-activated neutral proteinase. ImagesFig. 1.Fig. 2.Fig. 3. PMID:534501

  16. Immunodiagnosis of Fasciola hepatica infection (fascioliasis) in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase.

    PubMed

    O'Neill, S M; Parkinson, M; Strauss, W; Angles, R; Dalton, J P

    1998-04-01

    Cathepsin L1 (CL1), an immunogenic cysteine proteinase secreted by juvenile and adult Fasciola hepatica, was assessed for its potential as a diagnostic agent for the serologic detection of human fascioliasis. Using ELISAs, we compared the ability of liver fluke homogenates (LFH), excretory/secretory (ES) products, and CL1 to discriminate between seropositive (infected) and seronegative (noninfected) individuals within a population of 95 patients from the Bolivian Altiplano. A high prevalence of human fascioliasis has been reported in this region. The division between the seropositive and seronegative individuals was poorly defined when LFH was used as the antigen. A greater discrimination between these populations was achieved with both ES and CL1. A K-means cluster analysis using the combined ES and CL1 ELISA data identified a cluster of seropositive individuals. Cathepsin L1 detected a subset (20) of these seropositive individuals while ES detected all 26; however, ES detected nine additional individuals that were in the seronegative cluster. The ratio of the mean absorbance readings between seropositive and seronegative individuals was markedly improved by using conjugated second antibodies to IgG4, the predominant isotype elicited by infection. In these IgG4-ELISAs, CL1 again identified fewer individuals as seropositive than did ES, but improved the discrimination between the seropositive and seronegative individuals and thus provided a more conclusive diagnosis. Sera obtained from patients infected with schistosomiasis mansoni, cysticercosis, hydatidosis, and Chagas' disease were negative in these assays, which demonstrated the specificity of the IgG4-ELISA for detecting fascioliasis. Twenty of the 95 patients (21%) were seropositive for fascioliasis by the CL1 IgG4-ELISA, confirming the earlier reports of the high prevalence of disease in this region. A standardized diagnostic test for human fascioliasis, based on an ELISA that detects IgG4 responses to CL1

  17. AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor

    PubMed Central

    Gaczynska, Maria; Karpowicz, Przemyslaw; Stuart, Christine E.; Norton, Malgorzata G.; Teckman, Jeffrey H.; Marszal, Ewa; Osmulski, Pawel A.

    2016-01-01

    α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found in WT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo

  18. AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor

    DOE PAGESBeta

    Gaczynska, Maria; Karpowicz, Przemyslaw; Stuart, Christine E.; Norton, Malgorzata G.; Teckman, Jeffrey H.; Marszal, Ewa; Osmulski, Pawel A.

    2016-03-23

    α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemicalmore » data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found inWT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo

  19. Alpha 1-proteinase inhibitor is more sensitive to inactivation by cigarette smoke than is leukocyte elastase

    SciTech Connect

    Janoff, A.; Dearing, R.

    1982-10-01

    Aqueous solutions of gas phase cigarette smoke were incubated with pure human leukocyte elastase or with crude human leukocyte granule extract, and the effects on enzyme activity were determined using a synthetic amide substrate. Simultaneously, the same smoke solutions were incubated with 10% human serum under identical conditions, and the effects on serum inhibition of purified or crude leukocyte elastase were similarly measured. In addition, aqueous solutions of unfractionated cigarette smoke were incubated with leukocyte elastase or serum, and the abilities of the smoke-treated enzyme to digest elastin and of the smoke-treated serum to inhibit elastin digestion were determined. Both experimental protocols showed that serum elastase-inhibiting capacity (primarily caused by alpha 1-proteinase inhibitor) is more susceptible to inactivation by aqueous solutions of cigarette smoke than is leukocyte elastase, suggesting that elastase inhibition (rather than elastase activity) may be predominantly suppressed by cigarette smoke inhalation in vivo.

