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Sample records for plant cysteine proteinases

  1. Cysteine proteinases and cystatins

    Scientific Electronic Library Online (English)

    Adeliana S., Oliveira; José, Xavier-Filho; Maurício P., Sales.

    2003-01-01

    Full Text Available SciELO Brazil | Language: English Abstract in portuguese Nesta revisão foram descritas definições, localizações, funções e exemplos de proteinases cisteínicas e suas proteinas inibidoras em animais vertebrados e invertebrados e plantas. Tratamos principalmente com aqueles inibidores que são relatados com o mecanismo de defesa da planta contra pestes. Em a [...] dição, comentamos sobre recentes trabalhos que contribuíram para uma melhor compreenção dos fatores envolvidos na interação específica proteinase cisteínica-cistatina. Por outro lado, chamamos atenção para o alto grau de afinidade e grande especificidade na interação que não são apenas representadas pela compatibilidade entre os residuos de aminoácidos do sítio ativo envolvidos na catalise, mas também de todos os resíduos de aminoácidos que participam da interação enzima-inibidor. Abstract in english This review describeds the definition, localization, functions and examples of cysteine proteinases and their protein inhibitors in vertebrate, non-vertebrate animals and plants. These inhibitors are related with defense mechanisms of plant against pests. It also describes the factors involved in th [...] e specific cysteine proteinase-cystatin interaction and high degree of affinity and large specificity in this interaction which are not only represented by the compatibility between amino acid residues of the active site involved in catalysis, but also of all amino acid residues that participante in the enzyme-inhibitor interaction.

  2. Cysteine proteinases and cystatins

    Directory of Open Access Journals (Sweden)

    Adeliana S. Oliveira

    2003-01-01

    Full Text Available This review describeds the definition, localization, functions and examples of cysteine proteinases and their protein inhibitors in vertebrate, non-vertebrate animals and plants. These inhibitors are related with defense mechanisms of plant against pests. It also describes the factors involved in the specific cysteine proteinase-cystatin interaction and high degree of affinity and large specificity in this interaction which are not only represented by the compatibility between amino acid residues of the active site involved in catalysis, but also of all amino acid residues that participante in the enzyme-inhibitor interaction.Nesta revisão foram descritas definições, localizações, funções e exemplos de proteinases cisteínicas e suas proteinas inibidoras em animais vertebrados e invertebrados e plantas. Tratamos principalmente com aqueles inibidores que são relatados com o mecanismo de defesa da planta contra pestes. Em adição, comentamos sobre recentes trabalhos que contribuíram para uma melhor compreenção dos fatores envolvidos na interação específica proteinase cisteínica-cistatina. Por outro lado, chamamos atenção para o alto grau de afinidade e grande especificidade na interação que não são apenas representadas pela compatibilidade entre os residuos de aminoácidos do sítio ativo envolvidos na catalise, mas também de todos os resíduos de aminoácidos que participam da interação enzima-inibidor.

  3. In vitro anthelmintic effects of cysteine proteinases from plants against intestinal helminths of rodents.

    Science.gov (United States)

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

    2007-12-01

    Infections with gastrointestinal (GI) nematodes are amongst the most prevalent worldwide, especially in tropical climates. Control of these infections is primarily through treatment with anthelmintic drugs, but the rapid development of resistance to all the currently available classes of anthelmintic means that alternative treatments are urgently required. Cysteine proteinases from plants such as papaya, pineapple and fig are known to be substantially effective against three rodent GI nematodes, Heligmosomoides polygyrus, Trichuris muris and Protospirura muricola, both in vitro and in vivo. Here, based on in vitro motility assays and scanning electron microscopy, we extend these earlier reports, demonstrating the potency of this anthelmintic effect of plant cysteine proteinases against two GI helminths from different taxonomic groups - the canine hookworm, Ancylostoma ceylanicum, and the rodent cestode, Rodentolepis microstoma. In the case of hookworms, a mechanism of action targeting the surface layers of the cuticle indistinguishable from that reported earlier appears to be involved, and in the case of cestodes, the surface of the tegumental layers was also the principal location of damage. Hence, plant cysteine proteinases have a broad spectrum of activity against intestinal helminths (both nematodes and cestodes), a quality that reinforces their suitability for development as a much-needed novel treatment against GI helminths of humans and livestock. PMID:18005461

  4. Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro.

    Science.gov (United States)

    Stepek, G; Buttle, D J; Duce, I R; Lowe, A; Behnke, J M

    2005-02-01

    We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock. PMID:15727070

  5. Investigations into the effects of plant derived cysteine proteinases on tapeworms (cestoda)

    OpenAIRE

    Mansur, Fadlul Azim Fauzi Bin

    2013-01-01

    Gastrointestinal (GI) helminths pose a significant threat to the livestock industry and are a recognized cause of global morbidity in humans. Control relies principally on chemotherapy but in the case of nematodes is rapidly losing efficacy through widespread development and spread of resistance to conventional anthelmintics and hence the urgent need for novel classes of anthelmintics. Cysteine proteinases (CPs) from papaya latex have been shown to be effective against three murine nematodes ...

  6. The relative anthelmintic efficacy of plant-derived cysteine proteinases on intestinal nematodes.

    Science.gov (United States)

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

    2015-03-01

    We examined the in vitro and in vivo efficacy of plant cysteine proteinases (CPs) derived from pineapple (Ananas comosus) and kiwi fruit (Actinidia deliciosa), and compared their efficacy as anthelmintics to the known effects of CPs from the latex of papaya (Carica papaya) against the rodent intestinal nematode, Heligmosomoides bakeri. Both fruit bromelain and stem bromelain had significant in vitro detrimental effects on H. bakeri but in comparison, actinidain from kiwi fruit had very little effect. However, in vivo trials indicated far less efficacy of stem bromelain and fruit bromelain than that expected from the in vitro experiments (24.5% and 22.4% reduction in worm burdens, respectively) against H. bakeri. Scanning electron microscopy revealed signs of cuticular damage on worms incubated in fruit bromelain, stem bromelain and actinidain, but this was far less extensive than on those incubated in papaya latex supernatant. We conclude that, on the basis of presently available data, CPs derived from pineapples and kiwi fruits are not suitable for development as novel anthelmintics for intestinal nematode infections. PMID:24176056

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

    Science.gov (United States)

    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. PMID:24001183

  8. Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases

    OpenAIRE

    Gruden, K.; Kuipers, A. G. J.; Guncar, G.; Slapar, N.; Strukelj, B.; Jongsma, M. A.

    2004-01-01

    Potato synthesises high levels of proteinase inhibitors in response to insect attack. This can adversely affect protein digestion in the insects, leading to reduced growth, delayed development and lowered fecundity. Colorado potato beetle overcomes this defence mechanism by changing the composition of its digestive proteinases. The induced cysteine proteinases in the adapted gut sustain a normal rate of protein hydrolysis either by inactivating the inhibitors by cleavage or by insensitivity t...

  9. Caiman kininogen-like cysteine proteinase inhibitor.

    Science.gov (United States)

    Araujo, M S; Andreotti, R; Chudzinski, A M; Sampaio, C A; Sampaio, M U

    1992-01-01

    Kininogens are the major mammalian plasma cysteine proteinase inhibitors; a kininogen-like protein was also found in the snake Bothrops jararaca plasma. This communication describes a kininogen-like protein in plasma of Caiman crocodilus vacare. Caiman crude plasma, unlike snake plasma, contains a detectable cysteine proteinase inhibitor. The inhibitor was purified by DEAE-Sephadex ion-exchange chromatography and chromatography on carboxy-methylated-papain-Sepharose. The estimated molecular weight of Caiman cysteine proteinase inhibitor is 70,000. Caiman plasma also hydrolyzes plasma kallikrein synthetic substrates and inhibits trypsin. Reptilian kininogen may lack the site for interaction with plasma prokallikrein, and the sequence of the released kinin may be distinct from bradykinin. The poor effectiveness of bradykinin on reptile smooth muscle shows that the reptile kinin receptors may be adapted to a specific kinin. PMID:1466283

  10. Cathepsin D inactivates cysteine proteinase inhibitors, cystatins.

    Science.gov (United States)

    Lenarcic, B; Kos, J; Dolenc, I; Lucovnik, P; Krizaj, I; Turk, V

    1988-07-29

    The formation of inactive complexes in excess molar amounts of human cathepsins H and L with their protein inhibitors human stefin A, human stefin B and chicken cystatin at pH 5.6 has been shown by measurement of enzyme activity coupled with reverse-phase HPLC not to involve covalent cleavage of the inhibitors. Inhibition must be the direct result of binding. On the contrary the interaction of cystatins with aspartic proteinase cathepsin D at pH 3.5 for 60 min followed by HPLC resulted in their inactivation accompanied by peptide bond cleavage at several sites, preferentially those involving hydrophobic amino acid residues. The released peptides do not inhibit papain and cathepsin L. These results explain reported elevated levels of cysteine proteinases and lead to the proposal that cathepsin D exerts an important function, through inactivation of cystatins, in the increased activities of cysteine proteinases in human diseases including muscular distrophy. PMID:3261170

  11. Structural characterization of the papaya cysteine proteinases at low pH.

    Science.gov (United States)

    Huet, Joëlle; Looze, Yvan; Bartik, Kristin; Raussens, Vincent; Wintjens, René; Boussard, Paule

    2006-03-10

    Current control of gastrointestinal nematodes relies primarily on the use of synthetic drugs and encounters serious problems of resistance. Oral administration of plant cysteine proteinases, known to be capable of damaging nematode cuticles, has recently been recommended to overcome these problems. This prompted us to examine if plant cysteine proteinases like the four papaya proteinases papain, caricain, chymopapain, and glycine endopeptidase that have been investigated here can survive acidic pH conditions and pepsin degradation. The four papaya proteinases have been found to undergo, at low pH, a conformational transition that instantaneously converts their native forms into molten globules that are quite unstable and rapidly degraded by pepsin. As shown by activity measurements, the denatured state of these proteinases which finally results from acid treatment is completely irreversible. It is concluded that cysteine proteinases from plant origin may require to be protected against both acid denaturation and proteolysis to be effective in the gut after oral administration. PMID:16434027

  12. Cystatins may confer viral resistance in plants by inhibition of a virus-induced cell death phenomenon in which cysteine proteinases are active: cloning and molecular characterization of a cDNA encoding cysteine-proteinase inhibitor (celostatin) from Celosia cristata (crested cock's comb).

    Science.gov (United States)

    Gholizadeh, Ashraf; Santha, Ittiaparambu Mana; Kohnehrouz, Bahram Baghban; Lodha, Madan Lal; Kapoor, Harish Chander

    2005-12-01

    Cystatins (cysteine proteinase inhibitors) have been recently used in plants as antiviral strategy against those viruses whose replication involves cysteine proteinase activity. We proposed an idea that cystatins may confer resistance by inhibition of a virus-induced cell-death phenomenon in which cysteine proteinases are active. To test this idea, a full-length cDNA library was constructed from the preflowering stage of Celosia cristata (crested cock's comb) leaves, and a cDNA clone with cystatin domain was isolated using an oligonucleotide probe designed on the basis of the conserved peptide of plant cystatins. It was expressed in an Escherichia coli expression system as a fusion protein. The purified recombinant product, termed 'celostatin' (Celosia cystatin), inhibited the enzymatic activity of papain indicating its cystatin activity and prevented TMV (tobacco mosaic virus)-induced hypersensitive-response cell death in Nicotiana glutinosa (a wild species of tobacco) leaves by 65-70% at the concentration of approx. 50 ng/ml. It also offered resistance against TMV and caused normal growth of the test plant. Since the activity of cysteine proteinases is not involved in the TMV replication process, we speculated that inhibition of the hypersensitive response by celostatin may be due to the inactivation of proteolysis involved in the plant cell death programme, a phenomenon that has already been reported in animal systems. PMID:15842197

  13. Computational analysis of Ancylostoma ceylanicum cysteine proteinase.

    Science.gov (United States)

    Baska, Piotr; Wísniewski, Marcin; Mieszczanek, Juliusz; Wedrychowicz, Halina

    2006-01-01

    The potential tertiary structure of Ancylostoma ceylanicum cysteine proteinase was obtained by Automatic Program 3D-JIGSAW and used for finding homologues of known structure by VAST program. The results of computational analysis showed the presence of domains recognizing host immunoglobulins. Based on this analysis we suggest that this protein is involved in cleaving of host antibodies and therefore it may be promising vaccine candidate. In this paper we present the computational analysis of parasitic antigen which is very helpful in evaluation of the potential role of this protein. PMID:17432618

  14. The anthelmintic efficacy of natural plant cysteine proteinases against two rodent cestodes Hymenolepis diminuta and Hymenolepis microstoma in vitro.

    Science.gov (United States)

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

    2014-03-17

    Little is known about the efficacy of cysteine proteinases (CP) as anthelmintics for cestode infections. We examined the effects of CPs on two rodent cestodes, Hymenolepis diminuta and H. microstoma in vitro. Our data showed that naturally occurring mixtures of CPs, such as those found in papaya latex, and relatively pure preparations of fruit bromelain, papain and stem bromelain, were active in vitro against both juvenile, artificially excysted scoleces, as well as against adult worms of both rodent cestodes. Significant dose-dependent reduction in motility, ultimately leading to death of the worms, was observed with both species, and against both freshly excysted scoleces and 14-day old pre-adult worms. The most effective was fruit bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50=63 and 74 ?M, respectively, and for pre-adult worms=199 and 260 ?M, respectively). The least effective was stem bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50=2855 and 2772 ?M, respectively, and for pre-adult worms=1374 and 1332 ?M, respectively) and the efficacies of papaya latex supernatant and papain were between these extremes. In all cases these values are higher than those reported previously for efficacy of CPs against intestinal nematodes, and in contrast to nematodes, all CPs were effective against cestodes in the absence of exogenous cysteine in incubation media. The CPs appeared to attack the tegument resulting in generalised erosion mainly on the strobila. The scolex was more resistant to CP attack but nevertheless some damage to the tegument on the scolex was detected. PMID:24462509

  15. Purification and characterization of cysteine proteinase from a baculovirus gene.

    Science.gov (United States)

    Takahashi, S; Ushiyama, S; Suzuki, T; Ogawa, K; Oda, K

    1997-09-01

    To analyze the degradation of product proteins at the late stage of virus infection in the baculovirus expression system, a cysteine proteinase was purified from hemolymph of Bombyx mori infected with wild-type B. mori nuclear polyhedorosis virus (BmNPV). The purified cysteine proteinase preparation had two protein bands (major 35-kDa active protein and 28-kDa inactive protein) on SDS-PAGE. Based on the N-terminal amino acid sequences of them, it was found that both proteins originated in the cysteine proteinase gene of BmNPV. The purified cysteine proteinase had an optimum pH at 4.0, and also had activities at neutral pHs. When recombinant luciferase was used as a natural substrate, it was degraded rapidly by the cysteine proteinase at the physiological pH of hemolymph. These results suggest that the cysteine proteinase from a BmNPV gene participates in the degradation of foreign protein expressed by the baculovirus system. PMID:9339553

  16. Use of phage display to select novel cystatins specific for Acanthoscelides obtectus cysteine proteinases.

    Science.gov (United States)

    Melo, Francislete R; Mello, Márcia O; Franco, Octávio L; Rigden, Daniel J; Mello, Luciane V; Genú, Aline M; Silva-Filho, Márcio C; Gleddie, Steve; Grossi-de-Sá, Maria Fátima

    2003-09-23

    Cysteine proteinases from larvae of the common bean weevil, Acanthoscelides obtectus (Coleoptera: Bruchidae), were isolated by ion exchange affinity chromatography on a CM-Cellulose column and used to select mutant cystatins from a library made with the filamentous M13 phage display system. The library contained variant cystatins derived from the nematode Onchocerca volvulus cystatin through mutagenesis of loop 1, which contains the QVVAG motif that is involved in binding to proteinases. After three rounds of selection, the activity of variant cystatins against papain and cysteine proteinases from A. obtectus was assayed by ELISA. Two different variant cystatins (presenting amino acids DVVSA and NTSSA at positions 65-69) bound to A. obtectus cysteine proteinases more tightly than to papain. In contrast, the wild type had similar affinity for A. obtectus proteinases and for papain. These two selected variants cystatins have greater specificity towards A. obtectus cysteine proteinases than the original sequence and could represent good candidate genes for the production of transgenic plants resistant to this insect pest. PMID:14499599

  17. Porphyromonas gingivalis Cysteine Proteinase Inhibition by ?-Casein Peptides ?

    OpenAIRE

    Toh, Elena C. Y.; Dashper, Stuart G.; Huq, N. Laila; Attard, Troy J.; O Brien-simpson, Neil M.; Chen, Yu-yen; Cross, Keith J.; Stanton, David P.; Paolini, Rita A.; Reynolds, Eric C.

    2010-01-01

    Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The Arg-specific (RgpA/B) and Lys-specific (Kgp) cysteine proteinases of P. gingivalis are major virulence factors for the bacterium. In this study ?-casein(109-137) was identified in a chymosin digest of casein as an inhibiting peptide of the P. gingivalis proteinases. The peptide was synthesized and shown to inhibit proteolytic activity associa...

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

    Science.gov (United States)

    Chojnacka, Magdalena; Szewi?ska, Joanna; Mielecki, Marcin; Nykiel, Ma?gorzata; Imai, Ryozo; Bielawski, Wies?aw; Orzechowski, S?awomir

    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

  19. A Surface Amebic Cysteine Proteinase Inactivates Interleukin-18

    OpenAIRE

    Que, Xuchu; Kim, Soo-hyun; Sajid, Mohammed; Eckmann, Lars; Dinarello, Charles A.; Mckerrow, James H.; Reed, Sharon L.

    2003-01-01

    Amebiasis is a major cause of morbidity and mortality worldwide. Invasion by Entamoeba histolytica trophozoites causes secretion of proinflammatory cytokines from host epithelial cells, leading to a local acute inflammatory response, followed by lysis of colonic cells. Extracellular cysteine proteinases from amebic trophozoites are key virulence factors and have a number of important interactions with host defenses, including cleavage of immunoglobulin G (IgG), IgA, and complement components ...

  20. Porphyromonas gingivalis Cysteine Proteinase Inhibition by ?-Casein Peptides ?

    Science.gov (United States)

    Toh, Elena C. Y.; Dashper, Stuart G.; Huq, N. Laila; Attard, Troy J.; O'Brien-Simpson, Neil M.; Chen, Yu-Yen; Cross, Keith J.; Stanton, David P.; Paolini, Rita A.; Reynolds, Eric C.

    2011-01-01

    Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The Arg-specific (RgpA/B) and Lys-specific (Kgp) cysteine proteinases of P. gingivalis are major virulence factors for the bacterium. In this study ?-casein(109-137) was identified in a chymosin digest of casein as an inhibiting peptide of the P. gingivalis proteinases. The peptide was synthesized and shown to inhibit proteolytic activity associated with P. gingivalis whole cells, purified RgpA-Kgp proteinase-adhesin complexes, and purified RgpB proteinase. The peptide ?-casein(109-137) exhibited synergism with Zn(II) against both Arg- and Lys-specific proteinases. The active region for inhibition was identified as ?-casein(117-137) using synthetic peptides. Kinetic studies revealed that ?-casein(109-137) inhibits in an uncompetitive manner. A molecular model based on the uncompetitive action and its synergistic ability with Zn(II) was developed to explain the mechanism of inhibition. Preincubation of P. gingivalis with ?-casein(109-137) significantly reduced lesion development in a murine model of infection. PMID:21173178

  1. Porphyromonas gingivalis cysteine proteinase inhibition by kappa-casein peptides.

    Science.gov (United States)

    Toh, Elena C Y; Dashper, Stuart G; Huq, N Laila; Attard, Troy J; O'Brien-Simpson, Neil M; Chen, Yu-Yen; Cross, Keith J; Stanton, David P; Paolini, Rita A; Reynolds, Eric C

    2011-03-01

    Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The Arg-specific (RgpA/B) and Lys-specific (Kgp) cysteine proteinases of P. gingivalis are major virulence factors for the bacterium. In this study ?-casein(109-137) was identified in a chymosin digest of casein as an inhibiting peptide of the P. gingivalis proteinases. The peptide was synthesized and shown to inhibit proteolytic activity associated with P. gingivalis whole cells, purified RgpA-Kgp proteinase-adhesin complexes, and purified RgpB proteinase. The peptide ?-casein(109-137) exhibited synergism with Zn(II) against both Arg- and Lys-specific proteinases. The active region for inhibition was identified as ?-casein(117-137) using synthetic peptides. Kinetic studies revealed that ?-casein(109-137) inhibits in an uncompetitive manner. A molecular model based on the uncompetitive action and its synergistic ability with Zn(II) was developed to explain the mechanism of inhibition. Preincubation of P. gingivalis with ?-casein(109-137) significantly reduced lesion development in a murine model of infection. PMID:21173178

  2. The nematicidal effect of cysteine proteinases on the root knot nematode Meloidogne incognita

    OpenAIRE

    Gorny, Samuel Victor

    2013-01-01

    Despite current control measures, plant parasitic nematodes are estimated to be responsible for > $100 billion of damage to worldwide crop production per annum. Current nematicides are highly toxic, and due to health and environmental safety concerns, many are being withdrawn from the market under directive 914/414/EEC. Alternative control strategies are urgently required. The cysteine proteinases papain, actinidain and recombinant endoproteinase B isoform 2 (R.EP-B2) have been demonstrate...

  3. [Cloning and sequence analysis of a new cathepsin L-like cysteine proteinase gene from Ditylenchus destructor].

    Science.gov (United States)

    Wang, Gaofeng; Peng, Deliang; Sun, Jianhua; Huang, Wenkun; Peng, Huan; Long, Haibo

    2011-01-01

    The Cathepsin L-like cysteine proteinase genes (cpls) are multifunction genes related to the parasitic abilities of plant parasitic nematodes. A new cathepsin L-like cysteine proteinase gene (Dd-cpl-1) (GenBank Accession GQ 180107) was cloned from Ditylenchus destructor by RT-PCR and RACE. The cDNA sequence consisted of a 1 131 bp open reading frame (ORF) encoding 376 amino acid residues that were franked by a 29 bp 5'-untranslated region (UTR) and a 159 bp 3'-UTR. Genomic sequence analysis showed that Dd-cpl-1 contained 7 introns, obeyed the GT/AG rule in the splice-site junctions. Homology analysis showed that the identity was 77% between Dd-cpl-1 deduced protein Dd-CPL-1 and cathepsin L-like cysteine proteinase of Bursaphelenchus xylophilus. Multi-sequence alignment indicated that there were the catalytic triad (Cys183, His322 and Asn343) and two motifs ERFNIN motif and GNFD motif in deduced protein Dd-CPL-1. Cysteine proteinases phylogenetic analysis showed that Dd-cpl-1 belonged to the sub-clade of cathepsin L-like cysteine proteinases. PMID:21553491

  4. Purification and characterization of the cysteine proteinases in the latex of Vasconcellea spp.

    Science.gov (United States)

    Kyndt, Tina; Van Damme, Els J M; Van Beeumen, Jozef; Gheysen, Godelieve

    2007-01-01

    Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. In the present study, we show that this higher activity is correlated with a higher concentration of enzymes in the latex of Vasconcellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases present in papaya latex, which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. In this investigation, several cDNA sequences coding for cysteine proteinases in Vasconcellea x heilbornii and Vasconcellea stipulata were determined using primers based on conserved sequences. In silico translation showed that they hold the characteristic features of all known papain-class cysteine proteinases, and a phylogenetic analysis revealed the existence of several papain and chymopapain homologues in these species. Ion-exchange chromatography and gel filtration procedures were applied on latex of V. x heilbornii in order to characterize its cysteine proteinases at the protein level. Five major protein fractions (VXH-I-VXH-V) revealing very high amidase activities (between 7.5 and 23.3 nkat x mg protein(-1)) were isolated. After further purification, three of them were N-terminally sequenced. The observed microheterogeneity in the N-terminal and cDNA sequences reveals the presence of several distinct cysteine proteinase isoforms in the latex of Vasconcellea spp. PMID:17229150

  5. Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene.

    Science.gov (United States)

    Suzuki, T; Kanaya, T; Okazaki, H; Ogawa, K; Usami, A; Watanabe, H; Kadono-Okuda, K; Yamakawa, M; Sato, H; Mori, H; Takahashi, S; Oda, K

    1997-12-01

    Infection by a baculovirus (Bombyx mori nuclear polyhedrosis virus, BmNPV) in silkworm (Bombyx mori) larvae is highly efficient as an expression system for the production of useful proteins. However, the amount of the protein of interest expressed tends to decrease in the later stages of infection presumably due, in part, to a proteinase produced in the larval haemolymph. The N-terminal amino acid sequence of a proteinase purified from the haemolymph of BmNPV-infected larvae was identical to the internal amino acid sequence of the viral cysteine proteinase gene of BmNPV, suggesting that the cysteine proteinase in the haemolymph originated from the BmNPV gene. We constructed a mutant virus (CPd) which had a deletion in the cysteine proteinase gene. No proteinase activity corresponding to this proteinase was detected in the haemolymph of silkworm larvae infected with CPd. The firefly luciferase and the human growth hormone genes were separately introduced into CPd under control of the polyhedrin promoter. These constructs produced these proteins very efficiently, because of a greatly reduced degree of degradation of these proteins. A BmNPV vector system using CPd enhances the stability of foreign expressed proteins, especially for those that are cysteine proteinase-sensitive. PMID:9400955

  6. Characterization of the cathepsin-like cysteine proteinases of Schistosoma mansoni.

    OpenAIRE

    Dalton, J. P.; Clough, K. A.; Jones, M. K.; Brindley, P. J.

    1996-01-01

    Adult Schistosoma mansoni parasites synthesize and secrete both cathepsin L and cathepsin B cysteine proteinases. These cysteine proteinase activities, believed to be involved in hemoglobin digestion by adult schistosomes, were characterized by using specific fluorogenic peptide substrates and zymography. Both cathepsin L- and B-like activities with pH optima of 5.2 and 6.2, respectively, predominated in soluble extracts of worms, and both these activities were secreted by adult worms into th...

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

    Directory of Open Access Journals (Sweden)

    Lepelley Maud

    2012-03-01

    Full Text Available Abstract 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 in the programmed cell death associated with post-germination of the coffee grain. Expression analysis of the cysteine proteinase inhibitor genes suggests that CcCPI-1 could primarily be involved in modulating the activity of grain CP activity; while CcCPI-4 may play roles modulating grain CP activity and in the protection of the young coffee seedlings from insects and pathogens. CcCPI-2 and CcCPI-3, having lower and more widespread expression, could be more general "house-keeping" CPI genes.

  8. Factors affecting the anthelmintic efficacy of cysteine proteinases against GI nematodes and their formulation for use in ruminants

    OpenAIRE

    Luoga, Wenceslaus

    2013-01-01

    Gastrointestinal (GI) nematodes are important helminth pathogens responsible for severe losses to livestock industries and human health throughout the world. Control of these infections relies primarily on chemotherapy; however there is rapid development of resistance to all available classes of anthelmintic drugs, and therefore new alternative treatments are urgently required. Plant cysteine proteinases (CPs) from papaya latex, pineapple fruit and stem extracts have been demonstrated to b...

  9. Th1 Cell Development Induced by Cysteine Proteinases A and B in Localized Cutaneous Leishmaniasis Due to Leishmania guyanensis

    OpenAIRE

    Pascalis, Herve?; Lavergne, Anne; Bourreau, Eliane; Pre?vot-linguet, Ghislaine; Kariminia, Amina; Pradinaud, Roger; Rafati, Sima; Launois, Pascal

    2003-01-01

    The cysteine proteinases CPA and CPB from Leishmania major induced Th1 responses in patients with leishmaniasis due to Leishmania guyanensis. Furthermore, cysteine proteinases induced neither interleukin 4 (IL-4) nor IL-13 and low levels of IL-10 in controls and patients. The results suggest that CPs would be quite good candidates for a vaccine against different Leishmania species.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Recombinant Cysteine Proteinase from Leishmania (Leishmania) chagasi Implicated in Human and Dog T-Cell Responses

    OpenAIRE

    Da Costa Pinheiro, Paulo Henrique; Souza Dias, Suzana; Eula?lio, Kelsen Dantas; Mendonc?a, Ivete L.; Katz, Simone; Barbie?ri, Clara Lu?cia

    2005-01-01

    High in vitro lymphoproliferative responses were induced in humans and dogs by a recombinant Leishmania (Leishmania) chagasi cysteine proteinase, with secretion of IFN-? in asymptomatic subjects or of IFN-?, interleukin 4 (IL-4), and IL-10 in oligosymptomatic subjects. In contrast, responses of symptomatic patients and dogs were lower, with production of IL-4 and IL-10.

  12. Identification of a lymphocyte-produced cysteine proteinase based on its binding to ?-macroglobulin (?M)

    International Nuclear Information System (INIS)

    Proteinases play a role in collagen vascular diseases. However, the role of lymphocytes in the production of these proteinases is poorly understood. Rabbit lymph node cells (LNC) were cultured with 35S-methionine or 14C-leucine. When culture supernatants were analyzed by radioimmunoelectrophoresis using an anti-rabbit ?M antiserum, radio-active ?M-precipitin arcs developed. LNC biosynthetically labeled in serum-free medium generated supernatants that, upon incubation with normal rabbit serum, yielded radio-active ?M precipitin arcs. Purified B and T cells as well as human T and B cell lines were labeled separately under identical conditions. Analysis revealed the presence of radiolabeled ?M molecules. T cells usually generated less intense radioactive ?M precipitin arcs than B cells. Thoracic duct lymphocytes also produced radioactive ?M precipitin arcs. They treated radiolabeled supernatants with a variety of proteinase inhibitors before reacting them with rabbit serum. Only inhibitors specific for cysteine or thiol proteinases were effective in abolishing radioactivity associated with ?M. It appears that lymphocytes actively produce a cysteine or thiol proteinase which, theoretically, is capable of degrading collagen

  13. Miltpain, new cysteine proteinase from the milt of chum salmon, Oncorhynchus keta.

    Science.gov (United States)

    Kawabata, C; Ichishima, E

    1997-07-01

    A new cysteine proteinase, salmon miltpain, was isolated and purified from the milt of chum salmon (Oncorhynchus keta). Native molecular mass was estimated as 67,000 by gel filtration column chromatography (Shodex WS2003) and 22,300 by SDS-polyacrylamide gel electrophoresis. Isoelectoric point was determined to be 3.9 by isoelectric focusing. The first 15 amino acid residues in the N-terminal region were LPSFLY-AEMVGYNIL. The cysteine proteinase, which had a pH optimum of 6.0 for Z-Arg-Arg-MCA hydrolysis, required a thiol-reducing reagent for activation and was inhibited by E-64, iodacetamide, CA-074 Me, TLCK, TPCK and ZPCK. The cysteine proteinase exhibited unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg at the subsites of P2-P1 and had a K(m) of 16.3 microM and kcat of 20.3 s-1 with Z-Arg-Arg-MCA as substrate and a K(m) of 52.9 microM and kcat of 1.79 s-1 with Z-Phe-Arg-MCA. This proteinase was found to considerably hydrolyze basic proteins such as histone, salmine and clupaine but not milk casein. PMID:9253183

  14. Functional Properties of a Cysteine Proteinase from Pineapple Fruit with Improved Resistance to Fungal Pathogens in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Wei Wang

    2014-02-01

    Full Text Available 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.

  15. Electrophoretic analysis of the "cross-class" interaction between novel inhibitory serpin, squamous cell carcinoma antigen-1 and cysteine proteinases.

    Science.gov (United States)

    Nawata, S; Nakamura, K; Tanaka, T; Numa, F; Suminami, Y; Tsunaga, N; Kakegawa, H; Katunuma, N; Kato, H

    1997-05-01

    We investigated the "cross-class" interaction between cysteine proteinases and a novel inhibitory serpin, recombinant squamous cell carcinoma (rSCC) antigen-1, which inhibits a serine proteinase, chymotrypsin. rSCC antigen-1 inhibited the cysteine proteinases, papain, papaya proteinase IV and cathepsin L. Interestingly, although rSCC antigen-1 formed sodium dodecyl sulfate (SDS)- and heat-stable complexes with chymotrypsin, rSCC antigen-1 gave the 40 kDa fragment and small molecular mass peptide by incubation with papain without forming an SDS- and heat-stable complex. The cleavage was observed between the Gly353-Ser354 bond, indicating that rSCC antigen-1 interacts with cysteine proteinases not at the predicted reactive site P1-P1' portion (Ser354-Ser355), but at the Gly353-Ser354 of the P2-P1 portion. These findings promote understanding of the "suicide inhibition" mechanism of SCC antigen-1 against cysteine proteinases. PMID:9194607

  16. Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases

    OpenAIRE

    Pol, Ewa; Bjo?rk, Ingemar

    2001-01-01

    Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affini...

  17. Immunoprotective effect of cysteine proteinase fractions from two Haemonchus contortus strains adapted to sheep and goats.

    Science.gov (United States)

    Molina, J M; Martín, S; Hernández, Y I; González, J F; Ferrer, O; Ruiz, A

    2012-08-13

    A preliminary analysis of the significance of genetic diversity in cysteine proteinase genes has been performed simultaneously in sheep and goats, with regard to the immunological control using these enzymes against haemonchosis. For this purpose, we have studied the cross-immunoprotective effect of cysteine protease-enriched protein fractions (CPFs) in adult worms of two Haemonchus contortus strains from North America and Spain that are adapted to sheep and goats, respectively. Previous genetic analysis of cysteine proteinase genes in both strains has shown that some of loci are polymorphic and these differences are translated into changes in the amino acid sequences. However, our results show that CPFs from H. contortus adult worms have a protective effect against the parasite in both sheep and goats. These results are similar regardless of whether they were obtained from sheep or goat-adapted H. contortus strains, which could be very important in case H. contortus CPFs were commercially used in different countries, as vaccines to prevent the negative effects of this parasite. Interestingly, this experimental inoculation of both species with a heterologous strain of H. contortus contributes to the idea shown in previous studies about how difficult is the interpretation and the comparison of vaccination where strains not adapted to a specific host are used. Therefore, the challenger of using heterologous strains could provide similar results to those observed in immunised animals. This study suggests the possibility of exploring the mechanisms involved in natural protection against non-adapted strains, in order to develop strategies to control haemonchosis. PMID:22487211

  18. Role of cysteine proteinase inhibitors in preference of Japanese beetles (Popillia japonica) for soybean (Glycine max) leaves of different ages and grown under elevated CO2.

    Science.gov (United States)

    Zavala, Jorge A; Casteel, Clare L; Nabity, Paul D; Berenbaum, May R; DeLucia, Evan H

    2009-08-01

    Elevated levels of CO(2), equivalent to those projected to occur under global climate change scenarios, increase the susceptibility of soybean foliage to herbivores by down-regulating the expression of genes related to the defense hormones jasmonic acid and ethylene; these in turn decrease the gene expression and activity of cysteine proteinase inhibitors (CystPIs), the principal antiherbivore defenses in foliage. To examine the effects of elevated CO(2) on the preference of Japanese beetle (JB; Popillia japonica) for leaves of different ages within the plant, soybeans were grown at the SoyFACE facility at the University of Illinois at Urbana-Champaign. When given a choice, JB consistently inflicted greater levels of damage on older leaves than on younger leaves, and there was a trend for a greater preference for young leaves grown under elevated CO(2) compared to those grown under ambient CO(2). More heavily damaged older leaves and those grown under elevated CO(2) had reduced CystPI activity, and JB that consumed leaves with lower CystPI activity had correspondingly greater gut proteinase activity. Younger leaves with higher CystPI activity and photosynthetic rates may contribute disproportionately to plant fitness and are more protected against herbivore attack than older foliage. Cysteine proteinase inhibitors are potent defenses against JB, and the effectiveness of this defense is modulated by growth under elevated CO(2) as well as leaf position. PMID:19418071

  19. Inhibition of cysteine proteinases by Carica papaya cystatin produced in Escherichia coli.

    Science.gov (United States)

    Song, I; Taylor, M; Baker, K; Bateman, R C

    1995-09-11

    A papaya cystatin (Cst)-encoding cDNA clone was isolated from a papaya leaf cDNA library and the active protein produced in Escherichia coli. The amino-acid sequence reveals a protein of 11,262 Da with over 40% identity to other published plant Cst. Unique features of the papaya Cst include a single Cys residue, variation in the papain-binding region, and the first reported inhibition of papaya proteinase IV by a Cst. PMID:7557432

  20. Identification of a lymphocyte-produced cysteine proteinase based on its binding to. cap alpha. -macroglobulin (. cap alpha. M)

    Energy Technology Data Exchange (ETDEWEB)

    Schlesinger, C.; Hagen, K.; Borth, W.; Teodorescu, M.

    1986-03-01

    Proteinases play a role in collagen vascular diseases. However, the role of lymphocytes in the production of these proteinases is poorly understood. Rabbit lymph node cells (LNC) were cultured with /sup 35/S-methionine or /sup 14/C-leucine. When culture supernatants were analyzed by radioimmunoelectrophoresis using an anti-rabbit ..cap alpha..M antiserum, radio-active ..cap alpha..M-precipitin arcs developed. LNC biosynthetically labeled in serum-free medium generated supernatants that, upon incubation with normal rabbit serum, yielded radio-active ..cap alpha..M precipitin arcs. Purified B and T cells as well as human T and B cell lines were labeled separately under identical conditions. Analysis revealed the presence of radiolabeled ..cap alpha..M molecules. T cells usually generated less intense radioactive ..cap alpha..M precipitin arcs than B cells. Thoracic duct lymphocytes also produced radioactive ..cap alpha..M precipitin arcs. They treated radiolabeled supernatants with a variety of proteinase inhibitors before reacting them with rabbit serum. Only inhibitors specific for cysteine or thiol proteinases were effective in abolishing radioactivity associated with ..cap alpha..M. It appears that lymphocytes actively produce a cysteine or thiol proteinase which, theoretically, is capable of degrading collagen.

  1. Primary structure of CC-III, the glycosylated cysteine proteinase from the latex of Carica candamarcensis Hook.

    Science.gov (United States)

    Jaziri, M; Kleinschmidt, T; Walraevens, V; Schnek, A G; Looze, Y

    1994-06-01

    The amino acid sequence of the cysteine proteinase CC-III from the latex of the subtropical species Carica candamarcensis Hook has been determined with the exception of seven residues (pos. 180-186). It was deduced from the sequence analysis of the whole chain and peptides obtained by tryptic, chymotryptic, peptic and thermolysinolytic hydrolysis. CC-III consists of 214 amino acid residues. Out of a total of eight cysteine residues, six are located at positions involved in the formation of the three disulfide bridges stabilizing the structure of papain related enzymes. CC-III from Carica candamarcensis is a glycoprotein with the carbohydrate moiety bound to asparagine at position 44. Out of 210 residues compared with the sequences of the four cysteine proteinases of Carica papaya L., CC-III shares 125 identical ones (59.5%) with papain, 142 (67.6%) with papaya proteinase IV, 146 (69.5%) with papaya proteinase III and 156 (74.3%) with chymopapain. All amino acid residues constituting the active site and subsite S2 in chymopapain are conserved in CC-III with the exception of the substitution Leu157--> Val in the latter. This fact as well as the highest degree of identity between CC-III and chymopapain point to a similar specificity of both enzymes and thus CC-III might be a suitable substitute for chymopapain as a chemonucleolytic agent. PMID:7980869

  2. Development of an in vitro cleavage assay system to examine vaccinia virus I7L cysteine proteinase activity

    Directory of Open Access Journals (Sweden)

    Hruby Dennis E

    2005-08-01

    Full Text Available Abstract Through the use of transient expression assays and directed genetics, the vaccinia virus (VV I7L gene product has been implicated as the major maturational proteinase required for viral core protein cleavage to occur during virion assembly. To confirm this hypothesis and to enable a biochemical examination of the I7L cysteine proteinase, an in vitro cleavage assay was developed. Using extracts of VV infected cells as the source of enzyme, reaction conditions were developed which allowed accurate and efficient cleavage of exogenously added core protein precursors (P4a, P4b and P25K. The cleavage reaction proceeded in a time-dependent manner and was optimal when incubated at 25°C. I7L-mediated cleavage was not affected by selected inhibitors of metalloproteinases, aspartic acid proteinases or serine proteinases (EDTA, pepstatin, and PMSF, respectively, but was sensitive to several general cysteine proteinase inhibitors (E-64, EST, Iodoacetic acid, and NEM as well as the I7L active site inhibitor TTP-6171 [C. Byrd et al., J. Virol. 78:12147–12156 (2004]. Finally, in antibody pull down experiments, it could be demonstrated that monospecific ?I7L serum depleted the enzyme activity whereas control sera including ?G1L, directed against the VV metalloproteinase, did not. Taken together, these data provide biochemical evidence that I7L is a cysteine proteinase which is directly involved in VV core protein cleavage. Furthermore, establishment of this I7L-mediated in vitro cleavage assay should enable future studies into the enzymology and co-factor requirements of the proteolysis reaction, and facilitate antiviral drug development against this essential target.

  3. Cysteine proteinases of Trypanosoma cruzi: from digestive enzymes to programmed cell death

    Scientific Electronic Library Online (English)

    Gregor, Kosec; Vanina, Alvarez; Juan J., Cazzulo.

    2006-12-01

    Full Text Available SciELO Argentina | Language: English Abstract in english Trypanosoma cruzi, the parasite causing Chagas disease, contains a number of proteolytic enzymes. The recent completion of the genome sequence of the T. cruzi CL Brener clone suggests the presence of 70 cysteine peptidases, 40 serine peptidases (none of them from the chymotrypsin family), about 250 [...] metallopeptidases (most leishmanolysin homologues), 25 threonine peptidases, and only two aspartyl peptidases, none of them from the pepsin family. The cysteine peptidases belong to 7 families of Clan CA, 3 families of Clan CD, and one each of Clans CE and CF. In Clan CA, the C1 family is represented by cruzipains 1 and 2, biochemically well characterized, as well as cathepsin B and two other cathepsins. There are a number of homologues to calpains (family C2), probably non-functional, lacking the Ca-binding domain. Family C54 includes the Atg4 proteinases (autophagins), which seem to be involved in the autophagic process. Clan CD includes family C14, the metacaspases. We have expressed the metacaspases TcMCA3 and TcMCA5, and obtained indirect evidence of their participation in programmed cell death induced by fresh human serum in the parasite. More experiments are required to better define their role in apoptosis.

  4. Cysteine proteinases of Trypanosoma cruzi: from digestive enzymes to programmed cell death

    Directory of Open Access Journals (Sweden)

    Gregor Kosec

    2006-12-01

    Full Text Available Trypanosoma cruzi, the parasite causing Chagas disease, contains a number of proteolytic enzymes. The recent completion of the genome sequence of the T. cruzi CL Brener clone suggests the presence of 70 cysteine peptidases, 40 serine peptidases (none of them from the chymotrypsin family, about 250 metallopeptidases (most leishmanolysin homologues, 25 threonine peptidases, and only two aspartyl peptidases, none of them from the pepsin family. The cysteine peptidases belong to 7 families of Clan CA, 3 families of Clan CD, and one each of Clans CE and CF. In Clan CA, the C1 family is represented by cruzipains 1 and 2, biochemically well characterized, as well as cathepsin B and two other cathepsins. There are a number of homologues to calpains (family C2, probably non-functional, lacking the Ca-binding domain. Family C54 includes the Atg4 proteinases (autophagins, which seem to be involved in the autophagic process. Clan CD includes family C14, the metacaspases. We have expressed the metacaspases TcMCA3 and TcMCA5, and obtained indirect evidence of their participation in programmed cell death induced by fresh human serum in the parasite. More experiments are required to better define their role in apoptosis.

  5. Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

    OpenAIRE

    Cb, Toaldo; Steindel M; Ma, Sousa; Cc, Tavares

    2001-01-01

    The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarit...

  6. Putrescine-Dependent Re-Localization of TvCP39, a Cysteine Proteinase Involved in Trichomonas vaginalis Cytotoxicity

    OpenAIRE

    Carvajal-gamez, Bertha Isabel; Quintas-granados, Laura Itzel; Arroyo, Rossana; Va?zquez-carrillo, Laura Isabel; Ramo?n-luing, Lucero Los Angeles; Carrillo-tapia, Eduardo; Alvarez-sa?nchez, Mari?a Elizbeth

    2014-01-01

    Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP) involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB) (an inhibitor of putrescine biosynthesis), diminished the amount and proteolytic activity...

  7. Proregion of Acanthoscelides obtectus cysteine proteinase: a novel peptide with enhanced selectivity toward endogenous enzymes.

    Science.gov (United States)

    Silva, F B; Monteiro, A C S; Del Sarto, R P; Marra, B M; Dias, S C; Figueira, E L Z; Oliveira, G R; Rocha, T L; Souza, D S L; da Silva, M C M; Franco, O L; Grossi-de-Sa, M F

    2007-06-01

    Acanthoscelides obtectus is a devastating storage insect pest capable of causing severe bean crop losses. In order to maintain their own development, insect pest larvae feed continuously, synthesizing efficient digestive enzymes. Among them, cysteine proteinases (CPs) are commonly produced as inactive precursors (procysteines), requiring a cleavage of the peptide proregion to become active. The proregion fits tightly into the active site of procysteines, efficiently preventing their activity. In this report, a CP cDNA (cpao) was isolated from A. obtectus midgut larvae. In silico studies indicated that the complete CP sequence contains a hydrophobic signal peptide, a prodomain and a conserved catalytic region. Moreover, the encoding cDNA contains 963bp translating into a 321 residue protein, CPAo, which was expressed in E. coli, fused with thioredoxin. Enzymatic assays using the recombinant protein revealed that the enzyme was catalytically active, being able to cleave the synthetic substrate Z-Phe-Arg-7-AMC. Additionally, this report also focuses the cpao propeptide (PCPAo) subcloning and expression. The expressed propeptide efficiently inhibited CPAo, as well as digestive CP of other bean bruchids. Little or no activity was found against proteolytic enzymes of two other coleopterans: Rhyzopertha dominica and Anthonomus grandis. The data reported here indicate the possibility of endogenous propeptides as a novel strategy on bruchids control, which could be applicable to bean improvement programs. PMID:17485144

  8. Action of plant proteinase inhibitors on enzymes of physiopathological importance

    Scientific Electronic Library Online (English)

    Maria Luiza V., Oliva; Misako U., Sampaio.

    2009-09-01

    Full Text Available SciELO Brazil | Language: English Abstract in portuguese Obtidas de sementes leguminosas, várias proteínas inibem proteinases de origem animal, incluindo humanas, e podem ser consideradas para o desenvolvimento de compostos com atividade biológica. Inibidores da família Bowman-Birk e da família Kunitz vegetal tem sido caracterizados em relação a especific [...] idade para proteinase, estrutura primária e sitio reativo. O nosso grupo majoritariamente vem estudando o gênero Bauhinia, principalmente as espécies bauhinioides, rufa, ungulatae variegata. Em algumas espécies, mais de um inibidor com propriedades diferentes foi caracterizado. Embora tais proteínas apresentem alta similaridade estrutural, diferem quanto à inibição de proteinases, e foram exploradas em estudos utilizando diversos modelos biológicos. Abstract in english Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary [...] structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.

  9. Action of plant proteinase inhibitors on enzymes of physiopathological importance

    Directory of Open Access Journals (Sweden)

    Maria Luiza V. Oliva

    2009-09-01

    Full Text Available Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.Obtidas de sementes leguminosas, várias proteínas inibem proteinases de origem animal, incluindo humanas, e podem ser consideradas para o desenvolvimento de compostos com atividade biológica. Inibidores da família Bowman-Birk e da família Kunitz vegetal tem sido caracterizados em relação a especificidade para proteinase, estrutura primária e sitio reativo. O nosso grupo majoritariamente vem estudando o gênero Bauhinia, principalmente as espécies bauhinioides, rufa, ungulatae variegata. Em algumas espécies, mais de um inibidor com propriedades diferentes foi caracterizado. Embora tais proteínas apresentem alta similaridade estrutural, diferem quanto à inibição de proteinases, e foram exploradas em estudos utilizando diversos modelos biológicos.

  10. Serine and threonine beta-lactones: a new class of hepatitis A virus 3C cysteine proteinase inhibitors.

    Science.gov (United States)

    Lall, Manjinder S; Ramtohul, Yeeman K; James, Michael N G; Vederas, John C

    2002-03-01

    Hepatitis A virus (HAV) 3C enzyme is a cysteine proteinase essential for viral replication and infectivity and represents a target for the development of antiviral drugs. A number of serine and threonine beta-lactones were synthesized and tested against HAV 3C proteinase. The D-N-Cbz-serine beta-lactone 5a displays competitive reversible inhibition with a K(i) value of 1.50 x 10(-6) M. Its enantiomer, L-N-Cbz-serine beta-lactone 5b is an irreversible inactivator with k(inact) = 0.70 min(-1), K(Iota) = 1.84 x 10(-4) M and k(inact)/K(Iota) = 3800 M(-1) min(-1). Mass spectrometry and HMQC NMR studies using (13)C-labeled 5b show that inactivation of the enzyme occurs by nucleophilic attack of the cysteine thiol (Cys-172) at the beta-position of the oxetanone ring. Although the N-Cbz-serine beta-lactones 5a and 5b display potent inhibition, other related analogues with an N-Cbz side chain, such as the five-membered ring homoserine gamma-lactones 14a and 14b, the four-membered ring beta-lactam 33, 2-methylene oxetane 34, cyclobutanone 36, and 3-azetidinone 39, fail to give significant inhibition of HAV 3C proteinase, thus demonstrating the importance of the beta-lactone ring for binding. PMID:11871884

  11. Development of an in vitro cleavage assay system to examine vaccinia virus I7L cysteine proteinase activity

    OpenAIRE

    Hruby Dennis E; Byrd Chelsea M

    2005-01-01

    Abstract Through the use of transient expression assays and directed genetics, the vaccinia virus (VV) I7L gene product has been implicated as the major maturational proteinase required for viral core protein cleavage to occur during virion assembly. To confirm this hypothesis and to enable a biochemical examination of the I7L cysteine proteinase, an in vitro cleavage assay was developed. Using extracts of VV infected cells as the source of enzyme, reaction conditions were developed which all...

  12. Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

    Directory of Open Access Journals (Sweden)

    CB Toaldo

    2001-01-01

    Full Text Available The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70 and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

  13. Isolation and primary structure of the CCI papain-like cysteine proteinases from the latex of Carica candamarcensis hook.

    Science.gov (United States)

    Walraevens, V; Vandermeers-Piret, M C; Vandermeers, A; Gourlet, P; Robberecht, P

    1999-04-01

    The dried latex of the mountain papaya, Carica candamarcensis, was chromatographed on CM-Sephadex C50, giving rise to three peaks (CCI, CCII and CCIII) with amidase activity on N-alpha-benzoyl-DL-arginine-4-nitroanilide. The less basic, most active, peak, CCI, was separated into two components, CCIa and CCIb, by reverse-phase HPLC under denaturing conditions. The primary structures of CCIa and CCIb are presented. They were deduced from sequence analysis of the whole proteins and peptides resulting from enzymatic digestions. Both proteinases are made of 213 amino acid residues, CCIb sharing 88-89% similarity with the three subvariants (G90/R212, E90/R212, E90/K212) of CCIa. 139-140 amino acid residues (65.8%) of CCIa and 141 residues (66.5%) of CCIb are common to papain. The seven cysteine residues are aligned with those of papain and the catalytic triad (Cys25, His159, Asn175) of all cysteine peptidases of the papain family is conserved. The similarity with the other cysteine proteases from Carica papaya is discussed. PMID:10355634

  14. Effective pretreatment by cysteine proteinase inhibitor for improved analysis of protein components of trematodes on SDS-PAGE.

    Science.gov (United States)

    Itoh, M; Sato, S; Moriyama, A; Sasaki, M

    1990-09-01

    Since Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinesis, when their extracts were treated with E-64. PMID:2230026

  15. Antigen genes for molecular epidemiology of leishmaniasis: polymorphism of cysteine proteinase B and surface metalloprotease glycoprotein 63 in the Leishmania donovani complex

    OpenAIRE

    Quispe Tintaya, K. W.; Ying, X.; Dedet, J. P.; Rijal, S.; Bolle, X.; Dujardin, J. C.

    2004-01-01

    BACKGROUND: Efficient monitoring of endemic and resurgent visceral leishmaniasis (VL) requires discriminatory molecular tools that allow direct characterization of etiological agents (i.e., the Leishmania donovani complex) in host tissues. This characterization is possible through restriction fragment-length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified sequences (PCR-RFLP). METHODS: We present 2 new PCR-RFLP assays that target the gene locus of cysteine proteinase...

  16. Modulation of Major Histocompatibility Complex Protein Expression by Human Gamma Interferon Mediated by Cysteine Proteinase-Adhesin Polyproteins of Porphyromonas gingivalis

    OpenAIRE

    Yun, Peter L. W.; Decarlo, Arthur A.; Hunter, Neil

    1999-01-01

    Cysteine proteinases have been emphasized in the virulence of Porphyromonas gingivalis in chronic periodontitis. These hydrolases may promote the degradation of extracellular matrix proteins and disrupt components of the immune system. In this study it was shown that purified Arg-gingipain and Lys-gingipain inhibited expression of class II major histocompatibility complex (MHC) proteins in response to the stimulation of endothelial cells with human gamma interferon (IFN-?). Treatment with th...

  17. Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes.

    Science.gov (United States)

    Varghese, Anju; Raina, O K; Nagar, Gaurav; Garg, Rajat; Banerjee, P S; Maharana, B R; Kollannur, Justin D

    2012-02-10

    Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ? 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ? 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes. PMID:22055612

  18. Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus

    International Nuclear Information System (INIS)

    The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. DQ066227). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and Mr of 11kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with Ki value of 0.1 and 0.6nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extrt body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis

  19. Characterization of a mixture of lobster digestive cysteine proteinases by ionspray mass spectrometry and tryptic mapping with LC--MS and LC--MS--MS

    Science.gov (United States)

    Thibault, P.; Pleasance, S.; Laycock, M. V.; Mackay, R. M.; Boyd, R. K.

    1991-12-01

    An inseparable mixture of two cysteine proteinases, isolated from the digestive tract of the American lobster, was investigated by ionspray mass spectrometry (ISP-MS), using a combination of infusion of intact proteins with on-line liquid chromatography--mass spectrometry (LC--MS) and LC--MS--MS analyses of tryptic digests. These data were interpreted by comparisons with predictions from results of molecular cloning of cysteine-proteinase-encoding messenger RNA sequences previously isolated from the lobster hepatopancreas. Investigations of the numbers of free thiol groups and of disulfide bonds were made by measuring the molecular weights of the alkylated proteins with and without prior reduction of disulfide bonds, and comparison with the corresponding data for the native proteins. Identification of tyrptic fragment peptides containing cysteine residues was facilitated by comparing LC--MS analyses of tryptic digests of denatured and of denatured and alkylated proteins, since such tryptic peptides are subject to shifts in both mass and retention time upon reduction and alkylation. Confirmation of amino acid sequences was obtained from fragment ion spectra of each tryptic peptide (alkylated or not) as it eluted from the column. Acquisition of such on-line LC--MS data was possible through use of the entire effluent from a standard 1 mm high performance liquid chromatography (HPLC) column by an IonsSpray® LC--MS interface (pneumatically assisted electrospray).

  20. A barley cysteine-protease inhibitor reduces teh performance of two aphid species in artificial diets and transgenic arabidopsis plants

    OpenAIRE

    Carrillo Gil, Laura; Martinez Mun?oz, Manuel; Alvarez Alfageme, Fernando; Castan?era, Pedro; Smagghe, Guy; Diaz Rodriguez, Isabel; Ortego, Felix

    2011-01-01

    Cystatins from plants have been implicated in plant defense towards insects, based on their role as inhibitors of heterologous cysteine-proteinases. We have previously characterized thirteen genes encoding cystatins (HvCPI-1 to HvCPI-13) from barley (Hordeum vulgare), but only HvCPI-1 C68 ? G, a variant generated by direct-mutagenesis, has been tested against insects. The aim of this study was to analyze the effects of the whole gene family members of barley cystatins against two aphids, My...

  1. "Purification and evaluation of somatic, excretory-secretory and Cysteine proteinase antigens of Fasciola Hepatica using IgG-ELISA in diagnosing Fascioliasis "

    Directory of Open Access Journals (Sweden)

    "Rokni MB

    2001-08-01

    Full Text Available Fasciolosis, or liver fluke disease, caused by parasites of the genus Fasciola is emerging as an important disease in man and animals, in the world and Iran, particularly in nortern parts. The economical losses in domestic animals are considerable. In the recent decade there were two major outbreaks of human fasciolosis in the Caspian region, northern part of Iran with 7000-10000 infected cases. Sicne it is impossible to diagnose fasciolosis in acute phase using coprological methods and even in chronic phases its sensitivity is low, evaluating and establishing a reliable and cost-effetive test is indispensable and notewortly.In the present survey, we produced and examined the sensitivity and specificity of liver fluke homogenate (LFH , excretory-secetory (ES and cysteine proteinase (CP antigens of F. hepatica using IgG-ELISA test. A 25-27 kilo Dalton coomassie blue-stained band was observed and using of specific inhibitors indicated that this antigen belongs to the class of cysteine proteinase. The sensitivity of LFH, ES and CP antigen in IgG-ELISa was 100% for each, while their specificity was 97.8%, 98.8% and 98.8% respectively. There was a significant difference in mean OD values between cases of proven fasciolosis and other true negative cases, including healthy control individuals and patients with other parasitic diseases.This present report is the first to demonstrate the purification and evaluation of F. hepatica cysteine proteinase antigen by IgG-ELISA test for the diagnosis of fasciolosis in Iran. In conclusion, the IgG-ELISa using ES and CP show high sensitivity and specificity and would be a valuable tool to diagnose human fasciolosis in Iran, particularly in endemic areas.

  2. Changes in tissue and serum activity of cathepsin B-like cysteine proteinase during colorectal carcinogenesis by 1,2-dimethylhydrazine in mice.

    Science.gov (United States)

    Kolár, Z; Dufek, V; Krepela, E; Vicar, J; Král, V

    1985-01-01

    The activity of cathepsin B-like cysteine proteinase in the mice colon was studied during carcinogenesis induced by 1,2-dimethylhydrazine dihydrochloride (DMH). Before starting the treatment with DMH, the activity of the observed enzyme was very low in the colorectal area but the activity in the serum was rather high. In the course of carcinogenesis, the local activity markedly increased in the stroma equally as in the cells of developing tumors while the serum activity slightly decreased. Following the total ovariectomy, no significant changes in the local activity or in the serum activity were found. Possible causes of these variations are discussed. PMID:4069291

  3. Global proteome changes in larvae of Callosobruchus maculatus Coleoptera:Chrysomelidae:Bruchinae) following ingestion of a cysteine proteinase inhibitor.