  20. In Vitro Processing of Tomato Proteinase Inhibitor I by Barley Microsomal Membranes

    PubMed Central

    Osteryoung, Katherine W.; Sticher, Liliane; Jones, Russell L.; Bennett, Alan B.

    1992-01-01

    A plant-derived in vitro system for the study of cotranslational processing of plant endomembrane proteins has been developed and used to investigate cotranslational proteolytic processing of tomato proteinase inhibitor I. Translation of the inhibitor I precursor in wheat germ lysate supplemented with barley aleurone microsomal membranes resulted in cotranslational import of the protein into microsomal vesicles and cleavage of the signal sequence. NH2-terminal sequence analysis of the translocated inhibitor I processing intermediate showed that the signal sequence was cleaved between Ala23 and Arg24 of the precursor protein. Parallel experiments using dog pancreas microsomal membranes indicated an identical site of cleavage, suggesting that the substrate determinants for signal sequence processing are conserved across kingdoms. The plant-derived processing system used for this study may be valuable for analysis of cotranslational processing of other plant preproteins and for characterizing the components of the cotranslational import machinery in plants. ImagesFigure 1 PMID:16668894

  1. Proteinase-activated receptor 2 is involved in the behavioural changes associated with sickness behaviour.

    PubMed

    Abulkassim, Roua; Brett, Ros; MacKenzie, Scott M; Bushell, Trevor J

    2016-06-15

    Proteinase-activated receptor-2 (PAR2) is widely expressed in the CNS but whether it plays a key role in inflammation-related behavioural changes remains unknown. Hence, in the present study we have examined whether PAR2 contributes to behaviour associated with systemic inflammation using PAR2 transgenic mice. The onset of sickness behaviour was delayed and the recovery accelerated in PAR2(-/-) mice in the LPS-induced model of sickness behaviour. In contrast, PAR2 does not contribute to behaviour under normal conditions. In conclusion, these data suggest that PAR2 does not contribute to behaviour in the normal healthy brain but it plays a role in inflammation-related behavioural changes.

  2. Role of calcium-dependent proteinase in molt-induced claw muscle atrophy

    SciTech Connect

    Mykles, D.L.; Skinner, D.M.

    1984-01-01

    The claw closer muscle of the Bermuda land crab Gecarcinus lateralis undergoes a sequential atrophy and restoration during each intermolt cycle. Muscle protein decreases 40% during proecdysis and is restored following ecdysis. Amino acid incorporation into protein of postecdysial muscle is five times greater than that in anecdysial muscle. Since the rates of protein synthesis in anecdysial and proecdysial muscle are the same it appears that proecdysial muscle atrophy is caused primarily by an increase in protein degradation. A calcium-dependent proteinase (CDP) active at neutral pH has been implicated in the nonlysosomal hydrolysis of myofibrillar proteins. We have examined the role of a CDP in atrophy of the claw closer muscle. The many similarities between crustacean and vertebrate CDPs have established this crustacean system as a simple and convenient model for the role of Ca/sup 2 +/-dependent proteolysis in myofibrillar protein turnover and its manifestation in the structure of the sarcomere. 16 references, 8 figures. (ACR)

  3. A four-straight-line model for the proteinase-binding characteristics of human blood serum.

    PubMed Central

    Topping, R M; Seilman, S

    1979-01-01

    Kinetic evaluation of the capacity of human blood serum to form complexes with bovine trypsin generated partition profiles that may be approximated by a series of four intersecting straight lines. Such profiles are suggested to reflect the binding of trypsin to alpha 2-macroglobulin in a kinetically preferred mode (alpha-sites), followed by a subsidiary mode (beta-sites) and finally to alpha 1-antitrypsin. The form of the profile, in addition to revealing a hitherto unreported proteinase-binding capability of alpha 2-macroglobulin (beta-sites), also indicates that saturation of alpha-sites corresponds to a molar binding ratio of alpha 2-macroglobulin/trypsin of 1:2. Finally the profile provides, for certain pathological states, a clinically valuable characteristic. PMID:435247

  4. Plants that express a potyvirus proteinase gene are resistant to virus infection.

    PubMed Central

    Maiti, I B; Murphy, J F; Shaw, J G; Hunt, A G

    1993-01-01

    Transgenic tobacco plants that express the genome-linked protein/proteinase-coding region of the potyvirus tobacco vein mottling virus (TVMV) were produced and tested for their reaction to inoculation with TVMV and two other potyviruses. These plants did not develop disease symptoms after being inoculated with large doses of TVMV but were as susceptible to infection by the other potyviruses as were control plants. Lines of tobacco that express the coat protein- or the nonstructural cylindrical inclusion protein-coding regions were also produced. The coat protein transgenic plants were protected against all three potyviruses, and the cylindrical inclusion transgenic plants were susceptible to all three potyviruses. These results indicate that some, but not all, TVMV genes can be used to confer protection against potyviruses in plants. The results also suggest that combinations of viral genes in transgenic plants might improve protection against potyviruses. Images Fig. 2 PMID:8327491

  5. Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity

    PubMed Central

    Cárdenas-Guerra, Rosa Elena; Figueroa-Angulo, Elisa Elvira; Puente-Rivera, Jonathan; Zamudio-Prieto, Olga; Ortega-López, Jaime

    2015-01-01

    We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes. PMID:26090464

  6. Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity.

    PubMed

    Arroyo, Rossana; Cárdenas-Guerra, Rosa Elena; Figueroa-Angulo, Elisa Elvira; Puente-Rivera, Jonathan; Zamudio-Prieto, Olga; Ortega-López, Jaime

    2015-01-01

    We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes.

  7. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin activity from a case of vulvovaginitis.

    PubMed

    Kumar, Dharmendra; Banerjee, Tuhina; Pratap, Chandra Bhan; Tilak, Ragini

    2015-04-15

    Since the emergence of pathogenic non-albicans Candida species, a number of new isolates have been added to the list. One such unusual species is Candida auris (C. auris), recently isolated and studied in few reports. In this study, a case of vulvovaginitis caused by Candida auris incidentally identified by molecular methods using internal transcribed spacer polymerase chain reaction (ITS PCR) is described. Antifungal susceptibility testing revealed the isolate to be resistant to itraconazole (MIC ≥ 2 µg/ml) and expressed important virulence factors including phospholipase, proteinase and hemolysin activity. The patient was successfully treated with oral fluconazole and did not have any invasive fungemia. Very few cases of this emerging pathogen have been reported. However, its isolation from clinical specimens reveals the significance of non-albicans candida species over C. albicans and the diversity of Candida spp causing infections.

  8. [Effect of proteinaceous proteinase inhibitors from potato tubers on the growth and development of phytopathogenic microorganisms].

    PubMed

    Revina, T A; Gerasimova, N G; Kladnitskaia, G V; Chalenko, G I; Valueva, T A

    2008-01-01

    We studied the effect of two proteins, PSPI-21 and PKSI, on the growth and development of phytopathogenic microorganisms (Phytophthora infestans oomycete and Fusarium culmorum fungus). Both proteins were isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and served as inhibitors of serine proteinases. These proteins differed in the ability to inhibit growth of Phytophthora infestans oomycete and Fusarium culmorum fungus. PSPI-21 was the most potent in modulating the growth of oomycete mycelium. PKSI primarily affected the growth of the fungal mycelium. The proteins under study induced complete destruction of oomycete zoospores and partial destruction of fungal macroconidia. Our results suggest that these proteins are involved in the protection of potato plants from phytopathogenic microorganisms.