    Science.gov (United States)

    Nogueira, Fábio C S; Silva, Carlos P; Alexandre, Daniel; Samuels, Richard I; Soares, Emanoella L; Aragão, Francisco J L; Palmisano, Giuseppe; Domont, Gilberto B; Roepstorff, Peter; Campos, Francisco A P

    2012-08-01

    The seed-feeding beetle Callosobruchus maculatus is an important cowpea pest (Vigna unguiculata) as well as an interesting model to study insect digestive physiology. The larvae of C. maculatus rely on cysteine and aspartic peptidases to digest proteins in their diet. In this work, the global proteomic changes induced in the intestinal tract of larval C. maculatus challenged by the ingestion of cystatin, a cysteine peptidase inhibitor, was investigated by a nanoLC-MS/MS approach. The ingestion of cystatin caused a delay in the development of the larvae, but the mortality was not high, indicating that C. maculatus is able to adapt to this inhibitor. This proteomic strategy resulted in the identification of 752 and 550 protein groups in the midgut epithelia and midgut contents, respectively, and quantitative analyses allowed us to establish relative differences of the identified proteins. Ingestion of cystatin led to significant changes in the proteome of both the midgut epithelia and midgut contents. We have observed that proteins related to plant cell wall degradation, particularly the key glycoside hydrolases of the families GH5 (endo-?-1,4-mannanase) and GH 28 (polygalacturonase) were overexpressed. Conversely, ?-amylases were downexpressed, indicating that an increase in hemicelluloses digestion helps the larvae to cope with the challenge of cystatin ingestion. Furthermore, a number of proteins associated with transcription/translation and antistress reactions were among the cystatin-responsive proteins, implying that a substantial rearrangement in the proteome occurred in C. maculatus exposed to the inhibitor. PMID:22833537

  4. Global proteome changes in larvae of Callosobruchus maculatus Coleoptera:Chrysomelidae:Bruchinae) following ingestion of a cysteine proteinase inhibitor

    DEFF Research Database (Denmark)

    Nogueira, Fábio C S; Silva, Carlos P

    2012-01-01

    The seed-feeding beetle Callosobruchus maculatus is an important cowpea pest (Vigna unguiculata) as well as an interesting model to study insect digestive physiology. The larvae of C. maculatus rely on cysteine and aspartic peptidases to digest proteins in their diet. In this work, the global proteomic changes induced in the intestinal tract of larval C. maculatus challenged by the ingestion of cystatin, a cysteine peptidase inhibitor, was investigated by a nanoLC-MS/MS approach. The ingestion of cystatin caused a delay in the development of the larvae, but the mortality was not high, indicating that C. maculatus is able to adapt to this inhibitor. This proteomic strategy resulted in the identification of 752 and 550 protein groups in the midgut epithelia and midgut contents, respectively, and quantitative analyses allowed us to establish relative differences of the identified proteins. Ingestion of cystatin led to significant changes in the proteome of both the midgut epithelia and midgut contents. We have observed that proteins related to plant cell wall degradation, particularly the key glycoside hydrolases of the families GH5 (endo-?-1,4-mannanase) and GH 28 (polygalacturonase) were overexpressed. Conversely, ?-amylases were downexpressed, indicating that an increase in hemicelluloses digestion helps the larvae to cope with the challenge of cystatin ingestion. Furthermore, a number of proteins associated with transcription/translation and antistress reactions were among the cystatin-responsive proteins, implying that a substantial rearrangement in the proteome occurred in C. maculatus exposed to the inhibitor.

  5. Assessment of nematode resistance in wheat transgenic plants expressing potato proteinase inhibitor (PIN2) gene.

    Science.gov (United States)

    Vishnudasan, Dalia; Tripathi, M N; Rao, Uma; Khurana, Paramjit

    2005-10-01

    Serine proteinase inhibitors (IP's) are proteins found naturally in a wide range of plants with a significant role in the natural defense system of plants against herbivores. The question addressed in the present study involves assessing the ability of the serine proteinase inhibitor in combating nematode infestation. The present study involves engineering a plant serine proteinase inhibitor (pin2) gene into T. durum PDW215 by Agrobacterium-mediated transformation to combat cereal cyst nematode (Heterodera avenae) infestation. Putative T(0) transformants were screened and positive segregating lines analysed further for the study of the stable integration, expression and segregation of the genes. PCR, Southern analysis along with bar gene expression studies corroborate the stable integration pattern of the respective genes. The transformation efficiency is 3%, while the frequency of escapes was 35.71%. chi(2) analysis reveals the stable integration and segregation of the genes in both the T(1) and T(2) progeny lines. The PIN2 systemic expression confers satisfactory nematode resistance. The correlation analysis suggests that at p < 0.05 level of significance the relative proteinase inhibitor (PI) values show a direct positive correlation vis-à-vis plant height, plant seed weight and also the seed number. PMID:16245157

  6. Circular dichroism of cysteine proteinases from papaya latex. Evidence of differences in the folding of their polypeptide chains.

    Science.gov (United States)

    Solís-Mendiola, S; Arroyo-Reyna, A; Hernández-Arana, A

    1992-02-01

    Two forms of proteinase omega were isolated from a commercial preparation of chymopapain (EC 3.4.22.6) by means of cation-exchange liquid chromatography. Their circular dichroism (CD) spectra in the 182-320 nm region indicated that the two forms possess closely related structures. For comparison, we also recorded the CD spectra of chromatographically purified samples of papain (EC 3.422.2) and the most abundant form of chymopapain. According to the qualitative criteria proposed by Manavalan and Johnson (1983) Nature 305, 831-832), the spectral characteristics of papain correctly indicate that this protein belongs to the alpha + beta class. Proteinase omega is also placed in the alpha + beta category, while chymopapain seems to be an alpha/beta protein. Quantitative estimation of secondary structures yielded contents of helices and parallel beta-sheet that were higher in the case of chymopapain. Thus, the results of this work suggest that there are some differences in the folding pattern of chymopapain with respect to the other two proteinases. This proposal seems unexpected when the high amino acid sequence identity among these enzymes is considered. PMID:1737051

  7. Cysteine protease enhances plant-mediated bollworm RNA interference

    OpenAIRE

    Mao, Ying-bo; Xue, Xue-yi; Tao, Xiao-yuan; Yang, Chang-qing; Wang, Ling-jian; Chen, Xiao-ya

    2013-01-01

    Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in a...

  8. A computational analysis of SARS cysteine proteinase-octapeptide substrate interaction: implication for structure and active site binding mechanism

    OpenAIRE

    Watts Aaron; Sompornpisut Pornthep; Kyu Khin; Ratanakhanokchai Khanok; Phakthanakanok Krongsakda; Pinitglang Surapong

    2009-01-01

    Abstract Background SARS coronavirus main proteinase (SARS CoVMpro) is an important enzyme for the replication of Severe Acute Respiratory Syndrome virus. The active site region of SARS CoVMpro is divided into 8 subsites. Understanding the binding mode of SARS CoVMpro with a specific substrate is useful and contributes to structural-based drug design. The purpose of this research is to investigate the binding mode between the SARS CoVMpro and two octapeptides, especially in the region of the ...

  9. Cysteine–based redox regulation and signaling in plants

    OpenAIRE

    Couturier, Je?re?my; Chibani, Kamel; Jacquot, Jean-pierre; Rouhier, Nicolas

    2013-01-01

    Living organisms are subjected to oxidative stress conditions which are characterized by the production of reactive oxygen, nitrogen, and sulfur species. In plants as in other organisms, many of these compounds have a dual function as they damage different types of macromolecules but they also likely fulfil an important role as secondary messengers. Owing to the reactivity of their thiol groups, some protein cysteine residues are particularly prone to oxidation by these molecules. In the past...

  10. Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway

    OpenAIRE

    Weits, Daan A.; Giuntoli, Beatrice; Kosmacz, Monika; Parlanti, Sandro; Hubberten, Hans-michael; Riegler, Heike; Hoefgen, Rainer; Perata, Pierdomenico; Dongen, Joost T.; Licausi, Francesco

    2014-01-01

    In plant and animal cells, amino-terminal cysteine oxidation controls selective proteolysis via an oxygen-dependent branch of the N-end rule pathway. It remains unknown how the N-terminal cysteine is specifically oxidized. Here we identify plant cysteine oxidase (PCO) enzymes that oxidize the penultimate cysteine of ERF-VII transcription factors by using oxygen as a co-substrate, thereby controlling the lifetime of these proteins. Consequently, ERF-VII proteins are stabilized under hypoxia an...

  11. Antitumor Effects In Vitro and In Vivo and Mechanisms of Protection against Melanoma B16F10-Nex2 Cells By Fastuosain, a Cysteine Proteinase from Bromelia fastuosa1

    OpenAIRE

    Guimara?es-ferreira, Carla A.; Rodrigues, Elaine G.; Mortara, Renato A.; Cabral, Hamilton; Serrano, Fabiana A.; Ribeiro-dos-santos, Ricardo; Travassos, Luiz R.

    2007-01-01

    In the present work, the antitumor effect of fastuosain, a cysteine proteinase from Bromelia fastuosa, was investigated. In the intravenous model of lung colonization in C57Bl/6 mice, fastuosain and bromelain injected intraperitoneally were protective, and very few nodules of B16F10-Nex2 melanoma cells were detected. Tumor cells treated with fastuosain showed reduced expression of CD44 and decreased invasion through Matrigel, lost their cytoplasmic extensions and substrate adherence, and beca...

  12. In vitro phosphorylation of plant plasma membrane proteins in response to the proteinase inhibitor inducing factor

    OpenAIRE

    Farmer, Edward E.; Pearce, Gregory; Ryan, Clarence A.

    1989-01-01

    A polygalacturonide purified from a tomato leaf pectic polysaccharide that induces the systemic synthesis of proteinase inhibitors in tomato plants enhances the phosphorylation of specific proteins in plasma membrane fractions isolated from tomato and potato leaves. In tomato plasma membranes, two proteins of 34 and 29 kDa show enhanced phosphorylation in response to the polyuronide. In potato plasma membranes, only a protein of 34 kDa exhibited enhanced phosphorylation due to the polyuronide...

  13. Plant defensins: Defense, development and application

    Science.gov (United States)

    Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity, an...

  14. Antitumor Effects In Vitro and In Vivo and Mechanisms of Protection against Melanoma B16F10-Nex2 Cells By Fastuosain, a Cysteine Proteinase from Bromelia fastuosa

    Directory of Open Access Journals (Sweden)

    Carla A. Guimarães-Ferreira

    2007-09-01

    Full Text Available In the present work, the antitumor effect of fastuosain, a cysteine proteinase from Bromelia fastuosa, was investigated. In the intravenous model of lung colonization in C57BI/6 mice, fastuosain and bromelain injected intraperitoneally were protective, very few nodules of B16F10-Nex2 melanoma cells were detected. Tumor cells treated with fastuosain showed reduced expression of CD44 and decreased invasion through Matrigel, lost their cytoplasmic extensions and substrate adherence, became round and detached, forming strongly bound cell clusters in suspension. Peritoneal cells recruited and activated by fastuosain treatment (mainly monocytic cells and lymphocytes migrated to the lung, where pulmonary melanoma metastases grew. Adoptive transference of peritoneal cells recruited by fastuosain had no protective effect against lung metastases in recipient mice. Treatment of green fluorescent protein -chimeric animals with fastuosain did not change the number of cells that migrated to the lung, compared to PBSinjected control mice, but the number of positive major histocompatibility complex class II cells increased with fastuosain treatment. Murine antibodies against fastuosain, bromelain, cathepsins B and L crossreacted in ELISA and recognized surface and cytoplasmic components expressed on B16F10-Nex2 cells. Anti-fastuosain antibodies were cytotoxic/lytic to B16F10-Nex2 cells. Antitumor effects of fastuosain involve mainly the direct effect of the enzyme and elicitation of protective antibodies.

  15. A computational analysis of SARS cysteine proteinase-octapeptide substrate interaction: implication for structure and active site binding mechanism

    Science.gov (United States)

    Phakthanakanok, Krongsakda; Ratanakhanokchai, Khanok; Kyu, Khin Lay; Sompornpisut, Pornthep; Watts, Aaron; Pinitglang, Surapong

    2009-01-01

    Background SARS coronavirus main proteinase (SARS CoVMpro) is an important enzyme for the replication of Severe Acute Respiratory Syndrome virus. The active site region of SARS CoVMpro is divided into 8 subsites. Understanding the binding mode of SARS CoVMpro with a specific substrate is useful and contributes to structural-based drug design. The purpose of this research is to investigate the binding mode between the SARS CoVMpro and two octapeptides, especially in the region of the S3 subsite, through a molecular docking and molecular dynamics (MD) simulation approach. Results The one turn ?-helix chain (residues 47–54) of the SARS CoVMpro was directly involved in the induced-fit model of the enzyme-substrate complex. The S3 subsite of the enzyme had a negatively charged region due to the presence of Glu47. During MD simulations, Glu47 of the enzyme was shown to play a key role in electrostatic bonding with the P3Lys of the octapeptide. Conclusion MD simulations were carried out on the SARS CoVMpro-octapeptide complex. The hypothesis proposed that Glu47 of SARS CoVMpro is an important residue in the S3 subsite and is involved in binding with P3Lys of the octapeptide. PMID:19208150

  16. Characterization of cysteine proteases in Malian medicinal plants.

    Science.gov (United States)

    Bah, Sékou; Paulsen, Berit S; Diallo, Drissa; Johansen, Harald T

    2006-09-19

    Extracts form 10 different Malian medicinal plants with a traditional use against schistosomiasis were investigated for their possible content of proteolytic activity. The proteolytic activity was studied by measuring the hydrolysis of two synthetic peptide substrates Z-Ala-Ala-Asn-NHMec and Z-Phe-Arg-NHMec. Legumain- and papain-like activities were found in all tested crude extracts except those from Entada africana, with the papain-like activity being the strongest. Cissus quadrangularis, Securidaca longepedunculata and Stylosanthes erecta extracts showed high proteolytic activities towards both substrates. After gel filtration the proteolytic activity towards the substrate Z-Ala-Ala-Asn-NHMec in root extract of Securidaca longepedunculata appeared to have Mr of 30 and 97kDa, while the activity in extracts from Cissus quadrangularis was at 39kDa. Enzymatic activity cleaving the substrate Z-Phe-Arg-NHMec showed apparent Mr of 97 and 26kDa in extracts from roots and leaves of Securidaca longepedunculata, while in Cissus quadrangularis extracts the activity eluted at 39 and 20kDa, with the highest activity in the latter. All Z-Phe-Arg-NHMec activities were inhibited by E-64 but unaffected by PMSF. The legumain activity was unaffected by E-64 and PMSF. The SDS-PAGE analysis exhibited five distinct gelatinolytic bands for Cissus quadrangularis extracts (115, 59, 31, 22 and 20kDa), while two bands (59 and 30kDa) were detected in Securidaca longepedunculata extracts. The inhibition profile of the gelatinolytic bands and that of the hydrolysis of the synthetic substrates indicate the cysteine protease class of the proteolytic activities. Several cysteine protease activities with different molecular weights along with a strong variability of these activities between species as well as between plant parts from the same species were observed. PMID:16621376

  17. Aspartic proteinases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco.

    Science.gov (United States)

    An, Chung-Il; Fukusaki, Ei-ichiro; Kobayashi, Akio

    2002-03-01

    Carnivorous plants acquire significant amounts of nitrogen from insects. The tropical carnivorous plant Nepenthes accumulates acidic fluid containing aspartic proteinase (AP) in its trapping organs (pitchers), suggesting that the plant utilizes insect protein as a nitrogen source. Aspartic proteinases have been purified and characterized from sterile pitcher fluid of several species of Nepenthes; however, there is, as of yet, no information about sequence and expression of Nepenthes AP genes. To identify the pitcher AP, we cloned plant AP homologs from N. alata and examined their expressions. Five AP homologs ( NaAP1-NaAP5) were obtained by reverse transcription-polymerase chain reaction with degenerate primers designed for the conserved sequences of plant APs. Alignment of deduced amino acid sequences with other plant APs demonstrated that NaAP1-NaAP4 contained a plant-specific insert (PSI), a unique sequence of plant AP. However, NaAP5 did not possess the insert, and had a shorter sequence (by >100 amino acids) than the other APs. Northern analysis using a part of the coding region of NaAP1 as a probe showed that bands of approx. 1.8 kb corresponding to the sizes of NaAP1-NaAP4 mRNA were present in roots, stems, leaves, tendrils, and lower part of the pitchers, but a band of approx. 1.3 kb corresponding to the size of NaAP5 mRNA was not observed in any organs. In pitchers, highest expressions of NaAP1-NaAP4 were seen in the lower part of open pitchers containing natural prey, suggesting that the expressions of NaAP1-NaAP4 are coupled with prey capture. Transcripts of NaAP2 and NaAP4 were detected in the digestive glands, where AP secretion may occur. This result suggests that NaAP2 and NaAP4 are the possible APs secreted into the pitcher of N. alata. PMID:11882933

  18. Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis

    OpenAIRE

    Bozhkov, Peter V.; Suarez, Maria F.; Filonova, Lada H.; Daniel, Geoffrey; Zamyatnin, Andrey A.; Rodriguez-nieto, Salvador; Zhivotovsky, Boris; Smertenko, Andrei

    2005-01-01

    Programmed cell death (PCD) is indispensable for eukaryotic development. In animals, PCD is executed by the caspase family of cysteine proteases. Plants do not have close homologues of caspases but possess a phylogenetically distant family of cysteine proteases named metacaspases. The cellular function of metacaspases in PCD is unknown. Here we show that during plant embryogenesis, metacaspase mcII-Pa translocates from the cytoplasm to nuclei in terminally differentiated cells that are destin...

  19. FRACTIONATION OF DIGESTIVE PROTEINASES FROM TENEBRIO MOLITOR (COLEOPTERA: TENEBRIONIDAE) LARVAE AND ROLE IN PROTEIN DIGESTION

    Science.gov (United States)

    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 chro...

  20. Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway.

    Science.gov (United States)

    Weits, Daan A; Giuntoli, Beatrice; Kosmacz, Monika; Parlanti, Sandro; Hubberten, Hans-Michael; Riegler, Heike; Hoefgen, Rainer; Perata, Pierdomenico; van Dongen, Joost T; Licausi, Francesco

    2014-01-01

    In plant and animal cells, amino-terminal cysteine oxidation controls selective proteolysis via an oxygen-dependent branch of the N-end rule pathway. It remains unknown how the N-terminal cysteine is specifically oxidized. Here we identify plant cysteine oxidase (PCO) enzymes that oxidize the penultimate cysteine of ERF-VII transcription factors by using oxygen as a co-substrate, thereby controlling the lifetime of these proteins. Consequently, ERF-VII proteins are stabilized under hypoxia and activate the molecular response to low oxygen while the expression of anaerobic genes is repressed in air. Members of the PCO family are themselves targets of ERF-VII transcription factors, generating a feedback loop that adapts the stress response according to the extent of the hypoxic condition. Our results reveal that PCOs act as sensor proteins for oxygen in plants and provide an example of how proactive regulation of the N-end rule pathway balances stress response to optimal growth and development in plants. PMID:24599061

  1. SMALL CYSTEINE-RICH PEPTIDES RESEMBLING ANTIMICROBIAL PEPTIDES HAVE BEEN UNDER-PREDICTED IN PLANTS

    Science.gov (United States)

    Multicellular organisms produce small cysteine-rich anti-microbial peptides as an innate defense against pathogens. While defensins, a well-known class of such peptides, are common among eukaryotes, there are classes restricted to the plant kingdom. These include thionins, lipid transfer proteins,...

  2. Subclassification and biochemical analysis of plant papain-like cysteine proteases displays subfamily-specific characteristics.

    OpenAIRE

    Richau, Kh; Kaschani, F.; Verdoes, M.; Pansuriya, Tc; Niessen, S.; Stu?ber, K.; Colby, T.; Overkleeft, Hs; Bogyo, M.; Hoorn, Ra

    2012-01-01

    Papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes associated with development, immunity, and senescence. Although many properties have been described for individual proteases, the distribution of these characteristics has not been studied collectively. Here, we analyzed 723 plant PLCPs and classify them into nine subfamilies that are present throughout the plant kingdom. Analysis of these subfamilies revealed previously unreported distinct subfamily-specific func...

  3. Cysteine proteases from the Asclepiadaceae plants latex exhibited thrombin and plasmin like activities.

    Science.gov (United States)

    Shivaprasad, H V; Riyaz, M; Venkatesh Kumar, R; Dharmappa, K K; Tarannum, Shaista; Siddesha, J M; Rajesh, R; Vishwanath, B S

    2009-10-01

    In the present study we evaluated the presence of cysteine protease from the latex of four plants Asclepias curassavica L., Calotropis gigantea R.Br., Pergularia extensa R.Br. and Cynanchum puciflorum R.Br. belongs to the family Asclepiadaceae. Cysteine proteases from these plants latex exhibited both thrombin and plasmin like activities. Latex enzyme fraction in a concentration dependent manner induced the formation of clot in citrated blood plasma. Direct incubation of fibrinogen with latex enzyme fraction resulted in the formation of fibrin clot similar to thrombin enzyme. However prolonged incubation resulted in degradation of the formed fibrin clot suggesting plasmin like activity. Latex enzyme fraction preferentially hydrolyzed Aalpha and Bbeta chains of fibrinogen to form fibrin clot. Latex enzyme fraction also hydrolyzed the subunits of fully cross linked fibrin efficiently, the order of hydrolysis was alpha-polymer > alpha-chains > beta-chain and gamma-gamma dimer. Cysteine proteases from all the four Asclepiadaceae plants latex exhibited similar action on fibrinogen and fibrin. This study scientifically validate the use of plant latex in stop bleeding and wound healing by traditional healers all over the world. PMID:18979066

  4. Post-translational regulation and evolution of plant gamma-glutamate cysteine ligase

    OpenAIRE

    Gromes, Roland

    2007-01-01

    Glutamate cysteine ligase (GCL) is catalyzing the rate-limiting step in glutathione (GSH) synthesis. A complex regulation of this enzyme is required to integrate various signals as GSH is fulfilling a plethora of functions in housekeeping metabolism, stress defence, and in the regulation of development. In this thesis the post-translational redox regulation of plant GCL and closely related proteobacterial enzymes was studied. The crystal structure of Brassica juncea GCL (BjGCL) revealed the p...

  5. Fasciola hepatica: isolation and characterisation of a cathepsin L proteinase

    OpenAIRE

    Smith, Angela M.

    1994-01-01

    Fasciola hepatica, a parasitic trematode, is the causative agent of liver fluke disease. It has been shown previously, that both the migratory and adult worm stage of the parasite secrete multiple cysteine proteinases when they are cultured overnight (Dalton & Heffernan, 1989). In this study, one of these proteinases has been purified by standard chromatographic techniques. The purified enzyme was characterised as a cathepsin L-like proteinase using synthetic substrates, inhibition studies, N...

  6. Gastrointestinal absorption and biological activities of serine and cysteine proteases of animal and plant origin: review on absorption of serine and cysteine proteases

    OpenAIRE

    Lorkowski, Gerhard

    2012-01-01

    Research has confirmed that peptides and larger protein molecules pass through the mucosal barrier of the gastrointestinal tract. Orally administered serine and cysteine proteases of plant and animal origin also reach blood and lymph as intact, high molecular weight and physiologically active protein molecules. Their absorption may be supported by a self-enhanced paracellular transport mechanism resulting in sub-nanomolar concentration of transiently free protease molecules or, in a complex w...

  7. Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease.

    Science.gov (United States)

    Cantú, M D; Mariano, A G; Palma, M S; Carrilho, E; Wulff, N A

    2008-10-01

    Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected root-stock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Down-regulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted. PMID:18943454

  8. Tumor cell proteinase visualization and quantification using a fluorescent transition-state analog probe.

    OpenAIRE

    Kozlowski, K. A.; Wezeman, F. H.; Schultz, R. M.

    1984-01-01

    The fluorescent proteinase transition-state analog inhibitor, dansyl-L-argininal (DnsArgH), may be a selective probe of cysteine and serine-type proteinases in a fibrosarcoma tumor cell line (HSDM1C1). DnsArgH binds with high affinity to proteinases because of its transition-state analog properties, and on association it gives a dramatically increased fluorescent yield. The DnsArgH binding is inhibited by the serine proteinase inhibitor diisopropyl fluorophosphate and by the cysteine proteina...

  9. The toxic effects of l-Cysteine-capped cadmium sulfide nanoparticles on the aquatic plant Spirodela polyrrhiza

    International Nuclear Information System (INIS)

    Plants play an important role in the fate of nanoparticles in the environment through their uptake, bioaccumulation, and transfer to trophic chains. However, the impacts of nanoparticles on plants as essential components of all ecosystems are not well documented. In the present study, the toxic effects of l-Cysteine-capped CdS nanoparticles on Spirodela polyrrhiza as an aquatic higher plant species were studied. l-Cysteine-capped CdS nanoparticles were synthesized using hydrothermal method and their characteristics were determined by XRD, SEM, HR-TEM, and FT-IR techniques. The diameter of majority of synthesized nanoparticles was about 15–20 nm. Subsequently, the uptake of l-Cysteine-capped CdS nanoparticles by the plant species was confirmed using epifluorescence microscopy. The activity of peroxidase and superoxide dismutase as antioxidant enzymes was assayed and the relative frond number was calculated in the presence of different concentrations of l-Cysteine-capped CdS nanoparticles. The obtained results revealed the toxic effects of the synthesized nanoparticles on S. polyrrhiza, leading to growth reduction and significant changes in antioxidant enzymes’ activity.Graphical Abstract

  10. Papain-like cysteine proteases: key players at molecular battlefields employed by both plants and their invaders.

    OpenAIRE

    Shindo, T.; Hoorn, Ra

    2008-01-01

    Papain-like cysteine proteases (PLCPs) play crucial roles in plant-pathogen/pest interactions. During these parasitic interactions, PLCPs act on non-self substrates, provoking the selection of counteracting inhibitors and other means to evade proteolysis. We review examples of PLCPs acting on molecular battlefields in the extracellular space, plant cytoplasm and herbivore gut. Examples are maize Mir1 (Maize inbred resistance 1), tomato Rcr3 (Required for Cladosporium resistance-3), Pseudomona...

  11. Purification of a proteinase (Ac5-proteinase) and characterization of hemorrhagic toxins from the venom of the hundred-pace snake (Agkistrodon acutus).

    Science.gov (United States)

    Mori, N; Nikai, T; Sugihara, H

    1984-01-01

    Ac5-Proteinase (15.2 mg) was isolated from Agkistrodon acutus venom (1 g) by column chromatography on Sephadex G-75, CM-Sephadex C-50 and CM-Cellulose. Ac5-Proteinase was homogeneous by disc electrophoresis on polyacrylamide gel at pH 4.3 and also by SDS-disc polyacrylamide gel electrophoresis. Ac1-, Ac2-, Ac3- and Ac5-proteinases possessed lethal and hemorrhagic activities, but Ac4-proteinase had no lethal activity. These activities were inhibited completely by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or cysteine. The molecular weights of Ac1-, Ac2-, Ac3-, Ac4- and Ac5-proteinases were approximately 24,500, 25,000, 57,000, 33,000 and 24,000, respectively. Ac1-, Ac2-, Ac4- and Ac5-proteinases did not contain any carbohydrates, but Ac3-proteinase contained 0.1% carbohydrate by weight. PMID:6433514

  12. Selective adsorption of plant cysteine peptidases onto TiO2.

    Science.gov (United States)

    Llerena-Suster, C R F; Foresti, M L; Briand, L E; Morcelle, S R

    2009-08-01

    A crude extract rich in plant cysteine peptidases was obtained from the latex of the fruits of Araujia hortorum, a South American climbing plant. The highly concentrated extract was immobilized onto titanium dioxide to produce biocatalysts through a simple adsorption procedure. Absorbance measurement at 280 nm and Bradford's method for protein quantification revealed that the protein content of the crude extract was selectively adsorbed onto the titanium dioxide surface at a very high rate. In 5 min of contact with the support all protein present in the crude extract was selectively withdrawn from the solution, leading to an immobilized biocatalyst with a high protein concentration. Caseinolytic assays indicated that, except for the catalyst obtained with the highest crude amount contacted with the support, all the proteolytic activity present in the crude extract was adsorbed onto TiO(2). The amidasic activity of the immobilized catalysts (Ah/TiO(2)) was tested in the hydrolysis of a synthetic chromogenic substrate (PFLNA) showing partial deactivation with respect to the native enzyme. In amidasic activity assays the ionic strength of the buffer medium showed to be a key feature to consider in order to avoid protease desorption from the support, indicating the importance of electrostatic interactions between the enzymes and TiO(2). Reuse of the produced biocatalysts with PFLNA as substrate revealed that after five successive uses Ah/TiO(2) retained more than 20% of its initial activity. PMID:19394803

  13. PAPAIN, A PLANT ENZYME OF BIOLOGICAL IMPORTANCE: A REVIEW

    OpenAIRE

    Ezekiel Amri; Florence Mamboya

    2012-01-01

    Papain is a plant proteolytic enzyme for the cysteine proteinase family cysteine protease enzyme in which enormous progress has been made to understand its functions. Papain is found naturally in papaya (Carica papaya L.) manufactured from the latex of raw papaya fruits. The enzyme is able to break down organic molecules made of amino acids, known as polypeptides and thus plays a crucial role in diverse biological processes in physiological and pathological states, drug designs, industrial us...

  14. The preparation of fully active chymopapain free of contaminating proteinases.

    Science.gov (United States)

    Buttle, D J; Dando, P M; Coe, P F; Sharp, S L; Shepherd, S T; Barrett, A J

    1990-11-01

    Chymopapain (EC 3.4.22.6) was purified from commercially available dried latex of papaya (Carica papaya) by extraction at acidic pH, cation-exchange chromatography and active site-directed affinity chromatography on immobilized alanyl-phenyl-alaninaldehyde semicarbazone, with elution by mercuric chloride. The product was found by immunoassay to be essentially free of the other cysteine proteinases from papaya, including papaya proteinase IV, and was fully active. The rate of alkylation of the active site cysteine of chymopapain by iodoacetate was found to be sufficiently rapid and selective for this reagent to be used as an active-site titrant. PMID:2085414

  15. Proteinase activity in latex of three plants of the family Euphorbiaceae

    Scientific Electronic Library Online (English)

    Andréa Michel, Sobottka; Fabiana, Tonial; Sonja, Sytwala; Matthias, Melzig.

    2014-09-01

    Full Text Available Dentro da família Euphorbiaceae, os gêneros Euphorbia e Sapium são conhecidos por incluírem basicamente espécies produtoras de látex. No presente estudo, o látex das plantas Euphorbia selloi (Klotzsch & Garcke) Boiss., Euphorbia papillosa A.St.-Hil. e Sapium glandulosum (L.) Morong, espécies nativas [...] do Brasil, foi analisado em relação à atividade proteolítica. Todas as amostras analisadas possuem proteínas com significativa atividade, sendo que o látex da espécie E. papillosa apresenta a maior atividade específica. Com o objetivo de analisar quais os tipos de proteases responsáveis pela atividade proteolítica, realizaram-se ensaios com diferentes inibidores. Nas três plantas testadas a atividade foi inibida significativamente pelo cloridrato de 4-(fluoreto de 2-aminoetilbenzenossulfonil) (AEBSF), um inibidor de serino-proteases. Utilizando técnicas de eletroforese em gel de poliacrilamida (SDS-PAGE), as subunidades das proteínas foram separadas de acordo com sua massa molecular e, através da zimografia, a atividade proteolítica pode ser detectada visualmente. Abstract in english In the family of Euphorbiaceae, the genera Euphorbia and Sapium are known to contain essentially latex-bearing species. In the present study, the latex of Euphorbia selloi (Klotzsch & Garcke) Boiss., Euphorbia papillosa A.St.-Hil., and Sapium glandulosum (L.) Morong, plants native from Brazil, were [...] examined concerning proteolytic activity. All studied species have proteins with significant proteolytic activity and E. papillosa has the greatest specific activity. Aiming to verify the type of protease present, an assay with different inhibitors was performed. In the three tested plants, the proteolytic activity was significantly inhibited by a serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). Using techniques of electrophoresis with polyacrylamide gels (SDS-PAGE), the subunits of proteins were separated according to their molecular masses, and the protein activity was visually detected by zymography.

  16. Transglutaminase factor XIII uses proteinase-like catalytic triad to crosslink macromolecules.

    OpenAIRE

    Pedersen, L. C.; Yee, V. C.; Bishop, P. D.; Le Trong, I.; Teller, D. C.; Stenkamp, R. E.

    1994-01-01

    The X-ray crystal structure of human transglutaminase factor XIII has revealed a cysteine proteinase-like active site involved in a crosslinking reaction and not proteolysis. This is among the first observations of similar active sites in 2 different enzyme families catalyzing a similar reaction in opposite directions. Although the size and overall protein fold of factor XIII and the cysteine proteinases are quite different, the active site and the surrounding protein structure share structur...

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

    Science.gov (United States)

    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:15035659

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

    OpenAIRE

    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 act...

  19. [Transformation of wheat with insecticide gene of arrowhead proteinase inhibitor by pollen tube pathway and analysis of transgenic plants] [In Process Citation

    Science.gov (United States)

    Mu; Liu; Zhou; Wen; Zhang; Wei

    1999-01-01

    Arrowhead Proteinase Inhibitor(API), one kind of pure natural material, was derived from storage organ of Sagittaria trifolia. It belongs to serine proteinase inhibitor, and can inhibit trypsin, chemotrypsin and kallikrein. Furthermore, API is toxical to some species of insects such as lepidotera, Coleoptera and Diptrea etc. By means of pollen tube pathway, plasmid pBIAH-A(B) containing insect-resistant genes of API-A, API-B and selective marker gene of NPT-II were transferred into three lines of local winter wheat--JD-1, 8866, 866554. Then, Kanamycin-resistant screening and PCR analysis of genetic transformed plants showed that three of Kmr green plants (two from 866554, one from JD-1) were PCR positive with the positive rate of 0.29%. When the fragment of API gene was used as probe to hybrid with genomic DNA of Kmr green plants separately, all of three PCR positive ones displayed a single strong hybridizing band. Such results demonstrated that foreign target gene had been integrated into wheat genome already. Simultaneously, PCR analysis and Southern hybridization were carried out among selfiedoffsprings of transformed positive plant of the line 899554-3, some of them were PCR and Southern blotting positive, indicating that foreign gene integrated into wheat genome could stably transmitted into next generation. In addition, the expression level of NPT-II gene was checked via ELISA in our study, all of three PCR and Southern blot positive plants could yield high level of NPT-II. This data provided a more powerful evidence for integration of insecticide gene into wheat genome. PMID:10876664

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

    Science.gov (United States)

    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

  1. Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. Revealed by De Novo Transcriptome Analysis.

    Science.gov (United States)

    Kwon, Chang Woo; Park, Kyung-Min; Kang, Byoung-Cheorl; Kweon, Dae-Hyuk; Kim, Myoung-Dong; Shin, Sang Woon; Je, Yeon Ho; Chang, Pahn-Shick

    2015-01-01

    Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding. PMID:25786229

  2. Proteinase activity regulation by glycosaminoglycans

    Scientific Electronic Library Online (English)

    I.L.S., Tersariol; D.C., Pimenta; J.R., Chagas; P.C., Almeida.

    2002-02-01

    Full Text Available SciELO Brazil | Language: English Abstract in english There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also [...] to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.

  3. Proteinase activity regulation by glycosaminoglycans

    Directory of Open Access Journals (Sweden)

    Tersariol I.L.S.

    2002-01-01

    Full Text Available There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.

  4. Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus.

    Science.gov (United States)

    Silva, C P; Terra, W R; Lima, R M

    2001-05-01

    Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of epsilon-aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, and was activated by 1.5 mM DTT in both insects, while hydrolysis of the substrate Z-ArgArg-MCA was inhibited by aprotinin and E-64, which suggests that it is being hydrolysed by serine and cysteine proteinases. Gel assays showed that the proteolytic activity in larval midgut of C. maculatus was due to five major cysteine proteinases. However, based on the pattern of E-64 and aprotinin inhibition, proteolytic activity in larval midgut of Z. subfasciatus was not due only to cysteine proteinases. Fractionation of the larval midgut homogenates of both bruchids through ion-exchange chromatography (DEAE-Sepharose) revealed two peaks of activity against Z-ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characteristics of cysteine proteinases, while Z. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCl. The proteolytic activity of the retained peak is higher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 microM aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes alpha-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch. PMID:11317332

  5. Activation of cysteine proteases in cowpea plants during the hypersensitive response--a form of programmed cell death.

    Science.gov (United States)

    D'Silva, I; Poirier, G G; Heath, M C

    1998-12-15

    There is increasing evidence that the hypersensitive response during plant-pathogen interactions is a form of programmed cell death. In an attempt to understand the biochemical nature of this form of programmed cell death in the cowpea-cowpea rust fungus system, proteolytic activity in extracts of fungus-infected and uninfected cowpea plants was investigated, using exogenously added poly(ADP-ribose) polymerase as a marker. Unlike the proteolytic cleavage pattern of endogenous poly(ADP-ribose) polymerase in apoptotic animal cells, exogenously added poly(ADP-ribose) polymerase in extracts of fungus-infected plants was proteolytically cleaved into fragments of molecular masses 77, 52, 47, and 45 kDa. In vitro and in vivo protease inhibitor experiments revealed the activation of cysteine proteases, and possibly a regulatory role, during the hypersensitive response. PMID:9851880

  6. Influence of air temperature on proteinase activity and beverage quality in Coffea arabica

    Scientific Electronic Library Online (English)

    Hellen Marília Couto de, Abreu; Paula Macedo, Nobile; Milton Massao, Shimizu; Paula Yuri, Yamamoto; Emerson Alves, Silva; Carlos Augusto, Colombo; Paulo, Mazzafera.

    Full Text Available Fruits were collected from trees of Coffea arabica cv. Obatã grown at Mococa and Adamantina in São Paulo State, Brazil, which are regions with marked differences in air temperature that produce coffee with distinct qualities. Mococa is a cooler location that produces high-quality coffee, whereas cof [...] fee from Adamantina is of lower quality. The amino acid and protein contents, amino acid profile, and proteinase activity and type in endosperm protein extracts were analysed. Proteinase genes were identified, and their expression was assayed. All results indicate that temperature plays a role in controlling proteinase activity in coffee endosperm. Proteinase activity was higher in the endosperm of immature fruits from Adamantina, which was correlated with higher amino acid content, changes in the amino acid profile, and increased gene expression. Cysteine proteinases were the main class of proteinases in the protein extracts. These data suggest that temperature plays an important role in coffee quality by altering nitrogen compound composition.

  7. Nucleotide sequence of human preprocathepsin H, a lysosomal cyteine proteinase

    Energy Technology Data Exchange (ETDEWEB)

    Fuchs, R.; Gassen, H.G. (Institute fuer Biochemie, Darmstadt (West Germany))

    1989-11-25

    The lysosomal cystein proteinase cathepsin H is one of the most active proteinases in the human body. Until now only partial gene sequence information was available. A {lambda}gt10 cDNA library constructed from a human U937 monocyte cell line was screened with a gene probe derived from a partial kidney cathespin H clone coding for the mature part of the protein. From 4.5 {times} 10{sup 5} plaques two full-length clones were isolated and characterized. The article shows the previously unknown coding sequence of the prepro part of human preprocathespin H and the derived protein sequence.

  8. Inibidores de proteases de hospedeiros nativos e exóticos e sua ação em intestinos de lagartas de Thyrinteina leucoceraea / Proteinase inhibitors of novel and native host plants and their action in midgut of Thyrinteina leucoceraea caterpillars

    Scientific Electronic Library Online (English)

    Jeanne Scardini, Marinho; Maria Goreti Almeida, Oliveira; Raul Narciso Carvalho, Guedes; Angelo, Pallini; Claudinei Lima, Oliveira.

    1125-11-01

    Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese Os insetos podem causar perdas consideráveis aos seus hospedeiros, entretanto alguns deles habitam em plantas sem causar-lhes danos. Por exemplo, Thyrinteina leucoceraea, herbívoro da entomofauna brasileira, pode ser encontrado na goiabeira, hospedeiro nativo da família Myrtaceae, sem que cause dano [...] s severos a essa planta. Os Eucalyptus ssp., entretanto, são hospedeiros exóticos (também da família Myrtaceae) no Brasil, vindos da Austrália, os quais sofrem ataques das lagartas de T. leucoceraea, que se tornaram pragas severas dessas plantas. Sabe-se que as plantas podem se defender contra o ataque de herbívoros e que um dos seus mecanismos de defesa pode ser a produção de inibidores de proteases, que possuem a capacidade de diminuir o desenvolvimento dos insetos e podem levá-los à morte. Baseado no desempenho da lagarta de T. leucoceraea nesses dois hospedeiros e na possibilidade de defesa da planta, o objetivo deste trabalho foi verificar a produção de inibidores de proteases por plantas de eucalipto e de goiaba quando atacadas por essas lagartas, bem como observar a resposta bioquímica no intestino das lagartas a esses inibidores. Notou-se que as plantas de eucalipto produzem mais inibidores de proteases que as goiabeiras. O bom desenvolvimento de T. leucoceraea em plantas de eucalipto, apesar da alta concentração de inibidores de proteases, pode ser devido ao aumento da atividade enzimática nos intestinos das lagartas quando alimentadas com essa planta. Os dados evidenciaram que T. leucoceraea desenvolveu uma adaptação aos inibidores de proteases produzidos pelo eucalipto, por meio do aumento das atividades de serino-proteases e cisteíno-proteases. Abstract in english Insects may cause considerable losses to plants, but some insects inhabit plants without causing any damages. For example, Thyrinteina leucoceraea, found in the guava plants, and a native Myrtaceae family host, does not cause any serious damage. However, Eucalyptus ssp., novel hosts (also Myrtaceae) [...] in Brazil and introduced from Australia, suffer attacks by T. leucoceraea, which became a severe pest of this plant. Plants can defend themselves against herbivores using proteinase inhibitors which reduce insect development and lead them to death. Thus, based on studies on the development of T. leucoceraea caterpillars on these two hosts and plant defense, this work aimed to verify the production of proteinase inhibitors by guava and eucalyptus plants upon T. leucoceraea attack, and to observe the biochemical response of the midgut of the caterpillars to these inhibitors. Eucalyptus plants produced more proteinase inhibitors than guava plants. The good development of T. leucoceraea in eucalyptus plants despite the high concentration of proteinase inhibitors may be due to an increase of enzyme activity in the caterpillars' midgut. Our data suggest that T. leucoceraea developed an adaptation to the proteinase inhhibitor produced by eucalyptus plants, by increasing serine-proteinase and cys-proteinase activities.

  9. Inibidores de proteases de hospedeiros nativos e exóticos e sua ação em intestinos de lagartas de Thyrinteina leucoceraea Proteinase inhibitors of novel and native host plants and their action in midgut of Thyrinteina leucoceraea caterpillars

    Directory of Open Access Journals (Sweden)

    Jeanne Scardini Marinho

    2008-12-01

    Full Text Available Os insetos podem causar perdas consideráveis aos seus hospedeiros, entretanto alguns deles habitam em plantas sem causar-lhes danos. Por exemplo, Thyrinteina leucoceraea, herbívoro da entomofauna brasileira, pode ser encontrado na goiabeira, hospedeiro nativo da família Myrtaceae, sem que cause danos severos a essa planta. Os Eucalyptus ssp., entretanto, são hospedeiros exóticos (também da família Myrtaceae no Brasil, vindos da Austrália, os quais sofrem ataques das lagartas de T. leucoceraea, que se tornaram pragas severas dessas plantas. Sabe-se que as plantas podem se defender contra o ataque de herbívoros e que um dos seus mecanismos de defesa pode ser a produção de inibidores de proteases, que possuem a capacidade de diminuir o desenvolvimento dos insetos e podem levá-los à morte. Baseado no desempenho da lagarta de T. leucoceraea nesses dois hospedeiros e na possibilidade de defesa da planta, o objetivo deste trabalho foi verificar a produção de inibidores de proteases por plantas de eucalipto e de goiaba quando atacadas por essas lagartas, bem como observar a resposta bioquímica no intestino das lagartas a esses inibidores. Notou-se que as plantas de eucalipto produzem mais inibidores de proteases que as goiabeiras. O bom desenvolvimento de T. leucoceraea em plantas de eucalipto, apesar da alta concentração de inibidores de proteases, pode ser devido ao aumento da atividade enzimática nos intestinos das lagartas quando alimentadas com essa planta. Os dados evidenciaram que T. leucoceraea desenvolveu uma adaptação aos inibidores de proteases produzidos pelo eucalipto, por meio do aumento das atividades de serino-proteases e cisteíno-proteases.Insects may cause considerable losses to plants, but some insects inhabit plants without causing any damages. For example, Thyrinteina leucoceraea, found in the guava plants, and a native Myrtaceae family host, does not cause any serious damage. However, Eucalyptus ssp., novel hosts (also Myrtaceae in Brazil and introduced from Australia, suffer attacks by T. leucoceraea, which became a severe pest of this plant. Plants can defend themselves against herbivores using proteinase inhibitors which reduce insect development and lead them to death. Thus, based on studies on the development of T. leucoceraea caterpillars on these two hosts and plant defense, this work aimed to verify the production of proteinase inhibitors by guava and eucalyptus plants upon T. leucoceraea attack, and to observe the biochemical response of the midgut of the caterpillars to these inhibitors. Eucalyptus plants produced more proteinase inhibitors than guava plants. The good development of T. leucoceraea in eucalyptus plants despite the high concentration of proteinase inhibitors may be due to an increase of enzyme activity in the caterpillars' midgut. Our data suggest that T. leucoceraea developed an adaptation to the proteinase inhhibitor produced by eucalyptus plants, by increasing serine-proteinase and cys-proteinase activities.

  10. The proteinases of Psoroptes ovis, the sheep scab mite--their diversity and substrate specificity.

    Science.gov (United States)

    Kenyon, Fiona; Knox, David

    2002-05-10

    The sheep scab mite, Psoroptes ovis, causes severe dermatitis in infected sheep with severe welfare and production implications. The dermatitis has the characteristics of an immediate hypersensitivity type reaction which, by analogy to other mite species, including the house dust mites (Dermatophagoides spp.), is likely to be invoked by a variety of allergens including mite-derived proteinases. Here, the proteinases in P. ovis extracts were characterised using substrate gel analysis, inhibitor sensitivity and their ability to degrade a variety of potential natural protein substrates. These analyses showed that mites contain several proteinases which could be differentiated on the basis of molecular size and inhibitor sensitivity with cysteine, metalloproteinases and aspartyl proteinases predominating. These proteinases degraded collagen and fibronectin, possibly indicative of a role in lesion initiation, they degraded several blood proteins, a property which may aid mite feeding and they degraded immunoglobulin G, possibly aiding immuno-evasion. Because proteinases, particularly the cysteine class, are demonstrably allergenic in other mite infestations, these proteinases clearly merit further immunological and biochemical definition. PMID:11983306

  11. Structure and function of invertebrate Kazal-type serine proteinase inhibitors.

    Science.gov (United States)

    Rimphanitchayakit, Vichien; Tassanakajon, Anchalee

    2010-04-01

    Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. The proteinase inhibitors function as modulators for controlling the extent of deleterious proteinase activity. The Kazal-type proteinase inhibitors (KPIs) in family I1 are among the well-known families of proteinase inhibitors, widely found in mammals, avian and a variety of invertebrates. Like those classical KPIs, the invertebrate KPIs can be single or multiple domain proteins containing one or more Kazal inhibitory domains linked together by peptide spacers of variable length. All invertebrate Kazal domains of about 40-60 amino acids in length share a common structure which is dictated by six conserved cysteine residues forming three intra-domain disulfide cross-links despite the variability of amino acid sequences between the half-cystines. Invertebrate KPIs are strong inhibitors as shown by their extremely high association constant of 10(7)-10(13)M(-1). The inhibitory specificity of a Kazal domain varies widely with a different reactive P(1) amino acid. Different invertebrate KPI domains may arise from gene duplication but several KPI proteins can also be derived from alternative splicing. The invertebrate KPIs function as anticoagulants in blood-sucking animals such as leech, mosquitoes and ticks. Several KPIs are likely involved in protecting host from microbial proteinases while some from the parasitic protozoa help protecting the parasites from the host digestive proteinase enzymes. Silk moths produce KPIs to protect their cocoon from predators and microbial destruction. PMID:19995574

  12. Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana.

    Science.gov (United States)

    Romero, Luis C; Aroca, M Ángeles; Laureano-Marín, Ana M; Moreno, Inmaculada; García, Irene; Gotor, Cecilia

    2014-02-01

    Cysteine occupies a central position in plant metabolism because it is a reduced sulfur donor molecule involved in the synthesis of essential biomolecules and defense compounds. Moreover, cysteine per se and its derivative molecules play roles in the redox signaling of processes occurring in various cellular compartments. Cysteine is synthesized during the sulfate assimilation pathway via the incorporation of sulfide to O-acetylserine, catalyzed by O-acetylserine(thiol)lyase (OASTL). Plant cells contain OASTLs in the mitochondria, chloroplasts, and cytosol, resulting in a complex array of isoforms and subcellular cysteine pools. In recent years, significant progress has been made in Arabidopsis, in determining the specific roles of the OASTLs and the metabolites produced by them. Thus, the discovery of novel enzymatic activities of the less-abundant, like DES1 with L-cysteine desulfhydrase activity and SCS with S-sulfocysteine synthase activity, has provided new perspectives on their roles, besides their metabolic functions. Thereby, the research has been demonstrated that cytosolic sulfide and chloroplastic S-sulfocysteine act as signaling molecules regulating autophagy and protecting the photosystems, respectively. In the cytosol, cysteine plays an essential role in plant immunity; in the mitochondria, this molecule plays a central role in the detoxification of cyanide, which is essential for root hair development and plant responses to pathogens. PMID:24285094

  13. Proteinase inhibitor I accumulation in tomato suspension cultures : induction by plant and fungal cell wall fragments and an extracellular polysaccharide secreted into the medium.

    Science.gov (United States)

    Walker-Simmons, M; Ryan, C A

    1986-01-01

    Suspension-cultured cells of tomato accumulate proteinase Inhibitor I as the sucrose is depleted from 1% to less than 0.1% in the culture medium. Inhibitor I can be prematurely induced to accumulate in the cells by the addition to the medium of the proteinase inhibitor inducing factor, trigalacturonic acid, ethylene glycol chitin, or chitosan. In cultures grown in 0.6% initial sucrose with no inducers added, a uronic acid-rich extracellular polysaccharide appears in the medium during growth of the cells. This extracellular polysaccharide apparently contains an ;endogenous inducer' of Inhibitor I synthesis. When the partially purified polysaccharide is added to the culture medium, Inhibitor I accumulation is induced. Proteinase inhibitors also accumulate in tobacco and alfalfa suspension-cultured cells as the cell cultures age. As with the tomato cultures, a uronic acid-rich component(s) appears in the media prior to inhibitor accumulation. These data suggest that an endogenous inducer may be activating proteinase inhibitor genes through a similar mechanism in all three types of cells. PMID:16664609

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

    Science.gov (United States)

    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

  15. Complete amino acid sequence of ananain and a comparison with stem bromelain and other plant cysteine proteases.

    Science.gov (United States)

    Lee, K L; Albee, K L; Bernasconi, R J; Edmunds, T

    1997-10-01

    The amino acid sequences of ananain (EC3.4.22.31) and stem bromelain (3.4.22.32), two cysteine proteases from pineapple stem, are similar yet ananain and stem bromelain possess distinct specificities towards synthetic peptide substrates and different reactivities towards the cysteine protease inhibitors E-64 and chicken egg white cystatin. We present here the complete amino acid sequence of ananain and compare it with the reported sequences of pineapple stem bromelain, papain and chymopapain from papaya and actinidin from kiwifruit. Ananain is comprised of 216 residues with a theoretical mass of 23464 Da. This primary structure includes a sequence insert between residues 170 and 174 not present in stem bromelain or papain and a hydrophobic series of amino acids adjacent to His-157. It is possible that these sequence differences contribute to the different substrate and inhibitor specificities exhibited by ananain and stem bromelain. PMID:9355753

  16. A Naturally Occurring Plant Cysteine Protease Possesses Remarkable Toxicity against Insect Pests and Synergizes Bacillus thuringiensis Toxin

    OpenAIRE

    Mohan, Srinidi; Ma, Peter W. K.; Williams, W. Paul; Luthe, Dawn S.

    2008-01-01

    When caterpillars feed on maize (Zea maize L.) lines with native resistance to several Lepidopteran pests, a defensive cysteine protease, Mir1-CP, rapidly accumulates at the wound site. Mir1-CP has been shown to inhibit caterpillar growth in vivo by attacking and permeabilizing the insect's peritrophic matrix (PM), a structure that surrounds the food bolus, assists in digestion and protects the midgut from microbes and toxins. PM permeabilization weakens the caterpillar defenses by facilitati...

  17. Evolutionary patterns of proteinase activity in attine ant fungus gardens

    DEFF Research Database (Denmark)

    Semenova, Tatyana; Hughes, David Peter

    2011-01-01

    Background: Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved. Results: We determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian species of fungus-growing ants, representing eight genera. We mapped these activity profiles on an independently obtained molecular phylogeny of the symbionts and show that total proteinase activity in lower attine symbionts peaks at ca. pH 6. The higher attine symbionts that have no known free-living relatives had much higher proteinase activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH optimum of fungal proteinases may have changed after the irreversible domestication of evolutionary more derived fungal symbionts. This notion is also supported by buffering capacities of fungus gardens at pH 5.2 being remarkably high, and suggests that the fungal symbiont actively helps to maintain garden acidity at this specific level. Metalloproteinases dominated the activity profiles of lower attine gardens and may thus represent the ancestral type of proteinase production, whereas serine proteinase activity dominated the activity profiles of the higher attine gardens reared by Trachymyrmex and Sericomyrmex, suggesting that there may be trade-offs in the production of these enzyme classes. Remarkably, the single symbiont that is shared by species of the crown group of Atta and Acromyrmex leaf-cutting ants mostly showed metalloproteinase activity, suggesting that recurrent changes in enzyme production may have occurred throughout the domestication history of fungus-garden symbionts. Conclusions: Proteinase pH optima and buffering capacities of fungal symbionts appear to have evolved remarkable adaptations to living in obligate symbiosis with farming ants. Although the functional roles of serine and metalloproteinases in fungus gardens are unknown, the differential production of these classes of proteolytic enzymes suggest that substrate specificity may be important and that trade-offs may prevent the simultaneous upregulation of both classes of enzymes.

  18. Evolutionary patterns of proteinase activity in attine ant fungus gardens

    Directory of Open Access Journals (Sweden)

    Hughes David P

    2011-01-01

    Full Text Available Abstract Background Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved. Results We determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian species of fungus-growing ants, representing eight genera. We mapped these activity profiles on an independently obtained molecular phylogeny of the symbionts and show that total proteinase activity in lower attine symbionts peaks at ca. pH 6. The higher attine symbionts that have no known free-living relatives had much higher proteinase activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH optimum of fungal proteinases may have changed after the irreversible domestication of evolutionary more derived fungal symbionts. This notion is also supported by buffering capacities of fungus gardens at pH 5.2 being remarkably high, and suggests that the fungal symbiont actively helps to maintain garden acidity at this specific level. Metalloproteinases dominated the activity profiles of lower attine gardens and may thus represent the ancestral type of proteinase production, whereas serine proteinase activity dominated the activity profiles of the higher attine gardens reared by Trachymyrmex and Sericomyrmex, suggesting that there may be trade-offs in the production of these enzyme classes. Remarkably, the single symbiont that is shared by species of the crown group of Atta and Acromyrmex leaf-cutting ants mostly showed metalloproteinase activity, suggesting that recurrent changes in enzyme production may have occurred throughout the domestication history of fungus-garden symbionts. Conclusions Proteinase pH optima and buffering capacities of fungal symbionts appear to have evolved remarkable adaptations to living in obligate symbiosis with farming ants. Although the functional roles of serine and metalloproteinases in fungus gardens are unknown, the differential production of these classes of proteolytic enzymes suggest that substrate specificity may be important and that trade-offs may prevent the simultaneous upregulation of both classes of enzymes.

  19. Molecular cloning of a mitogenic proteinase from Carica candamarcensis: its potential use in wound healing.

    Science.gov (United States)

    Corrêa, Natássia C R; Mendes, Isabela C; Gomes, Marco Túlio R; Kalapothakis, Evanguedes; Chagas, Brisa C A; Lopes, Miriam T P; Salas, Carlos E

    2011-11-01

    Cysteine proteinases from the Caricaceae belong to the C1 family of the CA clan and display papain-like structured, the archetype enzyme for this group of proteins. Carica candamarcensis, also named Vasconcellea cundinamarcensis, a member of Caricaceae family common to many areas in South America, contains cysteine proteinases with proteolytic activity five to eight-fold higher than those from latex of Carica papaya. The cysteine protease CMS2MS2 from C. candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In this study, the cDNA cloning, expression and evaluation of biological activity of a CMS2MS2-like protein from C. candamarcensis is reported. The 650 bp fragment was cloned in bacteria and the DNA sequence confirmed a cysteine-proteinase similar to CMS2MS2. The recombinant protein is 30 kDa, induces a mitogenic response, and enhances ERK1/2 phosphorylation, like the non-recombinant enzyme, but lacks either amidase or caseinolytic activity. The mitogenic activity of this protein and its lack of proteolytic activity underscore a potential for use in wound healing treatment. PMID:21798567

  20. A secreted protein with plant-specific cysteine-rich motif functions as a mannose-binding lectin that exhibits antifungal activity.

    Science.gov (United States)

    Miyakawa, Takuya; Hatano, Ken-ichi; Miyauchi, Yumiko; Suwa, You-ichi; Sawano, Yoriko; Tanokura, Masaru

    2014-10-01

    Plants have a variety of mechanisms for defending against plant pathogens and tolerating environmental stresses such as drought and high salinity. Ginkbilobin2 (Gnk2) is a seed storage protein in gymnosperm that possesses antifungal activity and a plant-specific cysteine-rich motif (domain of unknown function26 [DUF26]). The Gnk2-homologous sequence is also observed in an extracellular region of cysteine-rich repeat receptor-like kinases that function in response to biotic and abiotic stresses. Here, we report the lectin-like molecular function of Gnk2 and the structural basis of its monosaccharide recognition. Nuclear magnetic resonance experiments showed that mannan was the only yeast (Saccharomyces cerevisiae) cell wall polysaccharide that interacted with Gnk2. Gnk2 also interacted with mannose, a building block of mannan, with a specificity that was similar to those of mannose-binding legume lectins, by strictly recognizing the configuration of the hydroxy group at the C4 position of the monosaccharide. The crystal structure of Gnk2 in complex with mannose revealed that three residues (asparagine-11, arginine-93, and glutamate-104) recognized mannose by hydrogen bonds, which defined the carbohydrate-binding specificity. These interactions were directly related to the ability of Gnk2 to inhibit the growth of fungi, including the plant pathogenic Fusarium spp., which were disrupted by mutation of arginine-93 or the presence of yeast mannan in the assay system. In addition, Gnk2 did not inhibit the growth of a yeast mutant strain lacking the ?1,2-linked mannose moiety. These results provide insights into the molecular basis of the DUF26 protein family. PMID:25139159

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

    International Nuclear Information System (INIS)

    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 [3H]-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 og 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. Aspartic proteinase in Dugesia tigrina (Girard) planaria.