  9. Quantification of the degree of biotinylation of proteins using proteinase K digestion and competition ELISA.

    PubMed

    Rispens, Theo; Ooijevaar-de Heer, Pleuni

    2016-03-01

    Quantification of the degree of biotinylation of proteins is useful to achieve and maintain a high degree of consistency of reagents used in research and diagnostic setting. Unfortunately, existing protocols and commercial kits suffer from a number of shortcomings that limit their usefulness. Here, we describe a simple protocol that overcomes the limitations of current assays. A robust competition ELISA was developed that is easy to carry out, uses no specialized equipment other than a standard plate reader for absorbance measurements and only reagents that are commonly available. The protocol uses a proteinase K digestion step of a sample of biotinylated protein to eliminate multivalency issues and sterical hindrance from bulky proteins. Furthermore, the use of an anti-biotin antibody instead of streptavidin results in a convenient range of sensitivity, avoiding million-fold dilutions that may impair precision. The resulting assay typically consumes about 1 μg of biotinylated protein.

  10. Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity.

    PubMed

    Arroyo, Rossana; Cárdenas-Guerra, Rosa Elena; Figueroa-Angulo, Elisa Elvira; Puente-Rivera, Jonathan; Zamudio-Prieto, Olga; Ortega-López, Jaime

    2015-01-01

    We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes. PMID:26090464

  11. Quantification of the degree of biotinylation of proteins using proteinase K digestion and competition ELISA.

    PubMed

    Rispens, Theo; Ooijevaar-de Heer, Pleuni

    2016-03-01

    Quantification of the degree of biotinylation of proteins is useful to achieve and maintain a high degree of consistency of reagents used in research and diagnostic setting. Unfortunately, existing protocols and commercial kits suffer from a number of shortcomings that limit their usefulness. Here, we describe a simple protocol that overcomes the limitations of current assays. A robust competition ELISA was developed that is easy to carry out, uses no specialized equipment other than a standard plate reader for absorbance measurements and only reagents that are commonly available. The protocol uses a proteinase K digestion step of a sample of biotinylated protein to eliminate multivalency issues and sterical hindrance from bulky proteins. Furthermore, the use of an anti-biotin antibody instead of streptavidin results in a convenient range of sensitivity, avoiding million-fold dilutions that may impair precision. The resulting assay typically consumes about 1 μg of biotinylated protein. PMID:26795634

  12. Selective killing of B-cell hybridomas targeting proteinase 3, Wegener's autoantigen

    PubMed Central

    Reiners, Katrin S; Hansen, Hinrich P; Krüssmann, Anne; Schön, Gisela; Csernok, Elena; Gross, Wolfgang L; Engert, Andreas; von Strandmann, Elke Pogge

    2004-01-01

    Wegener's granulomatosis (WG) is a rare disease characterized by granulomatous lesions, small vessel vasculitis and the presence of anti-neutrophil cytoplasmic autoantibodies (C-ANCAs) in the sera of affected patients. Their main target antigen is proteinase 3 (PR3), a neutrophil and monocyte-derived neutral serine protease. Since the standard treatment of this severe autoimmune disease, with cyclophosphamide and corticosteroids, is associated with potential side-effects, the development of a more specific immunotherapeutic agent is warranted. The key role of ANCA in the pathogenesis of vasculitis and the effectiveness of anti-CD20 antibodies in patients with refractory WG points towards the importance of B cells in WG. We thus evaluated a new approach to selectively eliminate PR3-specific autoreactive B cells by targeting the B-cell receptor. For this purpose we used a bifunctional recombinant fusion protein consisting of the antigen PR3 and a toxin. The cytotoxic component of this novel fusion protein was the ribonuclease angiogenin, a human toxin with low immunogenicity. The toxin was stabilized by exchanging the catalytically relevant histidine in position 44 with glutamine to eliminate the autoproteolytic activity. PR3H44Q was fused either to the N terminus or to the C terminus of angiogenin. The recombinant proteins were expressed in 293T cells. Binding assays demonstrated the appropriate size and recognition by anti-PR3 antibodies. Using TUNEL technology, we demonstrated that these autoantigen toxins kill proteinase 3-specific B-cell hybridomas selectively by inducing apoptosis. The data indicate that autoantigen-toxins are promising tools in the treatment or co-treatment of autoimmune diseases in which the antigen is known. PMID:15147566

  13. [Inhibitors of acrosomal proteinase as antifertility agents. A problem of acrosomal membrane permeability (author's transl)].