    Science.gov (United States)

    Zamora-Veyl, Fanny B; Guedes, Herbert L M; Giovanni-De-Simone, Salvatore

    2002-01-01

    A proteolytic activity was identified in Dugesia tigrina planaria using the chromogenic substrate Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu-O4MP. The activity of the enzyme increased four times during the regeneration and presented a maximum at 120 hr being higher in tail than head regenerating segments. The protease that displays this activity was purified from worms by a single step on pepstatin-agarose followed by gel-filtration high performance liquid chromatography. The purification resulted in a 34-fold increase in specific activity and the final yield was 10%. The active D. tigrina hydrolase appears to be a dimeric protein composed of identical subunits with 34 kDa associated by disulphide bridges similar to vertebrate cathepsin D. By SDS-PAGE several bands were detected but upon gel filtration HPLC one proteolytically active component, termed Asp-68, was detected and isolated. The maximal activity was observed in a range between pH 3.5-5.0 and the enzyme became inactivated at a pH value above 7.2. The purified enzyme was not inhibited by inhibitors from serine (aprotinin, TPCK, PMSF and TLCK), metallo (EDTA) and cysteine proteinase (E-64) classes. In contrast, inhibitors such as pepstatin, EPNP, and 4-beta-PMA efficiently inhibited the activity of the 68-kDa protease. PMID:12132699

  3. [Aspergillus ochraceus myxomycetes produce extracellular proteinases--protein C activators of blood plasma].

    Science.gov (United States)

    Osmolovski?, A A; Kre?er, V G; Kurakov, A V; Baranova, N A; Egorov, N S

    2012-01-01

    Natural isolates of Aspergillus ochraceus myxomycetes from soil and plant remains from various regions have been screened. The isolated strains were characterized by similar cultural and morphological features and an identical nucleotide sequence in the ITS1-5,8S-ITS2 region of rDNA. The ability of the extracellular proteinases of A. ochraceus myxomycetes to activate protein C of blood plasma has been established. Differences are revealed in the accumulation of proteinases activating protein C and proteinases with thrombin- and plasmin-like activities in the growth dynamics of producers. PMID:23101392

  4. Introgression of leginsulin, a cysteine-rich protein, and high-protein trait from an asian soybean plant introduction genotype into a north american experimental soybean line.

    Science.gov (United States)

    Krishnan, Hari B; Kim, Won-Seok; Oehrle, Nathan W; Alaswad, Alaa A; Baxter, Ivan; Wiebold, William J; Nelson, Randall L

    2015-03-25

    Soybean is an important protein source for both humans and animals. However, soybean proteins are relatively poor in the sulfur-containing amino acids, cysteine and methionine. Improving the content of endogenous proteins rich in sulfur-containing amino acids could enhance the nutritive value of soybean meal. Leginsulin, a cysteine-rich peptide, predominantly accumulates in Asian soybean accessions but not in most North American cultivars. By screening diverse soybean accessions from the USDA Soybean Germplasm Collection, we were able to identify one plant introduction, PI 427138, as a high-protein line with relatively high amounts of both elemental sulfur and leginsulin. We introgressed these desirable traits from PI 427138 into an experimental line with the aim of improving the overall protein content and quality of seed proteins. Biochemical characterization of inbred progenies from the cross of LD00-3309 with PI 427138 grown at six locations revealed stable ingression of high protein, high elemental sulfur, and high leginsulin accumulation. Comparison of soybean seed proteins resolved by high-resolution 2-D gel electrophoresis in combination with Delta2D image analysis software revealed preferential accumulation of a few glycinin subunits contributed to the increased protein content in the introgressed lines. Amino acid analysis revealed that even though the leginsulin introgressed lines had higher protein, leginsulin, and elemental sulfur, the overall concentration of sulfur-containing amino acids was not significantly altered when compared with the parental lines. The experimental soybean lines developed during this study (Leg-3, Leg-7, and Leg-8) lack A5, A4, and B3 glycinin subunits and could be utilized in breeding programs to develop high-quality tofu cultivars. PMID:25756929

  5. [Carboxylic proteinase from Trichoderma lignorum].

    Science.gov (United States)

    Rudenskaia, G N; Osterman, A L; Stepanov, V M

    1980-04-01

    A carboxylic proteinase has been isolated from a commercial preparation of Trichoderma lignorum used as a source of cellulolytic enzymes. The purification procedure included precipitation by (NH4)2SO4 (65% saturation), gel-filtration through Acrylex P-10, affinity chromatography on gramicidin S bound to an inorganic matrix, gel-filtration through Acrylex P-10, affinity chromatography on bacitracin-Sepharose and separation on Ultrogel AcA 54 followed by gel-filtration through Sephadex G-50. A 400-fold purification of enzyme was achieved, the enzyme yield being 7,2%. The molecular weight of carboxylic proteinase as determined by gel-filtration is 33 000; its amino acid composition is found to be similar to that of carboxylic proteinases isolated from other fungal species. The enzyme is stable within the pH range of 3,0-6,0. The enzyme was fully inhibited by the specific inhibitors of carboxyliec proteinases-N-diazoacetyl-N'-2,4-dinitrophenylethylenediamine and pepstatin. PMID:6991003

  6. Formation of biogenic amines in raw milk Hispánico cheese manufactured with proteinases and different levels of starter culture.

    Science.gov (United States)

    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 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. PMID:11079699

  7. 'Pergularain e I'--a plant cysteine protease with thrombin-like activity from Pergularia extensa latex.

    Science.gov (United States)

    Shivaprasad, Holenarasipura V; Rajaiah, Rajesh; Frey, Brigitte M; Frey, Felix J; Vishwanath, Bannikuppe S

    2010-03-01

    Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing approximately 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependent and time-dependent manner. At 10 microg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues. PMID:19853890

  8. The Cysteine Protease–Cysteine Protease Inhibitor System Explored in Soybean Nodule Development

    Directory of Open Access Journals (Sweden)

    Marian Dorcas Quain

    2013-08-01

    Full Text Available Almost all protease families have been associated with plant development, particularly senescence, which is the final developmental stage of every organ before cell death. Proteolysis remobilizes and recycles nitrogen from senescent organs that is required, for example, seed development. Senescence-associated expression of proteases has recently been characterized using large-scale gene expression analysis seeking to identify and characterize senescence-related genes. Increasing activities of proteolytic enzymes, particularly cysteine proteases, are observed during the senescence of legume nodules, in which a symbiotic relationship between the host plant and bacteria (Rhizobia facilitate the fixation of atmospheric nitrogen. It is generally considered that cysteine proteases are compartmentalized to prevent uncontrolled proteolysis in nitrogen-fixing nodules. In addition, the activities of cysteine proteases are regulated by endogenous cysteine protease inhibitors called cystatins. These small proteins form reversible complexes with cysteine proteases, leading to inactivation. However, very little is currently known about how the cysteine protease-cysteine protease inhibitor (cystatin system is regulated during nodule development. Moreover, our current understanding of the expression and functions of proteases and protease inhibitors in nodules is fragmented. To address this issue, we have summarized the current knowledge and techniques used for studying proteases and their inhibitors including the application of “omics” tools, with a particular focus on changes in the cysteine protease-cystatin system during nodule development.

  9. Maize rayado fino virus-like particles expressed in tobacco plants: a new platform for cysteine selective bioconjugation peptide display

    Science.gov (United States)

    The ability of plant virus coat proteins to self-assemble into virus-like particles (VLPs), coupled with unique properties including three-dimensional structures, orthogonal reactivities, suitability for genetic manipulation and chemical bio-conjugation, provide potential utility in nanotechnology a...

  10. Endogenous inhibitors of lysosomal proteinases.

    OpenAIRE

    Pontremoli, S.; Melloni, E.; Salamino, F.; Sparatore, B.; Michetti, M.; Horecker, B. L.

    1983-01-01

    Specific inhibitors of three lysosomal proteinases are present in the cytosolic and lysosomal compartments of rabbit liver. The cytosolic inhibitors, purified by chromatography on DEAE-Trisacryl and Sephadex G-75, show specificities toward cathepsin M, cathepsins B and L, and fructose 1,6-bisphosphatase converting enzyme (CE), respectively, and are designated IM, IB/L, and ICE. Inhibitors with similar specificities have been isolated from the intralysosomal compartment. Two of these inhibitor...

  11. Allium micronucleus (MNC) assay to assess bioavailability, bioconcentration and genotoxicity of mercury from solid waste deposits of a chloralkali plant, and antagonism of L-cysteine.

    Science.gov (United States)

    Panda, K K; Lenka, M; Panda, B B

    1989-02-01

    Samples of solid waste from a chloralkali plant were bioassayed employing the Allium micronucleus (MNC) assay. The endpoints measured were root length, root mercury and frequency of root meristematic cells with MNC. Chemical extraction methods such as 10% HNO3, ammonium acetate-EDTA and 0.05 or 0.1 M CaCl2 were used to assess the bioavailability of mercury from soil contaminated with solid waste. Analysis of mercury was by cold vapour atomic absorption spectrophotometry. The frequency of MNC induced by contaminated soil, which followed a concentration-response curve, was statistically correlated with soil mercury, extractable mercury and root mercury. The antagonism of L-cysteine against the suppression of root growth and induction of MNC by solid waste suggested the involvement of mercury as well as thiol groups in the process. The threshold assessment values, such as the median effective concentration (EC50) for root growth, gross toxicity concentration tested (GTCT) as indicated by complete inhibition of sprouting of roots, lowest effective concentration tested (LECT) and highest ineffective concentration tested (HICT) for induction of MNC, were determined. These assessment values may be useful for environmental management and regulatory purposes. Furthermore, the detection limit of the Allium MNC assay for solid waste mercury as indicated by LECT was 9.6 mg kg-1 which corresponded to 0.13 mg kg-1 of 0.05 M CaCl2 extractable or 8.3 mg kg-1 dry weight bioconcentrated mercury. PMID:2928769

  12. Specificity of Binding of the Low Density Lipoprotein Receptor-related Protein to Different Conformational States of the Clade E Serpins Plasminogen Activator Inhibitor-1 and Proteinase Nexin-1*

    OpenAIRE

    Jensen, Jan K.; Dolmer, Klavs; Gettins, Peter G. W.

    2009-01-01

    The low density lipoprotein receptor-related protein (LRP) is the principal clearance receptor for serpins and serpin-proteinase complexes. The ligand binding regions of LRP consist of clusters of cysteine-rich ?40-residue complement-like repeats (CR), with cluster II being the principal ligand-binding region. To better understand the specificity of binding at different sites within the cluster and the ability of LRP to discriminate in vivo between uncomplexed and proteinase-complexed serpi...

  13. Proteinases and their inhibitors in liver cancer

    OpenAIRE

    Verena Puxbaum, Lukas Mach

    2009-01-01

    Proteinases are known to be involved in many cancer-related processes, particularly in the breakdown of extracellular matrix barriers in the course of tumor invasion and metastasis. In this review we summarize the current knowledge about the role of the most important matrix-degrading proteinases (cathepsins, matrix metalloproteinases, plasmin/plasminogen activators) and their respective inhibitors in liver cancer progression and metastasis.

  14. Structural insights into the substrate specificity and activity of ervatamins, the papain-like cysteine proteases from a tropical plant, Ervatamia coronaria.

    Science.gov (United States)

    Ghosh, Raka; Chakraborty, Sibani; Chakrabarti, Chandana; Dattagupta, Jiban Kanti; Biswas, Sampa

    2008-02-01

    Multiple proteases of the same family are quite often present in the same species in biological systems. These multiple proteases, despite having high homology in their primary and tertiary structures, show deviations in properties such as stability, activity, and specificity. It is of interest, therefore, to compare the structures of these multiple proteases in a single species to identify the structural changes, if any, that may be responsible for such deviations. Ervatamin-A, ervatamin-B and ervatamin-C are three such papain-like cysteine proteases found in the latex of the tropical plant Ervatamia coronaria, and are known not only for their high stability over a wide range of temperature and pH, but also for variations in activity and specificity among themselves and among other members of the family. Here we report the crystal structures of ervatamin-A and ervatamin-C, complexed with an irreversible inhibitor 1-[l-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane (E-64), together with enzyme kinetics and molecular dynamic simulation studies. A comparison of these results with the earlier structures helps in a correlation of the structural features with the corresponding functional properties. The specificity constants (k(cat)/K(m)) for the ervatamins indicate that all of these enzymes have specificity for a branched hydrophobic residue at the P2 position of the peptide substrates, with different degrees of efficiency. A single amino acid change, as compared to ervatamin-C, in the S2 pocket of ervatamin-A (Ala67-->Tyr) results in a 57-fold increase in its k(cat)/K(m) value for a substrate having a Val at the P2 position. Our studies indicate a higher enzymatic activity of ervatamin-A, which has been subsequently explained at the molecular level from the three-dimensional structure of the enzyme and in the context of its helix polarizibility and active site plasticity. PMID:18167146

  15. PAPAIN, A PLANT ENZYME OF BIOLOGICAL IMPORTANCE: A REVIEW

    Directory of Open Access Journals (Sweden)

    Ezekiel Amri

    2012-01-01

    Full Text Available Papain is a plant proteolytic enzyme for the cysteine proteinase family cysteine protease enzyme in which enormous progress has been made to understand its functions. Papain is found naturally in papaya (Carica papaya L. manufactured from the latex of raw papaya fruits. The enzyme is able to break down organic molecules made of amino acids, known as polypeptides and thus plays a crucial role in diverse biological processes in physiological and pathological states, drug designs, industrial uses such as meat tenderizers and pharmaceutical preparations. The unique structure of papain gives it the functionality that helps elucidate how proteolytic enzymes work and also makes it valuable for a variety of purposes. In the present review, its biological importance, properties and structural features that are important to an understanding of their biological function are presented. Its potential for production and market opportunities are also discussed.

  16. Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): molecular characterization and transcriptional response upon immune stimulation.

    Science.gov (United States)

    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. PMID:23859879

  17. Revisiting the enzymes stored in the laticifers of Carica papaya in the context of their possible participation in the plant defence mechanism.

    Science.gov (United States)

    El Moussaoui, A; Nijs, M; Paul, C; Wintjens, R; Vincentelli, J; Azarkan, M; Looze, Y

    2001-04-01

    In the tropical species Carica papaya, the articulated and anastomosing laticifers form a dense network of vessels displayed in all aerial parts of the plant. Damaging the papaya tree inevitably severs its laticifers, eliciting an abrupt release of latex. Besides the well-known cysteine proteinases, papain, chymopapain, caricain and glycyl endopeptidase, papaya latex is also a rich source of other enzymes. Together, these enzymes could provide an important contribution to plant defence mechanisms by sanitising and sealing the wounded areas on the tree. PMID:11361091

  18. Pest Protection Conferred by a Beta vulgaris Serine Proteinase Inhibitor Gene

    OpenAIRE

    Smigocki, Ann C.; Ivic-haymes, Snezana; Li, Haiyan; Savic?, 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 3...

  19. Differential antibiosis against Helicoverpa armigera exerted by distinct inhibitory repeat domains of Capsicum annuum proteinase inhibitors.

    Science.gov (United States)

    Joshi, Rakesh S; Gupta, Vidya S; Giri, Ashok P

    2014-05-01

    Plant defensive serine proteinase inhibitors (PIs) are known to have negative impact on digestive physiology of herbivore insects and thus have a crucial role in plant protection. Here, we have assessed the efficacy and specificity of three previously characterized inhibitory repeat domain (IRD) variants from Capsicum annuum PIs viz., IRD-7, -9 and -12 against gut proteinases from Helicoverpa armigera. Comparative study of in silico binding energy revealed that IRD-9 possesses higher affinity towards H. armigera serine proteinases as compared to IRD-7 and -12. H. armigera fed on artificial diet containing 5 TIU/g of recombinant IRD proteins exhibited differential effects on larval growth, survival rate and other nutritional parameters. Major digestive gut trypsin and chymotrypsin genes were down regulated in the IRD fed larvae, while few of them were up-regulated, this indicate alterations in insect digestive physiology. The results corroborated with proteinase activity assays and zymography. These findings suggest that the sequence variations among PIs reflect in their efficacy against proteinases in vitro and in vivo, which also could be used for developing tailor-made multi-domain inhibitor gene(s). PMID:24559910

  20. Proteinase inhibitors in Brazilian leguminosae

    Scientific Electronic Library Online (English)

    C. A. M., Sampaio; M. L. V., Oliva; A. S., Tanaka; M. U., Sampaio.

    Full Text Available SciELO Brazil | Language: English Abstract in english Serine proteinase inhitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, a digestive enzyme, Factor XIIa and human plasma Kallikrein, two blood clotting factors. The inhibitors were purified from Enterolobium contortisiliquum (Mr=23,0 [...] 00), Torresea cearensis (Mr = 13,000), Bauhinia pentandra (Mr = 20,000) and Bauhinia bauhinioides (Mr = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XSSa, but does nor affect plasma kallikrein; both Bauhinia inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the Bpentandra inhibitor affects Factor XIIa. Ki values were calculated between 10 [raised to the power of] -7 and 10 [raised to the power of] -8 M.

  1. [Production and properties of recombinant glutenin-cleaving proteinases from Eurygaster integriceps Put].

    Science.gov (United States)

    2014-01-01

    cDNAs coding for a mature form of glutenin-cleaving trypsin-like proteinase (referred to as glutenin-hydrolyzing proteinase 3 or GHP3) from the insect pest-Eurygaster integriceps Put. and a zymogen of this proteinase containing a signal peptide required for protein secretion were cloned into vectors pPIC9 and pPIC3.5, respectively. The constructs were used for protein expression in cells of the methylotrophic yeast Pichia pastoris. The recombinant protein corresponding to the mature form of the proteinase was secreted into the culture medium and possessed proteolytic activity, while the zymogen acquired activity after trypsin, treatment. Both recombinant enzymes hydrolyzed high-molecular weight glutenin subunits from wheat of the variety Ege-88 and a range of other soft and durum wheat varieties. Chymotrypsin inhibitor I from potatoes and related inhibitors from seeds of plants of the subclass Asteridae, the Kunitz-type trypsin inhibitor from soybeans, and bovine aprotinin had a weak inhibitory effect on the recombinant proteinases, while the Bowman-Birk trypsin and chymotrypsin inhibitor from soybeans did not interact with these enzymes: PMID:25707103

  2. Insect resistance to sugar beet pests mediated by a Beta vulgaris proteinase inhibitor transgene

    Science.gov (United States)

    We transformed sugar beet (Beta vulgaris) hairy roots and Nicotiana benthamiana plants with a Beta vulgaris root gene (BvSTI) that codes for a serine proteinase inhibitor. BvSTI is a root gene cloned from the F1016 breeding line that has moderate levels of resistance to the sugar beet root maggot ...

  3. An L-cysteine Dependent Nitrate Reductase Inactivating Factor in Synchronous Chlorella sorokiniana.

    Science.gov (United States)

    Tischner, R; Schmidt, A

    1984-12-01

    Nitrate reductasee activity of cell-free Chlorella systems is inactivated in the presence of specific thiols. Out of 14 different thiols tested only L-cysteine and to a certain extent D-cysteine catalyzed an inactivation of the Chlorella nitrate reductase. This systeme was active only with reduced L-cysteine, since oxidized cystine had no effect, demonstrating that the reduced thiol group is necessary. A factor not identical with the nitrate reductase itself seems involved in this inactivation system. Evidence for this is that purified nitrate reductase is not inactivated by L-cysteine; however, when purified nitrate reductase was added to extracts inactivated by L-cysteine it was inactivated as well. The highest rate of degradation was found at a pH around 8. The data suggest that the inactivation factor from Chlorella is a proteinase, which has to be activated by L-cysteine. These results are discussed in relation to the regulation of assimilatory nitrate reduction. PMID:23195713

  4. Autocatalytic processing of pro-papaya proteinase IV is prevented by crowding of the active-site cleft.

    Science.gov (United States)

    Baker, K C; Taylor, M A; Cummings, N J; Tuñón, M A; Worboys, K A; Connerton, I F

    1996-06-01

    The DNA coding for pro-papaya proteinase IV (PPIV) has been cloned and expressed in Escherichia coli. Heterologous expression of the protein, followed by refolding in vitro, yields an enzymatically active pro-enzyme which fails to autodigest to form the mature protein. Mutagenesis of the active site of papain to simulate that of PPIV yields a proenzyme which also fails to autoactivate. Complementary mutagenesis of the pro-region/mature boundary of PPIV, to introduce its own substrate recognition sequence, has, however, produced a pro-enzyme that will autocatalytically cleave. This is the first report of enzymatic activity in a recombinant pro-cysteine proteinase, and the first time that such a protein has been shown to fail to autocatalytically cleave because of its stringent substrate specificity. PMID:8862553

  5. Formation of cysteine-S-conjugates in the Maillard reaction of cysteine and xylose.

    Science.gov (United States)

    Cerny, Christoph; Guntz-Dubini, Renée

    2013-11-15

    Cysteine-S-conjugates (CS-conjugates) occur in foods derived from plant sources like grape, passion fruit, onion, garlic, bell pepper and hops. During eating CS-conjugates are degraded into aroma-active thiols by ?-lyases that originate from oral microflora. The present study provides evidence for the formation of the CS-conjugates S-furfuryl-l-cysteine (FFT-S-Cys) and S-(2-methyl-3-furyl)-l-cysteine (MFT-S-Cys) in the Maillard reaction of xylose with cysteine at 100°C for 2h. The CS-conjugates were isolated using cationic exchange and reversed-phase chromatography and identified by (1)H NMR, (13)C NMR and LC-MS(2). Spectra and LC retention times matched those of authentic standards. To the best of our knowledge, this is the first time that CS-conjugates are described as Maillard reaction products. Furfuryl alcohol (FFA) is proposed as an intermediate which undergoes a nucleophilic substitution with cysteine. Both FFT-S-Cys and MFT-S-Cys are odourless but produce strong aroma when tasted in aqueous solutions, supposedly induced by ? -lyases from the oral microflora. The perceived aromas resemble those of the corresponding aroma-active thiols 2-furfurylthiol (FFT) and 2-methyl-3-furanthiol (MFT) which smell coffee-like and meaty, respectively. PMID:23790889

  6. Cloning eleven midgut trypsin cDNAs and evaluating the interaction of proteinase inhibitors with Cry1Ac against the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Zhu, Yu Cheng; Guo, Zibiao; Abel, Craig

    2012-10-01

    Midgut trypsins are associated with Bt protoxin activation and toxin degradation. Proteinase inhibitors have potential insecticidal toxicity against a wide range of insect species. This study was conducted to evaluate the interaction of proteinase inhibitors with Bt toxin and to examine midgut trypsin gene profile of Heliothis virescens. A sublethal dose (15 ppb) of Cry1Ac, 0.75% soybean trypsin inhibitor, and 0.1% and 0.2% N-?-tosyl-L-lysine chloromethyl ketone significantly suppressed midgut proteinase activities, and resulted in reductions in larval and pupal size and mass. The treatment with inhibitor+Bt suppressed approximately 65% more larval body mass and 21% more enzymatic activities than the inhibitor-only or Bt-only. Eleven trypsin-like cDNAs were sequenced from the midgut of H. virescens. All trypsins contained three catalytic center residues (H(73), D(153), and S(231)), substrate specificity determinant residues (D(225), G(250), and G(261)), and six cysteines for disulfide bridges. These putative trypsins were separated into three distinct groups, indicating the diverse proteinases evolved in this polyphagous insect. These results indicated that the insecticidal activity of proteinase inhibitors may be used to enhance Bt toxicity and delay resistance development. PMID:22824002

  7. Stimulation of fibroblast proliferation by the plant cysteine protease CMS2MS2 is independent of its proteolytic activity and requires ERK activation.

    Science.gov (United States)

    Gomes, Marco Túlio R; Turchetti, Andréia P; Lopes, Miriam T P; Salas, Carlos E

    2009-12-01

    The cysteine protease CMS2MS2 from Carica candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In experiments with CMS2MS2 irreversibly inhibited by E-64, the proliferative effect on fibroblasts remains unaffected. ERK phosphorylation mediated by CMS2MS2 was abolished in the presence of PD 98059 or U0126, both MAPK cascade inhibitors. In addition, these inhibitors suppress the mitogenic activity of intact CMS2MS2 or CMS2MS2-E-64. Furthermore, ERK phosphorylation and the mitogenic effect are partially suppressed by a phospholipase C (PLC) inhibitor. These data suggest that the mitogenic effect of CMS2MS2 on fibroblasts is independent of its proteolytic activity, requires ERK phosphorylation, and involves activation of PLC. PMID:19747075

  8. Squash inhibitor family of serine proteinases

    Energy Technology Data Exchange (ETDEWEB)

    Otlewski, J.; Krowarsch, D. [Institute of Biochemistry, University of Wroclaw, Wroclaw (Poland)

    1996-12-31

    Squash inhibitors of serine proteinases form an uniform family of small proteins. They are built of 27-33 amino-acid residues and cross-linked with three disulfide bridges. The reactive site peptide bond (P1-P1`) is between residue 5 (Lys, Arg or Leu) and 6 (always Ile). High resolution X-ray structures are available for two squash inhibitors complexed with trypsin. NMR solution structures have also been determined for free inhibitors. The major structural motif is a distorted, triple-stranded antiparallel beta-sheet. A similar folding motif has been recently found in a number of proteins, including: conotoxins from fish-hunting snails, carboxypeptidase inhibitor from potato, kalata B1 polypeptide, and in some growth factors (e.g. nerve growth factor, transforming growth factor {beta}2, platelet-derived growth factor). Squash inhibitors are highly stable and rigid proteins. They inhibit a number of serine proteinases: trypsin, plasmin, kallikrein, blood clotting factors: X{sub a} and XII{sub a}, cathepsin G. The inhibition spectrum can be much broadened if specific amino-acid substitutions are introduced, especially at residues which contact proteinase. Squash inhibitors inhibit proteinases via the standard mechanism. According to the mechanism, inhibitors are substrates which exhibit at neutral pH a high k{sub cat}/K{sub m} index for hydrolysis and resynthesis of the reactive site, and a low value of the hydrolysis constant. (author) 81 refs, 4 figs

  9. Squash inhibitor family of serine proteinases

    International Nuclear Information System (INIS)

    Squash inhibitors of serine proteinases form an uniform family of small proteins. They are built of 27-33 amino-acid residues and cross-linked with three disulfide bridges. The reactive site peptide bond (P1-P1') is between residue 5 (Lys, Arg or Leu) and 6 (always Ile). High resolution X-ray structures are available for two squash inhibitors complexed with trypsin. NMR solution structures have also been determined for free inhibitors. The major structural motif is a distorted, triple-stranded antiparallel beta-sheet. A similar folding motif has been recently found in a number of proteins, including: conotoxins from fish-hunting snails, carboxypeptidase inhibitor from potato, kalata B1 polypeptide, and in some growth factors (e.g. nerve growth factor, transforming growth factor ?2, platelet-derived growth factor). Squash inhibitors are highly stable and rigid proteins. They inhibit a number of serine proteinases: trypsin, plasmin, kallikrein, blood clotting factors: Xa and XIIa, cathepsin G. The inhibition spectrum can be much broadened if specific amino-acid substitutions are introduced, especially at residues which contact proteinase. Squash inhibitors inhibit proteinases via the standard mechanism. According to the mechanism, inhibitors are substrates which exhibit at neutral pH a high kcat/Km index for hydrolysis and resynthesis of the reactive site, and a low value of the hydrolysis constant. (author)ysis constant. (author)

  10. The relevance of compartmentation for cysteine synthesis in phototrophic organisms.

    Science.gov (United States)

    Birke, Hannah; Müller, Stefanie J; Rother, Michael; Zimmer, Andreas D; Hoernstein, Sebastian N W; Wesenberg, Dirk; Wirtz, Markus; Krauss, Gerd-Joachim; Reski, Ralf; Hell, Rüdiger

    2012-06-01

    In the vascular plant Arabidopsis thaliana, synthesis of cysteine and its precursors O-acetylserine and sulfide is distributed between the cytosol, chloroplasts, and mitochondria. This compartmentation contributes to regulation of cysteine synthesis. In contrast to Arabidopsis, cysteine synthesis is exclusively restricted to chloroplasts in the unicellular green alga Chlamydomonas reinhardtii. Thus, the question arises, whether specification of compartmentation was driven by multicellularity and specified organs and tissues. The moss Physcomitrella patens colonizes land but is still characterized by a simple morphology compared to vascular plants. It was therefore used as model organism to study evolution of compartmented cysteine synthesis. The presence of O-acetylserine(thiol)lyase (OAS-TL) proteins, which catalyze the final step of cysteine synthesis, in different compartments was applied as criterion. Purification and characterization of native OAS-TL proteins demonstrated the presence of five OAS-TL protein species encoded by two genes in Physcomitrella. At least one of the gene products is dual targeted to plastids and cytosol, as shown by combination of GFP fusion localization studies, purification of chloroplasts, and identification of N termini from native proteins. The bulk of OAS-TL protein is targeted to plastids, whereas there is no evidence for a mitochondrial OAS-TL isoform and only a minor part of OAS-TL protein is localized in the cytosol. This demonstrates that subcellular diversification of cysteine synthesis is already initialized in Physcomitrella but appears to gain relevance later during evolution of vascular plants. PMID:22543690

  11. Proteins of circularly permuted sequence present within the same organism: The major serine proteinase inhibitor from Capsicum annuum seeds

    OpenAIRE

    Antcheva, Nikolinka; Pintar, Alessandro; Patthy, Andra?s; Simoncsits, Andra?s; Barta, Endre; Tchorbanov, Bojidar; Pongor, Sa?ndor

    2001-01-01

    The major serine proteinase inhibitor from bell pepper (Capsicum annuum, paprika) seeds was isolated, characterized, and sequenced, and its disulfide bond topology was determined. PSI-1.2 is a 52-amino-acid-long, cysteine-rich polypeptide that inhibits both trypsin (Ki = 4.6 × 10?9 M) and chymotrypsin (Ki = 1.1 × 10?8 M) and is a circularly permuted member of the potato type II inhibitor family. Mature proteins of this family are produced from precursor proteins containing two to eight ...

  12. Cloning and sequence analysis of serine proteinase of Gloydius ussuriensis venom gland

    International Nuclear Information System (INIS)

    Objective: To construct a cDNA library by using mRNA from Gloydius ussuriensis (G. Ussuriensis) venom gland, to clone and analyze serine proteinase gene from the cDNA library. Methods: Total RNA was isolated from venom gland of G. ussuriensis, mRNA was purified by using mRNA isolation Kit. The whole length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure, lately cDAN was cloned into vector pBluescrip-sk. The recombinant cDNA was transformed into E. coli DH5?. The cDNA of serine proteinase gene in the venom gland of G. ussuriensis was detected and amplified using the in situ hybridization. The cDNA fragment was inserted into pGEMT vector, cloned and its nucleotide sequence was determined. Results: The capacity of cDNA library of venom gland was above 2.3 x 106. Its open reading frame was composed of 702 nucleotides and coded a protein pre-zymogen of 234 amino acids. It contained 12 cysteine residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shared high identity with the thrombin-like enzyme genes of other snakes in the GenBank. the query sequence exhibited strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase I of D. acutus and serine protease catroxase II of C. atrox respectively. Based on the amino acid sequences of other thrombin-like enzymes, the catalytic residues and disulfide bridges catalytic residues and disulfide bridges of this thrombin-like enzyme were deduced as follows: catalytic residues, His41, Asp86, Ser180; and six disulfide bridges Cys7-Cys139, Cys26-Cys42, Cys74-Cys232, Cys118-Cys186, Cys150-Cys165, Cys176-Cys201. Conclusion: The capacity of cDNA library of venom gland is above 2.3 x 106, overtop the level of 105 capicity. The constructed cDNA library of G. ussuriensis venom gland would be helpful platform to detect new target genes and further gene manipulate. The cloned serine proteinase gene exhibits strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase I of D. acutus and serine protease catroxase II of C. atrox respectively. (authors)

  13. Differential expression of cysteine desulfurases in soybean

    Directory of Open Access Journals (Sweden)

    Heis Marta D

    2011-11-01

    Full Text Available Abstract Background Iron-sulfur [Fe-S] clusters are prosthetic groups required to sustain fundamental life processes including electron transfer, metabolic reactions, sensing, signaling, gene regulation and stabilization of protein structures. In plants, the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the cell. Many environmental factors affect plant development and limit productivity and geographical distribution. The impact of these limiting factors is particularly relevant for major crops, such as soybean, which has worldwide economic importance. Results Here we analyze the transcriptional profile of the soybean cysteine desulfurases NFS1, NFS2 and ISD11 genes, involved in the biogenesis of [Fe-S] clusters, by quantitative RT-PCR. NFS1, ISD11 and NFS2 encoding two mitochondrial and one plastid located proteins, respectively, are duplicated and showed distinct transcript levels considering tissue and stress response. NFS1 and ISD11 are highly expressed in roots, whereas NFS2 showed no differential expression in tissues. Cold-treated plants showed a decrease in NFS2 and ISD11 transcript levels in roots, and an increased expression of NFS1 and ISD11 genes in leaves. Plants treated with salicylic acid exhibited increased NFS1 transcript levels in roots but lower levels in leaves. In silico analysis of promoter regions indicated the presence of different cis-elements in cysteine desulfurase genes, in good agreement with differential expression of each locus. Our data also showed that increasing of transcript levels of mitochondrial genes, NFS1/ISD11, are associated with higher activities of aldehyde oxidase and xanthine dehydrogenase, two cytosolic Fe-S proteins. Conclusions Our results suggest a relationship between gene expression pattern, biochemical effects, and transcription factor binding sites in promoter regions of cysteine desulfurase genes. Moreover, data show proportionality between NFS1 and ISD11 genes expression.

  14. Keratinolytic proteinase from Bacillus thuringiensis AD-12.

    Science.gov (United States)

    Gegeckas, Audrius; Gudiukait?, 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. PMID:24857878

  15. Identification of trypsin-inhibitory site and structure determination of human SPINK2 serine proteinase inhibitor.

    Science.gov (United States)

    Chen, Ting; Lee, Tian-Ren; Liang, Wei-Guang; Chang, Wun-Shaing Wayne; Lyu, Ping-Chiang

    2009-10-01

    Human serine proteinase inhibitor Kazal-type 2 (SPINK2) functions as a trypsin/acrosin inhibitor and is synthesized mainly in the testis and seminal vesicle where its activity is engaged in fertility. The SPINK2 protein contains a typical Kazal domain composed by six cysteine residues forming three disulfide bridges. The expression of SPINK2 is closely related to cancer such as lymphomas, in that a high transcript level of SPINK2 in patients with primary cutaneous follicle center cell lymphomas have better prognosis with lower mortality. To clarify the role of SPINK2 in cancer, we performed quantitative real-time PCR and showed that the expression level of SPINK2 is significantly elevated in most leukemia cell lines except B-lymphoblast TK-6 cells. The molecular function and structural features of SPINK2 were also investigated by employing the recombinant active and mutant inactive SPINK2 proteins to determine its key P2-P2' (Pro(23)-Arg(24)-His(25)-Phe(26)) active site. The inhibition assay results demonstrated that Arg(24) at the P1 site is crucial for the specificity of SPINK2 on target enzyme. Although His(25) at the P1' and Phe(26) at the P2' residues are also involved in trypsin-SPINK2 interaction, Pro(23) at the P2 site may not be directly participated in interacting with trypsin. In addition, we determined the 3D solution structure of SPINK2 and used this structure to predict the SPINK2-proteinase complex structure and binding properties. These studies not only provide critical information about the structural properties and biophysical features of the SPINK2 proteinase inhibitor, but also suggest its important role in tumor progression and response to treatment. PMID:19422058

  16. The main proteinases in Dermatobia hominis second and third instars larvae are serine-proteinases.

    Science.gov (United States)

    Pires, F A; Moya-Borja, G E; Barreira, J D; Pinho, R T; Alves, C R

    2007-04-30

    We performed a combination of proteinase assay, either in solution or immobilized in sodium dodecyl sulfate-polyacrylamide gel copolymerized with gelatin, to detect and quantify proteinases of Dermatobia hominis second (L2) and third (L3) instar larvae. In the quantitative assay, we examined proteinase activity by hydrolysis of a panel of peptide bonds specific for the main proteinase classes. We verified that the pGlu-Phe-Leu p-nitroanilide substrate was hydrolyzed by crude extracts of L2 (3.0+/-0.2 nmol h(-1)mg of protein(-1)) and L3 (7.7+/-0.1 nmol h(-1)mg of protein(-1)) and that both activities were partially inhibited by trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane, 15% and 3%, respectively. Also, we demonstrated that the Nalpha-p-Tosyl-l-Arg methyl ester substrate was hydrolyzed by crude extracts of L2 (117+/-24 nmol h(-1)mg of protein(-1)) and L3 (111+/-10 nmol h(-1)mg of protein(-1)), suggesting a predominance of esterase activity in the crude larval preparation. Interestingly, the specific activity of serine-proteinases was totally inhibited by phenylmethylsulphonyl fluoride in the L3 crude extract, while only 10% of this enzyme class activity was inhibited in the L2 crude extract. The results of the qualitative assays with substrate gels suggested that L2 and L3 larvae express serine-proteinases with similar (13 and 22 kDa) and distinct (50 kDa in L2 and 30 kDa in L3) relative molecular masses. These findings contribute to the biochemical characterization of D. hominis L2 and L3 larvae. PMID:17293049

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

    Directory of Open Access Journals (Sweden)

    Craik Charles S

    2010-07-01

    Full Text Available Abstract 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.

  18. Stress inducible proteinase inhibitor diversity in Capsicum annuum

    Directory of Open Access Journals (Sweden)

    Mishra Manasi

    2012-11-01

    Full Text Available Abstract Background Wound-inducible Pin-II Proteinase inhibitors (PIs are one of the important plant serine PIs which have been studied extensively for their structural and functional diversity and relevance in plant defense against insect pests. To explore the functional specialization of an array of Capsicum annuum (L. proteinase inhibitor (CanPIs genes, we studied their expression, processing and tissue-specific distribution under steady-state and induced conditions. Inductions were performed by subjecting C. annuum leaves to various treatments, namely aphid infestation or mechanical wounding followed by treatment with either oral secretion (OS of Helicoverpa armigera or water. Results The elicitation treatments regulated the accumulation of CanPIs corresponding to 4-, 3-, and 2-inhibitory repeat domains (IRDs. Fourty seven different CanPI genes composed of 28 unique IRDs were identified in total along with those reported earlier. The CanPI gene pool either from uninduced or induced leaves was dominated by 3-IRD PIs and trypsin inhibitory domains. Also a major contribution by 4-IRD CanPI genes possessing trypsin and chymotrypsin inhibitor domains was specifically revealed in wounded leaves treated with OS. Wounding displayed the highest number of unique CanPIs while wounding with OS treatment resulted in the high accumulation of specifically CanPI-4, -7 and ?10. Characterization of the PI protein activity through two dimensional gel electrophoresis revealed tissue and induction specific patterns. Consistent with transcript abundance, wound plus OS or water treated C. annuum leaves exhibited significantly higher PI activity and isoform diversity contributed by 3- and 4-IRD CanPIs. CanPI accumulation and activity was weakly elicited by aphid infestation yet resulted in the higher expression of CanPI-26, -41 and ?43. Conclusions Plants can differentially perceive various kinds of insect attacks and respond appropriately through activating plant defenses including regulation of PIs at transcriptional and post-translational levels. Based on the differentially elicited CanPI accumulation patterns, it is intriguing to speculate that generating sequence diversity in the form of multi-IRD PIs is a part of elaborative plant defense strategy to obtain a diverse pool of functional units to confine insect attack.

  19. Cysteine sulfoxide derivatives in Petiveria alliacea.

    Science.gov (United States)

    Kubec, R; Musah, R A

    2001-11-01

    Two diastereomers of S-benzyl-L-cysteine sulfoxide have been isolated from fresh roots of Petiveria alliacea. Their structures and absolute configurations have been determined by NMR, MALDI-HRMS, IR and CD spectroscopy and confirmed by comparison with authentic compounds. Both the R(S) and S(S) diastereomers of the sulfoxide are present in all parts of the plant (root, stem, and leaves) with the latter diastereomer being predominant. Their total content greatly varied in different parts of the plant between 0.07 and 2.97 mg g(-1) fr. wt, being by far the highest in the root. S-Benzylcysteine has also been detected in trace amounts (<10 microg g(-1) fr. wt) in all parts of the plant. This represents the first report of the presence of S-benzylcysteine derivatives in nature. PMID:11684199

  20. Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues.

    OpenAIRE

    Kobayashi, M.; Izui, H.; Nagasawa, T.; Yamada, H.

    1993-01-01

    Indole-3-acetic acid is the major auxin in most plants. In Cruciferae, including Brassicaceae, indole-3-acetic acid is synthesized from indole-3-acetonitrile by nitrilase, after indole-3-acetonitrile is formed from tryptophan via indole-3-acetaldoxime or indole glycosinolates as the intermediate. We cloned and sequenced the gene for nitrilase (EC 3.5.5.1), which catalyzes the hydrolysis of indole-3-acetonitrile to indole-3-acetic acid, from Alcaligenes faecalis JM3. The amino acid sequence de...

  1. Modification of S1 subsite specificity in the cysteine protease cathepsin B.

    Science.gov (United States)

    Fox, T; Mason, P; Storer, A C; Mort, J S

    1995-01-01

    Cysteine proteases of the papain family generally exhibit broad P1 specificity. A notable exception is papaya proteinase IV (PPIV), which only accepts Gly at this position. In all other cysteine proteases the S1 subsite residues 23 and 65 (papain numbering) are absolutely conserved as Gly, while in PPIV they are replaced by Glu and Arg, respectively. These differences appear to underlie both PPIV specificity and its resistance to inhibition by cystatins. To test this hypothesis, the equivalent residues (Gly27 and Gly73) in the mammalian cysteine protease cathepsin B were changed to Glu and Arg, respectively. Relative to the wild-type enzyme, the Gly27Glu and Gly73Arg mutants showed a drastic reduction in activity with substrates containing a P1 Arg. In contrast, substrates having a Gly residue in P1 were hydrolyzed effectively. The double mutant (Gly27Glu:Gly73Arg) exhibited no detectable activity against any substrate studied. Inhibition of the Gly73Arg mutant by E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane] was found to be similar to that of the wild-type enzyme. In contrast, inhibition by cystatin C exhibited a 20,000-fold reduction. These results demonstrate the dramatic influence of side chains at sequence locations 27 and 73 on the S1 subsite specificity of cysteine proteases. PMID:7770453

  2. Analysis of the solvent accessibility of cysteine residues on Maize rayado fino virus virus-like particles produced in Nicotiana benthamiana plants and cross-linking of peptides to VLPs.

    Science.gov (United States)

    Natilla, Angela; Hammond, Rosemarie W

    2013-01-01

    Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials, vaccines, electronic materials, chemical tools, and molecular electronic containers. In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus, Carnation mottle virus, Cowpea chlorotic mottle virus, Tobacco mosaic virus, Turnip yellow mosaic virus, and MRFV. PMID:23439009

  3. Modulation of cysteine biosynthesis in chloroplasts of transgenic tobacco overexpressing cysteine synthase [O-acetylserine(thiol)-lyase].

    Science.gov (United States)

    Saito, K; Kurosawa, M; Tatsuguchi, K; Takagi, Y; Murakoshi, I

    1994-11-01

    Cysteine synthase [O-acetyl-L-serine(thiol)-lyase, EC 4.2.99.8] (CSase), which is responsible for the terminal step of cysteine biosynthesis, catalyzes the formation of L-cysteine from O-acetyl-L-serine (OAS) and hydrogen sulfide. Three T-DNA vectors carrying a spinach (Spinacia oleracea) cytoplasmic CSase A cDNA (K. Saito, N. Miura, M. Yamazaki, H. Horano, I. Murakoshi [1992] Proc Natl Acad Sci USA 89: 8078-8082) were constructed as follows: pCSK3F, cDNA driven by the cauliflower mosaic virus (CaMV) 35S RNA promoter with a sense orientation; pCSK3R, cDNA driven by the CaMV 355 promoter with an antisense orientation; pCSK4F, cDNA fused with the sequence for chloroplast-targeting transit peptide of pea ribulose-1,5-biphosphate carboxylase small subunit driven by the CaMV 35S promoter with a sense orientation. These chimeric genes were transferred into tobacco (Nicotiana tabacum) with Agrobacterium-mediated transformation, and self-fertilized progeny were obtained. CSase activities in cell-free extracts of pCSK3F and pCSK4F transformants were 2- to 3-fold higher than those of control and pCSK3R plants. CSase activities in chloroplasts of pCSK4F transformants were severalfold higher than those of control and pCSK3F plants, indicating that the foreign CSase protein is transported and accumulated in a functionally active form in chloroplasts of pCSK4F plants. Isolated chloroplasts of a pCSK4F transformant had a more pronounced ability to form cysteine in response to addition of OAS and sulfur compounds than those of a control plant. In particular, feeding of OAS and sulfite resulted in enhanced cysteine formation, which required photoreduction of sulfite in chloroplasts. The enhanced cysteine formation in a pCSK4F plant responding to sulfite was also observed in leaf discs. In addition, these leaf discs were partially resistant to sulfite toxicity, possibly due to metabolic detoxification of sulfite by fixing into cysteine. These results suggested that overaccumulated foreign CSase in chloroplasts could modulate biosynthetic flow of cysteine in response to sulfur stress. PMID:7824657

  4. Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development.

    Science.gov (United States)

    Hendriks, T; Vreugdenhil, D; Stiekema, W J

    1991-09-01

    A highly efficient and synchronous in vitro tuberization system is described. One-node stem pieces from potato (Solanum tuberosum cv. Bintje) plants grown under short day-light conditions containing an axillary bud were cultured in the dark on a tuber-inducing medium. After 5 or 6 days all axillary buds started to develop tubers. To study gene expression during tuber development, RNA isolated from tuberizing axillary buds was used for both in vitro translation and northern blot hybridizations. The genes encoding the proteinase inhibitors I and II (PI-I and PI-II), a Kunitz- and a Bowman-Birk-type proteinase inhibitor were already expressed in uninduced axillary buds. The length of the day-light conditions differently influenced the expression level of the individual genes. In addition, the expression of each of these genes changed specifically during the development of the axillary bud to tuber. In contrast to the expression of these proteinase inhibitor genes, patatin gene expression was only detectable from the day tuberization was manifested as a radial expansion of the axillary bud. These results are discussed with respect to the regulation of the expression of the genes studied in relation to the regulation of tuber development. PMID:1715784

  5. Evaluation of the effect of staphylococcal serine proteinase on phagocytosis.

    Science.gov (United States)

    Miedzobrodzki, J; Tadeusiewicz, R; Porwit-Bóbr, Z

    1987-01-01

    Correlations between values of phagocytosis index and values of concentrations of staphylococcal serine proteinase were analysed. Preincubation of granulocytes with the proteinase stimulated phagocytosis of three bacterial strains: S. saprophyticus, S. aureus VS and S. aureus Smith diffuse. Significant correlations were also observed for S. saprophyticus strain in phagocytosis performed with or without bovine serum. Specific rabbit IgG anti-serine proteinase effected the increase of phagocytosis index only in the case of S. aureus Smith diffuse. Summary statistical analysis for all experimental conditions exhibits significant correlations also for S. aureus VS strain. No significant correlations were noted for the three remaining strains taken from patients. PMID:3508044

  6. Degradation of humoral host defense by Candida albicans proteinase.

    OpenAIRE

    Kaminishi, H.; Miyaguchi, H.; Tamaki, T.; Suenaga, N.; Hisamatsu, M.; Mihashi, I.; Matsumoto, H.; Maeda, H.; Hagihara, Y.

    1995-01-01

    The effect of an extracellular proteinase from the pathogenic yeast Candida albicans on the bactericidal and opsonizing activities of human serum was studied. The ability of human polymorphonuclear leukocytes to kill Staphylococcus aureus was greatly reduced when the bacteria were opsonized with human serum treated with the proteinase. The reduction in the opsonizing activity of human serum was attributed to degradation of the Fc portion of immunoglobulin G by the action of C. albicans protei...

  7. The Roles of ADAMs Family Proteinases in Skin Diseases

    OpenAIRE

    Kawaguchi, Masakazu; Hearing, Vincent J.

    2011-01-01

    A disintegrin and metalloproteinases (ADAMs) are members of a new gene family of transmembrane and secreted proteins, which belong to the zinc proteinase superfamily. These molecules are involved in various biological events such as cell adhesion, cell fusion, cell migration, membrane protein shedding, and proteolysis. Growing evidence now attests to the potential involvement of ADAMs proteinases in diverse processes such as skin wound healing, inflammation, pigmentation, tumor development, c...

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

    International Nuclear Information System (INIS)

    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 3H-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

  9. [Subtilisin-like proteinase SSPB from Streptomyces spheroides, strain 35].

    Science.gov (United States)

    Kre?er, V G; Rudenskaia, G N; Landau, N S; Pokrovskaia, S S; Stepanov, V M

    1983-08-01

    A serine proteinase possessing a fibrinolytic activity was isolated from a culture filtrate of Streptomyces spheroides, strain 35. A consecutive use of affinity chromatography on bacillichin-silochrome and bacitracin-sepharose and ion-exchange chromatography on anionie PAP and cationic KMT resulted in a homogeneous proteinase with 1060-fold purification and 19% yield. The enzyme has a molecular weight of 28000; its amino acid composition is Asp31, Ser28, Thr29, Glu9, Pro14, Gly35, Ala42, Val26, Ile14, Leu13, Met2, Tyr9, Phe4, Trp3, His6, Lys4, Arg10. The enzyme has a pI at pH greater than 10 and the activity optimum against Z-L-Ala-L-Ala-L-Leu-pNA at pH 10-11. The enzyme is stable within the pH range of 4-11 and in 6 M guanidinium chloride pH 8.0 in the presence of Ca2+. The enzyme is inhibited by diisopropylfluorophosphate and benzylsulfofluoride, specific inhibitors of serine proteinases as well as by potato proteinase inhibitor. The serine proteinase SSPB isolated from Str. spheroides, strain 35 can be related to subtilisin-like serine proteinase, especially to those of SGPD and SGPE of Str. griseus. PMID:6354275

  10. Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants.

    Science.gov (United States)

    Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Choi, HyungWon; Sobreira, Tiago J P; Nohara, Lilian L; Wermelinger, Luciana S; Almeida, Igor C; Zingali, Russolina B; Fernandes, Patricia M B

    2012-06-18

    Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses. PMID:22465191

  11. Comparison of proteinase inhibitor-inducing activities and phytoalexin elicitor activities of a pure fungal endopolygalacturonase, pectic fragments, and chitosans.

    Science.gov (United States)

    Walker-Simmons, M; Jin, D; West, C A; Hadwiger, L; Ryan, C A

    1984-11-01

    Rhizopus stolonifer endopolygalacturonase, an elicitor of casbene synthetase activity in castor bean seedlings, was found to be a potent elicitor of the phytoalexin pisatin in pea pods and of proteinase Inhibitor I in tomato leaves. The enzyme was an active elicitor or inducer only in its active native state; heat-denatured enzyme was inactive in all three systems. The activities of (a) the tomato pectic polysaccharide proteinase inhibitor-inducing factor, (b) a partially acid hydrolyzed proteinase inhibitor-inducing factor, (c) citrus pectic fragments, and (d) chitosan, were also compared in the three bioassay systems. The four oligosaccharide preparations were active in all three systems, but with different degrees of potency. In tomato leaves and pea pods, chitosans were most active, whereas in castor beans, the citrus pectic fragments were the best elicitors. The data presented support the hypothesis that plant and fungal cell wall fragments are important signals in mobilizing a wide variety of biochemically different types of plant defense responses, and that endopolygalacturonases play a key role in releasing the plant cell wall fragments during pest attacks. PMID:16663934

  12. Recombinant pro-regions from papain and papaya proteinase IV-are selective high affinity inhibitors of the mature papaya enzymes.

    Science.gov (United States)

    Taylor, M A; Baker, K C; Briggs, G S; Connerton, I F; Cummings, N J; Pratt, K A; Revell, D F; Freedman, R B; Goodenough, P W

    1995-01-01

    Proteolytic enzymes require the presence of their pro-regions for correct folding. Of the four proteolytic enzymes from Carica papaya, papain and papaya proteinase IV (PPIV) have 68% sequence identity. We find that their pro-regions are even more similar, exhibiting 73.6% identity. cDNAs encoding the pro-regions of these two proteinases have been expressed in Escherichia coli independently from their mature enzymes. The recombinant pro-regions of papain and PPIV have been shown to be high affinity inhibitors of all four of the mature native papaya cysteine proteinases. Their inhibition constants are in the range 10(-6) - 10(-9) M. PPIV was inhibited two to three orders of magnitude less effectively than papain, chymopapain and caricain. The pro-region of PPIV, however, inhibited its own mature enzyme more effectively than did the pro-region of papain. Alignment of the sequences of the four papaya enzymes shows that there is a highly variable section towards the C-terminal of the pro-region. This region may therefore confer selectivity to the pro-regions for the individual proteolytic enzymes. PMID:7770454

  13. The maize cystatin CC9 interacts with apoplastic cysteine proteases

    OpenAIRE

    Linde, Karina; Mueller, Andre? N.; Hemetsberger, Christoph; Kashani, Farnusch; Hoorn, Renier A. L.; Doehlemann, Gunther

    2012-01-01

    In a recent study we identified corn cystain9 (CC9) as a novel compatibility factor for the interaction of the biotrophic smut fungus Ustilago maydis with its host plant maize. CC9 is transcriptionally induced during the compatible interaction with U. maydis and localizes in the maize apoplast where it inhibits apoplastic papain-like cysteine proteases. The proteases are activated during incompatible interaction and salicylic acid (SA) treatment and, in turn, are sufficient to induce SA signa...

  14. Overexpression of a Weed (Solanum americanum Proteinase Inhibitor in Transgenic Tobacco Results in Increased Glandular Trichome Density and Enhanced Resistance to Helicoverpa armigera and Spodoptera litura

    Directory of Open Access Journals (Sweden)

    Zeng-Fu Xu

    2009-04-01

    Full Text Available In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm(Helicoverpa armigera and tobacco cutworm(Spodoptera litura larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants.

  15. Overexpression of a weed (Solanum americanum) proteinase inhibitor in transgenic tobacco results in increased glandular trichome density and enhanced resistance to Helicoverpa armigera and Spodoptera litura.

    Science.gov (United States)

    Luo, Ming; Wang, Zhaoyu; Li, Huapeng; Xia, Kuai-Fei; Cai, Yinpeng; Xu, Zeng-Fu

    2009-04-01

    In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura) larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants. PMID:19468345

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

    Science.gov (United States)

    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. PMID:24237606

  17. Neutrophil elastase and proteinase-3 trigger G protein-biased signaling through proteinase-activated receptor-1 (PAR1).

    Science.gov (United States)

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

    2013-11-15

    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 G?i-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

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

    Science.gov (United States)

    Alpi, A; Beevers, H

    1981-10-01

    Leupeptin, a 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 crystalloidstorage 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. PMID:16662011

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  20. Papain protects papaya trees from herbivorous insects: role of cysteine proteases in latex.

    Science.gov (United States)

    Konno, Kotaro; Hirayama, Chikara; Nakamura, Masatoshi; Tateishi, Ken; Tamura, Yasumori; Hattori, Makoto; Kohno, Katsuyuki

    2004-02-01

    Many plants contain latex that exudes when leaves are damaged, and a number of proteins and enzymes have been found in it. The roles of those latex proteins and enzymes are as yet poorly understood. We found that papain, a cysteine protease in latex of the Papaya tree (Carica papaya, Caricaceae), is a crucial factor in the defense of the papaya tree against lepidopteran larvae such as oligophagous Samia ricini (Saturniidae) and two notorious polyphagous pests, Mamestra brassicae (Noctuidae) and Spodoptera litura (Noctuidae). Leaves of a number of laticiferous plants, including papaya and a wild fig, Ficus virgata (Moraceae), showed strong toxicity and growth inhibition against lepidopteran larvae, though no apparent toxic factors from these species have been reported. When the latex was washed off, the leaves of these lactiferous plants lost toxicity. Latexes of both papaya and the wild fig were rich in cysteine-protease activity. E-64, a cysteine protease-specific inhibitor, completely deprived the leaves of toxicity when painted on the surface of papaya and fig leaves. Cysteine proteases, such as papain, ficin, and bromelain, all showed toxicity. The results suggest that plant latex and the proteins in it, cysteine proteases in particular, provide plants with a general defense mechanism against herbivorous insects. PMID:14731257

  1. Inhibitory potency of Erythrina variegata proteinase inhibitors toward serine proteinases in the blood coagulation and fibrinolytic systems.

    Science.gov (United States)

    Nakagaki, T; Shibuya, Y; Kouzuma, Y; Yamasaki, N; Kimura, M

    1996-08-01

    The Erythrina variegata Kunitz family trypsin inhibitors, ETIa and ETIb, prolonged the activated partial thromboplastin time (APTT) and also the prothrombin time (PT) of human plasma, but the Kunitz family chymotrypsin inhibitor, ECI, and Bowman-Birk family inhibitor, EBI, from E. variegata hardly prolonged these times. Trypsin inhibitors ETIa and ETIb inhibited the amidolytic activity of factor Xa, and ETIb but not ETIA inhibited plasma kallikrein. Neither ETIa nor ETIb exhibited any inhibitory activity toward beta-factor XIIa and thrombin. Furthermore, trypsin inhibitors ETIa and ETIb inhibited plasmin, a serine proteinase in the fibrinolytic system, whereas ECI and EBI did not. These results indicate that Erythrina Kunitz proteinase inhibitors possess different potency toward serine proteinases in the blood coagulation and fibrinolytic systems, in spite of their high similarity in amino acid sequence. PMID:8987561

  2. Regulatory Protein-Protein Interactions in Primary Metabolism: The Case of the Cysteine Synthase Complex

    Science.gov (United States)

    Sulfur is an essential nutrient for plant growth and development. In plant sulfur assimilation, cysteine biosynthesis plays a central role in fixing inorganic sulfur from the environment into the metabolic precursor for cellular thiol-containing compounds. A key regulatory feature of this process ...

  3. Getting a Knack for NAC: N-Acetyl-Cysteine

    OpenAIRE

    Sansone, Randy A.; Sansone, Lori A.

    2011-01-01

    N-acetyl-cysteine, N-acetylcysteine, N-acetyl cysteine, and N-acetyl-L-cysteine are all designations for the same compound, which is abbreviated as NAC. NAC is a precursor to the amino acid cysteine, which ultimately plays two key metabolic roles. Through its metabolic contribution to glutathione production, cysteine participates in the general antioxidant activities of the body. Through its role as a modulator of the glutamatergic system, cysteine influences the reward-reinforcement pathway....

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

    Science.gov (United States)

    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 ...

  5. DIVERSITY OF DIGESTIVE PROTEINASES IN TENEBRIO MOLITOR (COLEOPTERA: TENEBRIONIDAE) LARVAE

    Science.gov (United States)

    The spectrum of Tenebrio molitor larvae digestive proteinases was studied in the context of 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 ...

  6. Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

    Scientific Electronic Library Online (English)

    M.E., Pereira; F.A., Dörr; N.C., Peixoto; J.F., Lima-Garcia; F., Dörr; G.G., Brito.

    1633-16-01

    Full Text Available The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl an [...] d centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

  7. Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    M.E. Pereira

    2005-11-01

    Full Text Available The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

  8. Crystal structure of glycyl endopeptidase from Carica papaya: a cysteine endopeptidase of unusual substrate specificity.

    Science.gov (United States)

    O'Hara, B P; Hemmings, A M; Buttle, D J; Pearl, L H

    1995-10-10

    Glycyl endopeptidase is a cysteine endopeptidase of the papain family, characterized by specificity for cleavage C-terminal to glycyl residues only and by resistance to inhibition by members of the cystatin family of cysteine proteinase inhibitors. Glycyl endopeptidase has been crystallized from high salt with a substrate-like inhibitor covalently bound to the catalytic Cys 25. The structure has been solved by molecular replacement with the structure of papain and refined at 2.1 A to an R factor of 0.196 (Rfree = 0.258) with good geometry. The structure of the S1 substrate binding site of glycyl endopeptidase differs from that of papain by the substitution of glycines at residues 23 and 65 in papain, with glutamic acid and arginine, respectively, in glycyl endopeptidase. The side chains of these residues form a barrier across the binding pocket, effectively excluding substrate residues with large side chains from the S1 subsite. The constriction of this subsite in glycyl endopeptidase explains the unique specificity of this enzyme for cleavage after glycyl residues and is a major component of its resistance to inhibition by cystatins. PMID:7548082

  9. L-Cysteine inhibits root elongation through auxin/PLETHORA and SCR/SHR pathway in Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Zhen; Mao, Jie-Li; Zhao, Ying-Jun; Li, Chuan-You; Xiang, Cheng-Bin

    2015-02-01

    L-Cysteine plays a prominent role in sulfur metabolism of plants. However, its role in root development is largely unknown. Here, we report that L-cysteine reduces primary root growth in a dosage-dependent manner. Elevating cellular L-cysteine level by exposing Arabidopsis thaliana seedlings to high L-cysteine, buthionine sulphoximine, or O-acetylserine leads to altered auxin maximum in root tips, the expression of quiescent center cell marker as well as the decrease of the auxin carriers PIN1, PIN2, PIN3, and PIN7 of primary roots. We also show that high L-cysteine significantly reduces the protein level of two sets of stem cell specific transcription factors PLETHORA1/2 and SCR/SHR. However, L-cysteine does not downregulate the transcript level of PINs, PLTs, or SCR/SHR, suggesting that an uncharacterized post-transcriptional mechanism may regulate the accumulation of PIN, PLT, and SCR/SHR proteins and auxin transport in the root tips. These results suggest that endogenous L-cysteine level acts to maintain root stem cell niche by regulating basal- and auxin-induced expression of PLT1/2 and SCR/SHR. L-Cysteine may serve as a link between sulfate assimilation and auxin in regulating root growth. PMID:24798139

  10. Cytolytic effects of neutrophils: role for a membrane-bound neutral proteinase

    International Nuclear Information System (INIS)

    A neutral serine proteinase, purified 250-fold from the plasma membrane fraction of human neutrophils, differs in its catalytic and molecular properties from the well-known neutral proteinases present in azurophil (primary) granules. Stimulation of neutrophils with low concentrations of phorbol 12-myristate 13-acetate (PMA) results in the release into the medium of the membrane-bound proteinase and the concomitant production of oxygen radicals. These concentrations of PMA also induce full cytolytic activity measured with 51Cr-labeled ox erythrocytes. A role for the neutral serine proteinase in the cytolytic activity of PMA-stimulated neutrophils is supported by the following observations: (i) the lytic activity of the stimulated neutrophils is correlated with the quantity of neutral proteinase present in the membranes; (ii) the extracellular medium from PMA-stimulated neutrophils causes the cytolysis of 51Cr-labeled erythrocytes that have been exposed to nonlytic concentrations of H2O2; (iii) cytolysis of H2O2-treated erythrocytes is also observed with the crude proteinase solubilized from neutrophil membranes or with the purified proteinase from the same source; and (iv) in each case the cytolytic activity is proportional to the proteinase activity present and is prevented by the addition of serine proteinase inhibitors. The authors conclude that cytolysis of target cells by PMA-activated neutrophils carget cells by PMA-activated neutrophils can result from the cooperative effects of oxygen radicals and the membrane-bound neutral serine proteinase

  11. The aspartic proteinase family of three Phytophthora species

    Directory of Open Access Journals (Sweden)

    ten Have Arjen

    2011-05-01

    Full Text Available Abstract Background Phytophthora species are oomycete plant pathogens with such major social and economic impact that genome sequences have been determined for Phytophthora infestans, P. sojae and P. ramorum. Pepsin-like aspartic proteinases (APs are produced in a wide variety of species (from bacteria to humans and contain conserved motifs and landmark residues. APs fulfil critical roles in infectious organisms and their host cells. Annotation of Phytophthora APs would provide invaluable information for studies into their roles in the physiology of Phytophthora species and interactions with their hosts. Results Genomes of Phytophthora infestans, P. sojae and P. ramorum contain 11-12 genes encoding APs. Nine of the original gene models in the P. infestans database and several in P. sojae and P. ramorum (three and four, respectively were erroneous. Gene models were corrected on the basis of EST data, consistent positioning of introns between orthologues and conservation of hallmark motifs. Phylogenetic analysis resolved the Phytophthora APs into 5 clades. Of the 12 sub-families, several contained an unconventional architecture, as they either lacked a signal peptide or a propart region. Remarkably, almost all APs are predicted to be membrane-bound. Conclusions One of the twelve Phytophthora APs is an unprecedented fusion protein with a putative G-protein coupled receptor as the C-terminal partner. The others appear to be related to well-documented enzymes from other species, including a vacuolar enzyme that is encoded in every fungal genome sequenced to date. Unexpectedly, however, the oomycetes were found to have both active and probably-inactive forms of an AP similar to vertebrate BACE, the enzyme responsible for initiating the processing cascade that generates the A? peptide central to Alzheimer's Disease. The oomycetes also encode enzymes similar to plasmepsin V, a membrane-bound AP that cleaves effector proteins of the malaria parasite Plasmodium falciparum during their translocation into the host red blood cell. Since the translocation of Phytophthora effector proteins is currently a topic of intense research activity, the identification in Phytophthora of potential functional homologues of plasmepsin V would appear worthy of investigation. Indeed, elucidation of the physiological roles of the APs identified here offers areas for future study. The significant revision of gene models and detailed annotation presented here should significantly facilitate experimental design.

  12. Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen

    OpenAIRE

    Zahedifard, Farnaz; Gholami, Elham; Taheri, Tahereh; Taslimi, Yasaman; Doustdari, Fatemeh; Seyed, Negar; Torkashvand, Fatemeh; Meneses, Claudio; Papadopoulou, Barbara; Kamhawi, Shaden; Valenzuela, Jesus G.; Rafati, Sima

    2014-01-01

    More than 98 countries are reported as endemic for leishmaniasis, a vector-borne disease transmitted by sand flies. Drug-resistant forms have emerged and there is an increased need to develop advanced preventive strategies. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The lizard protozoan parasite, L. tarentolae, is nonpathogenic to humans and has been used effectively as a ...

  13. The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut.

    Science.gov (United States)

    Beton, Daniela; Guzzo, Cristiane R; Ribeiro, Alberto F; Farah, Chuck S; Terra, Walter R

    2012-09-01

    Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. PMID:22659439

  14. Analysis of serine proteinase–inhibitor interaction by alanine shaving

    OpenAIRE

    Buczek, Olga; Koscielska-kasprzak, Katarzyna; Krowarsch, Daniel; Dadlez, Micha?; Otlewski, Jacek

    2002-01-01

    We analyzed the energetic importance of residues surrounding the hot spot (the P1 position) of bovine pancreatic trypsin inhibitor (BPTI) in interaction with two proteinases, trypsin and chymotrypsin, by a procedure called molecular shaving. One to eight residues of the structural epitope, composed of two extended and exposed loops, were mutated to alanine(s). Although truncation of the side chains of residues surrounding the P1 position to methyl groups caused a decrease in ?Gden values up ...

  15. Functional role of aspartic proteinase cathepsin D in insect metamorphosis

    OpenAIRE

    Seo Sook; Je Yeon; Kim Iksoo; Yoon Hyung; Kang Pil; Choo Young; Wei Ya; Choi Yong; Kim Bo; Lee Kwang; Gui Zhong; Lee Sang; Guo Xijie; Sohn Hung; Jin Byung

    2006-01-01

    Abstract Background Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis. Results Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expressio...

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

    OpenAIRE

    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...

  17. Aminotransferase, L-amino acid oxidase and beta-lyase reactions involving L-cysteine S-conjugates found in allium extracts. Relevance to biological activity?

    Science.gov (United States)

    Cooper, Arthur J L; Pinto, John T

    2005-01-15

    Several cysteine S-conjugates that occur in extracts of garlic and other plants of the allium family possess anti-oxidant properties, and many, including S-allyl-L-cysteine (SAC) and S-allylmercapto-L-cysteine (SAMC), are promising anti-cancer agents. To understand possible biochemical mechanisms contributing to the protective effects, the ability of selected allium-derived L-cysteine S-conjugates to undergo various enzyme-catalyzed transformations was investigated. SAC, SAMC, S-propylmercapto-L-cysteine and S-penta-1,3-dienylmercapto-L-cysteine were shown to be substrates of: (a) highly purified rat kidney glutamine transaminase K (GTK); (b) purified snake venom L-amino acid oxidase; and (c) a cysteine S-conjugate beta-lyase present in rat liver cytosol. S-Methylmercapto-L-cysteine was shown to be a substrate of GTK and L-amino acid oxidase, but not of the cysteine S-conjugate beta-lyase. Evidence is presented that a major enzyme responsible for the cysteine S-conjugate beta-lyase reactions in the rat liver cytosol is gamma-cystathionase. The possible role of gamma-cystathionase in generating sulfane sulfur from the disulfide-containing cysteine S-conjugates present in allium extracts, and the possible role of this sulfane sulfur in enzyme regulation, targeting of cancer cells and detoxification reactions is discussed. An interesting side finding of the present work is that rat liver mitochondria are more active than rat liver cytosol in catalyzing a cysteine S-conjugate beta-lyase reaction with the mitochondrial protoxicant S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) at physiological pH and at low substrate concentration. PMID:15627473

  18. A change in proteinase activity of liver nuclei of gamma-irradiated rats

    International Nuclear Information System (INIS)

    Activity of nuclear proteinases in rat liver was studied, by their capacity of splitting a casein substrate, 5-48 h following ?-irradiation. The proteinase actuivity of nuclei was shown to increase by more than two times in 2-5 h and to decrease by 3-4 times after 15-48 h compared to that of the non-irradiated controls. A sharp decrease in the proteinase activity was also noted in the nuclear matrix of irradiated rat liver

  19. Trichoderma harzianum transformant has high extracellular alkaline proteinase expression during specific mycoparasitic interactions

    Directory of Open Access Journals (Sweden)

    Goldman Maria Helena S.

    1998-01-01

    Full Text Available The mycoparasite Trichoderma harzianum produces an alkaline proteinase that may be specifically involved in mycoparasitism. We have constructed transformant strains of this fungus that overexpress this alkaline proteinase. Some of the transformants were assessed for alkaline proteinase activity, and those with higher activity than the wild type were selected for further studies. One of these transformant strains produced an elevated and constitutive pbr1 mRNA level during mycoparasitic interactions with Rhizoctonia solani.

  20. Production of a heterologous proteinase A by Saccharomyces kluyveri

    DEFF Research Database (Denmark)

    MØller, K; Tidemand, L D

    2001-01-01

    In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a reference, S. cerevisiae CEN.PK 113-5D was transformed with the same plasmid and the two strains were characterised in batch cultivations on glucose. The glucose metabolism was found to be less fermentative in S. kluyveri than in S. cerevisiae. The yield of ethanol on glucose was 0.11 g/g in S. kluyveri, compared to a yield of 0.40 g/g in S. cerevisiae. Overexpression of PEP4 led to the secretion of active proteinase A in both S. kluyveri and S. cerevisiae. The yield of active proteinase A during growth on glucose was found to be 3.6-fold higher in S. kluyveri than in the S. cerevisiae reference strain.

  1. Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

    Science.gov (United States)

    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

  2. cDNA Cloning and Molecular Modeling of Procerain B, a Novel Cysteine Endopeptidase Isolated from Calotropis procera

    OpenAIRE

    Singh, Abhay Narayan; Yadav, Prity; Dubey, Vikash Kumar

    2013-01-01

    Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimen...

  3. The role of proteinase enzymes in the process of conversion of muscle to meat

    Directory of Open Access Journals (Sweden)

    Dümen Emek

    2006-01-01

    Full Text Available Post mortem meat tenderization is a complex mechanism and unfortunately it has not been fully identified scientifically. It is known that endogenous proteinases have an important role in this mechanism. Detailed studies are being performed about the destructive effects of lysosomal proteinases and calcium dependent proteinases on the myofibrils and these are most common topics that are being investigated about meat tenderization processes by the scientists. The aim of this paper is to review the role of proteinase enzymes in the process of conversion of muscle to meat. .

  4. ?-1-antitrypsin variants and the proteinase/antiproteinase imbalance in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Sinden, Nicola J; Baker, Michael J; Smith, David J; Kreft, Jan-Ulrich; Dafforn, Timothy R; Stockley, Robert A

    2015-01-15

    The excessive activities of the serine proteinases neutrophil elastase and proteinase 3 are associated with tissue damage in chronic obstructive pulmonary disease. Reduced concentrations and/or inhibitory efficiency of the main circulating serine proteinase inhibitor ?-1-antitrypsin result from point mutations in its gene. In addition, ?-2-macroglobulin competes with ?-1-antitrypsin for proteinases, and the ?-2-macroglobulin-sequestered enzyme can retain its catalytic activity. We have studied how serine proteinases partition between these inhibitors and the effects of ?-1-antitrypsin mutations on this partitioning. Subsequently, we have developed a three-dimensional reaction-diffusion model to describe events occurring in the lung interstitium when serine proteinases diffuse from the neutrophil azurophil granule following degranulation and subsequently bind to either ?-1-antitrypsin or ?-2-macroglobulin. We found that the proteinases remained uninhibited on the order of 0.1 s after release and diffused on the order of 10 ?m into the tissue before becoming sequestered. We have shown that proteinases sequestered to ?-2-macroglobulin retain their proteolytic activity and that neutrophil elastase complexes with ?-2-macroglobulin are able to degrade elastin. Although neutrophil elastase is implicated in the pathophysiology of emphysema, our results highlight a potentially important role for proteinase 3 because of its greater concentration in azurophil granules, its reduced association rate constant with all ?-1-antitrypsin variants studied here, its greater diffusion distance, time spent uninhibited following degranulation, and its greater propensity to partition to ?-2-macroglobulin where it retains proteolytic activity. PMID:25416382

  5. Nonfouling property of zwitterionic cysteine surface.

    Science.gov (United States)

    Lin, Peter; Ding, Ling; Lin, Chii-Wann; Gu, Frank

    2014-06-10

    Applications of implantable bioelectronics for analytical and curative purposes are currently limited by their poor long-term biofunctionality in physiological media and nonspecific interactions with biomolecules. In an attempt to prolong in vivo functionality, recent advances in surface modifications have demonstrated that zwitterionic coatings can rival the performance of conventional poly(ethylene glycol) polymers in reducing nonspecific protein fouling. Herein, we report the fabrication of a very thin layer of nonfouling zwitterionic cysteine surface capable of protecting implantable bioelectronics from nonspecific adsorption of plasma proteins. This work is the first of its kind to fabricate, through solution chemistry, a cysteine surface exhibiting zwitterionic state as high as 88% and to demonstrate antibiofouling under the exposure of bovine serum albumin (BSA) and human serum. The fabricated surface utilized a minimal amount of gold substrate, approximately 10 nm, and an extremely thin antifouling layer at 1.14 nm verified by ellipsometry. X-ray photoelectron spectroscopy assessment of the nitrogen (N1s) and carbon (C1s) spectra conclude that 87.8% of the fabricated cysteine surface is zwitterionic, 2.5% is positively charged, and 9.6% is noncharged. Antibiofouling performance of the cysteine surface is quantitatively determined by bicinchoninic acid (BCA) protein assay as well as qualitatively confirmed using scanning electron spectroscopy. Cysteine surfaces demonstrated a BSA fouling of 3.9 ± 4.84% ?g/cm(2), which is 93.6% and 98.5% lower than stainless steel and gold surfaces, respectively. Surface plasmon resonance imaging analysis returned similar results and suggest that a thinner cysteine coating will enhance performance. Scanning electron microscopy confirmed the results of BCA assay and suggested that the cysteine surface demonstrated a 69% reduction to serum fouling. The results reported in this paper demonstrate that it is possible to achieve a highly zwitterionic surface through solution chemistry on a macroscopic level that is capable of improving biocompatibility of long-term implantable bioelectronics. PMID:24841849

  6. Crystal Structure of Mammalian Cysteine dioxygenase: A Novel Mononuclear Iron Center for Cysteine Thiol Oxidation

    International Nuclear Information System (INIS)

    Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteinesulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or to the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5 Angstroms resolution, and these results confirm the canonical cupin ?-sandwich fold and the rare cysteinyl-tyrosine intramolecular crosslink (between Cys93 and Tyr157) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His86, His88, and His140) and a water molecule. Attempts to acquire a structure with bound ligand using either co-crystallization or soaks with cysteine revealed the formation of a mixed disulfide involving Cys164 near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploring the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine catalytic role of the cysteinyl-tyrosine linkage

  7. Crystal Structure of Mammalian Cysteine dioxygenase: A Novel Mononuclear Iron Center for Cysteine Thiol Oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Simmons,C.; Liu, Q.; Huang, Q.; Hao, Q.; Begley, T.; Karplus, P.; Stipanuk, M.

    2006-01-01

    Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteinesulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or to the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5 Angstroms resolution, and these results confirm the canonical cupin {beta}-sandwich fold and the rare cysteinyl-tyrosine intramolecular crosslink (between Cys93 and Tyr157) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His86, His88, and His140) and a water molecule. Attempts to acquire a structure with bound ligand using either co-crystallization or soaks with cysteine revealed the formation of a mixed disulfide involving Cys164 near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploring the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine linkage.

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

    Science.gov (United States)

    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. PMID:8276753

  9. Molecular cloning, sequencing, and mapping of the gene encoding protease I and characterization of proteinase and proteinase-defective Escherichia coli mutants.

    OpenAIRE

    Ichihara, S.; Matsubara, Y.; Kato, C.; Akasaka, K.; Mizushima, S.

    1993-01-01

    Clones carrying the gene encoding a proteinase were isolated from Clarke and Carbon's collection, using a chromogenic substrate, N-benzyloxycarbonyl-L-phenylalanine beta-naphthyl ester. The three clones isolated, pLC6-33, pLC13-1, and pLC36-46, shared the same chromosomal DNA region. A 0.9-kb Sau3AI fragment within this region was found to be responsible for the overproduction of the proteinase, and the nucleotide sequence of the region was then determined. The proteinase was purified to homo...

  10. Effects of the Human Immunodeficiency Virus (HIV) Proteinase Inhibitors Saquinavir and Indinavir on In Vitro Activities of Secreted Aspartyl Proteinases of Candida albicans Isolates from HIV-Infected Patients

    OpenAIRE

    Korting, Hans C.; Schaller, Martin; Eder, Gabriele; Hamm, Gerald; Bo?hmer, Ursula; Hube, Bernhard

    1999-01-01

    The effects of therapeutically relevant concentrations of the human immunodeficiency virus (HIV) proteinase inhibitors saquinavir and indinavir on the in vitro proteinase activity of Candida albicans were investigated with isolates from HIV-infected and uninfected patients with oral candidiasis. After exposure to the HIV proteinase inhibitors, proteinase activity was significantly reduced in a dose-dependent manner. These inhibitory effects, which were similar to that of pepstatin A, and the ...

  11. Metabolism of L-cysteine in guinea pig liver.

    OpenAIRE

    Hosaki, Yasuhiro; Nishina, Hideo; Ubuka, Toshihiko

    1986-01-01

    The metabolism of L-cysteine in guinea pig liver was studied. Guinea pig liver contained 0.45 +/- 0.05 (mean +/- SD) mumol of cysteine, 0.180 +/- 0.080 mumol of 3-mercaptolactate-cysteine disulfide [S-(2-hydroxy-2-carboxyethylthio)cysteine, HCETC], and 8.082 +/- 0.516 mumol of reduced glutathione per g of fresh tissue. The taurine content was 0.912 +/- 0.158 mumol per g of fresh liver. Cysteine dioxygenase (EC 1.13.11.20) activity was several-fold lower than cysteine aminotransferase (EC 2.6....

  12. Nivulian-II a new milk clotting cysteine protease of Euphorbia nivulia latex.

    Science.gov (United States)

    Badgujar, Shamkant B; Mahajan, Raghunath T

    2014-09-01

    Nivulian-II, new milk clotting cysteine protease has been purified from the latex of Euphorbia nivulia Buch.-Ham. Nivulian-II is a monomeric protein with an apparent molecular mass 43670.846 Da. It presents its optimum activity at pH 6.3 and temperature of 50°C. The enzyme was strongly inhibited by common thiol-blocking reagents thereby indicating that it belongs to cysteine protease family. Nivulian-II is a type of glycoprotein and its pI is 3.4. The N-terminal amino acid sequence of Nivulian-II is DFPPNTCCCICC. This sequence showed relatively low homology with several other proteases of Euphorbian plants, suggesting that the isolated enzyme is a new cysteine protease. PMID:25043129

  13. S-Substituted cysteine derivatives and thiosulfinate formation in Petiveria alliacea-part II.

    Science.gov (United States)

    Kubec, Roman; Kim, Seokwon; Musah, Rabi A

    2002-11-01

    Three cysteine derivatives, (R)-S-(2-hydroxyethyl)cysteine, together with (R(S)R(C))- and (S(S)R(C))-S-(2-hydroxyethyl)cysteine sulfoxides, have been isolated from the roots of Petiveria alliacea. Furthermore, three additional amino acids, S-methyl-, S-ethyl-, and S-propylcysteine derivatives, were detected. They were present only in trace amounts (<3 microg g(-1) fr. wt), precluding determination of their absolute configurations and oxidation states. In addition, four thiosulfinates, S-(2-hydroxyethyl) (2-hydroxyethane)-, S-(2-hydroxyethyl) phenylmethane-, S-benzyl (2-hydroxyethane)- and S-benzyl phenylmethanethiosulfinates, have been found in a homogenate of the roots. The formation pathways of various benzyl/phenyl-containing compounds previously found in the plant were also discussed. PMID:12423888

  14. Induction of Tumor Necrosis Factor (TNF) Release from Subtypes of T Cells by Agonists of Proteinase Activated Receptors

    OpenAIRE

    Haiwei Yang; Tao Li; Jifu Wei; Huiyun Zhang; Shaoheng He

    2013-01-01

    Serine proteinases have been recognized as playing an important role in inflammation via proteinase activated receptors (PARs). However, little is known about the influence of serine proteinases and PARs on TNF secretion from highly purified T cells. We challenged T cells from human peripheral blood with serine proteinases and agonist peptides of PARs and measured the levels of TNF in culture supernatants by ELISA. The results showed that thrombin and trypsin, but not tryptase, stimulated app...

  15. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures.

    Science.gov (United States)

    Semashko, Tatiana A; Vorotnikova, Elena A; Sharikova, Valeriya F; Vinokurov, Konstantin S; Smirnova, Yulia A; Dunaevsky, Yakov E; Belozersky, Mikhail A; Oppert, Brenda; Elpidina, Elena N; Filippova, Irina Y

    2014-03-15

    This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp=pyroglutamyl; Xaa=Phe or Val; and Y=pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes. PMID:24388866

  16. Engineered multidomain cysteine protease inhibitors yield resistance against western flower thrips (Frankliniella occidentalis) in greenhouse trials

    OpenAIRE

    Outchkourov, N. S.; Kogel, W. J.; Wiegers, G. L.; Abrahamson, M.; Jongsma, M. A.

    2004-01-01

    Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), cause very large economic damage on a variety of field and greenhouse crops. In this study, plant resistance against thrips was introduced into transgenic potato plants through the expression of novel, custom-made, multidomain protease inhibitors. Representative classes of inhibitors of cysteine and aspartic proteases [kininogen domain 3 (K), stefin A (A), cystatin C (C), potato cystatin (P) and equistatin...

  17. Cysteine-proteases and cystatins from barley: molecular and functional characterization in housekeeping and defense processes

    OpenAIRE

    Martinez Mun?oz, Manuel; Cambra Marin, Ines; Gonzalez-melendi Leon, Pablo; Santamaria, Maria E.; Diaz Rodriguez, Isabel

    2011-01-01

    Plant cysteine-proteases (CysProt) represent a well-characterized type of proteolytic enzymes that fulfill tightly regulated physiological functions (senescence and seed germination among others) and defense roles. This article is focused on the group of papain-proteases C1A (family C1, clan CA) and their inhibitors, phytocystatins (PhyCys). In particular, the protease–inhibitor interaction and their mutual participation in specific pathways throughout the plant's life are reviewed. C1A Cys...

  18. Rapid purification of serine proteinases from Bothrops alternatus and Bothrops moojeni venoms.

    Science.gov (United States)

    Fernandes de Oliveira, Liliane Maria; Ullah, Anwar; Masood, Rehana; Zelanis, André; Spencer, Patrick J; Serrano, Solange M T; Arni, Raghuvir K

    2013-12-15

    Envenomation by Bothrops species results, among other symptoms, in hemostatic disturbances. These changes can be ascribed to the presence of enzymes, primarily serine proteinases some of which are structurally similar to thrombin and specifically cleave fibrinogen releasing fibrinopeptides. A rapid, three-step, chromatographic procedure was developed to routinely purify serine proteinases from the venoms of Bothrops alternatus and Bothrops moojeni. The serine proteinase from B. alternatus displays an apparent molecular mass of ~32 kDa whereas the two closely related serine proteinases from B. moojeni display apparent molecular masses of ~32 kDa and ~35 kDa in SDS-PAGE gels. The partial sequences indicated that these enzymes share high identity with serine proteinases from the venoms of other Bothrops species. These proteins coagulate plasma and possess fibrinogenolytic activity but lack fibrinolytic activity. PMID:24140922

  19. Molecular and immunological characterization of a wheat serine proteinase inhibitor as a novel allergen in baker's asthma.

    Science.gov (United States)

    Constantin, Claudia; Quirce, Santiago; Grote, Monika; Touraev, Alisher; Swoboda, Ines; Stoecklinger, Angelika; Mari, Adriano; Thalhamer, Josef; Heberle-Bors, Erwin; Valenta, Rudolf

    2008-06-01

    IgE-mediated sensitization to wheat flour belongs to the most frequent causes of occupational asthma. A cDNA library from wheat seeds was constructed and screened with serum IgE from baker's asthma patients. One IgE-reactive phage clone contained a full-length cDNA coding for an allergen with a molecular mass of 9.9 kDa and an isoelectric point of 6. According to sequence analysis it represents a member of the potato inhibitor I family, a group of serine proteinase inhibitors, and thus is the first allergen belonging to the group 6 pathogenesis-related proteins. The recombinant wheat seed proteinase inhibitor was expressed in Escherichia coli and purified to homogeneity. According to circular dichroism analysis, it represented a soluble and folded protein with high thermal stability containing mainly beta-sheets, random coils, and an alpha-helical element. The recombinant allergen showed allergenic activity in basophil histamine release assays and reacted specifically with IgE from 3 of 22 baker's asthma patients, but not with IgE from grass pollen allergic patients or patients suffering from food allergy to wheat. Allergen-specific Abs were raised to localize the allergen by immunogold electron microscopy in the starchy endosperm and the aleuron layer. The allergen is mainly expressed in mature wheat seeds and, despite an approximately 50% sequence identity, showed no relevant cross-reactivity with allergens from other plant-derived food sources such as maize, rice, beans, or potatoes. Recombinant wheat serine proteinase inhibitor, when used in combination with other specific allergens, may be useful for the diagnosis and therapy of IgE-mediated baker's asthma. PMID:18490745

  20. A sequential approach to risk assessment of transgenic plants expressing protease inhibitors: effects on nontarget herbivorous insects.

    Science.gov (United States)

    Cowgill, S E; Atkinson, H J

    2003-08-01

    Protease inhibitors expressed in transgenic plants can provide enhanced levels of resistance to important pest species. A sequential approach for testing the effects of protease inhibitor-expressing crops on nontarget herbivorous insects has been developed. The approach consists of five tiers. The first two tiers comprise the selection phase. In tier one, field surveys are used to characterise the nontarget invertebrate fauna of a crop. In tier 2, histochemical assays are used to identify the subset of herbivores with a particular class of digestive proteolytic enzymes. In the assessment phase a combination of laboratory 'worst-case scenario' studies (tier 3) and controlled environment or small-scale field trials (tier 4) are used to evaluate the impact of the protease inhibitor-expressing plants on the selected nontarget species. In the final tier, field trials are used to compare the relative effect of transgenic plants and current management practices, such as pesticide use, on selected species. The first four tiers of the approach are described using potatoes expressing cystatins, a family of cysteine proteinase inhibitors, as an example. Although the plants have enhanced levels of resistance to potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, the results establish that they have negligible impact on the nontarget herbivorous insect, Eupteryx aurata. PMID:12885165

  1. A VOLTAMMETRIC STUDY ON THE INTERACTION OF NOVOBIOCIN WITH CYSTEINE

    OpenAIRE

    ENDER BÇER; PAKZE QETNKAYA

    2009-01-01

    The interaction of novobiocin (NOV), an aminocoumarin antibiotic, with cysteine was studied by square-wave voltammetry technique on the hanging mercury drop electrode in different pH values. After the addition of NOV into the cysteine solution, the peak current of mercurous cysteine thiolate decreased and its voltammetric peak potential shifted to more positive values. Voltammetric results showed that NOV binds with cysteine forming 1:1 nonelectroactive molecular complex by means of electrost...

  2. Effects of soybean proteinase inhibitors on development of the soil mite Scheloribates praeincisus (Acari: Oribatida).

    Science.gov (United States)

    Simões, R A; Silva-Filho, M C; Moura, D S; Delalibera, I

    2008-03-01

    Proteinase inhibitors (PI) are present in plant tissues, especially in seeds, and act as a defense mechanism against herbivores and pathogens. Serine PI from soybean such as Bowman-Birk (BBPI) and Kunitz have been used to enhance resistance of sugarcane varieties to the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), the major pest of this crop. The use of these genetically-modified plants (GM) expressing PI requires knowledge of its sustainability and environmental safety, determining the stability of the introduced characteristic and its effects on non-target organisms. The objective of this study was to evaluate direct effects of ingestion of semi-purified and purified soybean PI and GM sugarcane plants on the soil-dwelling mite Scheloribates praeincisus (Berlese) (Acari: Oribatida). This mite is abundant in agricultural soils and participates in the process of organic matter decomposition; for this reason it will be exposed to PI by feeding on GM plant debris. Eggs of S. praeincisus were isolated and after larvae emerged, immatures were fed milled sugarcane leaves added to semi-purified or purified PI (Kunitz and BBPI) or immatures were fed GM sugarcane varieties expressing Kunitz and BBPI type PI or the untransformed near isogenic parental line variety as a control. Developmental time (larva-adult) and survival of S. praeincisus was evaluated. Neither Kunitz nor BBPI affected S. praeincisus survival. On the other hand, ingestion of semi-purified and purified Kunitz inhibitor diminished duration of S. praeincisus immature stages. Ingestion of GM senescent leaves did not have an effect on S. praeincisus immature developmental time and survival, compared to ingestion of leaves from the isogenic parental plants. These results indicate that cultivation of these transgenic sugarcane plants is safe for the non-target species S. praeincisus. PMID:18357504

  3. Diisopropyl fluorophosphate labeling of sperm-associated proteinases

    International Nuclear Information System (INIS)

    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 proacrosin-acrosin system in live motile sperm

  4. Polymorphism of the membrane proteinases of the mitochondria

    International Nuclear Information System (INIS)

    Three protein fractions capable of catalyzing the proteolysis of cytochrome c and three other fractions catalyzing the hydrolysis of N-?-benzoyl-L-arginine-p-nitroanilide (BAPA) and N-?-benzoyl-L-arginine-?-naphthylamide (BANA) were separated by electrophoresis in polyacrylamide gel in the absence of SDS detergent extracts from ultrasonic submitochondrial particles (SMP). The indicated fractions were isolated from gel and studied according to a series of parameters. It was shown that cytochrome c hydrolases have the same molecular weight (17,000) but different isoelectric points (4.0, 4.2, and 4.4). The total cytochrome c hydrolase activity of these enzymes was inhibited by phenylmethylsulfonyl fluoride but was insensitive to ethylenediaminetetraacetate and o-phenanthroline. The three BANA (BAPA) hydrolases also had similar molecular weights (? 17,500) and different isoelectric points (4.2, 4.3, and 4.7). In addition to the indicated hydrolases, minor components, possessing the same activities but differing in strength of the bond to the inner mitochondrial membrane, molecular weight, and sensitivity to proteinase inhibitors, were detected in the detergent extracts of the SMP. It was concluded that there is a polymorphism of the proteinases associated with the inner mitochondrial membrane

  5. In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida Atividade diferencial in vitro de fosfolipases e proteinases ácidas de isolados clínicos de Candida

    Directory of Open Access Journals (Sweden)

    Aurean D'Eça Júnior

    2011-06-01

    Full Text Available INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3% of isolates tested were phospholipase positive and 16 (44.4% were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%. Statistically significant differences were observed in relation to production of phospholipases among species (pINTRODUÇÃO: Candida são leveduras comensais, porém, se o equilíbrio da flora normal for interrompido ou as defesas imunitárias estiverem comprometidas, espécies de Candida podem causar manifestações de doença. Vários atributos contribuem na virulência e patogenicidade de Candida, inclusive a produção de enzimas extracelulares hidrolíticas, especialmente fosfolipases e proteinases. O objetivo deste estudo foi verificar a atividade in vitro de fosfolipases e proteinases ácidas em isolados clínicos de Candida spp. MÉTODOS: Oitenta e dois isolados provenientes de pacientes hospitalizados coletados a partir de sítios de origem diversos foram analisados. A produção de fosfolipase foi verificada em meio egg yolk e a de proteinase em meio contendo soro albumina bovina. O estudo foi feito em triplicata. RESULTADOS: Cinquenta e seis (68,3% dos isolados testados apresentaram atividade de fosfolipase positiva e 16 (44,4% foram positivos para atividade de proteinase. C. tropicalis foi a espécie que apresentou o maior número de isolados positivos para fosfolipases (91,7%. Diferenças estatisticamente significantes em relação à produção de fosfolipases entre as espécies e entre as cepas provenientes de diferentes sítios de origem foram detectadas. Quanto à produção de proteinases ácidas, os isolados de C. parapsilosis testados foram os maiores produtores (69,2%. Entre as espécies analisadas, a porcentagem de produção de proteinase entre os isolados não diferiu estatisticamente (?2=1.9 p=0.5901 (?2=1.9 p=0.5901. CONCLUSÕES: A maioria dos isolados de C. não-albicans, assim como os de C. albicans, foram grandes produtores de enzimas hidrolíticas e, consequentemente, podem ser capazes de causar infecção em condições adequadas.

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

    Science.gov (United States)

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

    2010-11-01

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

  7. The nightshade proteinase inhibitor IIb gene is constitutively expressed in glandular trichomes.

    Science.gov (United States)

    Liu, Jin; Xia, Kuai-Fei; Zhu, Jing-Chun; Deng, Yu-Ge; Huang, Xiao-Le; Hu, Bo-Lun; Xu, Xinping; Xu, Zeng-Fu

    2006-09-01

    The best known property of plant proteinase inhibitor II (PIN2) genes is their wound-inducible expression in leaves and constitutive expression in flowers. Here we show by promoter analysis in transgenic plants and in situ reverse transcription-PCR (RT-PCR) analysis that SaPIN2b, a member of the PIN2 gene family of nightshade (Solanum americanum), is also constitutively expressed in glandular trichomes. SaPIN2b promoter and its deletions were cloned and fused upstream of beta-glucuronidase (GUS) to transform the nightshade and tobacco (Nicotiana tabacum) plants. Histochemical staining assays indicated that SaPIN2b:GUS was expressed constitutively in glandular trichomes, predominantly in the gland cells, of both transgenic nightshade and tobacco plants. Constitutive expression of SaPIN2b in glandular trichomes was further confirmed by liquid phase in situ RT-PCR analysis of nightshade leaves. Deletion analysis from the 5' end of the SaPIN2b promoter revealed that separate regulatory elements control SaPIN2b expression in gland cells and stalk cells of glandular trichomes. Fluorometric GUS assays showed that SaPIN2b:GUS expression was significantly increased in transgenic plant leaves after mechanical wounding or methyl jasmonate treatment. The SaPIN2b promoter sequence contains six MYB-binding motifs and an L1 box that are involved in trichome differentiation and development. Overexpression of SaPIN2b in tobacco resulted in a significant increase in glandular trichome density and promotion of trichome branching. These results suggest that, as well as being an induced defensive protein of the well-known PIN2 family, SaPIN2b could also play roles in trichome-based defense by functioning as a constitutive component of trichome chemical defense and/or by regulating the development of glandular trichomes. PMID:16926166

  8. Binding modes of a new epoxysuccinyl-peptide inhibitor of cysteine proteases. Where and how do cysteine proteases express their selectivity?

    Science.gov (United States)

    Czaplewski, C; Grzonka, Z; Jaskólski, M; Kasprzykowski, F; Kozak, M; Politowska, E; Ciarkowski, J

    1999-05-18

    Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain-ZPACK, papain-E64c and papain-leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loop-prosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases. PMID:10350606

  9. Characterization of a Cell Envelope-Associated Proteinase Activity from Streptococcus thermophilus H-Strains

    OpenAIRE

    Shahbal, Samaha; Hemme, Denis; Renault, Pierre

    1993-01-01

    The production and biochemical properties of cell envelope-associated proteinases from two strains of Streptococcus thermophilus (strains CNRZ 385 and CNRZ 703) were compared. No significant difference in proteinase activity was found for strain CNRZ 385 when cells were grown in skim milk medium and M17 broth. Strain CNRZ 703 exhibited a threefold-higher proteinase activity when cells were grown in low-heat skim milk medium than when grown in M17 broth. Forty-one percent of the total activity...

  10. Anti-staphylococcal serine proteinase and other serum factors in phagocytosis.

    Science.gov (United States)

    Miedzobrodzki, J; Gorka, M; Wasniowska, A; Tadeusiewicz, R; Porwit-Bobr, Z

    1990-01-01

    The interactions between polymorphonuclear cells (PMN), Staphylococcus saprophyticus cells and rabbit antibodies against Staphylococcus aureus V8 serine proteinase or normal rabbit serum proteins were investigated. The effect of opsonization on phagocytosis due to human peripheral polymorphonuclear cells was measured. The results were as follows: phagocytosis index values were relatively increased after the incubation of PMN cells with anti-serine proteinase gamma-globulin serum fraction, anti-serine proteinase IgG, non-immunized rabbit serum or with complement. PMID:1698965

  11. The mitochondrial toxicity of cysteine-S-conjugates: Studies with pentachlorobutadienyl-L-cysteine

    International Nuclear Information System (INIS)

    Nephrotoxic cysteine conjugates, arising from mercapturate biosynthesis, can perturb the mitochondrial membrane potential and calcium homeostasis in renal epithelial cells. Activation of these cysteine conjugates to reactive species by mitochondrial ?-lyases results in covalent binding and mitochondrial damage. PCBC and related cysteine conjugates inhibit ADP-stimulated respiration in mitochondria respiring on alpha-ketoglutrate/malate and succinate indicating that both dehydrogenases may be targets. The respiratory inhibition is blocked by aminooxyacetic acid, an inhibitor of the ?-lyase. Hence, metabolic activation is required implying that covalent binding of reactive intermediates may be important to the mitochondrial injury. Binding of 35S-fragments has been found for 5 conjugates with varying degrees of mitochondrial toxicity. PCBC is more lipophilic and has a higher affinity for cellular membranes than other cysteine conjugates. PCBC rapidly depolarizes the inner membrane potential resulting in an inhibition of mitochondrial oxidative phosphorylation and calcium upon sequestration. Consequently, mitochondria and renal epithelial cells exposed to PCBC show a sudden release of calcium upon exposure to PCBC which is followed by a later increase in state 4 respiration leading to an inhibition of oxidative phosphorylation. The primary effect of other cysteine conjugates is an inhibition of the dehydrogenases, thus inhibiting state 3 respiration inhibiting state 3 respiration

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

    Science.gov (United States)

    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. PMID:1731082

  13. Specific cysteine protease inhibitors act as deterrents of western flower thrips, Frankliniella occidentalis (Pergande), in transgenic potato.

    Science.gov (United States)

    Outchkourov, Nikolay S; de Kogel, Willem Jan; Schuurman-de Bruin, Antje; Abrahamson, Magnus; Jongsma, Maarten A

    2004-09-01

    In this study, the effects of the accumulation of cysteine protease inhibitors on the food preferences of adult female western flower thrips, Frankliniella occidentalis (Pergande), were investigated. Representative members of the cystatin and thyropin gene families (stefin A, cystatin C, kininogen domain 3 and equistatin) were expressed in potato (Solanum tuberosum) cv. Impala, Kondor and Line V plants. In choice assays, a strong time- and concentration-dependent deterrence from plants expressing stefin A and equistatin was observed. Cystatin C and kininogen domain 3 were not found to be active. All tested inhibitors were equally or more active than stefin A at inhibiting the proteolytic activity of thrips, but, in contrast with stefin A, they were all expressed in potato as partially degraded proteins. The resistance of cysteine protease inhibitors against degradation in planta by endogenous plant proteases may therefore be relevant in explaining the observed differences in the deterrence of thrips. The results demonstrate that, when given a choice, western flower thrips will select plants with low levels of certain cysteine protease inhibitors. The novel implications of the defensive role of plant cysteine protease inhibitors as both deterrents and antimetabolic proteins are discussed. PMID:17168890

  14. Homology models of main proteinase from coronavirus associated with SARS

    Science.gov (United States)

    Liu, Hsuan-Liang; Lin, Jin-Chung; Ho, Yih; Chen, Chin-Wen

    2005-01-01

    In this study, two homology models of the main proteinase (M pro) from the novel coronavirus associated with severe acute respiratory syndrome (SARS-CoV) were constructed. These models reveal three distinct functional domains, in which an intervening loop connecting domains II and III as well as a catalytic cleft containing the substrate binding subsites S1 and S2 between domains I and II are observed. S2 exhibits structural variations more significantly than S1 during the 200 ps molecular dynamics simulations because it is located at the open mouth of the catalytic cleft and the amino acid residues lining up this subsite are least conserved. In addition, the higher structural variation of S2 makes it flexible enough to accommodate a bulky hydrophobic residue from the substrate.

  15. Crystal and solution structure of oxo rhenium(V) complexes with cysteine and cysteine methyl ester.

    Science.gov (United States)

    Kirsch, S; Jankowsky, R; Leibnitz, P; Spies, H; Johannsen, B

    1999-02-01

    The monooxo rhenium(V) complexes of cysteine (complex 1) and cysteine methyl ester (complex 2) were synthesised via a ligand exchange reaction starting from gluconatooxorhenium(V). Unexpectedly, the obtained oxorhenium(V) complex with cysteine methyl ester (2) was partially saponified. Both complexes were characterised by common analytical techniques in their solid state. Thus, an octahedral complex structure with 2(NH2,S) co-ordination in the equatorial plane and one carboxyl group bound trans to the oxo group is proven for complex 2 by X-ray diffraction. Furthermore, the existence of a dioxo species at higher pH was proven for the first time with this type of ligand by determining the nearest co-ordination sphere of the rhenium centre in solution at a pH of 12 using extended X-ray absorption fine structure spectroscopy. PMID:10499102

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

    International Nuclear Information System (INIS)

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

  17. Proteinase and Growth Factor Alterations Revealed by Gene Microarray Analysis of Human Diabetic Corneas

    OpenAIRE

    Saghizadeh, Mehrnoosh; Kramerov, Andrei A.; Tajbakhsh, Jian; Aoki, Annette M.; Wang, Charles; Chai, Ning-ning; Ljubimova, Julia Y.; Sasaki, Takako; Sosne, Gabriel; Carlson, Marc R. J.; Nelson, Stanley F.; Ljubimov, Alexander V.

    2005-01-01

    PURPOSE. To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I.

  18. Purification and characterization of cysteine protease from germinating cotyledons of horse gram

    OpenAIRE

    Rao Sridhar K; Ramakrishna Vadde; Jinka Rajeswari; Rao Ramakrishna P

    2009-01-01

    Abstract Background Proteolytic enzymes play central role in the biochemical mechanism of germination and intricately involved in many aspects of plant physiology and development. To study the mechanism of protein mobilization, undertaken the task of purifying and characterizing proteases, which occur transiently in germinating seeds of horse gram. Results Cysteine protease (CPRHG) was purified to homogeneity with 118 fold by four step procedure comprising Crude extract, (NH4)2SO4 fractionati...

  19. Benzoquinone Reveals a Cysteine-Dependent Desensitization Mechanism of TRPA1

    OpenAIRE

    Ibarra, Yessenia; Blair, Nathaniel T.

    2013-01-01

    The transient receptor potential ankyrin 1 (TRPA1) nonselective cation channel has a conserved function as a noxious chemical sensor throughout much of Metazoa. Electrophilic chemicals activate both insect and vertebrate TRPA1 via covalent modification of cysteine residues in the amino-terminal region. Although naturally occurring electrophilic plant compounds, such as mustard oil and cinnamaldehyde, are TRPA1 agonists, it is unknown whether arthropod-produced electrophiles activate mammalian...

  20. A structural model of picornavirus leader proteinases based on papain and bleomycin hydrolase

    OpenAIRE

    Skern, Tim; Fita, Ignasi; Guarne?, Alba

    1998-01-01

    The leader (L) proteinases of aphthoviruses (foot-and-mouth disease viruses) and equine rhinovirus serotypes 1 and 2 cleave themselves from the growing polyprotein. This cleavage occurs intramolecularly between the C terminus of the L proteinases and the N terminus of the subsequent protein VP4. The foot-and-mouth disease virus enzyme has been shown, in addition, to cleave at least one cellular protein, the eukaryotic initiation factor 4G. Mechanistically, inhibitor studies and sequence analy...

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

    OpenAIRE

    Witko-sarsat, V.; Descamps-latscha, B.

    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 w...

  2. Effect of retroviral proteinase inhibitors on Mason-Pfizer monkey virus maturation and transmembrane glycoprotein cleavage.

    OpenAIRE

    Sommerfelt, M. A.; Petteway, S. R.; Dreyer, G. B.; Hunter, E.

    1992-01-01

    Mason-Pfizer monkey virus (M-PMV) is the prototype type D retrovirus which preassembles immature intracytoplasmic type A particles within the infected cell cytoplasm. Intracytoplasmic type A particles are composed of uncleaved polyprotein precursors which upon release are cleaved by the viral proteinase to their constituent mature proteins. This results in a morphological change in the virion described as maturation. We have investigated the role of the viral proteinase in virus maturation an...

  3. Expression of a microbial serine proteinase inhibitor gene enhances the tobacco defense against oomycete pathogens

    OpenAIRE

    Silva, Yussuan; Portieles, Roxana; Pujol, Merardo; Terauchi, Ryohei; Matsumura, Hideo; Serrano, Mario; Borra?s-hidalgo, Orlando

    2014-01-01

    In order to identify Nicotiana megalosiphon genes for novel inhibitors of microbial serine proteinase involved in resistance to the oomycete Phytophthora parasitica var. nicotianae, SuperSAGE technology combined with next-generation sequencing were used to generate libraries in order to identify transcripts that are differentially up-regulated. We identified a N. megalosiphon inhibitor of microbial serine proteinase gene (NmIMSP) rapidly induced during the interaction. Silencing of NmIMSP gen...

  4. [Carboxylic proteinases from the microscopic fungi Trichoderma viride and Trichoderma lignorum].

    Science.gov (United States)

    Ga?da, A V; Osterman, A L; Rudenskaia, G N; Stepanov, V M

    1981-01-01

    Using ion-exchange chromatography on aminosilochrome and biospecific chromatography on Bacitracin-Sepharose, the carboxylic proteinases have been isolated for the first time from the microscopic fungi of the Trichoderma genus -- Trichoderma viride and Trichoderma lignorum, commonly used to produce cellulases. The proteinases are stable within the pH range of 3 to 6; pI is 4,3 and 4,5, the pH optimum -- 2,3 and 2,8, respectively. The molecular weight of the enzymes is 32000, the amino acid composition of T. viride proteinase is Lys5His2Arg6Asx27Thr39Ser38Glx27Pro13..Gly41Ala28Cys2Val37Ile11Leu17Tyr13Phe11Trp3, that of T. lignorum is Lys9His4Arg6Asx36Thr26Ser46Glx25Pro14Gly35Ala23Cys2Val28Ile26Leu19Tyr12..Phe14Trp4. Both enzymes are completely inactivated by specific inhibitors of carboxylic proteinase, i. e. pepstatin, diazoacetyl-D,L-norvaline methylester and N-diazoacetyl-N'-2,4-dinitrophenylethylenediamine. The molecular and enzymatic properties of the proteinases under study are close to those of carboxylic proteinases of microscopic fungi and in a lesser degree to those of porcine pepsin. PMID:7018591

  5. Cysteine-mediated redox signalling in the mitochondria.

    Science.gov (United States)

    Bak, D W; Weerapana, E

    2015-03-17

    The mitochondria are critical mediators of cellular redox homeostasis due to their role in the generation and dissipation of reactive oxygen/nitrogen species (ROS/RNS). Modulations in ROS/RNS levels in the mitochondria are often reflected through oxidation/nitrosation of highly redox-sensitive cysteine residues within this organelle. Oxidation/nitrosation of functional cysteines on mitochondrial proteins serves to modulate protein activity, localization, and complexation in response to cellular stress, thereby controlling critical processes such as oxidative phosphorylation, apoptosis, and redox signalling. In this review, we describe mitochondrial sources of ROS/RNS, cysteine modifications that are triggered by increased mitochondrial ROS/RNS, and examples of key mitochondrial proteins that are regulated through cysteine-mediated redox signalling. We highlight recent advancements in proteomic methods to study cysteine posttranslational modifications. These tools will further aid in illuminating the important role of cysteine in maintaining and transducing redox signals in the mitochondria. PMID:25519845

  6. Pulse photolysis of NADH in the presence of cysteine

    International Nuclear Information System (INIS)

    In the UV irradiation of NADH under anaerobic conditions, cysteine, which often acts as a radioprotective substance, has a sensitizing effect. With the aid of pulse photolysis, it was studied which reaction mechanisms in the presence or absence of cysteine are responsible for the damage to NADH in aqueous solution. In the absence of cysteine, the characteristic NADH absorption at 340 nm is reduced immediately after UV quanta have been absorbed by the adenine fraction of the molecules; in the presence of cysteine, a secondary reaction causes additional damage. The spectra of the intermediate products of NADH and cysteine have been recorded for different cysteine concentrations, and the reaction constants have been determined. These values suggest that the sensitizing effect is due to a reaction of NADH with radical anions produced by photolysis. (orig.)

  7. Palmitoylation of human proteinase-activated receptor-2 differentially regulates receptor-triggered ERK1/2 activation, calcium signalling and endocytosis.

    Science.gov (United States)

    Botham, Andrew; Guo, Xiaodan; Xiao, Yu Pei; Morice, Alyn H; Compton, Steven J; Sadofsky, Laura R

    2011-09-01

    hPAR(2) (human proteinase-activated receptor-2) is a member of the novel family of proteolytically activated GPCRs (G-protein-coupled receptors) termed PARs (proteinase-activated receptors). Previous pharmacological studies have found that activation of hPAR(2) by mast cell tryptase can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR(2) that can regulate function, we have explored the functional role of the intracellular cysteine residue Cys(361). We have demonstrated, using autoradiography, that Cys(361) is the primary palmitoylation site of hPAR(2). The hPAR(2)C361A mutant cell line displayed greater cell-surface expression compared with the wt (wild-type)-hPAR(2)-expressing cell line. hPAR(2)C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV. In stark contrast, hPAR(2)C361A triggered greater and more prolonged ERK (extracellular-signal-regulated kinase) phosphorylation compared with that of wt-hPAR(2) possibly through Gi, since pertussis toxin inhibited the ability of this receptor to activate ERK. Finally, flow cytometry was utilized to assess the rate and extent of receptor internalization following agonist challenge. hPAR(2)C361A displayed faster internalization kinetics following trypsin activation compared with wt-hPAR(2), whereas SLIGKV had a negligible effect on internalization for either receptor. In conclusion, palmitoylation plays an important role in the regulation of PAR(2) expression, agonist sensitivity, desensitization and internalization. PMID:21627585

  8. An engineered retroviral proteinase from myeloblastosis associated virus acquires pH dependence and substrate specificity of the HIV-1 proteinase.

    OpenAIRE

    Konvalinka, J.; Horejsi?, M.; Andrea?nsky, M.; Novek, P.; Pichova?, I.; Bla?ha, I.; Fa?bry, M.; Sedla?cek, J.; Foundling, S.; Strop, P.

    1992-01-01

    In an attempt to understand the structural reasons for differences in specificity and activity of proteinases from two retroviruses encoded by human immunodeficiency virus (HIV) and myeloblastosis associated virus (MAV), we mutated five key residues predicted to form part of the enzyme subsites S1, S2 and S3 in the substrate binding cleft of the wild-type MAV proteinase wMAV PR. These were changed to the residues occupying a similar or identical position in the HIV-1 enzyme. The resultant mut...

  9. Cysteine protease inhibitors as chemotherapy: Lessons from a parasite target

    OpenAIRE

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; Mckerrow, James H.

    1999-01-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasi...

  10. Gamma irradiation or hydrocortisone treatment of rats increases the proteinase activity associated with histones of thymus nuclei

    International Nuclear Information System (INIS)

    An increase in the activity of histone-associated rat thymus nucleus proteinases specific for histones H2A, H2B and H1 was shown after ? irradiation or hydrocortisone treatment of animals. Histone H1-specific proteinase activity is dependent on DNA and increases in the presence of denatured DNA, whereas proteinases specific for core histones are inhibited in the presence of denatured DNA. The increase in the activity of histone-associated proteinases depends on the radiation dose and the time after irradiation or hydrocortisone injection. In the presence of dithiothreitol and sodium dodecyl sulfate, these proteinases dissociate from histones. It was found by gel electrophoresis that several proteinases of various molecular masses are closely associated with histones obtained from thymus nuclei of irradiated or hydrocortisone-treated rats. 43 refs., 7 figs

  11. The significance of cysteine synthesis for acclimation to high light conditions

    Science.gov (United States)

    Speiser, Anna; Haberland, Stefan; Watanabe, Mutsumi; Wirtz, Markus; Dietz, Karl-Josef; Saito, Kazuki; Hell, Rüdiger

    2015-01-01

    Situations of excess light intensity are known to result in the emergence of reactive oxygen species that originate from the electron transport chain in chloroplasts. The redox state of glutathione and its biosynthesis contribute importantly to the plant's response to this stress. In this study we analyzed the significance of cysteine synthesis for long-term acclimation to high light conditions in Arabidopsis thaliana. Emphasis was put on the rate-limiting step of cysteine synthesis, the formation of the precursor O-acetylserine (OAS) that is catalyzed by serine acetyltransferase (SERAT). Wild type Arabidopsis plants responded to the high light condition (800 ?mol m?2 s?1 for 10 days) with synthesis of photo-protective anthocyanins, induction of total SERAT activity and elevated glutathione levels when compared to the control condition (100 ?mol m?2 s?1). The role of cysteine synthesis in chloroplasts was probed in mutant plants lacking the chloroplast isoform SERAT2;1 (serat2;1) and two knock-out alleles of CYP20-3, a positive interactor of SERAT in the chloroplast. Acclimation to high light resulted in a smaller growth enhancement than wild type in the serat2;1 and cyp20-3 mutants, less induction of total SERAT activity and OAS levels but similar cysteine and glutathione concentrations. Expression analysis revealed no increase in mRNA of the chloroplast SERAT2;1 encoding SERAT2;1 gene but up to 4.4-fold elevated SERAT2;2 mRNA levels for the mitochondrial SERAT isoform. Thus, lack of chloroplast SERAT2;1 activity or its activation by CYP20-3 prevents the full growth response to high light conditions, but the enhanced demand for glutathione is likely mediated by synthesis of OAS in the mitochondria. In conclusion, cysteine synthesis in the chloroplast is important for performance but is dispensable for survival under long-term exposure to high light and can be partially complemented by cysteine synthesis in mitochondria. PMID:25653656

  12. Cysteine-containing peptides having antioxidant properties

    Science.gov (United States)

    Bielicki, John K. (Castro Valley, CA)

    2009-10-13

    Cysteine containing amphipathic alpha helices of the exchangeable apolipoproteins, as exemplified by apolipoprotein (apo) A-I.sub.Milano (R173C) and apoA-I.sub.Paris, (R151C) were found to exhibit potent antioxidant activity on phospholipid surfaces. The addition of a free thiol, at the hydrophobic/hydrophilic interface of an amphipathic alpha helix of synthetic peptides that mimic HDL-related proteins, imparts a unique antioxidant activity to these peptides which inhibits lipid peroxidation and protects phospholipids from water-soluble free radical initiators. These peptides can be used as therapeutic agents to combat cardiovascular disease, ischemia, bone disease and other inflammatory related diseases.

  13. Molecular characterization, expression and function analysis of a five-domain Kazal-type serine proteinase inhibitor from pearl oyster Pinctada fucata.