    PubMed

    Schill, W B; Feifel, M; Fritz, H; Hammerstein, J

    1982-01-01

    In vitro studies were performed to investigate the accessibility of acrosin to various proteinase inhibitors inside the intact acrosome of testicular, ejaculated, and uterine human spermatozoa. As test system, the gelatin plate assay was used. For this assay, it was shown formerly that a correlation exists between the size of the digested lysis areas (halo formation) and acrosin activity estimated with synthetic substrates. In addition, saturation of the gelatin substrate membranes with acrosin inhibitors including highly specific ones before application of spermatozoa completely prevented halo formation indicating that the gelatinolytic activity of human spermatozoa is caused exclusively by acrosin. When human spermatozoa were incubated with various acrosin inhibitors (concentration: 1 mmol/1) prior to application to the gelatine membrane, reduction of halo formation could not be observed, however. This result indicates that most of the tested acrosin inhibitors (9 naturally occurring protein inhibitors, 2 microbial peptide inhibitors, 19 synthetic inhibitors) were unable to penetrate the acrosomal membranes of testicular, ejaculated, and uterine human spermatozoa. Only 2 inhibitors caused moderate to complete inhibition of the gelatinolytic activity of the spermatozoa if applied in concentrations between 1-10 mmol/l: the proteinase inhibitor aprotinin and the synthetic inhibitor NPGB (4-nitrophenyl 4-guanidinobenzoate). Obviously, human acrosomal membranes seem to be especially impenetrable to proteins, polypeptides, and synthetic agents. Those acrosin inhibitors penetrating the human sperm head membranes are either too toxic or the local concentration necessary for effective acrosin inhibition in vivo cannot be achieved within the male or female genital tract secretions. Therefore, acrosin inhitibors cannot be used for human contraception at present. Thus, it is mandatory to continue the search for suitable acrosin inhibitors with low toxicity easily penetrating

  14. Application of Asian pumpkin (Cucurbita ficifolia) serine proteinase for production of biologically active peptides from casein.

    PubMed

    Dąbrowska, Anna; Szołtysik, Marek; Babij, Konrad; Pokora, Marta; Zambrowicz, Aleksandra; Chrzanowska, Józefa

    2013-01-01

    The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (μmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.

  15. tvcp12: a novel Trichomonas vaginalis cathepsin L-like cysteine proteinase-encoding gene.

    PubMed

    León-Sicairos, Claudia R; León-Félix, Josefina; Arroyo, Rossana

    2004-05-01

    Trichomonas vaginalis is the causative agent of trichomoniasis, one of the most common sexually transmitted diseases in humans. This protozoan has multiple proteinases that are mainly of the cysteine proteinase (CP) type, some of which are known to be involved in the parasite's virulence. Here, a novel T. vaginalis CP-encoding gene, tvcp12, was identified and characterized. tvcp12 is 948 bp long and encodes a predicted 34.4 kDa protein that has the characteristics of the papain-like CP family. TvCP12 does not appear to have a signal peptide, suggesting that this is a cytoplasmic CP. By Southern blot assays, the tvcp12 gene was found as a single copy in the T. vaginalis genome. Remarkably, Northern blot experiments showed a single transcript band of approximately 1.3 kb in the mRNA obtained from parasites grown in low iron conditions and no transcript was observed in the mRNA from parasites grown in high iron conditions. By RT-PCR assays, a 270 bp band was amplified from the cDNA of parasites grown in low iron medium, which was very faint when cDNA from parasites grown in high iron conditions was used. Transcripts of the 3' region obtained in both iron conditions presented differences in their poly(A) tail length. These data suggest that tvcp12 is another gene that is negatively regulated by iron and that the length of the poly(A) tail may be one of the factors involved in the iron-modulated protein expression.

  16. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

    PubMed

    Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

    2016-01-01

    Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years. PMID:27194832

  17. Effects of trypsin, thrombin and proteinase-activated receptors on guinea pig common bile duct motility.