    Science.gov (United States)

    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-dependent manner, when the ratios of rpoKSPI to chymotrypsin and trypsin were 36:1 and 72:1, respectively, the proteinase activities of chymotrypsin and trypsin could be almost completely inhibited, but the rpoKSPI could not inhibit subtilisin. PMID:24378679

  14. Lipases and proteinases in milk : occurrence, heat inactivation, and their importance for the keeping quality of milk products

    OpenAIRE

    Driessen, F. M.

    1983-01-01

    The occurrence and heat inactivation of native and bacterial lipases and proteinases in milk were studied.Production of these enzymes by Gram-negative psychrotrophic bacteria in milk was found to take place towards the end of exponential growth and in the stationary growth phase.Kinetics of heat inactivation in milk of milk lipoprotein lipase, alkaline milk proteinase and lipases and proteinases of some Gram-negative bacteria are given.The effects of residual lipolytic and proteolytic activit...

  15. Comparative Study of Action of Cell Wall Proteinases from Various Strains of Streptococcus cremoris on Bovine ?s1-, ?-, and ?-Casein

    OpenAIRE

    Visser, Servaas; Exterkate, Fred A.; Slangen, Charles J.; Veer, Gerrie J. C. M.

    1986-01-01

    Partially purified cell wall proteinases of eight strains of Streptococcus cremoris were compared in their action on bovine ?s1-, ?-, and ?-casein, as visualized by starch gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin-layer chromatography. Characteristic degradation profiles could be distinguished, from which the occurrence of two proteinases, represented by strain HP and strain AM1, was concluded. The action of the HP-type proteinase P1 (also det...

  16. A double WAP domain-containing protein Es-DWD1 from Eriocheir sinensis exhibits antimicrobial and proteinase inhibitory activities.

    Science.gov (United States)

    Li, Shuang; Jin, Xing-Kun; Guo, Xiao-Nv; Yu, Ai-Qing; Wu, Min-Hao; Tan, Shang-Jian; Zhu, You-Ting; Li, Wei-Wei; Wang, Qun

    2013-01-01

    Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides, which are critical in the host immune response against microbial invasion. The common feature of these proteins is a single WAP domain maintained by at least one four-disulfide core (4-DSC) structure rich in cysteine residues. In this study, a double WAP domain (DWD)-containing protein, Es-DWD1, was first cloned from the Chinese mitten crab (Eriocheirsinensis). The full-length Es-DWD1cDNA was 1193 bp, including a 411 bp open reading frame (ORF) encoding 136 amino acids with a signal peptide of 22 amino acids in the N-terminus. A comparison with other reported invertebrate and vertebrate sequences revealed the presence of WAP domains characteristic of WAP superfamilies. As determined by quantitative real-time RT-PCR, Es-DWD1 transcripts were ubiquitously expressed in all tissues, but it was up-regulated in hemocytes post-challenge with pathogen-associated molecular patterns (PAMPs). The mature recombinant Es-DWD1 (rEs-DWD1) protein exhibited different binding activities to bacteria and fungus. Moreover, rEs-DWD1 could exert agglutination activities against Bacillus subtilis and Pichiapastoris and demonstrated inhibitory activities against the growth of Staphylococcus aureus, Aeromonas hydrophila and P. pastoris. Furthermore, rEs-DWD1 showed a specific protease inhibitory activity in B. subtilis. Coating of rEs-DWD1 onto agarose beads enhanced encapsulation of the beads by crab hemocytes. Collectively, the results suggest that Es-DWD1 is a double WAP domain containing protein with antimicrobial and proteinase inhibitory activities, which play significant roles in the immunity of crustaceans. PMID:23967346

  17. Isolation and characterization of a serine proteinase specific to human C3b from human erythrocyte membranes

    International Nuclear Information System (INIS)

    In a previous report, they have shown that human C3b bound through CR1 to human erythrocytes is cleaved by a membrane proteinase activity. Following the molecular analysis of this proteinase activity, they have purified it by a four step procedure: ammonium sulfate precipitation, biogel filtration, fluid phase electrophoresis and hydroxylapatite chromatography. The highly purified proteinase was labeled by 125I iodine or 3H-DFP and analyzed by gel electrophoresis: a single band membrane component was characterized by its apparent molecular weight of 57 K or 60 K, under non reducing or reducing conditions respectively and was called p 57. Its reactivity with 3H-DFP and the inhibition by PMSF of the proteinase activity indicate that p 57 is a serine proteinase. Moreover, it is sensitive to aprotinin and ?1-antitrypsin. This membrane proteinase presents a higher activity in the presence of detergent and cleaves both alpha and beta chains of human C3b. Polyclonal antibody prepared against this purified proteinase inhibits its activity. On the basis of its structure and its functions, i.e. molecular weight, antigenic properties, proteinase properties and proteinases inhibitors sensitivity, p57 is not related to CR1 or DAF, two others membrane components which react with human C3b and identified by others on human erythrocytes. These specific antibodies allow to analyze the presence of p57 on human cells

  18. Wound-induced proteinase inhibitor in Salix viminalis and its association with defence against insects

    Energy Technology Data Exchange (ETDEWEB)

    Saarikoski, P. [Swedish Univ. of Agricultural Sciences, Uppsala (Sweden). Dept. of Forest Genetics

    1997-09-01

    For successful traditional breeding, the plant material has to be screened for genetic variation for the desired traits. By screening Salix clones for wound-induced proteinase inhibitor (PI) activity and ethylene evolution, it was possible to identify variation for both characters among the Salix clones tested. However, no correlation was observed with insect and pathogen resistance. Since there was no simple relationship between wound-induced ethylene production, accumulation of PI and pest resistance, a more systematic investigation of Salix PIs was begun. A gene (swin1.1) encoding a 21 kDa trypsin inhibitor with characteristics of Kunitz-type of PI was sequenced. The trypsin inhibitor encoded by the isolated swin1.1 gene was shown to be functional in vitro and exhibit specificity for trypsin. It is therefore likely that this PI is involved in the plant defence in Salix, since many insects have trypsin as their major digestive protease. In further support of this view, in bio-tests with poplar the mortality of the first instar larvae (Lymantria dispar) was significantly increased, both after application of the trypsin inhibitor encoded by swin1.1 directly on poplar leaves and after feeding the larvae with transgenic poplar over-expressing the swin1.1 gene. In Salix, the swin1.1 gene was shown to be induced by mechanical wounding, insect feeding and by treatment with the signalling substances salicylic and jasmonic acid. The locally wound-induced response (mechanical and insect) was greater than the systemic response. Other swin1 gene family members were also differentially expressed after the inductive treatment. 187 refs., 3 figs., 2 tabs.

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

    Science.gov (United States)

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

    2014-10-01

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

  20. cDNA cloning and molecular modeling of procerain B, a novel cysteine endopeptidase isolated from Calotropis procera.

    Science.gov (United States)

    Singh, Abhay Narayan; Yadav, Prity; Dubey, Vikash Kumar

    2013-01-01

    Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimensional structure of active procerain B was modeled by homology modeling using X-ray crystal structure of actinidin (PDB ID: 3P5U), a cysteine protease from the fruits of Actinidia arguta. The structural aspect of the enzyme is also discussed. PMID:23527269

  1. Procerain, a stable cysteine protease from the latex of Calotropis procera.

    Science.gov (United States)

    Dubey, Vikash Kumar; Jagannadham, M V

    2003-04-01

    A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0-9.0 and temperature 55-60 degree C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala-p-nitroanilide but not -Ala-Ala-p-nitroanilide, -Ala p-nitroanilide and N-d-Benzoyl--Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (epsilon 1% 280 nm) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0-12.0 and temperatures up to 70 degree C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue. PMID:12591258

  2. An O-acetylserine (thiol) lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthase.

    Science.gov (United States)

    Yafuso, Jannai T; Negi, Vishal Singh; Bingham, Jon-Paul; Borthakur, Dulal

    2014-07-01

    In plants, the final step of cysteine formation is catalyzed by O-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume Leucaena leucocephala (leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from Escherichia coli and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the Km was calculated to be 1,850±414 ?M sulfide and the Vmax was 200.6±19.92 ?M cysteine min(-1). The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as ?-substituted alanine synthases. PMID:24777760

  3. Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases

    OpenAIRE

    Carrillo Gil, Laura; Martinez Mun?oz, Manuel; Ramessar, Koreen; Cambra Marin, Ines; Castan?era, Pedro; Ortego, Felix; Diaz Rodriguez, Isabel

    2011-01-01

    Phytocystatins are inhibitors of cysteine-proteases from plants putatively involved in plant defence based on their capability of inhibit heterologous enzymes. We have previously characterised the whole cystatin gene family members from barley (HvCPI-1 to HvCPI-13). The aim of this study was to assess the effects of barley cystatins on two phytophagous spider mites, Tetranychus urticae and Brevipalpus chilensis. The determination of proteolytic activity profile in both mite species showed the...

  4. Antimicrobial Nodule-Specific Cysteine-Rich Peptides Induce Membrane Depolarization-Associated Changes in the Transcriptome of Sinorhizobium meliloti

    OpenAIRE

    Tiricz, Hilda; Szu?cs, Attila; Farkas, Attila; Pap, Bernadett; Lima, Rui M.; Maro?ti, Gergely; Kondorosi, E?va; Kereszt, Attila

    2013-01-01

    Leguminous plants establish symbiosis with nitrogen-fixing alpha- and betaproteobacteria, collectively called rhizobia, which provide combined nitrogen to support plant growth. Members of the inverted repeat-lacking clade of legumes impose terminal differentiation on their endosymbiotic bacterium partners with the help of the nodule-specific cysteine-rich (NCR) peptide family composed of close to 600 members. Among the few tested NCR peptides, cationic ones had antirhizobial activity measured...

  5. Specific cysteine protease inhibitors act as deterrents of Western flower thrips Frankliniella occidentalis (Pergande) in transgenic potato

    OpenAIRE

    Outchkourov, N. S.; Kogel, J.; Bruin, A.; Abrahamson, M.; Jongsma, M. A.

    2004-01-01

    In this study, the effects of the accumulation of cysteine protease inhibitors on the food preferences of adult female western flower thrips, Frankliniella occidentalis (Pergande), were investigated. Representative members of the cystatin and thyropin gene families (stefin A, cystatin C, kininogen domain 3 and equistatin) were expressed in potato (Solanum tuberosum) cv. Impala, Kondor and Line V plants. In choice assays, a strong time- and concentration-dependent deterrence from plants expres...

  6. Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTxa

    International Nuclear Information System (INIS)

    Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTxa), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca2+ release from sarcoplasmic reticulum (SR). IpTxa increases the binding of ryanodine to ryanodine receptors (RyRs) and encourages reconstituted single channel to induce subconductance states

  7. Functional role of aspartic proteinase cathepsin D in insect metamorphosis

    Directory of Open Access Journals (Sweden)

    Seo Sook

    2006-10-01

    Full Text Available Abstract Background Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis. Results Cathepsin D of the silkworm Bombyx mori (BmCatD was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage. Conclusion Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.

  8. Functional role of aspartic proteinase cathepsin D in insect metamorphosis

    Science.gov (United States)

    Gui, Zhong Zheng; Lee, Kwang Sik; Kim, Bo Yeon; Choi, Yong Soo; Wei, Ya Dong; Choo, Young Moo; Kang, Pil Don; Yoon, Hyung Joo; Kim, Iksoo; Je, Yeon Ho; Seo, Sook Jae; Lee, Sang Mong; Guo, Xijie; Sohn, Hung Dae; Jin, Byung Rae

    2006-01-01

    Background Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis. Results Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage. Conclusion Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis. PMID:17062167

  9. Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

    OpenAIRE

    Hansen, Daiane; Macedo-ribeiro, Sandra; Veri?ssimo, Paula; Yoo Im, Sonia; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

    2007-01-01

    Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human ...

  10. A VOLTAMMETRIC STUDY ON THE INTERACTION OF NOVOBIOCIN WITH CYSTEINE

    Scientific Electronic Library Online (English)

    ENDER, BÇER; PAKZE, QETNKAYA.

    Full Text Available The interaction of novobiocin (NOV), an aminocoumarin antibiotic, with cysteine was studied by square-wave voltammetry technique on the hanging mercury drop electrode in different pH values. After the addition of NOV into the cysteine solution, the peak current of mercurous cysteine thiolate decreas [...] ed and its voltammetric peak potential shifted to more positive values. Voltammetric results showed that NOV binds with cysteine forming 1:1 nonelectroactive molecular complex by means of electrostatic and hydrogen-bonding interactions. The binding constants of NOV with cysteine at pHs 5, 7 and 10 were calculated to be 3.06x10³, 1.54x10(4) and 1.06x10(5) M-1, respectively. Furthermore, apossible mechanism of such interaction was also discussed.

  11. A VOLTAMMETRIC STUDY ON THE INTERACTION OF NOVOBIOCIN WITH CYSTEINE

    Directory of Open Access Journals (Sweden)

    ENDER BÇER

    2009-01-01

    Full Text Available The interaction of novobiocin (NOV, an aminocoumarin antibiotic, with cysteine was studied by square-wave voltammetry technique on the hanging mercury drop electrode in different pH values. After the addition of NOV into the cysteine solution, the peak current of mercurous cysteine thiolate decreased and its voltammetric peak potential shifted to more positive values. Voltammetric results showed that NOV binds with cysteine forming 1:1 nonelectroactive molecular complex by means of electrostatic and hydrogen-bonding interactions. The binding constants of NOV with cysteine at pHs 5, 7 and 10 were calculated to be 3.06x10³, 1.54x10(4 and 1.06x10(5 M-1, respectively. Furthermore, apossible mechanism of such interaction was also discussed.

  12. BINDING OF CHLOROFORM TO THE CYSTEINE OF HEMOGLOBIN

    Science.gov (United States)

    The products of the covalent binding of chloroform to rat hemoglobin during microsomal metabolism were isolated and identified by gas chromatography (GC) and mass spectroscopy (MS). After isolation by Proteinase K hydrolysis, amino acid analysis and cellulose thin-layer chromatog...

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

    Scientific Electronic Library Online (English)

    Leah Theresa, Sigle; Marcelo, Ramalho-Ortigao.

    2013-09-01

    Full Text Available SciELO Brazil | Language: English Abstract in english 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 inhibi [...] tors 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 ?-chymotrypsin to 9.4% residual activity and also inhibited ?-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.

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

    Science.gov (United States)

    Sigle, Leah Theresa; Ramalho-Ortigão, Marcelo

    2013-09-01

    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 ?-chymotrypsin to 9.4% residual activity and also inhibited ?-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

  15. Domain 15 of the serine proteinase inhibitor LEKTI blocks HIV infection in vitro

    Directory of Open Access Journals (Sweden)

    David Palesch

    2013-08-01

    Full Text Available Background: Lympho-epithelial Kazal-type-related inhibitor (LEKTI is a 15-domain serine proteinase inhibitor, parts of which have first been isolated from human blood filtrate. It is encoded by the gene SPINK5. In the past, different groups reported antiviral activities of certain serine proteinase inhibitors, such as mucous proteinase inhibitor and alpha1-proteinase inhibitor. The purpose of this study was to test two representative domains of the proteinase inhibitor LEKTI for anti-HIV activities.Methods: LEKTI domains 6 and 15 were recombinantly produced in E.coli. To test their inhibitory activity against HIV infection, the reporter cell line P4-R5 MAGI carrying an HIV-inducible reporter gene was infected by a CCR5-tropic HIV strain in the presence of different inhibitor concentrations. After three days, infection rates were determined by quantifying ß-galactosidase activities using the Galacto-Light Plus™ ß-Galactosidase Reporter Gene Assay.Results: In contrast to LEKTI domain 6, LEKTI domain 15 suppressed HIV-induced reporter gene activities with an IC50 value of approximately 29 µM.Conclusion: LEKTI domain 15 represents an inhibitor of HIV infection. (Med J Indones. 2013;22:131-5. doi: 10.13181/mji.v22i3.580Keywords: HIV, inhibition, LEKTI, P4-R5 MAGI

  16. Determination of germ tube, phospholipase, and proteinase production by bloodstream isolates of Candida albicans

    Scientific Electronic Library Online (English)

    Antonella Souza, Mattei; Sydney Hartz, Alves; Cecilia Bittencourt, Severo; Luciana da Silva, Guazzelli; Flavio de Mattos, Oliveira; Luiz Carlos, Severo.

    2013-06-01

    Full Text Available SciELO Brazil | Language: English Abstract in english Introduction Candida albicans is a commensal and opportunistic agent that causes infection in immunocompromised individuals. Several attributes contribute to the virulence and pathogenicity of this yeast, including the production of germ tubes (GTs) and extracellular hydrolytic enzymes, particularl [...] y phospholipase and proteinase. This study aimed to investigate GT production and phospholipase and proteinase activities in bloodstream isolates of C. albicans. Methods One hundred fifty-three C. albicans isolates were obtained from blood samples and analyzed for GT, phospholipase, and proteinase production. The assays were performed in duplicate in egg yolk medium containing bovine serum albumin and human serum. Results Detectable amounts of proteinase were produced by 97% of the isolates, and 78% of the isolates produced phospholipase. GTs were produced by 95% of the isolates. A majority of the isolates exhibited low levels of phospholipase production and high levels of proteinase production. Conclusions Bloodstream isolates of C. albicans produce virulence factors such as GT and hydrolytic enzymes that enable them to cause infection under favorable conditions.

  17. [Effect of Streptomyces spheroides proteinases on the mechanism of antibacterial protection in an experiment].

    Science.gov (United States)

    Egorov, N S; Ole?nikova, E A; Anoshkina, G B; Landau, N S

    1984-08-01

    The effect of Streptomyces spheroides proteinases on the process and character of the local reaction to intraperitoneal infection of mice with E. coli was studied experimentally. It was shown that administration of the proteinases promoted a decrease in the dissemination of the abdominal cavity and a more rapid elimination of the microorganisms from the infection foci. The proteinases potentiated the macrophagal component of the local reaction by accelerating migration of the macrophages and increasing their phagocytic activity and the activity of the lysosomal enzymes. Proteinases gave rise to a pronounced activation of the neutrophils, an increase in the phagocytic capacity of the young forms and a change in intracellular enzymes. It was demonstrated that proteinases changed interrelation between the cell elements in the infection foci, the character of interaction of the quantitative and functional parameters of the local reaction and interrelationship between the phagocytic activity and the enzymatic balance of the cells. They increased the effect of the macrophages on migration and the functional state of the neutrophiles. PMID:6385832

  18. CHARACTERIZATION OF DANSYLATED CYSTEINE, GLUTATHIONE DISULFIDE, CYSTEINE AND CYSTINE BY NARROW BORE LIQUID CHROMATOGRAPHY/ELECTROSPRAY IONIZATION MASS SPECTROMETRY

    Science.gov (United States)

    A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometric (RP-LC/ESI-MS) method has been developed to confirm the identity of dansylated derivatives of cysteine and glutathione, and their respective dimers. Cysteine, GSH, CSSC...

  19. Activity profiling of papain-like cysteine proteases in plants

    OpenAIRE

    Hoorn, R. A. L.; Leeuwenburgh, M. A.; Bogyo, M.; Joosten, M. H. A. J.; Peck, S. C.

    2004-01-01

    Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in pl...

  20. Reduction of guanosyl radical by cysteine and cysteine-glycine studied by time-resolved CIDNP.

    Science.gov (United States)

    Morozova, Olga B; Kaptein, Robert; Yurkovskaya, Alexandra V

    2012-07-19

    As a model for chemical DNA repair, reduction of guanosyl radicals in the reaction with cysteine or the dipeptide cysteine-glycine has been studied by time-resolved chemically induced dynamic nuclear polarization (CIDNP). Radicals were generated photochemically by pulsed laser irradiation of a solution containing the photosensitizer 2,2'-dipyridyl, guanosine-5'-monophosphate, and the amino acid or peptide. In neutral and basic aqueous solution, the neutral guanosyl radical is formed via electron or hydrogen atom transfer to the triplet excited dye. The rate constants for reduction of guanosyl radical were determined by quantitative analysis of the CIDNP kinetics, which are sensitive to the rates of fast radical reactions. The rate constants vary from (1.0 ± 0.3) × 10(7) M(-1) s(-1) for the thiol form of cysteine to (1.6 ± 0.2) × 10(8) M(-1) s(-1) for the thiolate anion. These values are comparable with corresponding rate constants for reduction of neutral guanosyl radical by tyrosine. PMID:22708799

  1. Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus.

    Science.gov (United States)

    Sanfaçon, Hélène; Wellink, Joan; Le Gall, Olivier; Karasev, Alexander; van der Vlugt, René; Wetzel, Thierry

    2009-01-01

    The order Picornavirales includes several plant viruses that are currently classified into the families Comoviridae (genera Comovirus, Fabavirus and Nepovirus) and Sequiviridae (genera Sequivirus and Waikavirus) and into the unassigned genera Cheravirus and Sadwavirus. These viruses share properties in common with other picornavirales (particle structure, positive-strand RNA genome with a polyprotein expression strategy, a common replication block including type III helicase, a 3C-like cysteine proteinase and type I RNA-dependent RNA polymerase). However, they also share unique properties that distinguish them from other picornavirales. They infect plants and use specialized proteins or protein domains to move through their host. In phylogenetic analysis based on their replication proteins, these viruses form a separate distinct lineage within the picornavirales branch. To recognize these common properties at the taxonomic level, we propose to create a new family termed "Secoviridae" to include the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus and Waikavirus. Two newly discovered plant viruses share common properties with members of the proposed family Secoviridae but have distinct specific genomic organizations. In phylogenetic reconstructions, they form a separate sub-branch within the Secoviridae lineage. We propose to create a new genus termed Torradovirus (type species, Tomato torrado virus) and to assign this genus to the proposed family Secoviridae. PMID:19350366

  2. Reference: EVENINGAT [PLACE

    Lifescience Database Archive (English)

    Full Text Available EVENINGAT Rawat R, Xu ZF, Yao KM, Chye ML. Identification of cis-elements for ethylene and circa ... dian regulation of the Solanum ... melongena gene encoding cysteine proteinase. Plant ...

  3. Reference: ERELEE4 [PLACE

    Lifescience Database Archive (English)

    Full Text Available ERELEE4 Rawat R, Xu ZF, Yao KM, Chye ML. Identification of cis-elements for ethylene and circadi ... an regulation of the Solanum ... melongena gene encoding cysteine proteinase. Plant ...

  4. Purification and properties of a proteinase from Vipera lebetina (snake) venom.

    Science.gov (United States)

    Mähar, A; Siigur, E; Siigur, J

    1987-09-11

    A proteinase from the venom of Vipera lebetina was purified by chromatography on Sephadex G-100 and CM-cellulose. The purified proteinase was homogeneous on SDS-polyacrylamide gel electrophoresis and consisted of a single chain with molecular weight of 37,000 +/- 1500. The isoelectric point of the proteinase was over 10. The enzyme was active on casein but not on esters and amides of arginine. It split the oxidized insulin B-chain at the peptide bonds of Tyr16-Leu17, Phe24-Phe25 and Phe25-Tyr26, and glucagon at the bonds Tyr10-Ser11, Leu14-Asp15 and Leu26-Met27. The enzyme was inhibited by DFP and PMSF, and partially by soybean trypsin inhibitor, but not with EDTA. PMID:3304428

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

    Science.gov (United States)

    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 ?M 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. PMID:24435659

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

    Science.gov (United States)

    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-?B activation through TLR2 and TLR4. Possession of multiple activators of Toll-like receptors may be the unique strategy of C. jejuni to trigger hyperimmunity. PMID:25530156

  7. Cytotoxicity induced by Erythrina variegata serine proteinase inhibitors in tumor hematopoietic stem cell lines.

    Science.gov (United States)

    Ohba, H; Nishikawa, M; Kimura, M; Yamasaki, N; Moriwaki, S; Itoh, K

    1998-06-01

    Based on the soluble MTT tetrazolium/formazan assay, we evaluated the cytotoxicity of Erythrina variegata proteinase inhibitors in some tumor hematopoietic stem cell lines. Among the proteinase inhibitors, EBI, which belongs to the Bowman-Birk family of inhibitors, was cytotoxic in relatively differentiated cells such as Molt4 and Jurkat derived from acute T lymphoblastic leukemia (T-ALL) cells specifically, but ETIa and ECI, which are classified into Kunitz family inhibitors, did not. It was suggested that the differences in the cytotoxicity might be due to the molecular size of the inhibitors. The succinylation of lysine residue(s) of EBI led to about 50% loss of the trypsin inhibitory activity as compared with the authentic EBI. When Molt4 cells were incubated with this derivative, no significant cytotoxicity was observed. This suggests that the proteinase inhibitory activity might be involved in the cytotoxicity in human tumor cell lines. PMID:9692201

  8. Analysis of serine proteinase-inhibitor interaction by alanine shaving.

    Science.gov (United States)

    Buczek, Olga; Koscielska-Kasprzak, Katarzyna; Krowarsch, Daniel; Dadlez, Micha?; Otlewski, Jacek

    2002-04-01

    We analyzed the energetic importance of residues surrounding the hot spot (the P(1) position) of bovine pancreatic trypsin inhibitor (BPTI) in interaction with two proteinases, trypsin and chymotrypsin, by a procedure called molecular shaving. One to eight residues of the structural epitope, composed of two extended and exposed loops, were mutated to alanine(s). Although truncation of the side chains of residues surrounding the P(1) position to methyl groups caused a decrease in Delta G(den) values up to 6.4 kcal mole(-1), it did not influence the overall conformation of the inhibitor. We found that the replacement of up to six residues with alanines was fully additive at the level of protein stability. To analyze the influence of the structural epitope on the association energy, we determined association constants for BPTI variants and both enzymes and applied the additivity analysis. Shaving of two binding loops led to a progressive drop in the association energy, more pronounced for trypsin (decrease up to 9.6 kcal mole(-1)) than chymotrypsin (decrease up to 3.5 kcal mole(-1)). In the case of extensively mutated variants interacting with chymotrypsin, the association energies agreed very well with the values calculated from single mutational effects. However, when P(1)-neighboring residues were shaved to alanine(s), their contribution to the association energy was not fully removed because of the presence of methyl groups and main chain-main chain intermolecular hydrogen bonds. Moreover, the hot spot had a different contribution to the complex stability in the fully shaved BPTI variant compared with the wild type, which was caused by perturbations of the P(1)-S(1) electrostatic interaction. PMID:11910024

  9. Analysis of serine proteinase–inhibitor interaction by alanine shaving

    Science.gov (United States)

    Buczek, Olga; Koscielska-Kasprzak, Katarzyna; Krowarsch, Daniel; Dadlez, Micha?; Otlewski, Jacek

    2002-01-01

    We analyzed the energetic importance of residues surrounding the hot spot (the P1 position) of bovine pancreatic trypsin inhibitor (BPTI) in interaction with two proteinases, trypsin and chymotrypsin, by a procedure called molecular shaving. One to eight residues of the structural epitope, composed of two extended and exposed loops, were mutated to alanine(s). Although truncation of the side chains of residues surrounding the P1 position to methyl groups caused a decrease in ?Gden values up to 6.4 kcal mole?1, it did not influence the overall conformation of the inhibitor. We found that the replacement of up to six residues with alanines was fully additive at the level of protein stability. To analyze the influence of the structural epitope on the association energy, we determined association constants for BPTI variants and both enzymes and applied the additivity analysis. Shaving of two binding loops led to a progressive drop in the association energy, more pronounced for trypsin (decrease up to 9.6 kcal mole?1) than chymotrypsin (decrease up to 3.5 kcal mole?1). In the case of extensively mutated variants interacting with chymotrypsin, the association energies agreed very well with the values calculated from single mutational effects. However, when P1-neighboring residues were shaved to alanine(s), their contribution to the association energy was not fully removed because of the presence of methyl groups and main chain–main chain intermolecular hydrogen bonds. Moreover, the hot spot had a different contribution to the complex stability in the fully shaved BPTI variant compared with the wild type, which was caused by perturbations of the P1–S1 electrostatic interaction. PMID:11910024

  10. Negative effects of a nonhost proteinase inhibitor of ~19.8?kDa from Madhuca indica seeds on developmental physiology of Helicoverpa armigera (Hübner).

    Science.gov (United States)

    Jamal, Farrukh; Singh, Dushyant; Pandey, Prabhash K

    2014-01-01

    An affinity purified trypsin inhibitor from the seed flour extracts of Madhuca indica (MiTI) on denaturing polyacrylamide gel electrophoresis showed that MiTI consisted of a single polypeptide chain with molecular mass of ~19.8?kDa. MiTI inhibited the total proteolytic and trypsin-like activities of the midgut proteinases of Helicoverpa armigera larvae by 87.51% and 76.12%, respectively, at concentration of 5?µg/mL with an IC50 of 1.75?µg/mL against trypsin like midgut proteinases. The enzyme kinetic studies demonstrated that MiTI is a competitive inhibitor with a K i value of 4.1 × 10(-10)?M for Helicoverpa trypsin like midgut proteinases. In vivo experiments with different concentrations of MiTI in artificial diet (0.5, 1.0, and 1.5%?w/w) showed an effective downfall in the larval body weight and an increase in larval mortality. The concentration of MiTI in the artificial diet to cause 50% mortality (LD50) of larvae was 1.5%?w/w and that to cause reduction in mass of larvae by 50% (ED50) was 1.0%?w/w. Nutritional indices observations suggest the toxic and adverse effects of MiTI on the growth and development of H. armigera larvae. The results suggest a strong bioinsecticidal potential of affinity purified MiTI which can be exploited in insect pest management of crop plants. PMID:25298962

  11. Coronavirus 3CL(pro) proteinase cleavage sites: Possible relevance to SARS virus pathology

    DEFF Research Database (Denmark)

    Kiemer, Lars; Lund, Ole

    2004-01-01

    Background: Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS), efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results: We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator ( CFTR), transcription factors CREB-RP and OCT-I, and components of the ubiquitin pathway. Conclusions: Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses.

  12. Effect of (L)-cysteine on acetaldehyde self-administration.

    Science.gov (United States)

    Peana, Alessandra T; Muggironi, Giulia; Fois, Giulia R; Zinellu, Manuel; Sirca, Donatella; Diana, Marco

    2012-08-01

    Acetaldehyde (ACD), the first metabolite of ethanol, has been implicated in several behavioural actions of alcohol, including its reinforcing effects. Recently, we reported that l-cysteine, a sequestrating agent of ACD, reduced oral ethanol self-administration and that ACD was orally self-administered. This study examined the effects of l-cysteine pre-treatment during the acquisition and maintenance phases of ACD (0.2%) self-administration as well as on the deprivation effect after ACD extinction and on a progressive ratio (PR) schedule of reinforcement. In a separate PR schedule of reinforcement, the effect of l-cysteine was assessed on the break-point produced by ethanol (10%). Furthermore, we tested the effect of l-cysteine on saccharin (0.2%) reinforcement. Wistar rats were trained to self-administer ACD by nose poking on a fixed ratio (FR1) schedule in 30-min daily sessions. Responses on an active nose-poke caused delivery of ACD solution, whereas responses on an inactive nose-poke had no consequences. l-cysteine reduced the acquisition (40 mg/kg), the maintenance and the deprivation effect (100 mg/kg) of ACD self-administration. Furthermore, at the same dose, l-cysteine (120 mg/kg) decreased both ACD and ethanol break point. In addition, l-cysteine was unable to suppress the different responses for saccharin, suggesting that its effect did not relate to an unspecific decrease in a general motivational state. Compared to saline, l-cysteine did not modify responses on inactive nose-pokes, suggesting an absence of a non-specific behavioural activation. Taken together, these results could support the hypotheses that ACD possesses reinforcing properties and l-cysteine reduces motivation to self-administer ACD. PMID:22440691

  13. Cloning and characterization of a novel cysteine protease gene (HbCP1) from Hevea brasiliensis.

    Science.gov (United States)

    Peng, Shi-Qing; Zhu, Jia-Hong; Li, Hui-Liang; Tian, Wei-Min

    2008-12-01

    The full-length cDNA encoding a cysteine protease,designated HbCP1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCP1 contained a 1371 bp open reading frame encoding 457 amino acids.The deduced HbCP1 protein,which showed high identity to cysteine proteases of other plant species,was predicted to possess a putative repeat in toxin (RTX) domain at the N-terminal and a granulin (GRAN) domain at the C-terminal.Southern blot analysis indicated that the HbCP1 gene is present as a single copy in the rubber tree.Transcription pattern analysis revealed that HbCP1 had high transcription in laticifer,and low transcription in bark and leaf.The transcription of HbCP1 in latex was induced by ethylene and tapping.Cloning of the HbCP1 gene will enable us to further understand the molecular characterization of cysteine protease and its possible function in the rubber tree. PMID:19179756

  14. Studies of a novel cysteine sulfoxide lyase from Petiveria alliacea: the first heteromeric alliinase.

    Science.gov (United States)

    Musah, Rabi A; He, Quan; Kubec, Roman; Jadhav, Abhijit

    2009-11-01

    A novel alliinase (EC 4.4.1.4) was detected and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The isolated enzyme is a heteropentameric glycoprotein composed of two alpha-subunits (68.1 kD each), one beta-subunit (56.0 kD), one gamma-subunit (24.8 kD), and one delta-subunit (13.9 kD). The two alpha-subunits are connected by a disulfide bridge, and both alpha- and beta-subunits are glycosylated. The enzyme has an isoelectric point of 4.78 and pH and temperature optima of 8.0 and approximately 52 degrees C, respectively. Its activation energy with its natural substrate S-benzyl-l-cysteine sulfoxide is 64.6 kJ mol(-1). Kinetic studies showed that both K(m) and V(max) vary as a function of substrate structure, with the most preferred substrates being the naturally occurring P. alliacea compounds S-benzyl-l-cysteine sulfoxide and S-2-hydroxyethyl-l-cysteine sulfoxide. The alliinase reacts with these substrates to produce S-benzyl phenylmethanethiosulfinate and S-(2-hydroxyethyl) 2-hydroxyethanethiosulfinate, respectively. PMID:19789290

  15. [Inhibitors of proteolytic enzymes under abiotic stresses in plants (review)].

    Science.gov (United States)

    Mosolov, V V; Valueva, T A

    2011-01-01

    Data on the role of proteolytic enzyme inhibitors in plant adaptation to various unfavorable environmental abiotic factors--water deficiency, salinization of soil, extreme temperatures, etc.--and also probable functions of proteinases inhibitors in natural plant senescense are considered. PMID:22232890

  16. The cell envelope subtilisin-like proteinase is a virulence determinant for Streptococcus suis

    Directory of Open Access Journals (Sweden)

    Gottschalk Marcelo

    2010-02-01

    Full Text Available Abstract Background Streptococcus suis is a major swine pathogen and zoonotic agent that mainly causes septicemia, meningitis, and endocarditis. It has recently been suggested that proteinases produced by S. suis (serotype 2 are potential virulence determinants. In the present study, we screened a S. suis mutant library created by the insertion of Tn917 transposon in order to isolate a mutant deficient in a cell surface proteinase. We characterized the gene and assessed the proteinase for its potential as a virulence factor. Results Two mutants (G6G and M3G possessing a single Tn917 insertion were isolated. The affected gene coded for a protein (SSU0757 that shared a high degree of identity with Streptococccus thermophilus PrtS (95.9% and, to a lesser extent, with Streptococcus agalactiae CspA (49.5%, which are cell surface serine proteinases. The SSU0757 protein had a calculated molecular mass of 169.6 kDa and contained the catalytic triad characteristic of subtilisin family proteinases: motif I (Asp200, motif II (His239, and motif III (Ser568. SSU0757 also had the Gram-positive cell wall anchoring motif (Leu-Pro-X-Thr-Gly at the carboxy-terminus, which was followed by a hydrophobic domain. All the S. suis isolates tested, which belonged to different serotypes, possessed the gene encoding the SSU0757 protein. The two mutants devoid of subtilisin-like proteinase activity had longer generation times and were more susceptible to killing by whole blood than the wild-type parent strain P1/7. The virulence of the G6G and M3G mutants was compared to the wild-type strain in the CD1 mouse model. Significant differences in mortality rates were noted between the P1/7 group and the M3G and G6G groups (p Conclusion In summary, we identified a gene coding for a cell surface subtilisin-like serine proteinase that is widely distributed in S. suis. Evidences were brought for the involvement of this proteinase in S. suis virulence.

  17. A preliminary neutron crystallographic study of proteinase K at pD 6.5

    International Nuclear Information System (INIS)

    A preliminary neutron crystallographic study of the proteolytic enzyme proteinase K is presented. Large hydrogenated crystals were prepared in deuterated crystallization buffer using the vapor-diffusion method. Data were collected to a resolution of 2.3 on the LADI-III diffractometer at the Institut Laue Langevin (ILL) in 2.5 days. The results demonstrate the feasibility of a full neutron crystallographic analysis of this structure aimed at providing relevant information on the location of H atoms, particularly at the active site. This information will contribute to further understanding of the molecular mechanisms underlying proteinase K's catalytic activity and to an enriched understanding of the subtilisin clan of serine proteases

  18. The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction.

    OpenAIRE

    Stubbs, M. T.; Laber, B.; Bode, W.; Huber, R.; Jerala, R.; Lenarcic, B.; Turk, V.

    1990-01-01

    A stoichiometric complex of human stefin B and carboxymethylated papain has been crystallized in a trigonal crystal form. Data to 2.37 A resolution were collected using the area detector diffractometer FAST. The crystal structure of the complex has been solved by Patterson search techniques using papain as search model. Starting from the structure of chicken cystatin, the stefin structure was elucidated through cycles of model building and crystallographic refinement. The current crystallogra...

  19. Expression of Murine Coronavirus Recombinant Papain-Like Proteinase: Efficient Cleavage Is Dependent on the Lengths of both the Substrate and the Proteinase Polypeptides

    OpenAIRE

    Teng, Henry; Pin?o?n, Josefina D.; Weiss, Susan R.

    1999-01-01

    Proteolytic processing of the replicase gene product of mouse hepatitis virus (MHV) is essential for viral replication. In MHV strain A59 (MHV-A59), the replicase gene encodes two predicted papain-like proteinase (PLP) domains, PLP-1 and PLP-2. Previous work using viral polypeptide substrates synthesized by in vitro transcription and translation from the replicase gene demonstrated both cis and trans cleavage activities for PLP-1. We have cloned and overexpressed the PLP-1 domain in Escherich...

  20. Hidden weapons of microbial destruction in plant genomes

    OpenAIRE

    Manners, John M.

    2007-01-01

    Recent bioinformatic analyses of sequenced plant genomes reveal a previously unrecognized abundance of genes encoding antimicrobial cysteine-rich peptides, representing a formidable and dynamic defense arsenal against plant pests and pathogens.

  1. A Serendipitous Formation of a Cysteine-bridged Disaccharide

    Scientific Electronic Library Online (English)

    Mbulelo G., Nokwequ; Comfort M., Nkambule; David W., Gammon.

    2014-01-01

    Full Text Available SciELO South Africa | Language: English Abstract in english N-acetyl-L-cysteine bearing free carboxylic acid and sulfhydryl groups was glycosylated with 1,2,3,4,6-Penta-O-acetyl-ß-D-glucopyranoside in the presence of SnCl4 as a promoter to give the S-glycosylated cysteine in 64 % yield. However, when excess donor was used, a previously unreported cysteine-br [...] idged disaccharide was isolated in 54 % yield. The acetamido group on cysteine, which lowers the pKa of the carboxylic acid group of the amino acid, plays no role in the formation of the bridged disaccharide since 3-mercaptopropionic acid reacts in a similar manner to give the 3-mercaptopropionic acid-bridged disaccharide in 52 % yield.

  2. Drosophila photoreceptors express cysteine peptidase tan.

    Science.gov (United States)

    Wagner, Stefanie; Heseding, Christiane; Szlachta, Kamila; True, John R; Prinz, Heino; Hovemann, Bernhard T

    2007-02-01

    The Drosophila mutant tan (t) shows reciprocal pigmentation defects compared with the ebony (e) mutant. Visual phenotypes, however, are similar in both flies: Electroretinogram (ERG) recordings lack "on" and "off" transients, an indication of impaired synaptic transmission to postsynaptic cells L1 and L2. Cloning of tan revealed transcription of the gene in the retina, apparently in photoreceptor cells. We expressed Tan in Escherichia coli and confirmed by Western blotting and mass spectroscopic analyses that Tan is expressed as preprotein, followed by proteolytic cleavage into two subunits at a conserved --Gly--Cys-- motif like its fungal ortholog isopenicillin-N N-acyltransferase (IAT). Tan thus belongs to the large family of cysteine peptidases. To discriminate expression of Tan and Ebony in retina and optic neuropils, we raised antisera against specific Tan peptides. Testing for colocalization with GMR-driven n-Syb-GFP labeling revealed that Tan expression is confined to the photoreceptor cells R1-R8. A close proximity of Tan and Ebony expression is evident in lamina cartridges, where three epithelial glia cells envelop the six photoreceptor terminals R1-R6. In the medulla, R7/R8 axonal terminals appeared lined up side by side with glial extensions. This local proximity supports a model for Drosophila visual synaptic transmission in which Tan and Ebony interact biochemically in a putative histamine inactivation and recycling pathway in Drosophila. PMID:17154266

  3. Reexamination of the cysteine residues in glucocerebrosidase.

    Science.gov (United States)

    Moharram, Ramy; Maynard, Dawn; Wang, Eric S; Makusky, Anthony; Murray, Gary J; Martin, Brian M

    2006-06-12

    Glucocerebrosidase, the deficient enzyme in Gaucher disease, catalyzes the cleavage of the beta-glycosidic linkage of glucosylceramide. A previous study on the enzyme identified three disulfide bridges and a single sulfhydryl [Lee, Y., Kinoshita, H., Radke, G., Weiler, S., Barranger, J.A. and Tomich, J.M. (1995) Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase. J. Protein Chem. 14(3), 127-137] but recent publication of the X-ray structure identifies only two disulfide bridges with three free sulfhydryls [Dvir, H., Harel, M., McCarthy, A.A., Toker, L., Silman, I., Futerman, A.H. and Sussman, J.L. (2003) X-ray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease. EMBO. 4(7), 704-709]. Using chemical modifications, acid cleavage and enzymatic digestion methods, we report that three free sulfhydryls exist and that the remaining four cysteines form two disulfide bonds located within the first 25 amino-terminal residues, supporting the X-ray structure. PMID:16712845

  4. Expression of human ?1-proteinase inhibitor in Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Punt Peter J

    2007-10-01

    Full Text Available Abstract Background Human ?1-proteinase inhibitor (?1-PI, also known as antitrypsin, is the most abundant serine protease inhibitor (serpin in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, ?1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant ?1-PI (r-?1-PI could provide an attractive alternative. Although r-?1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation. Results We have explored the possibility of expressing the gene for human ?1-PI in the filamentous fungus Aspergillus niger (A. niger, a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of ?1-PI with a strongly expressed, secreted leader protein (glucoamylase G2, separated by dibasic processing site (N-V-I-S-K-R that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and ?1-PI activity assays enabled us to select the transformant(s secreting a biologically active glycosylated r-?1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS analysis further confirmed that molecular mass of the r-?1-PI was similar to that of the pd-?1-PI. In vitro stability of the r-?1-PI from A. niger was tested in comparison with pd-?1-PI reference and non-glycosylated human r-?1-PI from E. coli. Conclusion We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for ?1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for ?1-PI in A. niger was successfully achieved to produce the secreted mature human r-?1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth.

  5. Effects of cysteine introduction into three homologous cytochromes C.

    Science.gov (United States)

    Kobayashi, Yoshiko; Sonoyama, Takafumi; Takeda, Taku; Sambongi, Yoshihiro

    2009-05-01

    A cysteine residue was systematically introduced into three homologous cytochromes c from Hydrogenobacter thermophilus, Hydrogenophilus thermoluteolus, and Pseudomonas aeruginosa at a conserved position. The H. thermoluteolus variant showed the most decreased thermal stability as compared with the wild type, which might have been due in part to crosslinked polymer formation. The effects of cysteine introduction differed even at the conserved position in these homologous proteins. PMID:19420693

  6. ESR studies of radiation protection effect by cysteine and cysteamine

    International Nuclear Information System (INIS)

    By means of ESR a large transfer of radiation induced spins from thymine or cytosine to cysteamine in these binary systems has been confirmed. Furthermore, by substituting paramethylbenzenesulfonate of cysteine for its hydrochloride, marked protection effect of cysteine on thymine or deoxythymidine-5'-monophosphate-(NH4)2.2H2O (dTMP) in the above binary systems was also observed. The mechanism of this effect is briefly discussed. (author)

  7. Differential processing of Arabidopsis ubiquitin-like Atg8 autophagy proteins by Atg4 cysteine proteases

    OpenAIRE

    Woo, Jongchan; Park, Eunsook; Dinesh-kumar, S. P.

    2013-01-01

    Autophagy is a highly regulated biological process for recycling or degrading cellular contents in response to environmental stimuli. Atg4 cysteine protease-mediated processing of Atg8 proteins is required for formation of intracellular vesicles, autophagosomes, which carry cellular cargoes to the vacuole/lysosome. Because the Arabidopsis plant contains nine AtAtg8 and two AtAtg4 proteins, we have developed unique AtAtg8 synthetic substrates to determine AtAtg4s cleavage specificity. We show ...

  8. Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata).

    Science.gov (United States)

    Wilson, K A; Tan-Wilson, A L

    1987-05-01

    The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI-E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p-chlorophenylsulfonate, but not by iodoacetate and CuCl(2). It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4 degrees C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies. PMID:16665413

  9. Aspartic proteinases as virulence factors of .I.Candida./I. Spp.

    Czech Academy of Sciences Publication Activity Database

    Dostál, Ji?í; Hrušková-Heidingsfeldová, Olga; Hamal, P.; Pichová, Iva

    Smolenice, 2003. s. 66. [Výro?ná konferencia o kvasinkách /31./. 19.05.2003-21.05.2003, Smolenice] R&D Projects: GA MZd NI6485 Institutional research plan: CEZ:AV0Z4055905 Keywords : aspartic proteinases * .I.Candida./I. Subject RIV: CE - Biochemistry

  10. Correlation of phospholipase and proteinase production of Candida with in vivo pathogenicity in Galleria mellonella

    Scientific Electronic Library Online (English)

    Rodnei Dennis, Rossoni; Júnia Oliveira, Barbosa; Simone Furgeri Godinho, Vilela; Jéssica Diane dos, Santos; Antonio Olavo Cardoso, Jorge; Juliana Campos, Junqueira.

    2013-09-01

    Full Text Available SciELO Brazil | Language: English Abstract in english An essential factor to the virulence of the genus Candida is the ability to produce enzymes and this may be crucial in the establishment of fungal infections. AIM:This study investigated in vitro enzymatic activities of Candida species and their virulence in an in vivo Galleria mellonella experiment [...] al model. METHODS: Twenty-four clinical strains of Candida spp. isolated from the human oral cavity were evaluated, including the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. norvegensis, C. lusitaniae and C. guilliermondii. All Candida strains were tested in vitro for production of proteinase and phospholipase. The Candida strains were also injected into Galleria mellonella larvae to induce experimental candidiasis, and after 24 hours, the survival rate was assessed. RESULTS: Phospholipase and proteinase activity were observed in 100% of the C. albicans strains. In the non-albicans species, proteinase and phospholipase activity were observed in 25 and 43% of the studied strains, respectively. The most pathogenic Candida species in G. mellonella were C. albicans, C. dubliniensis and C. lusitaniae, whereas C. glabrata was the least virulent species. Furthermore, a positive significant correlation was found between both enzymatic activities with virulence in G. mellonella. CONCLUSIONS: The virulence of Candida strains in G. mellonella is related to the quantity of proteinases and phospholipases production of each strain.

  11. [Isolation and comparative properties of serine proteinases of the microscopic fungi Trichoderma lignorum and Trichoderma koningii].

    Science.gov (United States)

    Ga?da, A V; Rudenskaia, G N; Stepanov, V M

    1981-11-01

    Using affinity chromatography on bacitracin-Sepharose combined with ion-exchange chromatography on aminosilochrome and isoelectrofocusing, individual serine proteases have been isolated for the first time from surface cultures of T. lignorum and T. koningii. The proteinases have pI values at 6.8 and 6.7 and pH optima of 10.5. Both proteinases are stable within the pH range of 4-11 and have molecular weight of 21 000. The amino acid composition of T. lignorum enzyme is Lys3His4Arg9Asx23Thr17Ser23Glx10Pro8Gly27Ala25Cys3Val13Met2Ile11Leu11Tyr7Phe6Tr p3, that of the T. koningii enzyme is Lys3His4 Arg9Asx23Thr16Ser26Glx10Pro9Gly29Ala26Cys3Val14Met2Ile9Leu11Tyr6Phe5Trp3. The enzymes are completely inhibited by phenylmethylsulfonylfluoride, diphenylcarbamoylchloride and trypsin inhibitors from beans, Actinomyces janthinus and potato tubers. The enzymatic and molecular properties of the enzymes are similar to those of subtilisins and previously described fungal serine proteinases, especially to those of proteinase K of the fungus Tritirachium album Limber. PMID:7032610

  12. Isolation and Properties of Stachyrase A, a Chymotrypsin-Like Serine Proteinase from Stachybotrys chartarum

    OpenAIRE

    Kordula, Tomasz; Banbula, Agnieszka; Macomson, Jeremy; Travis, James

    2002-01-01

    A strain of the common mold Stachybotrys chartarum has been isolated from the lung of a child with pulmonary hemorrhage. We report the purification of stachyrase A, a new serine chymotrypsin-like proteinase from S. chartarum. This enzyme cleaves major protease inhibitors, several biologically active peptides, and collagen, all of which are found in the lung.

  13. Non-infectious fluorimetric assay for phenotyping of drug-resistant HIV proteinase mutants.

    Czech Academy of Sciences Publication Activity Database

    Majerová, Ta?ána; Dantuma, N. P.; Lindsten, K.; Masucci, M. G.; Konvalinka, Jan

    2006-01-01

    Ro?. 36, ?. 1 (2006), s. 50-59. ISSN 1386-6532 R&D Projects: GA MŠk(CZ) 1M0508; GA MZd(CZ) NR8571 Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV proteinase * Hiv protease * HIV PR * green fluorescent prrotein Subject RIV: CE - Biochemistry Impact factor: 2.630, year: 2006

  14. Proteinases release 35S-labeled macromolecules from cultured airway epithelial cells

    International Nuclear Information System (INIS)

    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 35SO4 (50?ci/ml/24h) then changed the medium every 20 min and measured nondialyzable 35S released into the medium. Two h later, the rate of spontaneous release of 35S-labeled-macromolecules was 5700 +/- 1600 CPM/20 min (mean +/- SD). At this time trypsin, thermolysin, pseudomonas elastase and alkaline proteinase, each released 35S-labeled-macromolecules, whereas aspergillus acid proteinase did not. In more detailed studies, trypsin released 35S 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 ? 106), and a second, included fraction (MW 2-3 x 105). 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

  15. OZONE EFFECTS ON ALPHA-1-PROTEINASE INHIBITOR IN VIVO: BLOOD PLASMA INHIBITORY ACTIVITY IS UNCHANGED

    Science.gov (United States)

    The possible oxidative inactivation of human blood plasma alpha-1-proteinase inhibitor (PI) by inhaled ozone was assessed. Eleven male volunteers (non-smokers) were exposed to 0.5 ppm ozone for four hours on two consecutive days and ten control subjects were exposed to air under ...

  16. Cysteine- and glutathione-mediated uptake of lead and cadmium into Zea mays and Brassica napus roots

    International Nuclear Information System (INIS)

    This study examines a new mechanism for the uptake of Pb and Cd into Brassica napus and Zea mays roots. During hydroponic experiments, the uptake of Pb and Cd was enhanced in the presence of cysteine and glutathione, whereas no or very low uptake was observed in EDTA and penicillamine controls. Uptake rates were also enhanced after pre-exposure to cysteine or glutathione and inhibited in the presence of vanadate, suggesting a biological mechanism of uptake. Increasing concentrations of glutathione in solution resulted in decreasing Pb uptake rates, indicating competition for transport between free-glutathione and Pb-glutathione species. Pb uptake in the presence of increasing cysteine concentrations resulted in decreased uptake initially but linearly increasing uptake at higher concentrations. Experimentation showed concentration dependent Pb uptake rates. We speculate that there are specific transporters for these thiol ligands and describe what barriers remain for application of this novel transport mechanism in chelator-assisted phytoremediation. - Cysteine and glutathione mediate the transport of lead and cadmium into plant roots.

  17. Hordeum vulgare cysteine protease heterologous expressed in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe

    During germination of barley seeds, the mobilization of protein is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction of the active site cysteines and via removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. One of the key cysteine proteases in Barley, (Hordeum vulgare) endoprotease B2 (HvEPB2) was cloned with and without the 5 amino acid C-terminal sequence into the Pichia pastoris expression vector pPICZ A? and electrotransformed into Pichia pastoris strain SDM1163. Heterologous protein production was induced with 2% MeOH and the protein expression were monitered during induction by collecting 1 ml samples every hr for 24 hrs. After 4 days, the supernatant were harvested and analyzed by SDS-PAGE, activity assay and Western blot. A significant amount of functional, heterologous protein was produced and the protein production was highest after 4 days and the expression in the C-terminal mutant was slightly higher than for the full length protease.

  18. Transcriptional activation of a 37 kDa ethylene responsive cysteine protease gene, RbCP1, is associated with protein degradation during petal abscission in rose

    OpenAIRE

    Tripathi, Siddharth Kaushal; Singh, Amar Pal; Sane, Aniruddha P.; Nath, Pravendra

    2009-01-01

    Cysteine proteases play an important role in several developmental processes in plants, particularly those related to senescence and cell death. A cysteine protease gene, RbCP1, has been identified that encodes a putative protein of 357 amino acids and is expressed in the abscission zone (AZ) of petals in rose. The gene was responsive to ethylene in petals, petal abscission zones, leaves, and thalamus. The expression of RbCP1 increased during both ethylene-induced as well as natural abscissio...

  19. Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinases and with hemoglobin.

    OpenAIRE

    Dalton, J. P.; Mcgonigle, S.; Rolph, T. P.; Andrews, S. J.

    1996-01-01

    Two cathepsin L proteinases, cathepsin L1 and cathepsin L2, secreted by liver flukes may be involved in tissue penetration, nutrition, and protection from immune attack. To ascertain the immunoprophylactic potential of these proteinases, and of another molecule, liver fluke hemoglobin (Hb), we performed vaccine trials in cattle. In the first vaccine trial various doses of cathepsin L1 were tested. The mean protection level obtained was 53.7%. In a second vaccine trial cathepsin L1 and Hb elic...

  20. Binding properties of the regulatory domains in Manduca sexta hemolymph proteinase-14, an initiation enzyme of the prophenoloxidase activation system

    OpenAIRE

    Wang, Yang; Jiang, Haobo

    2009-01-01

    Pathogen recognition and rapid initiation of defense responses are essential for the survival of host insects. In Manduca sexta, hemolymph proteinase-14 precursor (proHP14) senses non-self presence and triggers a branched serine proteinase pathway which leads to prophenoloxidase activation and melanin formation around the invading organisms. To understand functions of individual domains in HP14, we have produced a series of HP14 domains and truncation mutants and studied their interactions wi...

  1. Manduca sexta prophenoloxidase activating proteinase-1 (PAP-1) gene: Organization, expression, and regulation by immune and hormonal signals

    OpenAIRE

    Zou, Zhen; Wang, Yang; Jiang, Haobo

    2005-01-01

    Insect phenoloxidase (PO) participates in melanotic encapsulation, wound healing, and cuticle sclerotization. It is converted from prophenoloxidase (proPO) by a proPO-activating proteinase (PAP). Manduca sexta PAP-1, the final component of a serine proteinase cascade, cleaves proPO to generate active PO. In an effort to understand the transcriptional regulation, we isolated a genomic clone of the PAP-1 gene, determined its nucleotide sequence, and elucidated its exon–intron organization. Co...

  2. Effects of Serine Protease Inhibitors on Growth and Development and Digestive Serine Proteinases of the Sunn Pest, Eurygaster integriceps

    OpenAIRE

    Saadati, Fatemeh; Bandani, Ali R.

    2011-01-01

    In the current study the effects of serine proteinase inhibitors (TLCK, TPCK, SBTI, and a combination of SBTI and TPCK) with concentrations of 1% and 4% of dietary protein in artificial diets were tested against growth of the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), development, and its gut serine proteinase targets. Analysis of variance indicated that protease inhibitors affected nymphal development time, adult weight, and survival. Mean development time of third i...

  3. Seven cysteine-deficient mutants depict interplay between thermal and chemical stabilities of individual cysteine residues in MAP kinase JNK1

    International Nuclear Information System (INIS)

    To characterize the role of cysteine residues on the structure, function and stability of JNK1, we prepared and evaluated the wild-type JNK1 and seven cysteine-deficient JNK1 proteins. The solvent exposed cysteine residues did not influence biological function and mutating these residues raised the thermal stability because of newly formed hydrogen bonds and of higher hydration as speculated by the mutant structures. The surface cysteine involved in the molecular-surface hydrophobic pocket did not affect biological function; although a moderate thermal destabilization was observed. Cysteines in the loosely-assembled hydrophobic environment moderately contributed to thermal stability and the mutations of these cysteines had negligible effect on enzyme activity. The other cysteines are involved in the tightly-filled hydrophobic core and mutation of these residues conferred the adverse effects on the thermal stability and enzyme activity. (author)

  4. The involvement of mast cells and mast cell proteinases in the intestinal response to equine cyathostomin infection.

    Science.gov (United States)

    du Toit, Nicole; McGorum, Bruce C; Pemberton, Alan D; Brown, Jeremy; Dacre, Kirstie J

    2007-01-15

    Cyathostomins (Cyathostominae) are regarded as the most pathogenic equine nematode worldwide. These nematodes are difficult to control in equine populations due to emerging anthelmintic resistance and evasion of encysted larval cyathostomins to regular modern anthelmintics. Mast cells and their proteinases have been shown to play a role in the mammalian immune response to nematode infections. Involvement of mast cells and mast cell proteinases in the equine immune response to cyathostomin infection is proposed. A technique was established to perform immunohistochemical staining using polyclonal rabbit anti-equine mast cell proteinase-1 (eqMCP-1) and anti-equine tryptase on formalin-fixed large intestinal sections, from horses classified as cyathostomin positive and negative at the time of death based upon larval enumeration. Quantitative analysis of antibody labelled mast cells was used to detect mast cell proteinases in equine large intestinal sections positive and negative for cyathostomin larvae. This demonstrated an increase in equine tryptase labelled mucosal and submucosal mast cells in cyathostomin positive horses. This study has established an immunohistochemical technique to demonstrate mast cell proteinases in formalin-fixed large intestinal sections. This technique may be used to determine possible involvement of mast cells and their proteinases in the equine immune response to cyathostomin larvae. Further studies are required to define a specific role. PMID:17118461

  5. Clinical effects of L-cystein combined with radiotherapy

    International Nuclear Information System (INIS)

    Twenty-eight patients with cancer of the cervix uteri, etc., who had had radiotherapy were administered L-cystein. L-cystein's ability to protect against leukopenia was studied. Teletherapy using 60Co was performed. A dose of 200 R (total dose, 6,000 R) was administered as a rule. Treatment was remarkably effective in 1 of 28 patients (no leukopenia was noted after irradiation), effective in 23 (leukopenia occurred but radiotherapy could be continued), unremarkable in 2 (marked leukopenia was observed but radiotherapy using other agents could be continued), and ineffective in 2 (radiotherapy was discontinued due to marked leukopenia). The rate of effectiveness was 85.7%, and L-cystein was found to have the ability to protect against leukopenia. (Nishio, M.)

  6. Arabidopsis cysteine-rich receptor-like kinase 45 positively regulates disease resistance to Pseudomonas syringae.

    Science.gov (United States)

    Zhang, Xiujuan; Han, Xiaomin; Shi, Rui; Yang, Guanyu; Qi, Liwang; Wang, Ruigang; Li, Guojing

    2013-12-01

    Arabidopsis cysteine-rich receptor-like protein kinase 45 (CRK45) was found to be involved in ABA signaling in Arabidopsis thaliana previously. Here, we reported that it also positively regulates disease resistance. The CRK45 overexpression plants increased expression of the defense genes, and enhanced resistance to Pseudomonas syringae whereas the crk45 mutant were more sensitive to P. syringae and weakened expression of the defense genes, compared to the wild type. We also found that treatment with P. syringae leads to a declined expression of CRK45 in the npr1 mutant and the NahG transgenic plants. At the same time, significantly decreased expression of CRK45 transcript in the wrky70 mutant than that in the wild type was also detected. Our results suggested that CRK45 acted as a positive regulator in Arabidopsis disease resistance, and was regulated downstream of NPR1 and WRKY70 at the transcriptional level. PMID:24215930

  7. A cysteine endopeptidase with a C-terminal KDEL motif isolated from castor bean endosperm is a marker enzyme for the ricinosome, a putative lytic compartment.

    Science.gov (United States)

    Schmid, M; Simpson, D; Kalousek, F; Gietl, C

    1998-10-01

    A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863-871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.). PMID:9763713

  8. When activity requires breaking up: LEKTI proteolytic activation cascade for specific proteinase inhibition.

    Science.gov (United States)

    Furio, Laetitia; Hovnanian, Alain

    2011-11-01

    Lymphoepithelial Kazal-type related inhibitor (LEKTI) is a multidomain proteinase inhibitor whose defective expression causes Netherton syndrome (NS). LEKTI is encoded by SPINK5, which is also a susceptibility gene for atopic disease. In this issue, Fortugno et al. report an elegant and thorough study of the LEKTI proteolytic activation process in which they identify the precise nature of the cleavage sites used and the bioactive fragments generated. They propose a proteolytic activation model in human skin and confirm differential inhibition of kallikrein (KLK) 5, 7, and 14 by the major physiological LEKTI fragments. They show that these bioactive fragments inhibit KLK-mediated proteolysis of desmoglein 1 (DSG1) and suggest a fine-tuned inhibition process controlling target serine proteinase (SP) activity. PMID:21997416

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

    Directory of Open Access Journals (Sweden)

    Wided Nouira

    2014-07-01

    Full Text Available 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.

  10. Suppression of collagen-induced arthritis with a serine proteinase inhibitor (serpin) derived from myxoma virus.

    Science.gov (United States)

    Brahn, Ernest; Lee, Sarah; Lucas, Alexandra; McFadden, Grant; Macaulay, Colin

    2014-08-01

    Many viruses encode virulence factors to facilitate their own survival by modulating a host's inflammatory response. One of these factors, secreted from cells infected with myxoma virus, is the serine proteinase inhibitor (serpin) Serp-1. Because Serp-1 had demonstrated anti-inflammatory properties in arterial injury models and viral infections, it was cloned and evaluated for therapeutic efficacy in collagen-induced arthritis (CIA). Clinical severity was significantly lower in the Serp-1 protocols (pSerp-1 group had significantly less erosions than the controls (pSerp-1 group but antibody titers to type II collagen were not significantly altered. Recipients had minimal histopathologic synovial changes and did not develop neutralizing antibodies to Serp-1. These results indicate that Serp-1 impedes the pathogenesis of CIA and suggests that the therapeutic potential of serine proteinase inhibitors in inflammatory joint diseases, such as rheumatoid arthritis, should be investigated further. PMID:24845791

  11. On a Bowman-Birk family proteinase inhibitor from Erythrina variegata seeds.

    Science.gov (United States)

    Kimura, M; Kouzuma, Y; Abe, K; Yamasaki, N

    1994-03-01

    A Bowman-Birk family proteinase inhibitor (EBI) was isolated from the seeds of Erythrina variegata. The protein was purified by ion-exchange column chromatography on DEAE-cellulose followed by gel filtration on Sephadex G-75. The stoichiometry with trypsin was estimated to be 1:1, while that with chymotrypsin was not obvious, as determined from the titration patterns of its inhibitory activities. The complete amino acid sequence of EBI was determined by sequencing tryptic and chymotryptic peptides. The EBI protein consists of 61 amino acid residues, which is the shortest among the Bowman-Birk family inhibitors sequenced to date, and has a M(r) of 6,689. Comparison of this sequence with those of other leguminous Bowman-Birk family inhibitors revealed that EBI could be classified as a group II inhibitor, showing the best homology (67%) to the Bowman-Birk proteinase inhibitor from soybeans. PMID:8056744

  12. A preliminary neutron crystallographic study of proteinase K at pD 6.5

    Energy Technology Data Exchange (ETDEWEB)

    Gardberg, Anna S [ORNL; Blakeley, Matthew P. [Institut Laue-Langevin (ILL); Myles, Dean A A [ORNL

    2009-01-01

    AbstractA preliminary neutron crystallographic study of the proteolytic enzyme proteinase K is presented. Large hydrogenated crystals were prepared in deuterated crystallization buffer using the vapour-diffusion method. Data were collected to a resolution of 2.3 on the LADI-III diffractometer at the Institut Laue Langevin (ILL) in 2.5 days. The results demonstrate the feasibility of a full neutron crystallographic analysis of this structure aimed at providing relevant information on the location of H atoms, particularly at the active site. This information will contribute to further understanding of the molecular mechanisms underlying proteinase K's catalytic activity and to an enriched understanding of the subtilisin clan of serine proteases.

  13. Purification and characterization of cysteine protease from germinating cotyledons of horse gram

    Directory of Open Access Journals (Sweden)

    Rao Sridhar K

    2009-11-01

    Full Text Available Abstract Background Proteolytic enzymes play central role in the biochemical mechanism of germination and intricately involved in many aspects of plant physiology and development. To study the mechanism of protein mobilization, undertaken the task of purifying and characterizing proteases, which occur transiently in germinating seeds of horse gram. Results Cysteine protease (CPRHG was purified to homogeneity with 118 fold by four step procedure comprising Crude extract, (NH42SO4 fractionation, DEAE-Cellulose and CM-sephacel chromatography from the 2 day germinating cotyledons of horse gram (Macrotyloma uniflorum (Lam. Verdc.. CPRHG is a monomer with molecular mass of 30 k Da, was determined by SDS-PAGE and gel filtration. The purified enzyme on IEF showed two isoforms having pI values of 5.85 and 6.1. CPRHG composed of high content of aspartic acid, glutamic acid and serine. The enzyme activity was completely inhibited by pCMB, iodoacetate and DEPC indicating cysteine and histidine residues at the active site. However, on addition of sulfhydryl reagents (cysteine, dithiothreitol, glutathione and beta-ME reverse the strong inhibition by pCMB. The enzyme is fairly stable toward pH and temperature. Immunoblot analysis shows that the enzyme synthesized as zymogen (preproenzyme with 81 kDa and processed to a 40 kDa proenzyme which was further degraded to give 30 kDa active enzyme. Conclusion It appears that the newly synthesized protease is inactive, and activation takes place during germination. CPRHG has a broad substrate specificity and stability in pH, temperature, etc. therefore, this protease may turn out to be an efficient choice for the pharmaceutical, medicinal, food, and biotechnology industry.

  14. SARS CoV Main Proteinase: The Monomer-Dimer Equilibrium Dissociation Constant

    Energy Technology Data Exchange (ETDEWEB)

    Graziano,V.; McGrath, W.; Yang, L.; Mangel, W.

    2006-01-01

    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, K{sub D}, have varied more than 650000-fold, from <1 nM to more than 200 {mu}M. 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 K{sub D} values of 5.8 {+-} 0.8 {mu}M (obtained from the entire scattering curve), 6.5 {+-} 2.2 {mu}M (obtained from the radii of gyration), and 6.8 {+-} 1.5 {mu}M (obtained from the forward scattering). The K{sub D} from chemical cross-linking was 12.7 {+-} 1.1 {mu}M, and from enzyme kinetics, it was 5.2 {+-} 0.4 {mu}M. While each of these three techniques can present different, potential limitations, they all yielded similar K{sub D} values.

  15. Enzymatic reduction of oxidized alpha-1-proteinase inhibitor restores biological activity.

    OpenAIRE

    Abrams, W. R.; Weinbaum, G.; Weissbach, L.; Weissbach, H.; Brot, N.

    1981-01-01

    The major serum inhibitor of proteolytic activity, alpha-1-proteinase inhibitor (alpha-1-PI), (or alpha-1-antitrypsin) can be readily inactivated by oxidation [Carp, H. & Janoff, A. (1978) Am. Rev. Resp. Dis. 118, 617-621]. This inactivation appears to be due to the oxidation of a critical methionine(s) in alpha-1-PI that is required for the inhibition of elastase activity. An enzyme from Escherichia coli that reduces methionine sulfoxide residues in protein [Brot, N., Weissbach, L., Werth, J...

  16. Secretory leukoprotease inhibitor: partnering alpha 1-proteinase inhibitor to combat pulmonary inflammation.

    OpenAIRE

    Bingle, L.; Tetley, T. D.

    1996-01-01

    Secretory leukoprotease inhibitor (SLPI) is a low molecular weight serine proteinase inhibitor, notably of neutrophil elastase (NE), which is synthesised and secreted by the pulmonary epithelium. SLPI plays an important role in limiting NE-induced pulmonary inflammation and, significantly, it also possesses anti-HIV activity. SLPI is a significant component of the anti-NE shield in the lung which has different reactivity from, and is therefore complementary to, the anti-NE action of alpha 1-p...

  17. Interaction of ?-1,3-Glucan with Its Recognition Protein Activates Hemolymph Proteinase 14, an Initiation Enzyme of the Prophenoloxidase Activation System in Manduca sexta*

    OpenAIRE

    Wang, Yang; Jiang, Haobo

    2006-01-01

    A serine proteinase pathway in insect hemolymph leads to prophenoloxidase activation, an innate immune response against pathogen infection. In the tobacco hornworm Manduca sexta, recombinant hemolymph proteinase 14 precursor (pro-HP14) interacts with peptidoglycan, autoactivates, and initiates the proteinase cascade (Ji, C., Wang, Y., Guo, X., Hartson, S., and Jiang, H. (2004) J. Biol. Chem. 279, 34101–34106). Here, we report the purification and characterization of pro-HP14 from the hemoly...

  18. Phage display selection of P1 mutants of BPTI directed against five different serine proteinases.

    Science.gov (United States)

    Kiczak, L; Koscielska, K; Otlewski, J; Czerwinski, M; Dadlez, M

    1999-01-01

    The P1 position of protein inhibitors and oligopeptide substrates determines, to a large extent, association energy with many serine proteinases. To test the agreement of phage display selection with the existing thermodynamic data, a small library of all 20 P1 mutants of basic pancreatic trypsin inhibitor (BPTI) was created, fused to protein III, and displayed on the surface of M13 phage. The wild type of displayed inhibitor monovalently and strongly inhibited trypsin with an association constant of Ka = 3 x 10(11) M(-1). The library was applied to select BPTI variants active against five serine proteinases of different specificity (bovine trypsin and chymotrypsin, human leukocyte and porcine pancreatic elastases, human azurocidin). The results of enrichment with four proteinases agreed well with the available thermodynamic data. In the case of azurocidin, the phage display selection allowed determination of the P1 specificity of this protein with the following frequencies for selected P1 variants: 43% Lys, 36% Leu, 7% Met, 7% Thr, 7% Gln. PMID:10064144

  19. Epithelial effects of proteinase-activated receptors in the gastrointestinal tract

    Scientific Electronic Library Online (English)

    Wallace K, MacNaughton.

    2005-03-01

    Full Text Available SciELO Brazil | Language: English Abstract in english The intestinal epithelium plays a crucial role in providing a barrier between the external environment and the internal milieu of the body. A compromised mucosal barrier is characteristic of mucosal inflammation and is a key determinant of the development of intestinal diseases such as Crohn's disea [...] se and ulcerative colitis. The intestinal epithelium is regularly exposed to serine proteinases and this exposure is enhanced in numerous disease states. Thus, it is important to understand how proteinase-activated receptors (PARs), which are activated by serine proteinases, can affect intestinal epithelial function. This review surveys the data which demonstrate the wide distribution of PARs, particularly PAR-1 and PAR-2, in the gastrointestinal tract and accessory organs, focusing on the epithelium and those cells which communicate with the epithelium to affect its function. PARs have a role in regulating secretion by epithelia of the salivary glands, stomach, pancreas and intestine. In addition, PARs located on subepithelial nerves, fibroblasts and mast cells have important implications for epithelial function. Recent data outline the importance of the cellular site of PAR expression, as PARs expressed on epithelia may have effects that are countered by PARs expressed on other cell types. Finally, PARs and their ability to promote epithelial cell proliferation are discussed in terms of colon cancer.

  20. [Characteristics of proteinase digestive function in invertebrates--inhabitants of cold seas].

    Science.gov (United States)

    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. PMID:18038635

  1. Biochemical characterization of VQ-VII, a cysteine peptidase with broad specificity, isolated from Vasconcellea quercifolia latex.

    Science.gov (United States)

    Torres, María José; Trejo, Sebastián Alejandro; Natalucci, Claudia Luisa; López, Laura María Isabel

    2013-06-01

    The latex from Vasconcellea quercifolia ("oak leaved papaya"), a member of the Caricaceae family, contains at least seven cysteine endopeptidases with high proteolytic activity, which helps to protect these plants against injury. In this study, we isolated and characterized the most basic of these cysteine endopeptidases, named VQ-VII. This new purified enzyme was homogeneous by bidimensional electrophoresis and MALDI-TOF mass spectrometry, and exhibited a molecular mass of 23,984 Da and an isoelectric point >11. The enzymatic activity of VQ-VII was completely inhibited by E-64 and iodoacetic acid, confirming that it belongs to the catalytic group of cysteine endopeptidases. By investigating the cleavage of the oxidized insulin B-chain to establish the hydrolytic specificity of VQ-VII, we found 13 cleavage sites on the substrate, revealing that it is a broad-specificity peptidase. The pH profiles toward p-Glu-Phe-Leu-p-nitroanilide (PFLNA) and casein showed that the optimum pH is about 6.8 for both substrates, and that in casein, it is active over a wide pH range (activity higher than 80 % between pH 6 and 9.5). Kinetic enzymatic assays were performed with the thiol peptidase substrate PFLNA (K m = 0.454 ± 0.046 mM, k cat = 1.57 ± 0.07 s(-1), k cat/K m = 3.46 × 10(3) ± 14 s(-1) M(-1)). The N-terminal sequence (21 amino acids) of VQ-VII showed an identity >70 % with 11 plant cysteine peptidases and the presence of highly conserved residues and motifs shared with the "papain-like" family of peptidases. VQ-VII proved to be a new latex enzyme of broad specificity, which can degrade extensively proteins of different nature in a wide pH range. PMID:23568402

  2. One of the three proteinase inhibitor genes newly identified in the Brassica napus genome codes for an inhibitor of glutamyl endopeptidase.

    Science.gov (United States)

    De Leo, Francesca; Volpicella, Mariateresa; Sciancalepore, Marta; Gallerani, Raffaele; Ceci, Luigi R

    2006-02-01

    Three proteinase inhibitor genes have been identified in the rapeseed (Brassica napus) genome. They are highly homologous to other genes of the mustard inhibitor (MSI) family of proteinase inhibitors characteristic of Cruciferae. In germinating seeds, only the transcript of one gene, coding for a trypsin inhibitor, is detectable by Northern analysis. The other two genes are transcribed at basal levels detectable only by reverse transcription PCR. One of the other two genes (rti-2) encodes a polypeptide with a glutamic residue in the P1 position, characteristic of glutamyl proteinase inhibitors. The recombinant RTI-2 protein strongly inhibits (Ki=44 nM) a glutamyl proteinase from Streptomyces griseus. PMID:16438970

  3. Plants

    Science.gov (United States)

    SRowley

    2006-04-28

    Children will learn a variety of themes that will teach children about spring and how to grow plants while incorporating core related material. Flowers, The children will learn about different qualities of flowers while learning shapes, counting, and colors. Flowers Gardens, The children will learn how to plant and take care of a garden. Gardens Rain, The children will learn that gardens need rain to grow. Students will also learn about evaporation. Rain Making Rain Story Time Flower Story ...

  4. Comparison of concentrations of two proteinase inhibitors, porcine pancreatic elastase inhibitory capacity, and cell profiles in sequential bronchoalveolar lavage samples.

    Science.gov (United States)

    Morrison, H M; Kramps, J A; Dijkman, J H; Stockley, R A

    1986-01-01

    Bronchoalveolar lavage is used to obtain cells and proteins from the lower respiratory tract for diagnosis and research. Uncertainity exists about which site in the lung is sampled by the lavage fluid and what effect different lavage volumes have on recovery of the constituents of lavage fluid. Dilution of alveolar lining fluid by lavage fluid is variable and results are usually expressed as protein ratios to surmount this problem. We have compared cell profiles and the concentrations of two proteinase inhibitors--the low molecular weight bronchial protease inhibitor antileucoprotease and alpha 1 proteinase inhibitor, together with alpha 1 proteinase inhibitor function and its relationship to the cell profile in sequential bronchoalveolar lavage fluid samples from patients undergoing bronchoscopy. There was no difference in total or differential cell counts or albumin or alpha 1 proteinase inhibitor concentrations between the first and second halves of the lavage. Both the concentration of antileucoprotease and the ratio of antileucoprotease to albumin were, however, lower in the second half of the lavage (2p less than 0.01 and 2p less than 0.05 respectively). There was no difference in the function of alpha 1 proteinase inhibitor (assessed by inhibition of porcine pancreatic elastase--PPE) between aliquots (0.28 mole PPE inhibited/mol alpha 1 proteinase inhibitor; range 0-1.19 for the first half and 0.37 mol PPE inhibited/mol alpha 1 proteinase inhibitor; range 0.10-0.80 for the second half). About 60-70% of alpha 1 proteinase inhibitor in each half of the lavage fluid was inactive as an inhibitor. The function of alpha 1 proteinase inhibitor did not differ between bronchitic smokers and ex-smokers. Alpha 1 proteinase inhibitor function was not related to the number of total white cells, macrophages, or neutrophils in the lavage fluid. Contamination of lavage by red blood cells was found to alter the concentration of alpha 1 proteinase inhibitor but not its function when aliquots with and without erythrocytes were compared. These results show that the only difference between the two halves of these lavage samples is in the amount of antileucoprotease present, suggesting that more proximal secretions are being harvested early in the lavage procedure. Much of the alpha 1 proteinase inhibitor present in the samples is functionally inactive, but this is not clearly related to any particular cell type or to smoking habits, and does not differ between different stages of the lavage procedure. Much of the alpha1 proteinase inhibitor present in the samples is functionally inactive, but this is not clearly related to any particular cell type or to smoking habits, and does not differ between different stages of the lavage procedure. Finally, the presence of erythrocytes probably does affect alpha(1) proteinase inhibitor concentration and such samples should be excluded from analysis. PMID:3491440

  5. Cysteine-Mediated Reductive Dissolution of Poorly Crystalline Iron(III)Oxides by Geobacter sulfurreducens

    OpenAIRE

    Doong, Ruey-an; Schink, Bernhard

    2002-01-01

    The reductive dissolution of poorly crystalline ferric oxides in the presence of cysteine was investigated to evaluate the potential of cysteine as a possible electron carrier to stimulate the reduction of iron(III) oxides by Geobacter sulfurreducens. The extent and rate of biotic and abiotic reduction of iron(III) oxides in the presence of cysteine at various concentrations were compared. Iron(III) oxides were reduced abiotically by cysteine. The initial rate and extent of iron(III) oxide re...

  6. Cysteine pKa values for the bacterial peroxiredoxin AhpC†‡

    OpenAIRE

    Nelson, Kimberly J.; Parsonage, Derek; Hall, Andrea; Karplus, P. Andrew; Poole, Leslie B.

    2008-01-01

    Salmonella typhimurium AhpC is a founding member of the peroxiredoxin family, a ubiquitous group of cysteine-based peroxidases with high reactivity toward hydrogen peroxide, organic hydroperoxides and peroxynitrite. For all of the peroxiredoxins, the catalytic cysteine, referred to as the peroxidatic cysteine (CP), acts as a nucleophile in attacking the peroxide substrate, forming a cysteine sulfenic acid at the active site. Because thiolates are far stronger nucleophiles than thiol groups, i...

  7. Transaminative metabolism of L-cysteine in guinea pig liver and kidney.

    OpenAIRE

    Taniguchi, Miyabi; Hosaki, Yasuhiro; Ubuka, Toshihiko

    1984-01-01

    Transaminative metabolism of L-cysteine was investigated using homogenates of guinea pig liver and kidney. L-Cysteine was transaminated in the presence of 2-oxoglutarate and the homogenate of either liver or kidney. S-(2-Hydroxy-2-carboxyethylthio)cysteine (HCETC) (3-mercaptolactate-cysteine disulfide) was formed by liver homogenate, but the amount was very small. On the other hand, a relatively large amount of HCETC was formed in the presence of kidney homogenate. Transamination between 3-me...

  8. Some peculiarities of synthesis of cysteine-containing peptides

    Energy Technology Data Exchange (ETDEWEB)

    Kudryavtseva, Elena V; Sidorova, Mariya V [Russian Cardiologycal Scientific Centre, Ministry of Health Care and Medical Industry of the Russian Federation, Moscow (Russian Federation); Evstigneeva, Rima P [M.V. Lomonosov Moscow State Academy of Fine Chemical Technology, Moscow (Russian Federation)

    1998-07-31

    Data on protective groups for the thiol function of cysteine and methods of disulfide bonds formation used in modern peptide chemistry are considered and systematised. The advantages and disadvantages of protective groups, of reagents used for cyclisation, and possible side reactions associated with them are described. The bibliography includes 119 references.

  9. Some peculiarities of synthesis of cysteine-containing peptides

    Science.gov (United States)

    Kudryavtseva, Elena V.; Sidorova, Mariya V.; Evstigneeva, Rima P.

    1998-07-01

    Data on protective groups for the thiol function of cysteine and methods of disulfide bonds formation used in modern peptide chemistry are considered and systematised. The advantages and disadvantages of protective groups, of reagents used for cyclisation, and possible side reactions associated with them are described. The bibliography includes 119 references.

  10. Some peculiarities of synthesis of cysteine-containing peptides

    International Nuclear Information System (INIS)

    Data on protective groups for the thiol function of cysteine and methods of disulfide bonds formation used in modern peptide chemistry are considered and systematised. The advantages and disadvantages of protective groups, of reagents used for cyclisation, and possible side reactions associated with them are described. The bibliography includes 119 references.

  11. Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes.

    Czech Academy of Sciences Publication Activity Database

    Kašperová, A.; Kunert, J.; Horynová, M.; Weigl, E.; Sebela, M.; Lenobel, René; Raška, M.

    2011-01-01

    Ro?. 54, ?. 5 (2011), E456-E462. ISSN 0933-7407 R&D Projects: GA ?R GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cysteine dioxygenase * dermatophytes * recombinant protein * keratinolytic fungi * cDNA Subject RIV: CE - Biochemistry Impact factor: 2.247, year: 2011

  12. Cysteine functionalized copper organosol: synthesis, characterization and catalytic application

    Energy Technology Data Exchange (ETDEWEB)

    Panigrahi, Sudipa [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Kundu, Subrata [Department of Chemical Engineering, University of Nebraska, Lincoln (United States); Basu, Soumen [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Praharaj, Snigdhamayee [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Jana, Subhra [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Pande, Surojit [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Ghosh, Sujit Kumar [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Pal, Anjali [Department of Civil Engineering, Indian Institute of Technology, Kharagpur-721302 (India); Pal, Tarasankar [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India)

    2006-11-14

    We herein report a facile one-pot synthesis, stabilization, redispersion and Cu-S interaction of L-cysteine and dodecanethiol (DDT) protected copper organosol in toluene from precursor copper stearate using sodium borohydride in toluene under a nitrogen atmosphere. Surface modification of the synthesized copper organosol with an amino acid L-cysteine and an alkanethiol (dodecanethiol, DDT) is accomplished by a thiolate bond between the used ligands and nanoparticle surface. The cysteine molecule binds the copper surface via a thiolate and amine linkage but not through electrostatic interaction with the carboxylate group due to the solvent polarity and dielectric medium. Fourier transform infrared (FTIR) analysis was performed to confirm the surface functionalization of the amino acid and DDT to the copper surface. Copper organosol has been characterized by optical spectroscopy (UV/vis), transmission electron microscopy (TEM), x-ray photoelectron spectroscopy (XPS) and x-ray diffraction (XRD). The as-synthesized particles are spherical in shape and exhibit a Mie scattering profile with an absorption maxima in the visible range. Copper nanoparticles capped by cysteine and/or DDT in non-aqueous media are found to represent an interesting catalytic approach for the synthesis of octylphenyl ether.

  13. Crystal structure of the catalytic domain of DESC1, a new member of the type II transmembrane serine proteinase family.

    Science.gov (United States)

    Kyrieleis, Otto J P; Huber, Robert; Ong, Edgar; Oehler, Ryan; Hunter, Mike; Madison, Edwin L; Jacob, Uwe

    2007-04-01

    DESC1 was identified using gene-expression analysis between squamous cell carcinoma of the head and neck and normal tissue. It belongs to the type II transmembrane multidomain serine proteinases (TTSPs), an expanding family of serine proteinases, whose members are differentially expressed in several tissues. The biological role of these proteins is currently under investigation, although in some cases their participation in specific functions has been reported. This is the case for enteropeptidase, hepsin, matriptase and corin. Some members, including DESC1, are associated with cell differentiation and have been described as tumor markers. TTSPs belong to the type II transmembrane proteins that display, in addition to a C-terminal trypsin-like serine proteinase domain, a differing set of stem domains, a transmembrane segment and a short N-terminal cytoplasmic region. Based on sequence analysis, the TTSP family is subdivided into four subfamilies: hepsin/transmembrane proteinase, serine (TMPRSS); matriptase; corin; and the human airway trypsin (HAT)/HAT-like/DESC subfamily. Members of the hepsin and matriptase subfamilies are known structurally and here we present the crystal structure of DESC1 as a first member of the HAT/HAT-like/DESC subfamily in complex with benzamidine. The proteinase domain of DESC1 exhibits a trypsin-like serine proteinase fold with a thrombin-like S1 pocket, a urokinase-type plasminogen activator-type S2 pocket, to accept small residues, and an open hydrophobic S3/S4 cavity to accept large hydrophobic residues. The deduced substrate specificity for DESC1 differs markedly from that of other structurally known TTSPs. Based on surface analysis, we propose a rigid domain association for the N-terminal SEA domain with the back site of the proteinase domain. PMID:17388811

  14. Autoxidation of cysteine generates hydrogen peroxide: cytotoxicity and attenuation by pyruvate.

    Science.gov (United States)

    Nath, K A; Salahudeen, A K

    1993-02-01

    The reactivity of cysteine presents a paradox: although regarded as an antioxidant, cysteine interacts with oxygen in a metal-catalyzed reaction to produce reactive species. Because ischemia provokes the appearance of millimolar amounts of cysteine and increased amounts of transition metals, we studied whether cysteine, in the presence of transition metals, consumes oxygen, generates hydrogen peroxide, and is toxic. Using fluorescence cytometry, we provide direct evidence that hydrogen peroxide is copiously generated during cysteine autoxidation. Pyruvate attenuates such generation of hydrogen peroxide and cytotoxicity. Cysteine oxidation is stimulated by an EDTA-chelatable diethyl-dithiocarbamate-chelatable constituent of kidney extract; this suggests that copper is the catalytically active metal. The toxicity resulting from cysteine oxidation is less than that induced by amounts of reagent hydrogen peroxide that produce comparable fluorescence. Cysteine also prevents hydrogen peroxide-induced toxicity. Thus, although cysteine generates hydrogen peroxide, it can guard against hydrogen peroxide toxicity, possibly by binding metals on which the toxicity of hydrogen peroxide is dependent. Thus the behavior of cysteine can be salutary or pernicious; the net effect of cysteine, within this wide ambit of actions, is decisively influenced by the conditions to which cysteine is exposed. PMID:8447440

  15. Effects of cysteine on growth, protease production, and catalase activity of Pseudomonas fluorescens.

    OpenAIRE

    Himelbloom, B. H.; Hassan, H. M.

    1986-01-01

    Cysteine inhibits growth of and protease production by Pseudomonas fluorescens NC3. Catalase activity in P. fluorescens NC3 was increased by cysteine. The addition of exogenous hydrogen peroxide did not increase catalase activity, thus suggesting a role for the endogenous generation of hydrogen peroxide via the autoxidation of cysteine.

  16. Electricity generation from cysteine in a microbial fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Logan, Bruce E. [Pennsylvania State Univ., Dept. of Civil and Environmental Engineering, University Park, PA (United States); Murano, Cassandro; Scott, Keith [Newcastle Univ., School of Chemical Engineering and Advanced Materials, Newcastle upon Tyne (United Kingdom); Gray, Neil D.; Head, Ian M. [Newcastle Univ., School of Civil Engineering and Geosciences, Newcastle upon Tyne (United Kingdom)

    2005-03-01

    In a microbial fuel cell (MFC), power can be generated from the oxidation of organic matter by bacteria at the anode, with reduction of oxygen at the cathode. Proton exchange membranes used in MFCs are permeable to oxygen, resulting in the diffusion of oxygen into the anode chamber. This could either lower power generation by obligate anaerobes or result in the loss in electron donor from aerobic respiration by facultative or other aerobic bacteria. In order to maintain anaerobic conditions in conventional anaerobic laboratory cultures, chemical oxygen scavengers such as cysteine are commonly used. It is shown here that cysteine can serve as a substrate for electricity generation by bacteria in a MFC. A two-chamber MFC containing a proton exchange membrane was inoculated with an anaerobic marine sediment. Over a period of a few weeks, electricity generation gradually increased to a maximum power density of 19 mW/m2 (700 or 1000{omega} resistor; 385 mg/L of cysteine). Power output increased to 39 mW/m2 when cysteine concentrations were increased up to 770 mg/L (493{omega} resistor). The use of a more active cathode with Pt- or Pt-Ru, increased the maximum power from 19 to 33 mW/m2 demonstrating that cathode efficiency limited power generation. Power was always immediately generated upon addition of fresh medium, but initial power levels consistently increased by ca. 30% during the first 24 h. Electron recovery as electricity was 14% based on complete cysteine oxidation, with an additional 14% (28% total) potentially lost to oxygen diffusion through the proton exchange membrane. 16S rRNA-based analysis of the biofilm on the anode of the MFC indicated that the predominant organisms were Shewanella spp. closely related to Shewanella affinis (37% of 16S rRNA gene sequences recovered in clone libraries). (Author)

  17. Electricity generation from cysteine in a microbial fuel cell.

    Science.gov (United States)

    Logan, Bruce E; Murano, Cassandro; Scott, Keith; Gray, Neil D; Head, Ian M

    2005-03-01

    In a microbial fuel cell (MFC), power can be generated from the oxidation of organic matter by bacteria at the anode, with reduction of oxygen at the cathode. Proton exchange membranes used in MFCs are permeable to oxygen, resulting in the diffusion of oxygen into the anode chamber. This could either lower power generation by obligate anaerobes or result in the loss in electron donor from aerobic respiration by facultative or other aerobic bacteria. In order to maintain anaerobic conditions in conventional anaerobic laboratory cultures, chemical oxygen scavengers such as cysteine are commonly used. It is shown here that cysteine can serve as a substrate for electricity generation by bacteria in a MFC. A two-chamber MFC containing a proton exchange membrane was inoculated with an anaerobic marine sediment. Over a period of a few weeks, electricity generation gradually increased to a maximum power density of 19 mW/m(2) (700 or 1000 Omega resistor; 385 mg/L of cysteine). Power output increased to 39 mW/m(2) when cysteine concentrations were increased up to 770 mg/L (493 Omega resistor). The use of a more active cathode with Pt- or Pt-Ru, increased the maximum power from 19 to 33 mW/m(2) demonstrating that cathode efficiency limited power generation. Power was always immediately generated upon addition of fresh medium, but initial power levels consistently increased by ca. 30% during the first 24 h. Electron recovery as electricity was 14% based on complete cysteine oxidation, with an additional 14% (28% total) potentially lost to oxygen diffusion through the proton exchange membrane. 16S rRNA-based analysis of the biofilm on the anode of the MFC indicated that the predominant organisms were Shewanella spp. closely related to Shewanella affinis (37% of 16S rRNA gene sequences recovered in clone libraries). PMID:15743641

  18. Mass Spectrometric Analysis of l-Cysteine Metabolism: Physiological Role and Fate of l-Cysteine in the Enteric Protozoan Parasite Entamoeba histolytica

    OpenAIRE

    Jeelani, Ghulam; Sato, Dan; Soga, Tomoyoshi; Watanabe, Haruo; Nozaki, Tomoyoshi

    2014-01-01

    l-Cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins, l-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such as Entamoeba histolytica, l-cysteine has been implicated in growth, attachment, survival, and protection from oxidative s...

  19. Plants

    Science.gov (United States)

    NRICH team

    2012-01-01

    In this logic activity, students must determine how to represent three quantities using a fixed amount of space (Venn diagram) and objects. The goal is to represent the siblings’ ages, 5,6, and 7, using only ten plants. This resource includes teacher notes with extension suggestions and possible support options.

  20. Plant caspase-like proteases in plant programmed cell death

    OpenAIRE

    Xu, Qixian; Zhang, Lingrui

    2009-01-01

    Programmed cell death (PCD) is a genetically-controlled disassembly of the cell. In animal systems, the central core execution switch for apoptotic PCD is the activation of caspases (Cysteine-containing Aspartate-specific proteases). Accumulating evidence in recent years suggests the existence of caspase-like activity in plants and its functional involvement in various types of plant PCD, although no functional homologs of animal caspases were identified in plant genome. In this mini-review, ...

  1. Purification, inhibitory properties and amino acid sequence of a new serine proteinase inhibitor from white mustard (Sinapis alba L.) seed.

    Science.gov (United States)

    Menegatti, E; Tedeschi, G; Ronchi, S; Bortolotti, F; Ascenzi, P; Thomas, R M; Bolognesi, M; Palmieri, S

    1992-04-13

    A new serine proteinase inhibitor, mustard trypsin inhibitor 2 (MTI-2), has been isolated from white mustard (Sinapis alba L.) seed by affinity chromatography and reverse phase HPLC. The protein inhibits the catalytic activity of bovine beta-trypsin and bovine alpha-chymotrypsin, with dissociation constants (Kd) of 1.6 x 10(-10) M and 5.0 x 10(-7) M, respectively, at pH 8.0 and 21 degrees C, the stoichiometry of both proteinase-inhibitor complexes being 1:1. The amino acid sequence of MTI-2, which was determined following S-pyridylethylation, is comprised of 63 residues, corresponding to a molecular weight of about 7 kDa, and shows only extremely limited homology to other serine proteinase inhibitors. PMID:1451776

  2. Chemical characterization of complexation behavior of pertechnetate with cysteine

    International Nuclear Information System (INIS)

    The labeling behavior of cysteine with 99TcO4- ion and/or 99mTcO4- ion at different cysteine concentrations reductant and pH values has been studied by chromatography, and the labeling yield was calculated. Three major Tc-complexes, yellow, reddish brown and green can be separated by gel filtration chromatography (GFC). Thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and ion-exchange chromatography (IC) were used to separate the complexes collected from GFC. The TLC, HPLC data show the pertechnetate accompanied with a yellow complex; the green and purple complex contain more than two complexes. Electrophoresis and IC data show that the complexes carry a negative charge. The conductivity, UV-VIS, flow beta-detector with HPLC and autoradiography are also applied to analyze complex formation. (author) 20 refs.; 13 figs.; 5 tabs

  3. Heterologous expression of Hordeum vulgare cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe

    Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned with and without the 5 amino acid C-terminal sequence into the Pichia pastoris expression vector pPICZ A? and electrotransformed into Pichia pastoris strain SDM1163. Heterologous protein production was induced with 2% MeOH. To monitor the protein expression during induction, 1 ml samples was collected every hr for 24 hrs. After 4 days, the supernatant were harvested and analyzed by SDS-PAGE, activity assay and Western blot. A significant amount of heterologous protein was produced and the protein production was highest after 4 days and the expression in the C-terminal mutant was slightly higher than for the full length protease.

  4. Proteinase and Growth Factor Alterations Revealed by Gene Microarray Analysis of Human Diabetic Corneas

    Science.gov (United States)

    Saghizadeh, Mehrnoosh; Kramerov, Andrei A.; Tajbakhsh, Jian; Aoki, Annette M.; Wang, Charles; Chai, Ning-Ning; Ljubimova, Julia Y.; Sasaki, Takako; Sosne, Gabriel; Carlson, Marc R. J.; Nelson, Stanley F.

    2005-01-01

    PURPOSE. To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I. METHODS. RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agi-lent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10. RESULTS. More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin ?4 chain, and thymosin ?4 genes were down-regulated. The data were corroborated by QPCR and immuno-histochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin ?3?1, whereas in normal corneas MMP-10 decreased laminin-10 and integrin ?3?1 expression. CONCLUSIONS. Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy. PMID:16186340

  5. Molecular weight alterations of alpha-1 proteinase inhibitor in equine bronchoalveolar lavage fluid.

    Science.gov (United States)

    Milne, E M; Pemberton, A D; McGorum, B C; Dixon, P M; Miller, H R

    1994-01-01

    The equine alpha-1 proteinase inhibitor (alpha 1PI) system differs from that of man in that the equine system consists of four closely-linked genes (Spi1-Spi4) whereas in man, a single gene encodes for alpha 1PI. We have previously found differences in the proportion of the Spi proteins in equine serum and bronchoalveolar lavage fluid (BALF). We therefore wished to determine whether, as reported in man, there was any molecular weight difference between the Spi proteins in serum and BALF. alpha 1PI and albumin from equine BALF migrated further towards the anode compared with serum alpha 1PI on native polyacrylamide gel electrophoresis (PAGE) although the difference was only significant for alpha 1PI. Sodium dodecyl sulphate-PAGE (SDS-PAGE) showed that a mean decrease in molecular weight of 1.5 kDa for alpha 1PI and 1.3 kDa for albumin had occurred in BALF. These findings were observed in control animals and in those with symptomatic or asymptomatic chronic obstructive pulmonary disease. The mechanism of this decrease in molecular weight of alpha 1PI is likely to differ from reports of alpha 1PI cleavage by bacterial proteinases in man since the molecular weight change was relatively small and loss of trypsin inhibitory activity did not occur. Nor, in our system, was there evidence of bacterial infection. Damage by endogenous proteinases or glycosidases at a site other than the reactive site may be involved but the resultant effect on the efficiency of the antiproteinase screen of the lower respiratory tract is uncertain. PMID:7859728

  6. Cysteine Peptidases as Schistosomiasis Vaccines with Inbuilt Adjuvanticity

    OpenAIRE

    El Ridi, Rashika; Tallima, Hatem; Selim, Sahar; Donnelly, Sheila; Cotton, Sophie; Gonzales Santana, Bibiana; Dalton, John P.

    2014-01-01

    Schistosomiasis is caused by several worm species of the genus Schistosoma and afflicts up to 600 million people in 74 tropical and sub-tropical countries in the developing world. Present disease control depends on treatment with the only available drug praziquantel. No vaccine exists despite the intense search for molecular candidates and adjuvant formulations over the last three decades. Cysteine peptidases such as papain and Der p 1 are well known environmental allergens that sensitize the...

  7. Subcellular distribution of glutathione and cysteine in cyanobacteria

    OpenAIRE

    Zechmann, Bernd; Tomas?ic?, Ana; Horvat, Lucija; Fulgosi, Hrvoje

    2010-01-01

    Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine,...

  8. Differential Expression of Cysteine Dioxygenase 1 in Complex Karyotype Liposarcomas

    OpenAIRE

    Mohammed Shaker; Pascarelli, Kara M.; Plantinga, Matthew J.; Love, Miles A.; Lazar, Alexander J.; Ingram, Davis R.; Margaret von Mehren; Dina Lev; David Kipling; Dominique Broccoli

    2014-01-01

    Altered cysteine dioxygenase 1 (CDO1) gene expression has been observed in several cancers but has not yet been investigated in liposarcomas. The aim of this study was to evaluate CDO1 expression in a cohort of liposarcomas and to determine its association with clinicopathological features. Existing microarray data indicated variable CDO1 expression in liposarcoma subtypes. CDO1 mRNA from a larger cohort of liposarcomas was quantified by real time-PCR, and CDO1 protein expression was determin...

  9. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    Energy Technology Data Exchange (ETDEWEB)

    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-1 and uPA were weakly induced, if at all, in hydrocortisonetreated mice. Furthermore, mRNA for membrane-type matrix metalloproteinase decreased after hydrocortisone treatment and paralleled the almost complete inhibition of activation of latent gelatinase A. Concomitantly, the gland filled with an overabundance of milk. Our data support the hypothesis that there are at least two distinct phases of involution: an initial phase, characterized by induction of the apoptosis-associated genes SGP-2 and ICE and apoptosis of fully differentiated mammary epithelial cells without visible degradation of the extracellular matrix, and a second phase, characterized by extracellular matrix remodeling and altered mesenchymal-epithelial interactions, followed by apoptosis of cells that are losing differentiated functions.

  10. Identification of Mouse Hepatitis Virus Papain-Like Proteinase 2 Activity

    OpenAIRE

    Kanjanahaluethai, Amornrat; Baker, Susan C.

    2000-01-01

    Mouse hepatitis virus (MHV) is a 31-kb positive-strand RNA virus that is replicated in the cytoplasm of infected cells by a viral RNA-dependent RNA polymerase, termed the replicase. The replicase is encoded in the 5?-most 22 kb of the genomic RNA, which is translated to produce a polyprotein of >800 kDa. The replicase polyprotein is extensively processed by viral and perhaps cellular proteinases to give rise to a functional replicase complex. To date, two of the MHV replicase-encoded protei...

  11. Pulmonary deposition and clearance of aerosolized alpha-1-proteinase inhibitor administered to dogs and to sheep.

    OpenAIRE

    Smith, R. M.; Traber, L. D.; Traber, D. L.; Spragg, R. G.

    1989-01-01

    Augmentation of lung antiprotease levels may be an important therapeutic intervention in the prevention of pulmonary emphysema. We have administered aerosols of plasma-derived human alpha 1 proteinase inhibitor (A1PI) to the lungs of dogs and sheep to investigate (a) delivery of the protein to the distal air spaces of the lung; (b) maintenance of functional activity of the protein; and (c) flux of the protein across the components of the alveolar-capillary membrane. A1PI (26.4 mg/kg body weig...

  12. Biochemical characterization of fibrinogenolytic serine proteinases from Vipera lebetina snake venom.

    Science.gov (United States)

    Samel, Mari; Subbi, Juhan; Siigur, Jüri; Siigur, Ene

    2002-01-01

    Two glycosylated serine fibrinogenases isolated from Vipera lebetina venom have homologous N-terminal sequences and antigenic determinants but can be clearly differentiated according to substrate specificity, glycosylation levels, molecular mass and fibrinogen degradation. alpha-Fibrinogenase has no homolog among known serine proteinases. It has N-terminal similarity with snake venom arginine esterases but does not hydrolyze the esters of arginine, lysine and tyrosine. The enzyme has strong proteolytic activity and degrades alpha-chain of fibrinogen altering its clottability by thrombin. beta-Fibrinogenase is a typical arginine esterase which hydrolyzes esters and amides of arginine and attacks the beta-chain of fibrinogen. PMID:11602278

  13. Classical Swine Fever Virus Leader Proteinase Npro Is Not Required for Viral Replication in Cell Culture

    OpenAIRE

    Tratschin, Jon-duri; Moser, Christian; Ruggli, Nicolas; Hofmann, Martin A.

    1998-01-01

    The sequence encoding the viral leader proteinase Npro was replaced by the murine ubiquitin gene in a full-length cDNA clone of the classical swine fever virus (CSFV) strain Alfort/187. The recombinant virus vA187-Ubi showed growth characteristics similar to those of the parent vA187-1 virus. At two occasions cells infected with vA187-Ubi exhibited a cytopathic effect and were found to contain a subgenomic viral RNA. This RNA lacked the same viral genes as the subgenomic RNA which has been fo...

  14. Purification of neutral lens endopeptidase: close similarity to a neutral proteinase in pituitary.

    OpenAIRE

    Ray, K.; Harris, H.

    1985-01-01

    A neutral endopeptidase (EC 3.4.24.5) that degrades alpha- and beta-crystallins occurs in mammalian lens. A procedure for purification of this enzyme from bovine lens is described. The enzyme appears to have a high molecular weight (Mr approximately equal to 700,000) and under denaturing conditions dissociates into at least eight polypeptide subunits with Mrs ranging from 24,000 to 32,000. A neutral proteinase in bovine pituitary has been reported previously to have similar structural charact...

  15. Saccharomyces cerevisiae can secrete Sapp1p proteinase of Candida parapsilosis but cannot use it for efficient nitrogen acquisition.

    Science.gov (United States)

    Vinterová, Zuzana; Bauerová, Václava; Dostál, Ji?í; Sychrová, Hana; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    2013-06-01

    Secreted aspartic proteinase Sapp1p of Candida parapsilosis represents one of the factors contributing to the pathogenicity of the fungus. The proteinase is synthesized as an inactive pre-pro-enzyme, but only processed Sapp1p is secreted into extracellular space. We constructed a plasmid containing the SAPP1 coding sequence under control of the ScGAL1 promoter and used it for proteinase expression in a Saccharomyces cerevisiae kex2? mutant. Because Sapp1p maturation depends on cleavage by Kex2p proteinase, the kex2? mutant secreted only the pro-form of Sapp1p. Characterization of this secreted proteinase form revealed that the Sapp1p signal peptide consists of 23 amino acids. Additionally, we prepared a plasmid with the SAPP1 coding sequence under control of its authentic CpSAPP1 promoter, which contains two GATAA motifs. While in C. parapsilosis SAPP1 expression is repressed by good low molecular weight nitrogen sources (e.g., ammonium ions), S. cerevisiae cells harboring this plasmid secreted a low concentration of active proteinase regardless of the type of nitrogen source used. Quantitative real-time PCR analysis of a set of genes related to nitrogen metabolism and uptake (GAT1, GLN3, STP2, GAP1, OPT1, and PTR2) obtained from S. cerevisiae cells transformed with either plasmid encoding SAPP1 under control of its own promoter or empty vector and cultivated in media containing various nitrogen sources also suggested that SAPP1 expression can be connected with the S. cerevisiae regulatory network. However, this regulation occurs in a different manner than in C. parapsilosis. PMID:23812814

  16. Cysteine peptidases as schistosomiasis vaccines with inbuilt adjuvanticity.

    Science.gov (United States)

    El Ridi, Rashika; Tallima, Hatem; Selim, Sahar; Donnelly, Sheila; Cotton, Sophie; Gonzales Santana, Bibiana; Dalton, John P

    2014-01-01

    Schistosomiasis is caused by several worm species of the genus Schistosoma and afflicts up to 600 million people in 74 tropical and sub-tropical countries in the developing world. Present disease control depends on treatment with the only available drug praziquantel. No vaccine exists despite the intense search for molecular candidates and adjuvant formulations over the last three decades. Cysteine peptidases such as papain and Der p 1 are well known environmental allergens that sensitize the immune system driving potent Th2-responses. Recently, we showed that the administration of active papain to mice induced significant protection (Pvaccines with inbuilt adjuvanticity to protect against these parasites. Here we demonstrate that sub-cutaneous injection of functionally active S. mansoni cathepsin B1 (SmCB1), or a cathepsin L from a related parasite Fasciola hepatica (FhCL1), elicits highly significant (Pprotection (up to 73%) against an experimental challenge worm infection. Protection and reduction in worm egg burden were further increased (up to 83%) when the cysteine peptidases were combined with other S. mansoni vaccine candidates, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) and peroxiredoxin (PRX-MAP), without the need to add chemical adjuvants. These studies demonstrate the capacity of helminth cysteine peptidases to behave simultaneously as immunogens and adjuvants, and offer an innovative approach towards developing schistosomiasis vaccines. PMID:24465551

  17. Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.

    OpenAIRE

    Bazan, J. F.; Fletterick, R. J.

    1988-01-01

    Proteases that are encoded by animal picornaviruses and plant como- and potyviruses form a related group of cysteine-active-center enzymes that are essential for virus maturation. We show that these proteins are homologous to the family of trypsin-like serine proteases. In our model, the active-site nucleophile of the trypsin catalytic triad, Ser-195, is changed to a Cys residue in these viral proteases. The other two residues of the triad, His-57 and Asp-102, are otherwise absolutely conserv...

  18. Brewer's spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3

    Directory of Open Access Journals (Sweden)

    Rodrigo Pires do Nascimento

    2011-12-01

    Full Text Available Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.

  19. The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.

    OpenAIRE

    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 ferm...

  20. The Tomato yellow leaf curl virus (TYLCV) V2 protein interacts with the host papain-like cysteine protease CYP1

    OpenAIRE

    Bar-ziv, Amalia; Levy, Yael; Hak, Hagit; Mett, Anahit; Belausov, Eduard; Citovsky, Vitaly; Gafni, Yedidya

    2012-01-01

    The V2 protein of Tomato yellow leaf curl geminivirus (TYLCV) is an RNA-silencing suppressor that counteracts the innate immune response of the host plant. However, this anti-host defense function of V2 may include targeting of other defensive mechanisms of the plant. Specifically, we show that V2 recognizes and directly binds the tomato CYP1 protein, a member of the family of papain-like cysteine proteases which are involved in plant defense against diverse pathogens. This binding occurred b...

  1. Michael-type addition of illudin S, a toxic substance from Lampteromyces japonicus, with cysteine and cysteine-containing peptides in vitro.

    Science.gov (United States)

    Tanaka, K; Inoue, T; Tezuka, Y; Kikuchi, T

    1996-02-01

    Reactions of illudin S with cysteine derivatives (cysteine methyl ester, glutathione and a peptide, Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg) were investigated. In the reaction with cysteine methyl ester, four products (P1, P2, P3, P4) were obtained and their structures were determined, on the basis of MS and NMR data, to be adducts of the mercapto group of cysteine methyl ester with the alpha,beta-unsaturated carbonyl group of illudin S. In the reactions with glutathione and the peptide, two addition products in each case were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and NMR analyses. The structures of these adducts also indicated that the alpha,beta-unsaturated carbonyl group in illudin S behaves as a Michael acceptor for the mercapto group in cysteine. PMID:8998835

  2. Random substitution of large parts of the propeptide of yeast proteinase A

    DEFF Research Database (Denmark)

    van den Hazel, H B; Kielland-Brandt, Morten

    1995-01-01

    The yeast aspartic protease, proteinase A, has a 54 amino-acid propeptide, which is removed during activation of the zymogen in the vacuole. Apart from being involved inhibition/activation, the propeptide has been shown to be essential for formation of a stable active enzyme (van den Hazel, H. B., Kielland-Brandt, M. C., and Winther, J. R. (1993) J. Biol. Chem. 268, 18002-18007). We have investigated the sequence requirements for function of the propeptide. The N-terminal half and the C-terminal half of the propeptide were replaced by random sequences at the genetic level, and collections of the mutants were subjected to a colony screen for ones exhibiting activity. A high frequency (around 1%) of active constructs was found, which indicates a very high tolerance for mutations in the propeptide. Thirty-nine functional mutant forms containing random sequence at either the N- or C-terminal half of the propeptide were characterized. Comparison of the propeptides of the active constructs suggests that a particular lysine residue is important for efficient biosynthesis of proteinase A.

  3. Kinetic constants for the hydrolysis of aggrecan by the papaya proteinases and their relevance for chemonucleolysis.

    Science.gov (United States)

    Dekeyser, P M; Buttle, D J; Devreese, B; Van Beeumen, J; Demeester, J; Lauwers, A

    1995-07-10

    The four known proteinases from papaya latex, namely papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30), and glycyl endopeptidase (EC 3.4.22.25), were purified to homogeneity and fully characterized by single radial immunodiffusion and active-site titration. A modified HPLC gel permeation assay was used to determine the kinetic constants for aggrecan hydrolysis by the papaya proteinases. The disappearance of intact aggrecan monomer was first-order, indicating that for the four enzymes studied the Km was much larger than 0.5 microM and that kcat/Km = 1.2 +/- 0.1 x 10(6) M-1 s-1 for chymopapain, 1.20 +/- 0.08 x 10(6) M-1 s-1 for caricain, 0.90 +/- 0.02 x 10(6) M-1 s-1 for papain, and 0.120 +/- 0.005 x 10(6) M-1 s-1 for glycyl endopeptidase. Chymodiactin, the chymopapain preparation used for chemonucleolysis, consists of a mixture of chymopapain (70%), caricain (20%), and glycyl endopeptidase (4%). The rate constant for the aggrecan hydrolysis by such a mixture was not significantly different from the rate constant for pure chymopapain. As a result of these observations, we predict that pure chymopapain could replace partially purified chymopapain preparations for chemonucleolysis. PMID:7625846

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

    Directory of Open Access Journals (Sweden)

    Emilia Marttila

    2014-07-01

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

  5. Modeling the growth and proteinase A production in continuous cultures of recombinant Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Carlsen, Morten; Jochumsen, Kirsten Væver

    1997-01-01

    Overexpression of the homologous protein proteinase A (PrA) in Saccharomyces cerevisiae has been achieved by inserting the PrA gene (PEP4) with its own promoter on a 2 mu multicopy plasmid. With this system the specific PrA production rate was found to be described well by a linear function of the oxidative glucose metabolism, the reductive glucose metabolism, and the oxidative ethanol metabolism, with a significant lower yield resulting from the reductive glucose metabolism compared with the oxidative glucose metabolism. To describe the experimental data, a simple mathematical model has been set up. The model is based on an assumption of a limited respiratory capacity as suggested by Sonnleitner and Kappeli but extended to describe production of an extracellular protein. The model predicts correctly the critical dilution rate to be between 0.15 and 0.16 h(-1), the decrease in the biomass yield above the critical dilution rate, and the production of proteinase A at different dilution rates. Both the experimental data and model simulations suggest that the optimum operating conditions for protein production is just at the critical dilution rate. (C) 1997 John Wiley & Sons, Inc.

  6. Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii.

    Science.gov (United States)

    Hwang, Sungmin; Cordova, Bryan; Chavarria, Nikita; Elbanna, Dina; McHugh, Stephen; Rojas, Jenny; Pfeiffer, Friedhelm; Maupin-Furlow, Julie A

    2014-10-28

    BackgroundThiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms.ResultsBased on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant.ConclusionsBased on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis. PMID:25348237

  7. Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a.

    Science.gov (United States)

    Xu, Zeng-Fu; Teng, Whei-Lan; Chye, Mee-Len

    2004-02-01

    SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements. To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation. Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R1 progeny. SaPIN2a mRNA was detected in both the R0 and R1 transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a. Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited. Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants. PMID:14574575

  8. The Cysteine Protease–Cysteine Protease Inhibitor System Explored in Soybean Nodule Development

    OpenAIRE

    Marian Dorcas Quain; Karl Kunert; Christine Helen Foyer; Ignatious Ncube; Matome Eugene Makgopa; Stefan van Wyk; Magdeleen du Plessis; Urte Schlüter; Barend Juan Vorster

    2013-01-01

    Almost all protease families have been associated with plant development, particularly senescence, which is the final developmental stage of every organ before cell death. Proteolysis remobilizes and recycles nitrogen from senescent organs that is required, for example, seed development. Senescence-associated expression of proteases has recently been characterized using large-scale gene expression analysis seeking to identify and characterize senescence-related genes. Increasing activities of...

  9. Proteinases in excretory-secretory products of Toxocara canis second-stage larvae: zymography and modeling insights.

    Science.gov (United States)

    González-Páez, Gonzalo Ernesto; Alba-Hurtado, Fernando; García-Tovar, Carlos Gerardo; Argüello-García, Raúl

    2014-01-01

    Components released in excretory-secretory products of Toxocara canis larvae (TES) include phosphatidylethanolamine-binding proteins (TES26), mucins (TES120, MUC2-5), and C-type lectins (TES32, TES70) and their biochemical, immunological, and diagnostic properties have been extensively studied albeit proteinase activities towards physiological substrates are almost unknown. Proteolytic activities in TES samples were first analyzed by gel electrophoresis with gelatin as substrate. Major activities of ~400, 120, and 32?kDa in TES were relatively similar over a broad pH range (5.5-9.0) and all these were of the serine-type as leupeptin abolished gelatinolysis. Further, the ~400?kDa component degraded all physiological substrates tested (laminin, fibronectin, albumin, and goat IgG) and the 120?kDa component degraded albumin and goat IgG while proteinases of lower MW (45, 32, and 26?kDa) only degraded laminin and fibronectin, preferentially at alkaline pH (9.0). By protein modeling approaches using the known sequences of TES components, only TES26 and MUC4 displayed folding patterns significantly related to reference serine proteinases. These data suggest that most of serine proteinase activities secreted in vitro by infective larvae of T. canis have intriguing nature but otherwise help the parasite to affect multiple components of somatic organs and bodily fluids within the infected host. PMID:25197645

  10. Evaluation of digestive proteinases from the Antarctic krill Euphasia superba as potential chemonucleolytic agents. In vitro and in vivo studies.

    Science.gov (United States)

    Melrose, J; Hall, A; Macpherson, C; Bellenger, C R; Ghosh, P

    1995-01-01

    Chemonucleolysis is a therapeutic procedure whereby a degradative enzyme is injected intradiscally to reduce disc height/width by depolymerisation of extracellular matrix components. This process is considered to diminish disc pressure on inflamed nerve roots, resulting in the alleviation of sciatic pain. In the present study two krill (Euphasia superba) enzyme preparations, a proteinase and an esterase preparation, were evaluated for their potential as chemonucleolytic agents. Initially, their ability to degrade several protein (azocoll, casein, proteoglycans, PGs) and peptide (CBZ-arg-4-nitroanilide, CBZ-lys-thiobenzyl ester) substrates was assessed in vitro. The krill proteinase preparation rapidly converted azocoll, casein and PGs to small peptides. Furthermore, when this degradative enzyme preparation was evaluated in vivo, a relatively low intradiscal dose (0.54 mg/disc) was found to reduce intervertebral disc widths in beagles to 48% +/- 10.5% (mean +/- SEM) of their pre-injection values within 2 weeks of administration. Moreover, the discs injected with this proteinase had reconstituted up to 80% +/- 9% (mean +/- SEM) of their pre-injection widths at the termination of the experiment (32 weeks). These data suggest that the krill protease preparation has potential as a chemonucleolytic agent which would allow disc matrix reconstitution. Conversely, the krill esterase preparation also degraded PGs, but into relatively large fragments. This limited digestion of PGs indicates that the krill esterase would be a less effective chemonucleolytic agent than the corresponding proteinase. PMID:7619635

  11. Cysteine-rich domains related to Frizzled receptors and Hedgehog-interacting proteins.

    Science.gov (United States)

    Pei, Jimin; Grishin, Nick V

    2012-08-01

    Frizzled and Smoothened are homologous seven-transmembrane proteins functioning in the Wnt and Hedgehog signaling pathways, respectively. They harbor an extracellular cysteine-rich domain (FZ-CRD), a mobile evolutionary unit that has been found in a number of other metazoan proteins and Frizzled-like proteins in Dictyostelium. Domains distantly related to FZ-CRDs, in Hedgehog-interacting proteins (HHIPs), folate receptors and riboflavin-binding proteins (FRBPs), and Niemann-Pick Type C1 proteins (NPC1s), referred to as HFN-CRDs, exhibit similar structures and disulfide connectivity patterns compared with FZ-CRDs. We used computational analyses to expand the homologous set of FZ-CRDs and HFN-CRDs, providing a better understanding of their evolution and classification. First, FZ-CRD-containing proteins with various domain compositions were identified in several major eukaryotic lineages including plants and Chromalveolata, revealing a wider phylogenetic distribution of FZ-CRDs than previously recognized. Second, two new and distinct groups of highly divergent FZ-CRDs were found by sensitive similarity searches. One of them is present in the calcium channel component Mid1 in fungi and the uncharacterized FAM155 proteins in metazoans. Members of the other new FZ-CRD group occur in the metazoan-specific RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) proteins that are putative tumor suppressors acting as inhibitors of matrix metalloproteases. Finally, sequence and three-dimensional structural comparisons helped us uncover a divergent HFN-CRD in glypicans, which are important morphogen-binding heparan sulfate proteoglycans. Such a finding reinforces the evolutionary ties between the Wnt and Hedgehog signaling pathways and underscores the importance of gene duplications in creating essential signaling components in metazoan evolution. PMID:22693159

  12. Emission of hydrogen sulfide by leaf tissue in response to L-cysteine

    International Nuclear Information System (INIS)

    Leaf discs and detached leaves exposed to L-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H2S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to L-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H2S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar L-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar L-cysteine emitted only 2% as much as did the discs exposed to 10 millimolar L-cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H2S emission was a specific consequence of exposure to L-cysteine; neither D-cysteine nor L-cysteine elicited H2S emissr L-cysteine elicited H2S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H2S formation and its release occurred in response to L-cysteine. Feeding experiments with [35S]t-cysteine showed that most of the sulfur in H2S was derived from sulfur in the L-cysteine supplied

  13. Cysteine-674 Oxidation and Degradation of SERCA in Diabetic Pig Aorta

    OpenAIRE

    Ying, Jia; Sharov, Victor; Xu, Shanqin; Jiang, Bingbing; Gerrity, Ross; Scho?neich, Christian; Cohen, Richard A.

    2008-01-01

    The sarcoplasmic reticulum Ca2+ ATPase (SERCA) is redox-regulated by post-translational thiol modifications of cysteine-674 to regulate smooth muscle relaxation and migration. To detect oxidation of cysteine-674 that irreversibly prevents redox-regulation, a polyclonal, sequence-specific antibody was developed towards a peptide containing cysteine-674 sulfonic acid. The antibody stained intact 110 kDa SERCA in pig cardiac SR that was oxidized in vitro by peroxynitrite in a sequence-specific m...

  14. Substitutions of a cysteine conserved among DNA cytosine methylases result in a variety of phenotypes.

    OpenAIRE

    Wyszynski, M. W.; Gabbara, S.; Bhagwat, A. S.

    1992-01-01

    The proposed mechanism for DNA (cytosine-5)-methyltransferases envisions a key role for a cysteine residue. It is expected to form a covalent link with carbon 6 of the target cytosine, activating the normally inactive carbon 5 for methyl transfer. There is a single conserved cysteine among all DNA (cytosine-5)-methyltransferases making it the candidate nucleophile. We have changed this cysteine to other amino acids for the EcoRII methylase; which methylates the second cytosine in the sequence...

  15. Flow injection spectrofluorimetric determination of cystine and cysteine

    Scientific Electronic Library Online (English)

    Ali A., Ensafi; B., Rezaei; S., Nouroozi.

    Full Text Available Um procedimento relativamente simples e sensível com detecção espectrofluorimétrica foi desenvolvido para a determinação de cistina e cisteína por sistema de injeção em fluxo com determinação seqüencial. Esse método é fundamentado na redução de Tl(III) com cisteína em meio ácido, produzindo o reagen [...] te fluorescente TlCl3(2-) (?ex = 227 nm, ?em = 419 nm). Antes da injeção, a solução da amostra foi dividida em dois fluxos. O primeiro fluxo foi tratado com coluna de redução de Cd e então refluxado com a solução do carregador para reagir em pH 5,0 com Tl(III), passado através de uma cela de reação de 100 cm e posteriormente para a cela fluxo do espectrofluorímetro, onde a intensidade de fluorescência foi medida (?ex = 227 nm, ?em = 419 nm). Esse sinal está relacionado às concentrações de cistina e cisteína. O segundo fluxo da solução de amostra foi injetado diretamente no fluxo carregador para reagir e, então, pela cela de reação e detector para medida da intensidade de fluorescência. O sinal nessa etapa é relacionado apenas à cisteína. Assim, o conteúdo de cistina foi determinado diretamente da diferença entre os dois sinais. Cistina e cisteína podem ser determinadas no intervalo de 0,10 a 5,50 µmol L-1 e 0,20 a 8,0 µmol L-1, respectivamente, em uma razão de 20 amostras por hora. O limite de detecção (3s/k) foi 0,10 µmol L-1 para os dois analitos. Os desvios padrões relativos para a determinação de dez replicatas de 4,0 e 3,5 µmol L-1 de cistina ou cisteína foram 1,1% e 1,8%, respectivamente. A influência de substâncias interferentes foi estudada. O método proposto foi aplicado com sucesso na determinação seqüencial de ambos analitos em amostras farmacêuticas. Abstract in english A relatively simple and sensitive procedure with spectrofluorimetric detection was developed for the determination of cystine and cysteine by flow injection system with sequential determination. This method is based on the reduction of Tl(III) with cysteine in acidic media, producing a fluorescence [...] reagent, TlCl3(2-) (?ex = 227 nm, ?em = 419 nm). Before injection, the sample solution was divided into two streams. The first stream was treated with Cd reduction column and then joined with the carrier to react with Tl(III) at pH 5.0 and then passed through a 100 cm reaction coil to the flow cell of the spectrofluorimeter, where the fluorescence intensity was measured (?ex = 227 nm, ?em = 419 nm). This signal is related to cystine and cysteine concentrations. The second stream of sample solution was injected directly into the carrier stream to react with the reagents and then passed through the reaction coil and detector for measuring the fluorescence intensity. The signal in this step is related only to cysteine. Thus, the cystine content was determined directly from difference of the two signals. Cystine and cysteine can be determined in the range of 0.10 to 5.50 µmol L-1 and 0.20 to 8.0 µmol L-1, respectively, at a rate of 20 samples per hour. The limit of detection (3s/k) was 0.10 µmol L-1 for both analytes. The relative standard deviations for ten replicates determination of 4.0 and 3.5 µmol L-1 cystine or cysteine were 1.1% and 1.8%, respectively. The influence of potential interfering substances was studied. The proposed method was successfully applied to the sequential determination of both analytes in pharmaceutical samples.

  16. Characterization, kinetics, and possible function of Kazal-type proteinase inhibitors of Chinese white shrimp, Fenneropenaeus chinensis.

    Science.gov (United States)

    Wang, Zong-Heng; Zhao, Xiao-Fan; Wang, Jin-Xing

    2009-06-01

    Serine proteinase inhibitor plays an essential role in arthropods by restraining the activities of endogenic or exogenic serine proteinases. Four Kazal-type serine proteinase inhibitors, Fcspi-1-4, from the hepatopancreas of Chinese white shrimp, Fenneropenaeus chinensis, were cloned and identified. The open reading frames (ORFs) of Fcspis are 1389, 1236, 1080, and 939 base pairs, encode the pre-proteins of 462, 411, 359, and 312 amino acids and form the 9, 8, 7, and 6 typical Kazal domains, respectively. When analyzing the amino acid sequences of the four inhibitors, it was found that they might have been derived from the same transcript, which was subjected to alternative splicing, and none of the Kazal domains were identical within each inhibitor. Multiple alignments showed that the Kazal inhibitors were homologous with a conserved motif of Cx(3)Cx(6)VCGSDGxTYx(3)CxLx(5)Cx(5)ITx(6)GC. The results from RT-PCR indicated that the expression of Fcspis as a whole was upregulated by bacterial challenge, no obvious change was noticed after viral challenge, and Fcspi-1 had a similar expression pattern with that of Fcspis. Recombinant FcSPIs were successfully expressed in bacteria and purified for further study. Recombinant FcSPI-1 was sensitive to DTT and had thermal stability. The inhibitory kinetics assay suggested that rFcSPI-1 was a mixed-type fast tight binding inhibitor with inhibitory activities against subtilisin A at a molar ratio of 1:1, 1:2 against proteinase K, and 2:1 against elastase. It can firmly bound to two Gram-positive and one Gram-negative bacteria but without anti-bacterial ability. In addition, it inhibited the activities of both bacterial-secreted proteinases and natural chymotrypsin of Chinese white shrimp, suggesting that FcSPI-1 may participate in the immune defence response by inhibition of bacterial pathogen proteinases and possibly be involved in the regulation of shrimp proteinase activity. PMID:19379816

  17. Effects of cysteine protease inhibitors on rabbit cathepsin D maturation.

    Science.gov (United States)

    Samarel, A M; Ferguson, A G; Decker, R S; Lesch, M

    1989-12-01

    To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed (24 h) to media containing leupeptin (0-10 mM), E 64 (0-10 mM), or chloroquine (0-50 microM). Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D (Mr 53,000) within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D (Mr 48,000). Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate (not detectable in control fibroblasts). Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation. PMID:2610247

  18. Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

    International Nuclear Information System (INIS)

    To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed to media containing leupeptin, E 64, or chloroquine. Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D. Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate. Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newdent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation

  19. Efficacy of N-Acetyl Cysteine in Traumatic Brain Injury

    OpenAIRE

    Eakin, Katharine; Baratz-goldstein, Renana; Pick, Chiam G.; Zindel, Ofra; Balaban, Carey D.; Hoffer, Michael E.; Lockwood, Megan; Miller, Jonathan; Hoffer, Barry J.

    2014-01-01

    In this study, using two different injury models in two different species, we found that early post-injury treatment with N-Acetyl Cysteine (NAC) reversed the behavioral deficits associated with the TBI. These data suggest generalization of a protocol similar to our recent clinical trial with NAC in blast-induced mTBI in a battlefield setting [1], to mild concussion from blunt trauma. This study used both weight drop in mice and fluid percussion injury in rats. These were chosen to simulate e...

  20. A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom.

    Science.gov (United States)

    Siigur, Ene; Tõnismägi, Külli; Trummal, Katrin; Samel, Mari; Vija, Heiki; Aaspõllu, Anu; Rönnholm, Gunilla; Subbi, Juhan; Kalkkinen, Nisse; Siigur, Jüri

    2011-02-01

    Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-L-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, A?-chain and more slowly B?-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms. PMID:20950666

  1. Purification and Properties of a Highly Thermostable, Sodium Dodecyl Sulfate-Resistant and Stereospecific Proteinase from the Extremely Thermophilic Archaeon Thermococcus stetteri

    OpenAIRE

    Klingeberg, M.; Galunsky, B.; Sjoholm, C.; Kasche, V.; Antranikian, G.

    1995-01-01

    The cultivation of the extremely thermophilic archaeon Thermococcus stetteri in a dialysis membrane reactor was paralleled by the production of an extremely heat-stable proteinase(s). By applying preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, an SDS-resistant proteinase was purified 67-fold in one step with a yield of 34%. The purified enzyme, which was composed of a single polypeptide chain with a molecular mass of 68 kDa, showed a broad temperature and pH profi...

  2. [Purification of cadmium ion binding metallothionein-3 by proteinase digestion on affinity chromatographic column].

    Science.gov (United States)

    Zheng, Weijuan; Yang, Feng; Wu, Fang; Lu, Chun; Hua, Zichun

    2006-05-01

    The gene encoding human metallothionein-3 (hMT-3) was synthesized and inserted into the poly-cloning sites of fusion expression vector pALEX, and fused downstream to its glutathione S-transferase (GST) fusion partner. Fusion protein GST-Cd2+-hMT-3 was expressed after isopropyl-beta-D-thiogalactopyranoside (IPTG) induction and addition of 0.1 mmol/L CdSO4 into the culture medium, and mainly existed in cellular soluble fraction as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Recombinant MT was purified by two purification procedures: "proteinase digestion after purification" method, e. g. by elution of GST-Cd2+-hMT-3 from GST affinity chromatography first, then proteinase digestion and GST affinity chromatographic purification again subsequently; and "proteinase digestion in situ" method, e. g. digestion of GST-Cd2+-hMT-3 directly on the column while its binding to the GST affinity chromatographic resin and collection of Cd2+-MT directly from the flow through fraction after the digestion. It was confirmed that the later procedure exhibited more effective and more convenient by avoiding the conventional elution, dialysis and lyophilization processes and increasing the purity, recovery or yield of the final product. After further purification by a Superdex 75 HR 10/30 column, finally 6-7 mg of Cd2+-hMT-3 was obtained from 3 L of flask culture with the recovery of about 1.8%. SDS-PAGE, amino acid composition and inductively coupled plasma atomic emission spectrometer (ICP-AES) analysis showed that the relative molecular mass of Cd2+-hMT-3 is about 7 000, with a purity above 90%. Its amino acid composition is consistent with the expected value of natural hMT-3, particularly no aromatic amino acid and histidine, and the atomic ratio of 21: (7.5 +/- 0.1) for S: Cd, is also consistent with the theoretical value of 21: 7. PMID:16929849

  3. Copper oxide assisted cysteine hierarchical structures for immunosensor application

    Science.gov (United States)

    Pandey, Chandra Mouli; Sumana, Gajjala; Tiwari, Ida

    2014-09-01

    The present work describes the promising electrochemical immunosensing strategy based on copper (II) assisted hierarchical cysteine structures (CuCys) varying from star to flower like morphology. The CuCys having average size of 10 ?m have been synthesised using L-Cysteine as initial precursor in presence of copper oxide under environmentally friendly conditions in aqueous medium. To delineate the synthesis mechanism, detailed structural investigations have been carried out using characterization techniques such as X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The electrochemical behaviour of self-assembled CuCys on gold electrode shows surface controlled electrode reaction with an apparent electron transfer rate constant of 3.38 × 10-4 cm s-1. This innovative platform has been utilized to fabricate an immunosensor by covalently immobilizing monoclonal antibodies specific for Escherichia coli O157:H7 (E. coli). Under the optimal conditions, the fabricated immunosensor is found to be sensitive and specific for the detection of E. coli with a detection limit of 10 cfu/ml.

  4. Copper oxide assisted cysteine hierarchical structures for immunosensor application

    International Nuclear Information System (INIS)

    The present work describes the promising electrochemical immunosensing strategy based on copper (II) assisted hierarchical cysteine structures (CuCys) varying from star to flower like morphology. The CuCys having average size of 10??m have been synthesised using L-Cysteine as initial precursor in presence of copper oxide under environmentally friendly conditions in aqueous medium. To delineate the synthesis mechanism, detailed structural investigations have been carried out using characterization techniques such as X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The electrochemical behaviour of self-assembled CuCys on gold electrode shows surface controlled electrode reaction with an apparent electron transfer rate constant of 3.38?×?10?4?cm s?1. This innovative platform has been utilized to fabricate an immunosensor by covalently immobilizing monoclonal antibodies specific for Escherichia coli O157:H7 (E. coli). Under the optimal conditions, the fabricated immunosensor is found to be sensitive and specific for the detection of E. coli with a detection limit of 10?cfu/ml.

  5. Copper oxide assisted cysteine hierarchical structures for immunosensor application

    Energy Technology Data Exchange (ETDEWEB)

    Pandey, Chandra Mouli [Biomedical Instrumentation Section, CSIR-National Physical Laboratory, New Delhi 110012 (India); Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005 (India); Sumana, Gajjala, E-mail: sumanagajjala@gmail.com [Biomedical Instrumentation Section, CSIR-National Physical Laboratory, New Delhi 110012 (India); Tiwari, Ida [Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005 (India)

    2014-09-08

    The present work describes the promising electrochemical immunosensing strategy based on copper (II) assisted hierarchical cysteine structures (CuCys) varying from star to flower like morphology. The CuCys having average size of 10??m have been synthesised using L-Cysteine as initial precursor in presence of copper oxide under environmentally friendly conditions in aqueous medium. To delineate the synthesis mechanism, detailed structural investigations have been carried out using characterization techniques such as X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The electrochemical behaviour of self-assembled CuCys on gold electrode shows surface controlled electrode reaction with an apparent electron transfer rate constant of 3.38?×?10{sup ?4?}cm s{sup ?1}. This innovative platform has been utilized to fabricate an immunosensor by covalently immobilizing monoclonal antibodies specific for Escherichia coli O157:H7 (E. coli). Under the optimal conditions, the fabricated immunosensor is found to be sensitive and specific for the detection of E. coli with a detection limit of 10?cfu/ml.

  6. Nutritional Requirements and Nitrogen-Dependent Regulation of Proteinase Activity of Lactobacillus helveticus CRL 1062

    OpenAIRE

    Hebert, Elvira M.; Raya, Raul R.; Giori, Graciela S.

    2000-01-01

    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...

  7. Emission of hydrogen sulfide by leaf tissue in response to L-cysteine

    Energy Technology Data Exchange (ETDEWEB)

    Sekiya, J.; Schmidt, A.; Wilson, L.G.; Filner, P.

    1982-08-01

    Leaf discs and detached leaves exposed to L-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H/sub 2/S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to L-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H/sub 2/S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar L-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar L-cysteine emitted only 2% as much as did the discs exposed to 10 millimolar L-cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H/sub 2/S emission was a specific consequence of exposure to L-cysteine; neither D-cysteine nor L-cysteine elicited H/sub 2/S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H/sub 2/S formation and its release occurred in response to L-cysteine. Feeding experiments with (/sup 35/S)t-cysteine showed that most of the sulfur in H/sub 2/S was derived from sulfur in the L-cysteine supplied.

  8. A competitive chemical-proteomic platform to identify zinc-binding cysteines.

    Science.gov (United States)

    Pace, Nicholas J; Weerapana, Eranthie

    2014-01-17

    Zinc ions (Zn(2+)) play vital catalytic, structural, and regulatory roles in protein function and are commonly chelated to cysteine residues within the protein framework. Current methods to identify Zn(2+)-binding cysteines rely on computational studies based on known Zn(2+)-chelating motifs, as well as high-resolution structural data. These available approaches preclude the global identification of putative Zn(2+)-chelating cysteines, particularly on poorly characterized proteins in the proteome. Herein, we describe an experimental platform that identifies metal-binding cysteines on the basis of their reduced nucleophilicity upon treatment with metal ions. As validation of our platform, we utilize a peptide-based cysteine-reactive probe to show that the known Zn(2+)-chelating cysteine in sorbitol dehydrogenase (SORD) demonstrates an expected loss in nucleophilicity in the presence of Zn(2+) ions and a gain in nucleophilicity upon treatment with a Zn(2+) chelator. We also identified the active-site cysteine in glutathione S-transferase omega-1 (GSTO1) as a potential Zn(2+)-chelation site, albeit with lower metal affinity relative to SORD. Treatment of recombinant GSTO1 with Zn(2+) ions results in a dose-dependent decrease in GSTO1 activity. Furthermore, we apply a promiscuous cysteine-reactive probe to globally identify putative Zn(2+)-binding cysteines across ?900 cysteines in the human proteome. This proteomic study identified several well-characterized Zn(2+)-binding proteins, as well as numerous uncharacterized proteins from functionally distinct classes. This platform is highly versatile and provides an experimental tool that complements existing computational and structural methods to identify metal-binding cysteine residues. PMID:24111988

  9. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin activity from a case of vulvovaginitis.

    Science.gov (United States)

    Kumar, Dharmendra; Banerjee, Tuhina; Pratap, Chandra Bhan; Tilak, Ragini

    2015-01-01

    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. PMID:25881537

  10. Protein degradation in Euglena gracilis: Purification and characterization of the major proteinase

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Y.J.

    1988-01-01

    Protolysis in a crude extract of Euglena gracilis was characterized by autolysis and the hydrolysis of {sup 125}I-labeled bovine serum albumin ({sup 125}I-BSA). Both procedures showed similar properties: stimulation by dithiothreitol, inhibition by leupeptin, and the same pH optima. Hydrolysis of {sup 125}I-BSA increased with growth stage and with the depletion of nutrient in the medium. The major proteolytic enzyme was purified to near homogeneity from extracts of dark-grown, stationary-phase Euglena gracilis by acid treatment, and by chromatography on CM-cellulose, DEAE-cellulose, Sephadex G-75, and hydroxyapatite using {sup 125}I-BSA as substrate. The molecular weight of the proteinase was 30,000 when determined by gel filtration on Sephadex G-75 and 15,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme therefore appears to be composed of two subunits.

  11. Protein degradation in Euglena gracilis: Purification and characterization of the major proteinase

    International Nuclear Information System (INIS)

    Protolysis in a crude extract of Euglena gracilis was characterized by autolysis and the hydrolysis of 125I-labeled bovine serum albumin (125I-BSA). Both procedures showed similar properties: stimulation by dithiothreitol, inhibition by leupeptin, and the same pH optima. Hydrolysis of 125I-BSA increased with growth stage and with the depletion of nutrient in the medium. The major proteolytic enzyme was purified to near homogeneity from extracts of dark-grown, stationary-phase Euglena gracilis by acid treatment, and by chromatography on CM-cellulose, DEAE-cellulose, Sephadex G-75, and hydroxyapatite using 125I-BSA as substrate. The molecular weight of the proteinase was 30,000 when determined by gel filtration on Sephadex G-75 and 15,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme therefore appears to be composed of two subunits

  12. Antioxidant activity of bovine casein hydrolysates produced by Ficus carica L.-derived proteinase.

    Science.gov (United States)

    Di Pierro, Giovanna; O'Keeffe, Martina B; Poyarkov, Alexey; Lomolino, Giovanna; FitzGerald, Richard J

    2014-08-01

    A Ficus carica L. latex proteinase preparation was investigated for its ability to produce antioxidant hydrolysates/peptides from bovine casein (CN). The Oxygen Radical Absorbance Capacity (ORAC) values for NaCN and ?-CN hydrolysates ranged from 0.06 to 0.18, and from 0.51 to 1.19?mol Trolox equivalents/mg freeze-dried sample, respectively. Gel permeation HPLC showed that the ?-CN hydrolysate with a degree of hydrolysis of 21% had 65% of peptide material with a molecular mass <500Da. The RP-UPLC profiles also indicated that ?-CN was substantially hydrolysed during the early stages of hydrolysis. Analysis of the 4h ?-CN hydrolysate by LC-ESI-MS/MS allowed identification of 8 peptide sequences with potential antioxidant properties. PMID:24629973

  13. Decrease in the alpha 1-proteinase inhibitor Spi3 in equine bronchoalveolar lavage fluid.

    Science.gov (United States)

    Milne, E M; Pemberton, A D; Dixon, P M; McGorum, B C; Scudamore, C L; Miller, H R

    1994-10-01

    The alpha 1-proteinase inhibitors of trypsin, Spi1, Spi3A, and Spi3B, in bronchoalveolar lavage fluid (BALF) and serum of horses were separated by electrophoresis, and their proportions were quantified in 12 control horses and 12 with chronic obstructive pulmonary disease (COPD). A significantly lower proportion of Spi3B (P < 0.05) and higher proportion of Spi1 (P < 0.02 to P < 0.01) were detected in BALF, compared with serum, in control and COPD-affected horses and appeared to be attributable to reduced Spi3 activity in BALF. There was no significant difference between the control and COPD groups in this respect, indicating that the decrease in Spi3 may be a physiologic phenomenon. The differences observed may be associated with proteolytic damage to or preferential complex formation by Spi3. PMID:7998693

  14. Production and administration to dogs of aerosols of alpha-1-proteinase inhibitor

    International Nuclear Information System (INIS)

    The feasibility of aerosol administration of alpha-1-proteinase inhibitor (human) (A1PI) was assessed. Of three different methods of aerosolizing A1PI that were evaluated, an ultrasonic nebulizer was found to be best suited to the present purpose, producing particles of a size that allowed them to reach the distal air spaces of the lung and that retained specific A1PI anti-elastase activity. Administration of 20 mg/kg of A1PI and 150 microCi of 131-iodine-A1PI to three dogs was accomplished without complications. Gamma camera scans documented a relatively homogenous distribution throughout the lungs. Bronchial lavage fluid that was recovered from the lungs of the dogs six hours after administration contained large amounts of human A1PI and showed a proportional elevation of anti-elastase activity. There was no evidence of acute toxicity

  15. Proteinases involved in the degradation of trypsin inhibitor in germinating mung beans.

    Science.gov (United States)

    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. PMID:6346766

  16. 046. Unusual pulmonary infection in a patient treated with a1-proteinase inhibitor

    Science.gov (United States)

    Renesis, Basileios; Tryfon, Stavros; Moulara, Evdokia; Konstantinou, Eleftheria; Siopi, Dimitra; Serasli, Evangelia; Tsara, Venetia

    2015-01-01

    Background Description of clinical worsening and radiological image characteristics in a patient under chronic treatment with a1-proteinase inhibitor (prolastine). Case report We present a case of 47 years old woman, ex-smoker (30 p/y) with pulmonary emphysema. She has been under treatment with a1-proteinase inhibitor—once per month intravenous, for a six months period. She was admitted to our hospital because of persistent febrile and hypoxic deterioration of her already known chronic respiratory failure. Blood tests were revealed elevated inflammatory markers (WBC, CRP). Radiological investigation (Rx and CT) revealed: (I) consolidation with many small sized cavitations in the right upper lobe lesion; (II) ground glass opacification in the same lobe and (III) consolidation without cavitations in the left lower lobe. The patient was further investigated bronchoscopically. The culture of bronchial washing yielded no positive result for common microbes, mycobacteria or fungi. The cytological examination of bronchial lavage was negative for malignancy but in the same specimen rare fungal hyphae were identified. Blood and sputum cultures were also negative. She was administered moxifloxacin and piperacillin/tazobactam but she remained febrile and then she was given linezolid and voriconazole but again without response. The fever waves resolved automatically within ten days. Respiratory function improved gradually, and radiographic findings resolved after a month. Conclusions Chronic treatment with prolastin predispose to the occurrence of viral respiratory infections—mainly the type of Parvovirus. The radiographic image shows simultaneous infiltrates in different lobes that vary from ground glass opacification to consolidation with or without cavitary lesions.

  17. Calcium protects a mesophilic xylanase from proteinase inactivation and thermal unfolding.

    Science.gov (United States)

    Spurway, T D; Morland, C; Cooper, A; Sumner, I; Hazlewood, G P; O'Donnell, A G; Pickersgill, R W; Gilbert, H J

    1997-07-11

    Crystal structure analysis of Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLA) indicated that the enzyme contained a single calcium binding site that did not exhibit structural features typical of the EF-hand motif. Isothermal titration calorimetry revealed that XYLA binds calcium with a Ka of 4.9 x 10(4) M-1 and a stoichiometry consistent with one calcium binding site per molecule of enzyme. Occupancy of the calcium binding domain with its ligand protected XYLA from proteinase and thermal inactivation and increased the melting temperature of the enzyme from 60.8 to 66.5 degrees C. However, the addition of calcium or EDTA did not influence the catalytic activity of the xylanase. Replacement of the calcium binding domain, which is located within loop 7 of XYLA, with the corresponding short loop from Cex (a Cellulomonas fimi xylanase/exoglucanase), did not significantly alter the biochemical properties of the enzyme. These data suggest that the primary function of the calcium binding domain is to increase the stability of the enzyme against thermal unfolding and proteolytic attack. To understand further the nature of the calcium binding domain of XYLA, four variants of the xylanase, D256A, N261A, D262A, and XYLA"', in which Asp-256, Asn-261, and Asp-262 had all been changed to alanine, were constructed. These mutated enzymes did not show any significant binding to Ca2+, indicating that Asp-256, Asn-261, and Asp-262 play a pivotal role in the affinity of XYLA for the divalent cation. In the presence or absence of calcium, XYLA"' exhibited thermal stability similar to that of the native enzyme bound to Ca2+ ions, although the variant was sensitive to proteinase inactivation. The role of the calcium binding domain in vivo and the possible mechanism by which the domain evolved are discussed. PMID:9211898

  18. SERINE proteinase like activity in apolipophorin III from the hemolymph of desert locust, Schistocerca gregaria.

    Science.gov (United States)

    Malik, Zulfiqar A; Amir, Sumaira; Venekei, István

    2012-06-01

    Apolipophorin III (apoLp-III) has been known as a lipid transport protein of insects. Recent studies indicated the involvement of apoLp-III in immune reactions and in the control of cell destruction, but no enzymatic activity has so far been detected. In the present study, a protease from the hemolymph of Schistocerca gregaria was purified to homogeneity and its enzymatic activity was examined. Identity as chymotrypsin-like proteinase was established by its high affinity toward bulky aromatic substrates and its catalytic specificity for amide or ester bonds on the synthetic substrates, Suc-Ala-Ala-Pro-Xaa-AMC (where Xaa was Phe, Tyr, Trp, and Lys, and AMC is 7-amino-4-methyl-coumarin) and thiolbenzyl ester substrate Suc-Ala-Ala-Pro-Phe-SBzl. The sensitivity for serine protease and chymotrypsin-specific covalent inhibitors, PMSF, TPCK, and noncovalent inhibitors SGCI, showed that it is a chymotrypsin-like proteinase. It showed its maximum activity at pH 8.0 and 55°C for the hydrolysis of Suc-Ala-Ala-Pro-Tyr-AMC. According to similarities in the amino terminal sequence, molar mass (19 kDa) and retention on reversed-phase analytical high-performance liquid chromatography (HPLC) column, this protein is S. gregaria homologue of Locusta migratoria apoLp-III. Our data suggest that apoLp-III also has an inherent proteolytic activity. Results indicated that S. gregaria apoLp-III is a good catalyst and could be used as a biotechnological tool in food processing and in agricultural biotechnology. PMID:22499434

  19. Slime production and proteinase activity of Candida species isolated from blood samples and the comparison of these activities with minimum inhibitory concentration values of antifungal agents

    Scientific Electronic Library Online (English)

    Semiha, Ozkan; Fatma, Kaynak; Ayse, Kalkanci; Ufuk, Abbasoglu; Semra, Kustimur.

    2005-05-01

    Full Text Available Slime and proteinase activity of 54 strains consisting of 19 Candida parapsilosis and 35 C. albicans strains isolated from blood samples were investigated in this study. Ketoconazole, amphothericin B, and fluconazole susceptibility of Candida species were compared with slime production and proteinas [...] e activity of these species. For both Candida species, no correlation was detected between the slime activity and minimum inhibitory concentration (MIC) values of the three antifungal agents. For both Candida species no correlation was detected between the proteinase activity and the MIC values of amphothericin B, and fluconazole however, statistically significant difference, was determined between the proteinase activity and MIC values of ketoconazole (p = 0.007). Slime production was determined by using modified Christensen macrotube method and proteinase activity was measured by the method of Staib. Antifungal susceptibility was determined through the guidelines of National Committee for Laboratory Standards (NCCLS M27-A).

  20. Interaction of L-cysteine with a human excitatory amino acid transporter.

    Science.gov (United States)

    Zerangue, N; Kavanaugh, M P

    1996-01-01

    1. The interaction of L-cysteine with three excitatory amino acid transporter subtypes cloned from human brain (EAAT1-3) was examined by measuring transporter-mediated electrical currents and radiolabelled amino acid flux in voltage-clamped Xenopus oocytes expressing the transporters. 2. L-Cysteine was transported by the neuronal subtype EAAT3 (EAAC1) with an affinity constant of 190 microM and a maximal rate of flux similar to that of L-glutamate; the relative efficacies (Vmax/K(m)) of the EAAT1 and EAAT2 subtypes for transporting L-cysteine were 10- to 20-fold lower. 3. Changing the ionization state of L-cysteine by raising the external pH did not significantly change the apparent affinity, transport rate, or magnitude of currents induced by L-cysteine, suggesting that both the neutral zwitterionic and anionic forms of the amino acid are transported with the same net charge stoichiometry. 4. In addition to competing with L-glutamate for uptake by the neuronal carrier, L-cysteine caused transporter-mediated release of transmitter by heteroexchange; both actions would elevate extracellular glutamate concentrations and may thus contribute to the known excitotoxic actions of L-cysteine in the brain. 5. Because the EAAT3 transporter is also expressed in tissues including kidney and intestine, the results suggest the possibility of a heretofore unrecognized mechanism of L-cysteine uptake in peripheral tissues as well as in brain. PMID:8782106

  1. Bio-functionalized silver nanoparticles: a novel colorimetric probe for cysteine detection.

    Science.gov (United States)

    Borase, Hemant P; Patil, Chandrashekhar D; Salunkhe, Rahul B; Suryawanshi, Rahul K; Kim, Beom S; Bapat, Vishwas A; Patil, Satish V

    2015-04-01

    Chemical interactions between nanoparticles and biomolecules are vital for applying nanoparticles in medicine and life science. Development of sensitive, rapid, low-cost, and eco-friendly sensors for the detection of molecules acting as disease indicator is need of an hour. In the present investigation, a green trend for silver nanoparticle synthesis was followed using leaf extract of Calotropis procera. Silver nanoparticles exhibited surface plasmon absorption peak at 421 nm, spherical shape with average size of 10 nm, and zeta potential of -22.4 mV. The as-synthesized silver nanoparticles were used for selective and sensitive detection of cysteine. Cysteine induces aggregation in stable silver nanoparticles owing to selective and strong interaction of -SH group of cysteine with silver nanoparticle surface. Cysteine-induced silver nanoparticle aggregation can be observed visually by change in color of silver nanoparticles from yellow to pink. Cysteine concentration was estimated colorimetrically by measuring absorption at surface plasmon wavelength. Limit of detection for cysteine using silver nanoparticles is ultralow, i.e., 100 nM. The mechanistic insight into cysteine detection by silver nanoparticles was investigated using FT-IR, TEM, DLS, and TLC analysis. Proposed method can be applied for the detection of cysteine in blood plasma and may give rise to a new insight into development of eco-friendly fabricated nanodiagnostic device in future. PMID:25637511

  2. An aryl-thioether substituted nitrobenzothiadiazole probe for the selective detection of cysteine and homocysteine.

    Science.gov (United States)

    Lee, Dayoung; Kim, Gyoungmi; Yin, Jun; Yoon, Juyoung

    2015-03-31

    An aryl-thioether substituted nitrobenzothiadiazole probe was synthesized and employed to detect cysteine and homocysteine selectively in living cells. Interestingly, both cysteine (Cys) and homocysteine (Hcy) promote an enhancement of the fluorescence intensity of the probe at pH 7.4 while only Cys gives rise to this enhancement under weakly acidic conditions (pH 6.0). PMID:25773705

  3. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

    Science.gov (United States)

    Cysteine peptidases are important in many biological processes. In this study, we describe the design, synthesis and use of selective peptide substrates for cysteine peptidases of the C1 papain family. The structure of the proposed substrates can be expressed by the general formula Glp-Xaa-Ala-Y, wh...

  4. Binding of Procollagen C-Proteinase Enhancer-1 (PCPE-1) to Heparin/Heparan Sulfate: PROPERTIES AND ROLE IN PCPE-1 INTERACTION WITH CELLS*

    OpenAIRE

    Weiss, Tali; Ricard-blum, Sylvie; Moschcovich, Laura; Wineman, Eitan; Mesilaty, Shlomit; Kessler, Efrat

    2010-01-01

    Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of...

  5. Short-term variability of biomarkers of proteinase activity in patients with emphysema associated with type Z alpha-1-antitrypsin deficiency

    OpenAIRE

    Nieuwenhuizen Willem; van Kuppevelt Toine H; Versteeg Elly M; Zanone Chiara; Annovazzi Laura; Veldhuisen Barbara; Stolk Jan; Iadarola Paolo; Luisetti Maurizio

    2005-01-01

    Abstract Background The burden of proteinases from inflammatory cells in the lung of subjects with type Pi ZZ of alpha-1-antitrypsin deficiency is higher than in those without the deficiency. Cross-sectional studies have shown increased levels of biomarkers of extracellular matrix degradation in vivo. Longitudinal variability of these biomarkers is unknown but desirable for clinical studies with proteinase inhibitors. Methods We measured three different types of biomarkers, including desmosin...

  6. Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs

    OpenAIRE

    Wang, Yang; Lu, Zhiqiang; Jiang, Haobo

    2014-01-01

    Melanization participates in various insect physiological processes including antimicrobial immune responses. Phenoloxidase (PO), a critical component of the enzyme system catalyzing melanin formation, is produced as an inactive precursor prophenoloxidase (proPO) and becomes active via specific proteolytic cleavage by proPO activating proteinase (PAP). In Manduca sexta, three PAPs can activate proPOs in the presence of two serine proteinase homologs (SPH1 and SPH2). While the hemolymph protei...

  7. Resolution of oxidative stress by thioredoxin reductase: Cysteine versus selenocysteine

    Directory of Open Access Journals (Sweden)

    Brian Cunniff

    2014-01-01

    Full Text Available Thioredoxin reductase (TR catalyzes the reduction of thioredoxin (TRX, which in turn reduces mammalian typical 2-Cys peroxiredoxins (PRXs 1–4, thiol peroxidases implicated in redox homeostasis and cell signaling. Typical 2-Cys PRXs are inactivated by hyperoxidation of the peroxidatic cysteine to cysteine-sulfinic acid, and regenerated in a two-step process involving retro-reduction by sulfiredoxin (SRX and reduction by TRX. Here transient exposure to menadione and glucose oxidase was used to examine the dynamics of oxidative inactivation and reactivation of PRXs in mouse C10 cells expressing various isoforms of TR, including wild type cytoplasmic TR1 (Sec-TR1 and mitochondrial TR2 (Sec-TR2 that encode selenocysteine, as well as mutants of TR1 and TR2 in which the selenocysteine codon was changed to encode cysteine (Cys-TR1 or Cys-TR2. In C10 cells endogenous TR activity was insensitive to levels of hydrogen peroxide that hyperoxidize PRXs. Expression of Sec-TR1 increased TR activity, reduced the basal cytoplasmic redox state, and increased the rate of reduction of a redox-responsive cytoplasmic GFP probe (roGFP, but did not influence either the rate of inactivation or the rate of retro-reduction of PRXs. In comparison to roGFP, which was reduced within minutes once oxidants were removed reduction of 2-Cys PRXs occurred over many hours. Expression of wild type Sec-TR1 or Sec-TR2, but not Cys-TR1 or TR2, increased the rate of reduction of PRXs and improved cell survival after menadione exposure. These results indicate that expression levels of TR do not reduce the severity of initial oxidative insults, but rather govern the rate of reduction of cellular factors required for cell viability. Because Sec-TR is completely insensitive to cytotoxic levels of hydrogen peroxide, we suggest TR functions at the top of a redox pyramid that governs the oxidation state of peroxiredoxins and other protein factors, thereby dictating a hierarchy of phenotypic responses to oxidative insults.

  8. Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

    International Nuclear Information System (INIS)

    A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

  9. Intramolecular synergistic effect of glutamic acid, cysteine and glycine against copper corrosion in hydrochloric acid solution

    International Nuclear Information System (INIS)

    The corrosion protection of copper by glutamic acid, cysteine, glycine and their derivative (glutathione) in 0.5 M hydrochloric acid solution has been studied by the electrochemical impedance spectroscopy and cyclic voltammetry. The inhibition efficiency of the organic inhibitors on copper corrosion increases in the order: glutathione > cysteine > cysteine + glutamic acid + glycine > glutamic acid > glycine. Maximum inhibition efficiency for cysteine reaches about 92.9% at 15 mM concentration level. The glutathione can give 96.4% inhibition efficiency at a concentration of 10 mM. The molecular structure parameters were obtained by PM3 (Parametric Method 3) semi-empirical calculation. The intramolecular synergistic effect of glutamic acid, cysteine and glycine moieties in glutathione is attributed to the lower energy of the lowest unoccupied molecular orbital (ELUMO) level and to the excess hetero-atom adsorption centers and the bigger coverage on the copper surface.

  10. Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong, E-mail: yerong24@fudan.edu.cn

    2012-06-05

    A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

  11. Intramolecular synergistic effect of glutamic acid, cysteine and glycine against copper corrosion in hydrochloric acid solution

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Daquan, E-mail: zhdq@sh163.net [Department of Environmental Engineering, Shanghai University of Electric Power, Shanghai 200090 (China); Xie Bin; Gao Lixin; Cai Qirui [Department of Environmental Engineering, Shanghai University of Electric Power, Shanghai 200090 (China); Joo, Hyung Goun; Lee, Kang Yong [Stress Analysis and Failure Design Laboratory, School of Mechanical Engineering, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2011-10-31

    The corrosion protection of copper by glutamic acid, cysteine, glycine and their derivative (glutathione) in 0.5 M hydrochloric acid solution has been studied by the electrochemical impedance spectroscopy and cyclic voltammetry. The inhibition efficiency of the organic inhibitors on copper corrosion increases in the order: glutathione > cysteine > cysteine + glutamic acid + glycine > glutamic acid > glycine. Maximum inhibition efficiency for cysteine reaches about 92.9% at 15 mM concentration level. The glutathione can give 96.4% inhibition efficiency at a concentration of 10 mM. The molecular structure parameters were obtained by PM3 (Parametric Method 3) semi-empirical calculation. The intramolecular synergistic effect of glutamic acid, cysteine and glycine moieties in glutathione is attributed to the lower energy of the lowest unoccupied molecular orbital (E{sub LUMO}) level and to the excess hetero-atom adsorption centers and the bigger coverage on the copper surface.

  12. Production of hydrogen sulfide from D-cysteine and its therapeutic potential

    Directory of Open Access Journals (Sweden)

    NorihiroShibuya

    2013-07-01

    Full Text Available Accumulating evidence shows that H2S has physiological functions in various tissues and organs. It includes regulation of neuronal activity, vascular tension, a release of insulin, and protection of the heart, kidney and brain from ischemic insult. H2S is produced by enzymes from L-cysteine; cystathionine ?-synthase (CBS, cystathionine ?-lyase (CSE, and 3-mercaptopyruvate sulfurtransferase (3MST along with cysteine aminotransferase (CAT. We recently discovered an additional pathway for the production of H2S from D-cysteine. D-Amino acid oxidase (DAO provides 3-mercaptopyruvate (3MP for 3MST to produce H2S. D-Cysteine protects cerebellar neurons from oxidative stress and attenuates ischemia-reperfusion injury caused in the kidney more effectively than L-cysteine. This review focuses on a novel pathway for the production of H2S and its therapeutic application especially to the renal diseases.

  13. Application of iTRAQ Reagents to Relatively Quantify the Reversible Redox State of Cysteine Residues

    Science.gov (United States)

    McDonagh, Brian; Martínez-Acedo, Pablo; Vázquez, Jesús; Padilla, C. Alicia; Sheehan, David; Bárcena, José Antonio

    2012-01-01

    Cysteines are one of the most rarely used amino acids, but when conserved in proteins they often play critical roles in structure, function, or regulation. Reversible cysteine modifications allow for potential redox regulation of proteins. Traditional measurement of the relative absolute quantity of a protein between two samples is not always necessarily proportional to the activity of the protein. We propose application of iTRAQ reagents in combination with a previous thiol selection method to relatively quantify the redox state of cysteines both within and between samples in a single analysis. Our method allows for the identification of the proteins, identification of redox-sensitive cysteines within proteins, and quantification of the redox status of individual cysteine-containing peptides. As a proof of principle, we applied this technique to yeast alcohol dehydrogenase-1 exposed in vitro to H2O2 and also in vivo to the complex proteome of the Gram-negative bacterium Bacillus subtilis. PMID:22844595

  14. In vitro digestibility of globulins from sapucaia (Lecythis pisonis Camb.) nuts by mammalian digestive proteinases / Digestibilidade in vitro de globulinas das amêndoas de sapucaia (Lecythis pisonis Camb.) por proteinases digestivas de mamíferos

    Scientific Electronic Library Online (English)

    Sandra Maria Silveira, Denadai; Priscila Aiko, Hiane; Sergio, Marangoni; Paulo Aparecido, Baldasso; Ana Maria Rauen de Oliveira, Miguel; Maria Lígia Rodrigues, Macedo.

    2007-09-01

    Full Text Available Amêndoas cruas de Sapucaia (Lecythis pisonis Camb.) colhidas no Brasil foram analisadas para se determinar a composição centesimal, o perfil de aminoácidos de suas proteínas, a digestibilidade protéica in vitro e a presença de fatores antinutricionais, para avaliar o seu potencial como complemento a [...] limentar protéico. As amêndoas apresentaram quantidades adequadas de aminoácidos essenciais, ácidos graxos e minerais; no entanto, baixo teor de fibra foi observado. No presente estudo, a presença de lectinas ou inibidores de proteinases, quando detectada, apresentou baixos níveis. A digestibilidade in vitro de globulinas, in natura ou aquecidas, por proteinases digestivas de mamíferos foi realizada utilizando-se tripsina + quimotripsina + peptidase, obtendo-se valores aproximados de 71,5 e 73,5%, respectivamente. Estes resultados sugerem que as amêndoas de sapucaia podem ser utilizadas como complemento alimentar de proteínas, sendo um potencial agente nutricional. Abstract in english Sapucaia (Lecythis pisonis Camb.) raw nuts collected from Brazil were analyzed to determine the proximate composition, amino acid profile of protein fractions, in vitro protein digestibility and antinutritional factors in order to evaluate their potential as a protein alimentary complement. The nuts [...] contained adequate amounts of essential amino acids, fatty acids and minerals. In the present study, no hemagglutinating or inhibitory activities were observed in any of the samples investigated, indicating low or non-detectable levels of proteinase inhibitors or lectins in the samples. In vitro digestibility of in natura and heated nut globulins by mammalian digestive proteinases was carried out using trypsin + chymotrypsin + peptidase, with resulting mean values of approximately 70.30 and 71.35%, respectively. Taken together, the results suggest that sapucaia nuts may provide a new source of protein to use as a potential nutritional agent.

  15. Utilização da fração semipurificada da proteinase do Trypanosoma cruzi no imunodiagnóstico da doença de Chagas The use of a semipurified fraction of Trypanosoma cruzi proteinase in immunodiagnosis of Chagas' disease

    OpenAIRE

    Ajax Mercês Atta; Angela Maria de Carvalho Pontes; Maria Luiza de Souza; Daria Repka; Rangel, Humberto A.

    1984-01-01

    Foram sensibilizadas hemácias humanas 0 Rh negativo com a fração semipurificada (Fp) da proteinase do Trypanosoma cruzi, e testadas quanto a antigenicidade com soros de pacientes portadores de tripanossomíase americana crônica e de outras doenças parasitárias não relacionadas. Reações de hemaglutinação positivas foram observadas com os soros de pacientes chagásicos e com alguns soros de indivíduos portadores de leishmaniose cutaneo-mucosa. Não foram observadas reações cruzada...

  16. In vitro digestibility of globulins from sapucaia (Lecythis pisonis Camb. nuts by mammalian digestive proteinases Digestibilidade in vitro de globulinas das amêndoas de sapucaia (Lecythis pisonis Camb. por proteinases digestivas de mamíferos

    Directory of Open Access Journals (Sweden)

    Sandra Maria Silveira Denadai

    2007-09-01

    Full Text Available Sapucaia (Lecythis pisonis Camb. raw nuts collected from Brazil were analyzed to determine the proximate composition, amino acid profile of protein fractions, in vitro protein digestibility and antinutritional factors in order to evaluate their potential as a protein alimentary complement. The nuts contained adequate amounts of essential amino acids, fatty acids and minerals. In the present study, no hemagglutinating or inhibitory activities were observed in any of the samples investigated, indicating low or non-detectable levels of proteinase inhibitors or lectins in the samples. In vitro digestibility of in natura and heated nut globulins by mammalian digestive proteinases was carried out using trypsin + chymotrypsin + peptidase, with resulting mean values of approximately 70.30 and 71.35%, respectively. Taken together, the results suggest that sapucaia nuts may provide a new source of protein to use as a potential nutritional agent.Amêndoas cruas de Sapucaia (Lecythis pisonis Camb. colhidas no Brasil foram analisadas para se determinar a composição centesimal, o perfil de aminoácidos de suas proteínas, a digestibilidade protéica in vitro e a presença de fatores antinutricionais, para avaliar o seu potencial como complemento alimentar protéico. As amêndoas apresentaram quantidades adequadas de aminoácidos essenciais, ácidos graxos e minerais; no entanto, baixo teor de fibra foi observado. No presente estudo, a presença de lectinas ou inibidores de proteinases, quando detectada, apresentou baixos níveis. A digestibilidade in vitro de globulinas, in natura ou aquecidas, por proteinases digestivas de mamíferos foi realizada utilizando-se tripsina + quimotripsina + peptidase, obtendo-se valores aproximados de 71,5 e 73,5%, respectivamente. Estes resultados sugerem que as amêndoas de sapucaia podem ser utilizadas como complemento alimentar de proteínas, sendo um potencial agente nutricional.

  17. Differential expression of cysteine dioxygenase 1 in complex karyotype liposarcomas.

    Science.gov (United States)

    Shaker, Mohammed; Pascarelli, Kara M; Plantinga, Matthew J; Love, Miles A; Lazar, Alexander J; Ingram, Davis R; von Mehren, Margaret; Lev, Dina; Kipling, David; Broccoli, Dominique

    2014-01-01

    Altered cysteine dioxygenase 1 (CDO1) gene expression has been observed in several cancers but has not yet been investigated in liposarcomas. The aim of this study was to evaluate CDO1 expression in a cohort of liposarcomas and to determine its association with clinicopathological features. Existing microarray data indicated variable CDO1 expression in liposarcoma subtypes. CDO1 mRNA from a larger cohort of liposarcomas was quantified by real time-PCR, and CDO1 protein expression was determined by immunohistochemistry (IHC) in more than 300 tumor specimens. Well-differentiated liposarcomas (WDLSs) had significantly higher CDO1 gene expression and protein levels than dedifferentiated liposarcomas (DDLSs) (P liposarcoma. Recurrent tumors did not show any difference in CDO1 expression when compared to primary tumors. CDO1 expression was upregulated as human mesenchymal stem cells (hMSCs) undergo differentiation into mature adipocytes. Our results suggest that CDO1 is a marker of liposarcoma progression and adipogenic differentiation. PMID:24741338

  18. Cysteine modified polyaniline films improve biocompatibility for two cell lines.

    Science.gov (United States)

    Yslas, Edith I; Cavallo, Pablo; Acevedo, Diego F; Barbero, César A; Rivarola, Viviana A

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using l-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV-visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86°±1 to 90°±1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. PMID:25842107

  19. Philibertain g I, the most basic cysteine endopeptidase purified from the latex of Philibertia gilliesii Hook. et Arn. (Apocynaceae).

    Science.gov (United States)

    Sequeiros, C; Torres, M J; Trejo, S A; Esteves, J L; Natalucci, C L; López, L M I

    2005-11-01

    A new papain-like cysteine peptidase isolated from latex of Philibertia gilliesii Hook. et Arn., Apocynaceae (formerly Asclepiadaceae) has been purified and characterized. The enzyme, named philibertain g I, is the most basic component present in latex extracts and was purified by acetone fractionation followed by cation exchange chromatography (SP-Sepharose HR) using FPLC system. Homogeneity was confirmed by SDS-PAGE and mass spectroscopy (MS). Molecular mass of the enzyme was 23,530 Da (MALDI-TOF MS), its isoelectric point was >10.25, and maximum proteolytic activity (casein) was achieved at pH 7-8. The new protease was inhibited by E-64 a cysteine peptidases inhibitor. Km was 0.15 mM, using PFLNA as substrate. The N-terminal sequence of philibertain g I (LPASVDWRKEGAVLPIRHQGQCG) was compared with those of twenty plant proteases. Philibertain g I showed the higher degree of identity (73%) with caricain, one of the Carica papaya endopepetidases. PMID:16328737

  20. Purification and characterization of a cysteine endopeptidase from Vasconcellea quercifolia A. St.-Hil. latex displaying high substrate specificity.

    Science.gov (United States)

    Torres, M José; Trejo, Sebastián A; Martin, M Inés; Natalucci, Claudia L; Avilés, Francesc X; López, Laura M I

    2010-10-27

    A new proteolytic preparation from Vasconcellea quercifolia ("oak leaved papaya") latex containing several cysteine endopeptidases with high proteolytic activity has been obtained. The specific activity of the new enzymatic preparation (VQ) was higher than that of Carica papaya latex. VQ was able to coagulate milk and to hydrolyze caseins and then could be used to produce cheeses and/or casein hydrolysates. Ion exchange chromatography of VQ allowed the isolation of a new protease, named quercifoliain I, homogeneous when analyzed by SDS-PAGE, IEF and MALDI-TOF-MS. Molecular mass was 24195 Da, and its isoelectric point was >9.3. The N-terminal sequence was determined (YPESVDWRQ). Insulin B-chain cleavage showed higher specificity than that of papain and was restricted to glycyl and alanyl residues at P1' position. The tryptic peptide mass fingerprint of quercifoliain I analyzed with the MASCOT search tool did not find a match with papain or any other plant cysteine proteases. PMID:20873836

  1. Up-regulation of a cysteine protease accompanies the ethylene-insensitive senescence of daylily (Hemerocallis) flowers.

    Science.gov (United States)

    Valpuesta, V; Lange, N E; Guerrero, C; Reid, M S

    1995-06-01

    The flowers of daylily (Hemerocallis x hybrida cv. Cradle Song) open at midnight, start to senesce 12 h later, and are completely senescent by the following midnight. Differential screening of a cDNA library constructed from tepals of flowers showing incipient senescence revealed 25 clones that were strongly up-regulated in senescent tepals. Re-screening and interactive Southern analysis of these clones revealed 3 families of up-regulated clones. Transcripts of one clone, SEN10, were not detectable at midnight, but increased dramatically as senescence proceeded. The derived amino acid sequence of the full-length cDNA (SEN102) has strong homology with cysteine proteases that have been reported from other plant tissues. The sequence contains a secretory signal peptide and a probable prosequence upstream of the mature protein. Amino acids critical to the active site and structure of cysteine proteases are conserved, and the C-terminus of the polypeptide has a unique putative endoplasmic reticulum retention signal -RDEL. PMID:7632925

  2. Brewer's spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3

    Scientific Electronic Library Online (English)

    Rodrigo Pires do, Nascimento; Nelson, Alves Junior; Rosalie Reed Rodrigues, Coelho.

    1384-13-01

    Full Text Available SciELO Brazil | Language: English Abstract in english Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands wer [...] e detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.

  3. Sequence diversity of Vipera lebetina snake venom gland serine proteinase homologs--result of alternative-splicing or genome alteration.

    Science.gov (United States)

    Siigur, E; Aaspõllu, A; Siigur, J

    2001-01-24

    Four clones encoding homologous protein(ase)s were isolated from the Vipera lebetina (snake) venom gland cDNA library. One of them represented DNA encoding factor V activating enzyme (Siigur et al., 1999), the other is homologous to VLFVA but has two principal discrepancies in the translated protein sequence in comparison with snake venom serine proteinase structures: in the active site triad Ser195 is replaced by Asn195 and His57 by Arg57. The third and the fourth clone represent combinations of the first two clones. The possibilities of generation of such clones via trans-splicing of the primary gene transcript, by exon shuffling or by unequal crossing-over on the genome level are discussed. PMID:11223258

  4. Complex of digestive proteinases of Galleria mellonella Caterpillars: composition, properties, and limited proteolysis of Bacillus thuringiensis endotoxins.

    Science.gov (United States)

    Bulushova, N V; Elpidina, E N; Zhuzhikov, D P; Lyutikova, L I; Ortego, F; Kirillova, N E; Zalunin, I A; Chestukhina, G G

    2011-05-01

    The complex of digestive proteinases in caterpillars of the greater wax moth Galleria mellonella was studied. Using chromogenic substrates and inhibitor analysis, it was found that serine proteinases play a key role in this complex. Three anionic and two cationic forms of trypsin and one anionic and one cationic form of chymotrypsin were identified by zymography in the midgut extract of G. mellonella. The most active trypsin was purified to electrophoretic homogeneity, and its N-terminal amino acid sequence was shown to be identical to that of mature trypsin from Plodia interpunctella. Midgut extract from G. mellonella was capable of processing Cry-proteins from Bacillus thuringiensis ssp. galleriae. Enzymes with tryptic and chymotryptic activities participate in this process, and activation of protoxin Cry9A is not the rate-limiting stage in the toxic action of this protein on the greater wax moth. PMID:21639838

  5. Proteinases from buckwheat (Fagopyrum esculentum moench seeds: Purification and properties of the 47 kDa enzyme

    Directory of Open Access Journals (Sweden)

    Timotijevi? Gordana S.

    2006-01-01

    Full Text Available Aspartic proteinases from buckwheat seeds are analyzed. Three forms of 47 kDa, 40 kDa and 28 kDa, were purified from mature buckwheat seeds, while two forms of 47 kDa and 28 kDa were detected in developing buckwheat seeds using pepstatin A affinity chromatography. A form of 47 kDa was selectively precipitated from other forms by ammonium sulfate precipitation. This enzyme resembles the chymosin-like pattern of proteolytic activity, as it was shown using BSA and k-casein as substrates, clarifying its ability for milk-clotting. The 47 kDa aspartic proteinase form is localized in the membrane fraction. .

  6. Interaction of cysteine and copper ions on the surface of iron: EIS, polarization and XPS study

    International Nuclear Information System (INIS)

    Highlights: ? The current study demonstrates a comprehensive study for Cysteine + Cu(II) ions as an efficient inhibitor as demonstrated by EIS, XPS and potentiodynamic polarization measurements, in addition to traditional weight loss measurements. ? The novelty of the current work originates from the combined use of an eco-friendly compound (i.e., cysteine) with a minute amount of copper ions (in the micro molar range) as a corrosion inhibitor for low carbon steel in acidic medium. To this end, cysteine shows only moderate inhibition ca. 60% for iron which jumps up to more than 95% in the presence of micro molar range of Cu(II) ions. ? Cysteine-Cu(II) blends are found superior to benzotriazole (BTAH)-Cu(II) blends in terms of their long-term stability in addition to the avoidance of the use of the well-reported highly toxic BTAH. - Abstract: This study addresses the enhancing effect of copper ions on the inhibition efficiency (IE) of cysteine (an eco-friendly compound) against the corrosion of iron in 0.5 M sulphuric acid. Electrochemical impedance spectroscopy (EIS) data revealed a significant increase in the polarization resistance (Rp) of the iron/solution interface in the presence of cysteine and Cu(II) ions instead of cysteine alone. That is, IE of 95% is obtained in the presence of 5 mM cysteine and 25 ?M Cu(II) ions, compared to 66% in absence of Cu(II) ions. Moreover, electrochemical polarization measurements indicate that cysteine and Cu(II)s indicate that cysteine and Cu(II) ions blends act as mixed-type inhibitors for the corrosion of iron. The formation of Cu(I)-cysteinate complex and/or cysteine SAM at Cu atop the iron surface (as evident from X-ray photoelectron spectroscopy (XPS)) blocks the underlying iron surface and imparts a pronounced protection against its corrosion. IE of cysteine-Cu(II) blend remains effectively unchanged with immersion time indicating its high stability in the used acidic medium.

  7. Emission of Hydrogen Sulfide by Leaf Tissue in Response to l-Cysteine.

    Science.gov (United States)

    Sekiya, J; Schmidt, A; Wilson, L G; Filner, P

    1982-08-01

    Leaf discs and detached leaves exposed to l-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H(2)S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to l-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H(2)S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar l-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar l-cysteine emitted only 2% as much as did the discs exposed to 10 millimolar l-cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H(2)S emission was a specific consequence of exposure to l-cysteine; neither d-cysteine nor l-cystine elicited H(2)S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H(2)S formation and its release occurred in response to l-cysteine. Feeding experiments with [(35)S]l-cysteine showed that most of the sulfur in H(2)S was derived from sulfur in the l-cysteine supplied and that the H(2)S emitted for 9 hours accounted for 7 to 10% of l-cysteine taken up. (35)S-labeled SO(3) (2-) and SO(4) (2-) were found in the tissue extract in addition to internal soluble S(2-). These findings suggest the existence of a sulfur cycle which converts l-cysteine to SO(4) (2-) through cysteine desulfhydration. PMID:16662510

  8. ADENOVIRUS-DRIVEN OVEREXPRESSION OF PROTEINASES IN ORGAN-CULTURED NORMAL HUMAN CORNEAS LEADS TO DIABETIC-LIKE CHANGES

    Science.gov (United States)

    Saghizadeh, Mehrnoosh; Kramerov, Andrei A.; Yaghoobzadeh, Yousha; Hu, Jinwei; Ljubimova, Julia Y.; Black, Keith L.; Castro, Maria G.; Ljubimov, Alexander V.

    2009-01-01

    Our previous data suggested the involvement of matrix metalloproteinase-10 (MMP-10) and cathepsin F (CTSF) in the basement membrane and integrin changes occurring in diabetic corneas. These markers were now examined in normal human organ-cultured corneas upon recombinant adenovirus (rAV)-driven transduction of MMP-10 and CTSF genes. Fifteen pairs of normal autopsy human corneas were used. One cornea of each pair was transduced with rAV expressing either CTSF or MMP-10 genes. 1–2 × 108 plaque forming units of rAV per cornea were added to cultures for 48 hr with or without sildenafil citrate. The fellow cornea of each pair received control rAV with vector alone. After 6–10 days incubation without rAV, corneas were analyzed by Western blot or immunohistochemistry, or tested for healing of 5-mm circular epithelial wounds caused by topical application of n-heptanol. Sildenafil significantly increased epithelial transduction efficiency, apparently through stimulation of rAV endocytosis through caveolae. Corneas transduced with CTSF or MMP-10 genes or their combination had increased epithelial immunostaining of respective proteins compared to fellow control corneas. Staining for diabetic markers integrin ?3?1, nidogen-1, nidogen-2, and laminin ?2 chain became weaker and irregular upon proteinase transduction. Expression of phosphorylated Akt was decreased in proteinase-transduced corneas. Joint overexpression of both proteinases led to significantly slower corneal wound healing that became similar to that observed in diabetic ones. The data suggest that MMP-10 and CTSF may be responsible for abnormal marker patterns and impaired wound healing in diabetic corneas. Inhibition of these proteinases in diabetic corneas may alleviate diabetic keratopathy symptoms. PMID:19828126

  9. Effects of serine protease inhibitors on growth and development and digestive serine proteinases of the Sunn pest, Eurygaster integriceps.

    Science.gov (United States)

    Saadati, Fatemeh; Bandani, Ali R

    2011-01-01

    In the current study the effects of serine proteinase inhibitors (TLCK, TPCK, SBTI, and a combination of SBTI and TPCK) with concentrations of 1% and 4% of dietary protein in artificial diets were tested against growth of the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), development, and its gut serine proteinase targets. Analysis of variance indicated that protease inhibitors affected nymphal development time, adult weight, and survival. Mean development time of third instar nymphs in control, SBTI (1%), TLCK (1%), and TPCK was 7.18, 9.74, 9.97, and 8.52 days, respectively. The highest mortality (100 % mortality) was observed when a combination of TPCK and SBTI, both at 4% of dietary protein, was used followed by TPCK (4%) that produced 95% mortality. There were significant differences in proteinase activity between treatments and controls when BApNA and SAAPFpNA were used as substrates for trypsin and chymotrypsin, respectively. Reduction of trypsin activity in insects fed with low doses of SBTI (1%), TLCK (1%), and both doses of TPCK (1% and 4%) was 40, 26, 23, and 17%, respectively. Inhibition of chymotrypsin activity was seen in the insects fed on SBTI (1%), TLCK (1%), and TPCK (4%) where inhibition was 14, 9, and 36%, respectively. Maximum inhibition of chymotrypsin activity was observed in the insects fed on diets containing high doses of TPCK (4%). In gel assays, the greatest effects were observed when E. integriceps were fed on high doses of SBTI and TPCK. Therefore, TPCK followed by SBTI proved to be the most effective proteinase inhibitors of E. integriceps. PMID:21867440

  10. Proteinase-activated receptors 1 and 4 counter-regulate endostatin and VEGF release from human platelets

    OpenAIRE

    Ma, Li; Perini, Rafael; Mcknight, Webb; Dicay, Michael; Klein, Andre; Hollenberg, Morley D.; Wallace, John L.

    2004-01-01

    The roles of proteinase-activated receptors (PARs) in platelet functions other than aggregation are not well understood. Among these is the release of factors that regulate the process of angiogenesis, such as endostatin and VEGF, which, respectively, inhibit and promote angiogenesis. PAR1 and PAR4 are expressed on the surface of human platelets and can be activated by thrombin. In the present study, we have attempted to determine the roles of PAR1 and PAR4 in regulating release of endostatin...

  11. Quantitation of alpha-1 proteinase inhibitor in the pulmonary epithelial lining fluid of horses with chronic obstructive pulmonary disease.

    Science.gov (United States)

    Milne, E M; Pemberton, A D; McGorum, B C; Dixon, P M; Scudamore, C L; Miller, H R

    1994-09-01

    The concentration of alpha-1 proteinase inhibitor (API) was measured in the pulmonary epithelial lining fluid (PELF) of horses with chronic obstructive pulmonary disease (COPD) while they had clinical signs and while they had none. The concentrations of total protein, albumin and API were significantly higher in the PELF of animals with clinical signs of COPD. The correlation between albumin and API in the PELF suggested that most of the API was derived from the serum. PMID:7817020

  12. Proteinases in Excretory-Secretory Products of Toxocara canis Second-Stage Larvae: Zymography and Modeling Insights

    OpenAIRE

    Gonzalo Ernesto González-Páez; Fernando Alba-Hurtado; Carlos Gerardo García-Tovar; Raúl Argüello-García

    2014-01-01

    Components released in excretory-secretory products of Toxocara canis larvae (TES) include phosphatidylethanolamine-binding proteins (TES26), mucins (TES120, MUC2-5), and C-type lectins (TES32, TES70) and their biochemical, immunological, and diagnostic properties have been extensively studied albeit proteinase activities towards physiological substrates are almost unknown. Proteolytic activities in TES samples were first analyzed by gel electrophoresis with gelatin as substrate. Major activi...

  13. Effect of sulphated polysaccharides on the alpha 1-antitrypsin inhibition of amidolysis catalysed by coagulation cascade proteinases.

    OpenAIRE

    Long, W. F.; Williamson, F. B.

    1981-01-01

    1 The ability of several sulphated polysaccharide anticoagulants to prevent alpha 1-antitrypsin inhibition of thrombin paralleled their ability to potentiate antithrombin III inhibition of thrombin. None of the compounds examined altered the ability of alpha 1-antitrypsin to inhibit activated coagulation factor X (Xa). 2 These results are consistent with the possibility that a direct polysaccharide-proteinase interaction may be involved in the sulphated polysaccharide-modulated inhibition of ...

  14. Inhibition of plasmin, urokinase, tissue plasminogen activator, and C1S by a myxoma virus serine proteinase inhibitor.

    OpenAIRE

    Lomas, D. A.; Evans, D. L.; Upton, C.; Mcfadden, G.; Carrell, R. W.

    1993-01-01

    The myxoma and malignant rabbit fibroma poxviruses are lethal tumorigenic viruses of rabbits whose virulence is modulated by the production of a virus-encoded secreted serine proteinase inhibitor, SERP-1. This viral protein was detected in medium harvested from myxoma and malignant rabbit fibroma virus-infected cells, and its inhibitory profile has been characterized by gel and kinetic analysis. SERP-1 forms complexes with and inhibits the human fibrinolytic enzymes plasmin, urokinase, and tw...

  15. Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: a bacteria-inducible protein similar to Drosophila easter.

    Science.gov (United States)

    Jiang, H; Wang, Y; Kanost, M R

    1998-10-13

    Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal "clip" domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle. PMID:9770467

  16. Extracellular serine-proteinases isolated from Streptomyces alboniger: Partial characterization and effect of aprotinin on cellular structure

    Directory of Open Access Journals (Sweden)

    Lopes Andréa

    1999-01-01

    Full Text Available Streptomyces alboniger ATCC 12461 grown in brain heart infusion (BHI medium produced two extracellular serine-proteinases, denoted SP I and SP II, which were purified by ammonium sulfate precipitation and aprotinin-agarose affinity chromatography. SP I was purified 88,9-fold and SP II 66,7- fold, with 33.4% and 10.4% yield, respectively. The optimum pH for the proteinases activity, using a-N-p-tosyl-L-arginine-methyl ester (TAME as substrate, was 9-10 and the optimum temperature was 37ºC. The proteolytic activity of SP I and SP II was inhibited by aprotinin and SP I was partially inhibited by leupeptin, both serine-proteinase inhibitors. S. alboniger growth in BHI-liquid medium decreased when 5 mg/ml, 10 mg/ml of aprotinin was used, being completely inhibited with 20 mg/ml and 40 mg/ml. At the ultrastructural level, aprotinin-treated S. alboniger cells showed swelling of the bacterial body and condensation of the genetic material, probably related to the inhibition of its growth.

  17. Molecular cloning of Soli E2: an elastase-like serine proteinase from the imported red fire ant (Solenopsis invicta).

    Science.gov (United States)

    Whitworth, S T; Kordula, T; Travis, J

    1999-03-01

    The 4th instar larva of the imported red fire ant, Solenopsis invicta, possesses several serine proteinases, one of which, referred to as Soli E2, has been found to contain elastase-like activity. A cDNA library was constructed in lambda gt10 using Poly(A)+RNA from these larvae and a synthetic degenerate oligonucleotide probe corresponding to a sequence encoding the N-terminal of Soli E2 was utilized to screen the library. Two clones were obtained covering the entire coding region of Soli E2, and the predicted structure of this proteinase indicated that it was synthesized as a preproenzyme of 249 amino acids, which was cleaved to give the mature form of 226 residues. In addition, both clones showed identity to the N-terminal amino acid sequence obtained from Edman degradation of the mature purified proteinase. Significantly, Northern blot analysis of the different developmental stages revealed that Soli E2 was selectively expressed in the 4th instar larvae. PMID:10319438

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

    Science.gov (United States)

    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 pGAPZ?A 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. PMID:23065363

  19. Small cysteine-rich antifungal proteins from radish: their role in host defense.

    Science.gov (United States)

    Terras, F R; Eggermont, K; Kovaleva, V; Raikhel, N V; Osborn, R W; Kester, A; Rees, S B; Torrekens, S; Van Leuven, F; Vanderleyden, J

    1995-05-01

    Radish seeds have previously been shown to contain two homologous, 5-kD cysteine-rich proteins designated Raphanus sativus-antifungal protein 1 (Rs-AFP1) and Rs-AFP2, both of which exhibit potent antifungal activity in vitro. We now demonstrate that these proteins are located in the cell wall and occur predominantly in the outer cell layers lining different seed organs. Moreover, Rs-AFPs are preferentially released during seed germination after disruption of the seed coat. The amount of released proteins is sufficient to create a microenvironment around the seed in which fungal growth is suppressed. Both the cDNAs and the intron-containing genomic regions encoding the Rs-AFP preproteins were cloned. Transcripts (0.55 kb) hybridizing with an Rs-AFP1 cDNA-derived probe were present in near-mature and mature seeds. Such transcripts as well as the corresponding proteins were barely detectable in healthy uninfected leaves but accumulated systemically at high levels after localized fungal infection. The induced leaf proteins (designated Rs-AFP3 and Rs-AFP4) were purified and shown to be homologous to seed Rs-AFPs and to exert similar antifungal activity in vitro. A chimeric Rs-AFP2 gene under the control of the constitutive cauliflower mosaic virus 35S promoter conferred enhanced resistance to the foliar pathogen Alternaria longipes in transgenic tobacco. The term "plant defensins" is proposed to denote these defense-related proteins. PMID:7780308

  20. Glutenase and collagenase activities of wheat cysteine protease Triticain-?: Feasibility for enzymatic therapy assays.

    Science.gov (United States)

    Savvateeva, Lyudmila V; Gorokhovets, Neonila V; Makarov, Vladimir A; Serebryakova, Marina V; Solovyev, Andrey G; Morozov, Sergey Yu; Reddy, V Prakash; Zernii, Evgeni Yu; Zamyatnin, Andrey A; Aliev, Gjumrakch

    2015-05-01

    Insufficient and/or improper protein degradation is associated with the development of various human pathologies. Enzymatic therapy with proteolytic enzymes aimed to improve insufficient proteolytic activity was suggested as a treatment of protease deficiency-induced disorders. Since in many cases human degradome is incapable of degrading the entire target protein(s), other organisms can be used as a source of proteases exhibiting activities distinct from human enzymes, and plants are perspective candidates for this source. In this study recombinant wheat cysteine protease Triticain-? was shown to refold in vitro into an autocatalytically activated proteolytic enzyme possessing glutenase and collagenase activities at acidic (or close to neutral) pH levels at the temperature of human body. Mass-spectrometry analysis of the products of Triticain-?-catalyzed gluten hydrolysis revealed multiple cleavage sites within the sequences of gliadin toxic peptides, in particular, in the major toxic 33-mer ?-gliadin-derived peptide initiating inflammatory responses to gluten in celiac disease (CD) patients. Triticain-? was found to be relatively stable in the conditions simulating stomach environment. We conclude that Triticain-? can be exploited as a basic compound for development of (i) pharmaceuticals for oral administration aimed at release of the active enzyme into the gastric lumen for CD treatment, and (ii) topically active pharmaceuticals for wound debridement applications. PMID:25765959

  1. The mechanism of cysteine detection in biological media by means of vanadium oxide nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Bezerra, A. G. [Universidade Tecnologica Federal do Parana, Departamento Academico de Fisica (Brazil); Barison, A. [Universidade Federal do Parana, Departamento de Quimica (Brazil); Oliveira, V. S. [Universidade Federal do Parana, Departamento de Fisica (Brazil); Foti, L.; Krieger, M. A. [Fundacao Oswaldo Cruz, Instituto de Biologia Molecular do Parana (Brazil); Dhalia, R.; Viana, I. F. T. [Fundacao Oswaldo Cruz, Centro de Pesquisas Aggeu Magalhaes (Brazil); Schreiner, W. H., E-mail: wido@fisica.ufpr.br [Universidade Federal do Parana, Departamento de Fisica (Brazil)

    2012-09-15

    We report on the interaction of vanadate nanoparticles, produced using the laser ablation in liquids synthesis, with cysteine in biological molecules. Cysteine is a very important amino acid present in most proteins, but also because cysteine and the tripeptide glutathione are the main antioxidant molecules in our body system. Detailed UV-Vis absorption spectra and dynamic light scattering measurements were done to investigate the detection of cysteine in large biological molecules. The intervalence band of the optical absorption spectra shows capability for quantitative cysteine sensing in the {mu}M range in biological macromolecules. Tests included cytoplasmic repetitive antigen and flagellar repetitive antigen proteins of the Trypanosoma cruzi protozoa, as well as the capsid p24 proteins from Human Immunodeficiency Virus type 1 and type 2. Detailed NMR measurements for hydrogen, carbon, and vanadium nuclei show that cysteine in contact with the vanadate looses hydrogen of the sulphydryl side chain, while the vanadate is reduced. The subsequent detachment of two deprotonated molecules to form cystine and the slow return to the vanadate complete the oxidation-reduction cycle. Therefore, the vanadate acts as a charge exchanging catalyst on cysteine to form cystine. The NMR results also indicate that the nanoparticles are not formed by the common orthorhombic V{sub 2}O{sub 5} form.

  2. Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Araki Hiromasa

    2007-04-01

    Full Text Available Abstract Background Proteinase-activated receptors (PARs; PAR1–4 that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, ?-smooth muscle actin (?-SMA for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells. Results Stimulation of PAR with thrombin (1 U/ml or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 ?M for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased ?-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-? (TGF-?. Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation.

  3. Identification and characterization of MOR-CP, a cysteine protease induced by ozone and developmental senescence in maize (Zea mays L.) leaves.

    Science.gov (United States)

    Ahmad, Rafiq; Zuily-Fodil, Yasmine; Passaquet, Chantal; Bethenod, Olivier; Roche, Romain; Repellin, Anne

    2014-08-01

    Among the different classes of endoproteases, cysteine proteases are consistently associated with senescence, defense signaling pathways and cellular responses to abiotic stresses. The objectives of this work were to study the effects of various concentrations of ozone on gene expression and enzymatic activity for papain-like cysteine proteases (PLCPs), in the leaves of maize plants grown under field conditions. Leaves from ranks 12 and 10 (cob leaf) were harvested regularly over a long-term artificial ozone fumigation experiment (50 d). Tissues were tested for transcriptional and activity changes concerning cysteine proteases, using qRT-PCR for the newly identified ozone-responsive PLCP gene (Mor-CP) and synthetic oligopeptide Boc-Val-Leu-Lys-AMC as a PLCP-specific substrate, respectively. Results showed that developmental senescence induced a significant and progressive rise in CP activity, only in the older leaves 10 and had no effect on Mor-CP gene expression levels. On the other hand, ozone dramatically enhanced Mor-CP mRNA levels and global PLCP enzymatic activity in leaves 12 and 10, particularly toward the end of the treatment. Ozone impact was more pronounced in the older leaves 10. Together, these observations concurred to conclude that ozone stress enhances natural senescence processes, such as those related to proteolysis. PMID:24594488

  4. Cysteine detection in water using an organic field-effect transistor with a gold extended-gate electrode

    Science.gov (United States)

    Minami, Tsuyoshi; Minamiki, Tsukuru; Fukuda, Kenjiro; Kumaki, Daisuke; Tokito, Shizuo

    2015-04-01

    We report on the detection of cysteine in water using an extended-gate-type organic field-effect transistor (OFET). The fabricated OFET device can be operated below 3 V. The portion of the designed device used for cysteine detection is the extended-gate electrode prepared by vacuum deposition of a gold (Au) layer. Cysteine can bind to the Au electrode surface through chemisorption. By this technique, we have successfully observed a shift in the threshold voltage of the OFET upon the addition of cysteine in an aqueous solution, whereby the detection limit for cysteine was found to be 39 ppb.

  5. Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

    OpenAIRE

    Hansen, M. S.; Barklis, E.

    1995-01-01

    We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking...

  6. Interaction of cysteine and copper ions on the surface of iron: EIS, polarization and XPS study

    Energy Technology Data Exchange (ETDEWEB)

    El-Deab, Mohamed S., E-mail: msaada68@yahoo.com [Department of Chemistry, Faculty of Science, Cairo University, Cairo (Egypt)

    2011-09-15

    Highlights: {yields} The current study demonstrates a comprehensive study for Cysteine + Cu(II) ions as an efficient inhibitor as demonstrated by EIS, XPS and potentiodynamic polarization measurements, in addition to traditional weight loss measurements. {yields} The novelty of the current work originates from the combined use of an eco-friendly compound (i.e., cysteine) with a minute amount of copper ions (in the micro molar range) as a corrosion inhibitor for low carbon steel in acidic medium. To this end, cysteine shows only moderate inhibition ca. 60% for iron which jumps up to more than 95% in the presence of micro molar range of Cu(II) ions. {yields} Cysteine-Cu(II) blends are found superior to benzotriazole (BTAH)-Cu(II) blends in terms of their long-term stability in addition to the avoidance of the use of the well-reported highly toxic BTAH. - Abstract: This study addresses the enhancing effect of copper ions on the inhibition efficiency (IE) of cysteine (an eco-friendly compound) against the corrosion of iron in 0.5 M sulphuric acid. Electrochemical impedance spectroscopy (EIS) data revealed a significant increase in the polarization resistance (R{sub p}) of the iron/solution interface in the presence of cysteine and Cu(II) ions instead of cysteine alone. That is, IE of 95% is obtained in the presence of 5 mM cysteine and 25 {mu}M Cu(II) ions, compared to 66% in absence of Cu(II) ions. Moreover, electrochemical polarization measurements indicate that cysteine and Cu(II) ions blends act as mixed-type inhibitors for the corrosion of iron. The formation of Cu(I)-cysteinate complex and/or cysteine SAM at Cu atop the iron surface (as evident from X-ray photoelectron spectroscopy (XPS)) blocks the underlying iron surface and imparts a pronounced protection against its corrosion. IE of cysteine-Cu(II) blend remains effectively unchanged with immersion time indicating its high stability in the used acidic medium.

  7. A simple and effective coumarin-based fluorescent probe for cysteine.

    Science.gov (United States)

    Dai, Xi; Wu, Qing-Hua; Wang, Peng-Chong; Tian, Jie; Xu, Yu; Wang, Sheng-Qing; Miao, Jun-Ying; Zhao, Bao-Xiang

    2014-09-15

    Acrylic acid 3-acetyl-2-oxo-2 H-chromen-7-yl ester (ACA) was rationally designed and synthesized as a simple and effective fluorescent probe for sensing cysteine with high selectivity and naked-eye detection. The probe can detect cysteine by fluorescence spectrometry with a detection limit of 0.657 ?M and can be used with calf serum and in live cell imaging. The conjugate addition/cyclization sequence mechanism of the reaction between ACA and cysteine was con?rmed by ESI-MS and fluorescence spectra. PMID:24690559

  8. Cysteine Metabolism-Related Genes and Bacterial Resistance to Potassium Tellurite? †

    OpenAIRE

    Fuentes, Derie E.; Fuentes, Eugenia L.; Castro, Miguel E.; Pe?rez, Jose? M.; Araya, Manuel A.; Chasteen, Thomas G.; Pichuantes, Sergio E.; Va?squez, Claudio C.

    2007-01-01

    Tellurite exerts a deleterious effect on a number of small molecules containing sulfur moieties that have a recognized role in cellular oxidative stress. Because cysteine is involved in the biosynthesis of glutathione and other sulfur-containing compounds, we investigated the expression of Geobacillus stearothermophilus V cysteine-related genes cobA, cysK, and iscS and Escherichia coli cysteine regulon genes under conditions that included the addition of K2TeO3 to the culture medium. Results ...

  9. Soilborne wheat mosaic virus (SBWMV 19K protein belongs to a class of cysteine rich proteins that suppress RNA silencing

    Directory of Open Access Journals (Sweden)

    Howard Amanda

    2005-03-01

    Full Text Available Abstract Amino acid sequence analyses indicate that the Soilborne wheat mosaic virus (SBWMV 19K protein is a cysteine-rich protein (CRP and shares sequence homology with CRPs derived from furo-, hordei-, peclu- and tobraviruses. Since the hordei- and pecluvirus CRPs were shown to be pathogenesis factors and/or suppressors of RNA silencing, experiments were conducted to determine if the SBWMV 19K CRP has similar activities. The SBWMV 19K CRP was introduced into the Potato virus X (PVX viral vector and inoculated to tobacco plants. The SBWMV 19K CRP aggravated PVX-induced symptoms and restored green fluorescent protein (GFP expression to GFP silenced tissues. These observations indicate that the SBWMV 19K CRP is a pathogenicity determinant and a suppressor of RNA silencing.

  10. Cloning and characterisation of a serine proteinase from the haemocytes of mud crab Scylla serrata.

    Science.gov (United States)

    Vaseeharan, Baskaralingam; Lin, Yong-Chin; Ko, Chi-Fong; Chen, Jiann-Chu

    2006-07-01

    A serine proteinase (SP) cDNA was cloned from the haemocytes of mud crab Scylla serrata using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1,131 bp encoding a protein of 376 amino acids. The calculated molecular mass of the SP mature protein is 39.54 kDa with an estimated pI of 5.37. The C-terminal half of S. serrata SP is composed of a trypsin-like domain, with a sequence similar to that of other invertebrate and vertebrate SP domain. The typical catalytic triad of SP required for functional activity (His150, Asp217 and Ser331) was conserved in the polypeptide sequence. Sequence comparison showed that SP deduced amino acid has an overall similarity of 55%, 51% and 50% to SP deduced amino acid from spiny lobster Panulirus argus, horseshoe crab Tachypleus tridentatus and crayfish Pacifastaus leniusculus, respectively. The SP was strongly expressed in haemocytes, but was weakly expressed in heart, eyestalk and antennules. The SP transcript decreased significantly for the S. serrata following 3 days exposure to pH 9.5. However, the SP transcript increased significantly 24 h post-zymosan injection. PMID:16326112

  11. Substitutions at the P(1) position in BPTI strongly affect the association energy with serine proteinases.

    Science.gov (United States)

    Grzesiak, A; Helland, R; Smalås, A O; Krowarsch, D; Dadlez, M; Otlewski, J

    2000-08-01

    The role of the S(1) subsite in trypsin, chymotrypsin and plasmin has been examined by measuring the association with seven different mutants of bovine pancreatic trypsin inhibitor (BPTI); the mutants contain Gly, Ala, Ser, Val, Leu, Arg, and Trp at the P(1) position of the reactive site. The effects of substitutions at the P(1) position on the association constants are very large, comprising seven orders of magnitude for trypsin and plasmin, and over five orders for chymotrypsin. All mutants showed a decrease of the association constant to the three proteinases in the same order: Ala>Gly>Ser>Arg>Val>Leu>Trp. Calorimetric and circular dichroism methods showed that none of the P1 substitutions, except the P1-Val mutant, lead to destabilisation of the binding loop conformation. The X-ray structure of the complex formed between bovine beta-trypsin and P(1)-Leu BPTI showed that the P(1)-Leu sterically conflicts with the side-chain of P(3)-Ile, which thereby is forced to rotate approximately 90 degrees. Ile18 (P(3)) in its new orientation, in turn interacts with the Tyr39 side-chain of trypsin. Introduction of a large side-chain at the P1' position apparently leads to a cascade of small alterations of the trypsin-BPTI interface that seem to destabilise the complex by it adopting a less optimized packing and by tilting the BPTI molecule up to 15 degrees compared to the native trypsin-BPTI complex. PMID:10926503

  12. Selective radioprotection of normal tissues by bowman-birk proteinase inhibitor (BBI) in mice

    Energy Technology Data Exchange (ETDEWEB)

    Dittmann, K.; Toulany, M.; Peter Rodemann, H. [Div. of Radiobiology and Environmental Research, Dept. of Radiation Oncology, Univ. of Tuebingen (Germany); Classen, J.; Heinrich, V. [Dept. of Radiation Oncology, Univ. of Tuebingen (Germany); Milas, L. [Dept. of Experimental Radiation Oncology, The Univ. of Texas, M.D. Anderson Cancer Center, Houston, TX (United States)

    2005-03-01

    Background and purpose: the efficacy of radiotherapy is limited by the response of normal tissues within the radiation field. The application of normal-tissue-specific radioprotectors may improve the therapeutic benefit of radiotherapy. The purpose of the present study was to explore the in vivo normal-tissue radioprotective potential of Bowman-Birk proteinase inhibitor (BBI), which acts as a normal-cell-specific radioprotector in vitro. Material and methods: the leg contracture assay in mice, a model system assessing radiation-induced fibrotic processes, was used. To determine whether BBI acts also as a radioprotector of tumors (i.e., FSA and FSAII), the tumor growth delay assay was used. Results: radiation induced leg contracture in mice with a maximum of about 8 mm at day 150 after irradiation. Treatment of mice with 100 mg/kg BBI before irradiation reduced leg contracture by > 4 mm, by about 50% (p < 0.05, t-test). Doses < 100 mg/kg were ineffective, and doses > 100 mg/kg did not further increase the degree of radioprotection. By contrast, BBI did not induce radioprotection of either TP53-mutated FSA or TP53-normal FSAII tumor xenografts in mice, which argues for normal-tissue-specific effect. Conclusion: BBI acts as a potent selective normal-tissue radioprotector in vitro and in vivo, apparently without protecting tumors, and thus has the potential to improve clinical radiotherapy. (orig.)

  13. Selective radioprotection of normal tissues by bowman-birk proteinase inhibitor (BBI) in mice

    International Nuclear Information System (INIS)

    Background and purpose: the efficacy of radiotherapy is limited by the response of normal tissues within the radiation field. The application of normal-tissue-specific radioprotectors may improve the therapeutic benefit of radiotherapy. The purpose of the present study was to explore the in vivo normal-tissue radioprotective potential of Bowman-Birk proteinase inhibitor (BBI), which acts as a normal-cell-specific radioprotector in vitro. Material and methods: the leg contracture assay in mice, a model system assessing radiation-induced fibrotic processes, was used. To determine whether BBI acts also as a radioprotector of tumors (i.e., FSA and FSAII), the tumor growth delay assay was used. Results: radiation induced leg contracture in mice with a maximum of about 8 mm at day 150 after irradiation. Treatment of mice with 100 mg/kg BBI before irradiation reduced leg contracture by > 4 mm, by about 50% (p 100 mg/kg did not further increase the degree of radioprotection. By contrast, BBI did not induce radioprotection of either TP53-mutated FSA or TP53-normal FSAII tumor xenografts in mice, which argues for normal-tissue-specific effect. Conclusion: BBI acts as a potent selective normal-tissue radioprotector in vitro and in vivo, apparently without protecting tumors, and thus has the potential to improve clinical radiotherapy. (orig.)

  14. Conformational changes of ovine ?-1-proteinase inhibitor: The influence of heparin binding

    Science.gov (United States)

    Gupta, Vivek Kumar; Gowda, Lalitha R.

    2008-11-01

    ?-1-Proteinase inhibitor (?-1-PI), the archetypal serpin causes rapid, irreversible stoichiometric inhibition of redundant circulating serine proteases and is associated with emphysema, inflammatory response and maintenance of protease-inhibitor equilibrium in vascular and peri-vascular spaces. A homogenous preparation of heparin octasaccharide binds to ovine and human ?-1-PI and enhances their protease inhibitory activity phenomenally. Size-exclusion chromatography and dynamic light scattering experiments reveal that ovine ?-1-PI undergoes a decrease in the Stokes' radius upon heparin binding. A strong binding; characterizes this ?-1-PI-heparin interaction as revealed by the binding constant ( K?) 1.98 ± 0.2 × 10 -6 M and 2.1 ± 0.2 × 10 -6 M determined by fluorescence spectroscopy and equilibrium dialysis, respectively. The stoichiometry of heparin binding to ovine ?-1-PI was 1.1 ± 0.2:1. The Stern-Volmer constants ( Ksv) for heparin activated ovine and human ?-1-PI were found to be 5.13 × 10 -6 M and 5.67 × 10 -6 M, respectively, significantly higher than the native inhibitors. FTIR and CD spectroscopy project the systematic structural reorientations that ?-1-PI undergoes upon heparin binding characterized by a decrease in ?-helical content and a concomitant increase in ?-turn and random coil elements. It is likely that these conformational changes result in the movement of the ?-1-PI reactive site loop into an extended structure that is better poised to combat the cognate protease and accelerate the inhibition.

  15. Proteinase 3 carries small unusual carbohydrates and associates with ?lpha-defensins

    DEFF Research Database (Denmark)

    Zoega, Morten; Ravnsborg, Tina

    2012-01-01

    The neutrophil granulocyte is an important first line of defense against intruding pathogens and it contains a range of granules armed with antibacterial peptides and proteins. Proteinase 3 (PR3) is one among several serine proteases of the azurophilic granules in neutrophil granulocytes. Here, we characterize the glycosylation of PR3 and its association with antimicrobial human neutrophil peptides (HNPs, ?-defensins) and the effect of these on the mechanism of inhibition of the major plasma inhibitor of PR3, ?1-antitrypsin. The glycosylation of purified, mature PR3 showed some heterogeneity with carbohydrates at Asn 102 and 147 carrying unusual small moieties indicating heavy processing. Mass spectrometric analysis and immuno blotting revealed strong association of highly purified PR3 with ?-defensins and oligomers hereof. Irreversible inhibition of PR3 by ?1-antitrypsin did not affect its association with defensins. Other proteins from neutrophil granules were also found to be associated with defensins, whereas purified plasma proteins did not carry defensins. These results point to a role of defensins in controlling and targeting the activity of neutrophil granule proteins.

  16. Activation of ion secretion via proteinase-activated receptor-2 in human colon.

    Science.gov (United States)

    Mall, Marcus; Gonska, Tanja; Thomas, Jörg; Hirtz, Stephanie; Schreiber, Rainer; Kunzelmann, Karl

    2002-02-01

    Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial cells and rat jejunum. With the use of a microUssing chamber, we demonstrate the role of PAR-2 for ion transport in native human colonic mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl(-) secretion when added to the basolateral but not luminal side of normal epithelia. Activation of Cl(-) secretion by trypsin was inhibited by indomethacin and was further increased by cAMP in normal tissues but was not present in CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl(-), by basolateral bumetanide, and in the presence of barium or clotrimazole, but not by tetrodotoxin. Furthermore, trypsin-induced secretion was inhibited by the Ca(2+)-ATPase inhibitor cyclopiazonic acid and in low-Ca(2+) buffer. The effects of trypsin were almost abolished by trypsin inhibitor. Thrombin, an activator of PAR types 1, 3, and 4, had no effects on equivalent short-circuit currents. The presence of PAR-2 in human colon epithelium was confirmed by RT-PCR and additional experiments with PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte secretion by release of mast cell tryptase and potentiation of PAR-2 expression by tumor necrosis factor-alpha may contribute to the hypersecretion observed in inflammatory processes such as chronic inflammatory bowel disease. PMID:11804840

  17. Oligomerization and the sensitivity of phosphoenolpyruvate carboxylase to inactivation by proteinases.

    Science.gov (United States)

    Wedding, R T; Black, M K

    1987-08-01

    Phosphenolpyruvate (PEP) carboxylase from leaves of Crassula argentea displays varying levels of sensitivity to inactivation by various proteolytic enzymes. In general, the native enzyme is sensitive to proteinases known to attack at the carbonyl end of lysine or arginine (trypsin, papain, or bromelain). The ineffective proteolytic enzymes are those which have low specificity or which attack at the N-terminal end of hydrophobic amino acids, or which cannot attack lysine. The lack of an effect of endoproteinase arginine C, which is specific for arginine, probably indicates that lysine is the critical residue. When the native enzyme, which is comprised of an equilibrium of dimers with tetramers in approximately equal quantities, is treated by preincubation with 5 millimolar PEP, the enzyme becomes much more resistant to proteolytic inactivation. When the preincubation is with 5 millimolar malate rather than buffer alone, the effect is to slightly increase (ca. 15%) the sensitivity of the enzyme to inactivation by trypsin as measured by estimates of the pseudo-first order rate constant for inactivation. PEP carboxylase from corn leaves appears to be relatively susceptible to inactivation by trypsin, but is unaffected by preincubation with malate or PEP. The sensitivity of this C(4) enzyme to inhibition by malate is also unaffected by preincubation with these ligands. PMID:16665631

  18. Oligomerization and the Sensitivity of Phosphoenolpyruvate Carboxylase to Inactivation by Proteinases 1

    Science.gov (United States)

    Wedding, Randolph T.; Black, M. Kay

    1987-01-01

    Phosphenolpyruvate (PEP) carboxylase from leaves of Crassula argentea displays varying levels of sensitivity to inactivation by various proteolytic enzymes. In general, the native enzyme is sensitive to proteinases known to attack at the carbonyl end of lysine or arginine (trypsin, papain, or bromelain). The ineffective proteolytic enzymes are those which have low specificity or which attack at the N-terminal end of hydrophobic amino acids, or which cannot attack lysine. The lack of an effect of endoproteinase arginine C, which is specific for arginine, probably indicates that lysine is the critical residue. When the native enzyme, which is comprised of an equilibrium of dimers with tetramers in approximately equal quantities, is treated by preincubation with 5 millimolar PEP, the enzyme becomes much more resistant to proteolytic inactivation. When the preincubation is with 5 millimolar malate rather than buffer alone, the effect is to slightly increase (ca. 15%) the sensitivity of the enzyme to inactivation by trypsin as measured by estimates of the pseudo-first order rate constant for inactivation. PEP carboxylase from corn leaves appears to be relatively susceptible to inactivation by trypsin, but is unaffected by preincubation with malate or PEP. The sensitivity of this C4 enzyme to inhibition by malate is also unaffected by preincubation with these ligands. PMID:16665631

  19. Metabolism and distribution of the virus-encoded serine proteinase inhibitor SERP-1 in healthy rabbits.

    Science.gov (United States)

    Hatton, M W; Ross, B; Southward, S M; Lucas, A

    2000-11-01

    SERP-1 is a secreted myxoma virus-encoded 55-kd protein of the serine proteinase inhibitor ("serpin") family that strongly inhibits the mitosis of medial arterial smooth muscle cells, thus preventing stenosis in injured rabbit and rat arteries. We have measured the fractional catabolic rate (FCR) and compartmental distribution of 1251-SERP-1 after injection of various doses into the circulation of healthy rabbits. The FCR within the intravascular space decreased from 2.99 d(-1) to 2.39 d(-1) and the whole-body FCR decreased from 0.66 d(-1) to 0.51 d(-1) as the dose was increased 35-fold from 0.11 microg/kg to 3.8 microg/kg. The fractional distribution of SERP-1 between the intravascular (0.21), noncirculating vascular wall (0.09), and extravascular compartments (0.70) at equilibrium did not change significantly over this dose range. SERP-1 did not appear to selectively accumulate in any organ in any of 11 rabbits studied over a 6-day interval. In comparison to other rabbit plasma serpins, the behavior of SERP-1 in vivo most closely resembled that of heparin cofactor II. PMID:11092510

  20. Original features of cell-envelope proteinases of Lactobacillus helveticus. A review.

    Science.gov (United States)

    Sadat-Mekmene, Leila; Genay, Magali; Atlan, Danièle; Lortal, Sylvie; Gagnaire, Valérie

    2011-03-15

    Lactobacillus helveticus is a lactic acid bacterium very used in fermented milks and cheese. The rapid growth of L. helveticus in milk is supported by an efficient cell envelope proteinase (CEP) activity, due to subtilisin-like serine proteases. These enzymes play also crucial roles in texture and flavor formation in dairy products as well as in generating in situ bioactive peptides. In L. helveticus, several genes encoding putative CEPs were detected and characterized by a large intraspecific diversity; little is known about regulation of expression of CEP-encoding genes. Anchored at the bacterial surface, CEPs are large-sized enzymes (> 150 kDa) hydrolyzing ?- and ?(s1)-casein as well. Substrate cleavages occur after almost all types of amino acids residues, but mass spectrometry analysis revealed L. helveticus strains with specific profiles of substrate hydrolysis, which could explain identification of strains associated with interesting technological properties. In this review, the most recent data regarding CEP-encoding genes, CEP activities toward caseins and L. helveticus strain diversity are discussed. PMID:21354644

  1. Purification and partial characterization of ?1-proteinase inhibitor in the common marmoset (Callithrix jacchus).

    Science.gov (United States)

    Parambeth, Joseph Cyrus; Suchodolski, Jan S; Steiner, Jörg M

    2015-04-01

    Fecal alpha1-proteinase inhibitor (?1-PI) concentration has been to diagnose enteric protein loss in dogs and cats. Chronic lymphocytic enteritis is commonly seen in the marmoset (Callithrix jaccus) and is characterized by hypoalbuminemia. As a prelude to immunoassay development for detecting enteric protein loss, marmoset serum ?1-PI was purified using immunoaffinity chromatography and ceramic hydroxyapatite chromatography. Partial characterization was performed by reducing gel electrophoresis and enzyme inhibitory assays. Protein identity was confirmed with peptide mass fingerprinting and N-terminal amino acid sequencing. Molecular mass, relative molecular mass, and isoelectric point for marmoset ?1-PI were 54?kDa, 51,677, and 4.8-5.4, respectively. Trypsin, chymotrypsin, and elastase inhibitory activity were observed. N-terminal amino acid sequence for marmoset ?1-PI was EDPQGDAAQKMDTSHH. In conclusion, marmoset ?1-PI was successfully purified from serum with an overall yield of 12% using a rapid and efficient method. Purified marmoset ?1-PI has characteristics similar to those of ?1-PI reported for other species. PMID:25745866

  2. Treponema denticola chymotrypsin-like proteinase (CTLP) integrates spirochaetes within oral microbial communities.

    Science.gov (United States)

    Cogoni, Valentina; Morgan-Smith, Alex; Fenno, J Christopher; Jenkinson, Howard F; Dymock, David

    2012-03-01

    Treponema denticola is found ubiquitously in the human oral cavity and is mainly associated with bacterial communities implicated in the establishment and development of periodontal disease. The ability to become integrated within biofilm communities is crucial to the growth and survival of oral bacteria, and involves inter-bacterial coaggregation, metabolic cooperation, and synergy against host defences. In this article we show that the chymotrypsin-like proteinase (CTLP), found within a high-molecular-mass complex on the cell surface, mediates adherence of T. denticola to other potential periodontal pathogens, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia and Parvimonas micra. Proteolytic activity per se did not appear to be required for the interactions, and expression of the major outer-sheath protein (Msp) was not necessary, except for binding Parv. micra. Biofilms of densely packed cells and matrix, up to 40 µm in depth, were formed between T. denticola and P. gingivalis on salivary pellicle, with T. denticola cells enriched in the upper layers. Expression of CTLP, but not Msp, was critical for dual-species biofilm formation with P. gingivalis. T. denticola did not form dual-species biofilms with any of the other three periodontal bacterial species under various conditions. Synergy between T. denticola and P. gingivalis was also shown by increased inhibition of blood clotting, which was CTLP-dependent. The results demonstrate the critical role of CTLP in interactions of T. denticola with other oral micro-organisms, leading to synergy in microbial community development and host tissue pathogenesis. PMID:22313692

  3. Compatibility in the Ustilago maydis-maize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2.

    Science.gov (United States)

    Mueller, André N; Ziemann, Sebastian; Treitschke, Steffi; Aßmann, Daniela; Doehlemann, Gunther

    2013-02-01

    The basidiomycete Ustilago maydis causes smut disease in maize, with large plant tumors being formed as the most prominent disease symptoms. During all steps of infection, U. maydis depends on a biotrophic interaction, which requires an efficient suppression of plant immunity. In a previous study, we identified the secreted effector protein Pit2, which is essential for maintenance of biotrophy and induction of tumors. Deletion mutants for pit2 successfully penetrate host cells but elicit various defense responses, which stops further fungal proliferation. We now show that Pit2 functions as an inhibitor of a set of apoplastic maize cysteine proteases, whose activity is directly linked with salicylic-acid-associated plant defenses. Consequently, protease inhibition by Pit2 is required for U. maydis virulence. Sequence comparisons with Pit2 orthologs from related smut fungi identified a conserved sequence motif. Mutation of this sequence caused loss of Pit2 function. Consequently, expression of the mutated protein in U. maydis could not restore virulence of the pit2 deletion mutant, indicating that the protease inhibition by Pit2 is essential for fungal virulence. Moreover, synthetic peptides of the conserved sequence motif showed full activity as protease inhibitor, which identifies this domain as a new, minimal protease inhibitor domain in plant-pathogenic fungi. PMID:23459172

  4. Cysteine-Free Proteins in the Immunobiology of Arthropod-Borne Diseases

    Directory of Open Access Journals (Sweden)

    J. Santiago Mejia

    2010-01-01

    Full Text Available One approach to identify epitopes that could be used in the design of vaccines to control several arthropod-borne diseases simultaneously is to look for common structural features in the secretome of the pathogens that cause them. Using a novel bioinformatics technique, cysteine-abundance and distribution analysis, we found that many different proteins secreted by several arthropod-borne pathogens, including Plasmodium falciparum, Borrelia burgdorferi, and eight species of Proteobacteria, are devoid of cysteine residues. The identification of three cysteine-abundance and distribution patterns in several families of proteins secreted by pathogenic and nonpathogenic Proteobacteria, and not found when the amino acid analyzed was tryptophan, provides evidence of forces restricting the content of cysteine residues in microbial proteins during evolution. We discuss these findings in the context of protein structure and function, antigenicity and immunogenicity, and host-parasite relationships.

  5. Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

    DEFF Research Database (Denmark)

    Baron, Caroline; Jacobsen, S.

    2004-01-01

    The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin molecule were identified by N-terminal sequencing of the different proteolytic fragments. Desmin incubated with either m-calpain or mu-calpain was primarily cleaved in the head and tail region leaving the rod domain relatively intact even after prolonged incubation. Incubation with cathepsin B produces a sequential C-terminal degradation pattern characteristic of this dipeptylpeptidase. The substrate primary structure was not found to be essential for regulation of the proteolytic activity of the cysteine peptidases studied. However, the degradation patterns obtained imply that calpains are involved in degradation of desmin early post-mortem, targeting the non-helical region of the desmin molecule and resulting in depolymerisation and initial disorganisation of the intermediate filament structures of the muscle cell.

  6. Identification and preliminary characterization of protein-cysteine farnesyltransferase

    International Nuclear Information System (INIS)

    Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus in order to exert their biological activity. Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group. Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2Valendash19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an onocogenic Ras p21 protein of human origin. These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, the authors describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue. Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa. Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway

  7. Efficacy of N-acetyl cysteine in traumatic brain injury.

    Science.gov (United States)

    Eakin, Katharine; Baratz-Goldstein, Renana; Pick, Chiam G; Zindel, Ofra; Balaban, Carey D; Hoffer, Michael E; Lockwood, Megan; Miller, Jonathan; Hoffer, Barry J

    2014-01-01

    In this study, using two different injury models in two different species, we found that early post-injury treatment with N-Acetyl Cysteine (NAC) reversed the behavioral deficits associated with the TBI. These data suggest generalization of a protocol similar to our recent clinical trial with NAC in blast-induced mTBI in a battlefield setting, to mild concussion from blunt trauma. This study used both weight drop in mice and fluid percussion injury in rats. These were chosen to simulate either mild or moderate traumatic brain injury (TBI). For mice, we used novel object recognition and the Y maze. For rats, we used the Morris water maze. NAC was administered beginning 30-60 minutes after injury. Behavioral deficits due to injury in both species were significantly reversed by NAC treatment. We thus conclude NAC produces significant behavioral recovery after injury. Future preclinical studies are needed to define the mechanism of action, perhaps leading to more effective therapies in man. PMID:24740427

  8. Rapid activation of glutamate cysteine ligase following oxidative stress.

    Science.gov (United States)

    Krejsa, Cecile M; Franklin, Christopher C; White, Collin C; Ledbetter, Jeffrey A; Schieven, Gary L; Kavanagh, Terrance J

    2010-05-21

    Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in the formation of the cellular antioxidant glutathione (GSH). The GCL holoenzyme consists of two separately coded proteins, a catalytic subunit (GCLC) and a modifier subunit (GCLM). Both GCLC and GLCM are controlled transcriptionally by a variety of cellular stimuli, including oxidative stress. This study addresses post-translational control of GCL activity, which increased rapidly in human lymphocytes following oxidative stress. Activation of GCL occurred within minutes of treatment and without any change in GCL protein levels and coincided with an increase in the proportion of GCLC in the holoenzyme form. Likewise, GCLM shifted from the monomeric form to holoenzyme and higher molecular weight species. Normal rat tissues also showed a distribution of monomeric and higher molecular weight forms. Neither GCL activation, nor the formation of holoenzyme, required a covalent intermolecular disulfide bridge between GCLC and GCLM. However, in immunoprecipitation studies, a neutralizing epitope associated with enzymatic activity was protected following cellular oxidative stress. Thus, the N-terminal portion of GCLC may undergo a change that stabilizes the GCL holoenzyme. Our results suggest that a dynamic equilibrium exists between low and high activity forms of GCL and is altered by transient oxidative stress. This provides a mechanism for the rapid post-translational activation of GCL and maintenance of cellular GSH homeostasis. PMID:20332089

  9. Cysteine protease activation and apoptosis in Murine norovirus infection

    Directory of Open Access Journals (Sweden)

    Ettayebi Khalil

    2009-09-01

    Full Text Available Abstract Background Noroviruses are the leading cause of viral gastroenteritis. Because a suitable in vitro culture system for the human virus has yet to be developed, many basic details of the infection process are unknown. Murine norovirus (MNV serves as a model system for the study of norovirus infection. Recently it was shown that infection of RAW 264.7 cells involved a novel apoptotic pathway involving survivin. Results Using a different set of approaches, the up-regulation of caspases, DNA condensation/fragmentation, and membrane blebbing, all of which are markers of apoptosis, were confirmed. Live cell imaging and activity-based protein profiling showed that activation of caspase-like proteases occurred within two hours of infection, followed by morphological changes to the cells. MNV infection in the presence of caspase inhibitors proceeded via a distinct pathway of rapid cellular necrosis and reduced viral production. Affinity purification of activity-based protein profiling targets and identification by peptide mass fingerprinting showed that the cysteine protease cathepsin B was activated early in infection, establishing this protein as an upstream activator of the intrinsic apoptotic pathway. Conclusion This work adds cathepsin B to the noncanonical programmed cell death induced by MNV, and provides data suggesting that the virus may induce apoptosis to expand the window of time for viral replication. This work also highlights the significant power of activity-based protein profiling in the study of viral pathogenesis.

  10. Antifungal defensins and their role in plant defense

    OpenAIRE

    PATRICIABARBOSAPELEGRINI; ArianeLacerda; Maria FatimaGrossi-de-Sa

    2014-01-01

    Since the beginning of the 90s lots of cationic plant, cysteine-rich antimicrobial peptides (AMP) have been studied. However, Broekaert et al. (1995) only coined the term “plant defensin,” after comparison of a new class of plant antifungal peptides with known insect defensins. From there, many plant defensins have been reported and studies on this class of peptides encompass its activity toward microorganisms and molecular features of the mechanism of action against bacteria and fungi. P...

  11. Chemical Issues Addressing the Construction of the Distal Ni[Cysteine-Glycine-Cysteine]2- Site of Acetyl CoA Synthase: Why Not Copper?

    OpenAIRE

    Green, Kayla N.; Brothers, Scott M.; Lee, Boram; Darensbourg, Marcetta Y.; Rockcliffe, David A.

    2009-01-01

    The discovery of the Ni(Cysteine-Glycine-Cysteine)2-, Ni(CGC)2-, in the A-cluster active site of Acetyl CoA Synthase has prompted the synthesis of many small molecule models which employ M(N2S2) complexes as metalloligands. In vitro studies have shown that nickel incorporates into the N2S2 binding pocket even when copper is in the enzyme growth medium, while copper is preferentially taken up in the proximal site, displacing the catalytically active nickel. (Darnault, C.; Volbeda, A.; Kim, E.J...

  12. Proteinase PrtP impairs lactococcin LcnB activity in Lactococcus lactis BGMN1-501: new insights into bacteriocin regulation

    Science.gov (United States)

    Vukotic, Goran; Mirkovic, Nemanja; Jovcic, Branko; Miljkovic, Marija; Strahinic, Ivana; Fira, Djordje; Radulovic, Zorica; Kojic, Milan

    2015-01-01

    Proteinases and bacteriocins are of great importance to the dairy industry, but their interactions have not been studied so far. Lactococcus lactis subsp. lactis BGMN1-5 is a natural isolate from homemade semi-hard cheese which produces two bacteriocins (Lactococcin B and LsbB), as well as proteinase PrtP. A medium-dependent increase in the bacteriocin LcnB activity of L. lactis BGMN1-501, a derivate of L. lactis subsp. lactis BGMN1-5, was shown to be accompanied by a decrease in its promoter activity. A similar effect of media components on gene expression was reported for proteinase PrtP, whose gene is co-localized on the same plasmid as the lcnB gene. Thus, the PrtP-LcnB interplay was investigated. Single gene knockout mutants were constructed with disrupted prtP or lcnB genes. PrtP- mutants showed higher bacteriocin activity that had lost its growth medium dependence, which was in contrast to the original strain. When LcnB from this mutant was combined with proteinase from the LcnB- mutant in vitro, its activity was rendered to the original level, suggesting that proteinase reduces bacteriocin activity. We propose a new model of medium dependent expression of these genes with regard to the effects of their interaction in vivo. PMID:25713574

  13. Removal of palladium ions from aqueous systems by chemically modified cysteine carbon powder

    OpenAIRE

    Abiman, P.; Wildgoose, Gg; Crossley, A.; Compton, Rg

    2008-01-01

    l-Cysteine methyl ester modified graphite powder (Cyscarbon) was used as a material to remove palladium ions from aqueous media. Cheap graphite powders (2-20 ?m in diameter) were surface functionalised with l-cysteine methyl ester. The removal of Pd(ii) ions was studied as a function of concentration of Pd(ii) ions, contact time with modified carbon and amount of modified carbon used. Determination of palladium ions was performed by adsorptive stripping voltammetry using a mercury nanodrople...

  14. A Parasite Cysteine Protease Is Key to Host Protein Degradation and Iron Acquisition*S?

    OpenAIRE

    O Brien, Theresa C.; Mackey, Zachary B.; Fetter, Richard D.; Choe, Youngchool; O Donoghue, Anthony J.; Zhou, Min; Craik, Charles S.; Caffrey, Conor R.; Mckerrow, James H.

    2008-01-01

    Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated...

  15. Mapping Inhibitor Binding Modes on an Active Cysteine Protease via NMR Spectroscopy

    OpenAIRE

    Lee, Gregory M.; Balouch, Eaman; Goetz, David H.; Lazic, Ana; Mckerrow, James H.; Craik, Charles S.

    2012-01-01

    Cruzain is a member of the papain/cathepsin-L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas’ disease. We report an auto-induction methodology that provides soluble-cruzain at high yields (> 30 mg per liter in minimal media). These increased yields provide sufficient quantities of active enzyme for use in NMR-based ligand mapping. Using CD and NMR spectroscopy, we also examined the solution-state structural dyn...

  16. Acetaldehyde Removal from Indoor Air through Chemical Absorption Using L-Cysteine

    OpenAIRE

    Miyuki Noguchi; Atsushi Mizukoshi; Kyoko Yamashita; Yukio Yanagisawa

    2010-01-01

    The irreversible removal of acetaldehyde from indoor air via a chemical reaction with amino acids was investigated. To compare effectiveness, five types of amino acid (glycine, L-lysine, L-methionine, L-cysteine, and L-cystine) were used as the reactants. First, acetaldehyde-laden air was introduced into aqueous solutions of each amino acid and the removal abilities were compared. Among the five amino acids, L-cysteine solution showed much higher removal efficiency, while the other amino acid...

  17. Comparative study of abiogenesis of cysteine and other amino acids catalyzed by various metal ions.

    Science.gov (United States)

    Bahadur, K; Sen, P

    1975-01-01

    The present work pertains to the study of the influence of nickel, cobalt, thorium, vanadium, molybdate, ferrous ions on the formation of cysteine which is synthesized abiogenically together with other amino acids in sterilized aqueous mixtures of ammonium thiocyanate, formaldehyde, potassium dihydrogen phosphate, calcium acetate, and biological minerals after irradiating by artificial light. The effect of these catalysts on cysteine formation was of the order: Fe++ greater than Mo++ greater than Th++++ greater than V++ greater than Co++ greater than Ni. PMID:1189468

  18. Mutation of Neuraminidase Cysteine Residues Yields Temperature-Sensitive Influenza Viruses

    OpenAIRE

    Basler, Christopher F.; Garci?a-sastre, Adolfo; Palese, Peter

    1999-01-01

    The influenza virus neuraminidase (NA) is a tetrameric, virus surface glycoprotein possessing receptor-destroying activity. This enzyme facilitates viral release and is a target of anti-influenza virus drugs. The NA structure has been extensively studied, and the locations of disulfide bonds within the NA monomers have been identified. Because mutation of cysteine residues in other systems has resulted in temperature-sensitive (ts) proteins, we asked whether mutation of cysteine residues in t...

  19. Production of Hydrogen Sulfide from D-Cysteine and Its Therapeutic Potential

    OpenAIRE

    NorihiroShibuya

    2013-01-01

    Accumulating evidence shows that H2S has physiological functions in various tissues and organs. It includes regulation of neuronal activity, vascular tension, a release of insulin, and protection of the heart, kidney and brain from ischemic insult. H2S is produced by enzymes from L-cysteine; cystathionine ?-synthase (CBS), cystathionine ?-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase (CAT). We recently discovered an additional pathway f...

  20. Protein topology determines cysteine oxidation fate: the case of sulfenyl amide formation among protein families.

    Science.gov (United States)

    Defelipe, Lucas A; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A; Turjanski, Adrián G

    2015-03-01

    Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function. PMID:25741692