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Berne L Jones - One of the best experts on this subject based on the ideXlab platform.
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purification and identification of barley hordeum vulgare l proteins that inhibit the Alkaline serine Proteinases of fusarium culmorum
Journal of Agricultural and Food Chemistry, 2003Co-Authors: Anja I Pekkarinen, Berne L JonesAbstract:: It has been proposed that microbial Proteinase inhibitors, which are present in abundance in cereal grains, protect the seed against plant pathogens. So far, however, very little is known about the interactions of those inhibitors with the Proteinases of phytopathogenic microbes. The increased Alkaline Proteinase activities of Fusarium head blight (FHB) diseased wheat and barley grain imply that the Fusarium fungi synthesize those enzymes during the colonization of the kernel. To study which barley proteins can inhibit Fusarium Proteinases, and hence, possibly protect the seed from FHB, the proteins of a grain extract have been separated and tested for their abilities to inhibit two Alkaline serine Proteinases that we previously isolated from F. culmorum. The proteins were separated by size exclusion, ion exchange, and reversed-phase-HPLC chromatographies. The purified inhibitors were identified by their molecular masses and N-terminal amino acid sequences. The proteins that inhibited the subtilisin-like Fusarium Proteinase were the chymotrypsin/subtilisin (CI) inhibitors 1A, 1B, and 2A and the barley alpha-amylase/subtilisin inhibitor (BASI). Only one of the purified proteins inhibited the trypsin-like Proteinase, the barley Bowman-Birk inhibitor (BBBI). No novel inhibitors were detected.
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purification and properties of an Alkaline Proteinase of fusarium culmorum
FEBS Journal, 2002Co-Authors: Anja I Pekkarinen, Berne L Jones, Marjaleena NikupaavolaAbstract:The disease Fusarium head blight (scab) causes severe problems for farmers and for the industries that use cereals. It is likely that the fungi that cause scab (Fusarium spp.) use various enzymes when they invade grains. We are studying enzymes that the fungi may use to hydrolyze grain proteins. To do this, Fusarium culmorum was grown in a gluten-containing medium from which an Alkaline serine Proteinase with a molecular mass of 28.7 kDa was purified by size-exclusion and cation exchange chromatographies. The enzyme was maximally active at pH 8.3–9.6 and 50 °C, but was unstable under these conditions. It hydrolyzed the synthetic substrates N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and, to a lesser extent, N-succinyl-Ala-Ala-Pro-Leu p-nitroanilide. It was inhibited by phenylmethanesulfonyl fluoride and chymostatin, but not by soybean trypsin or Bowman–Birk inhibitors. Parts of the amino-acid sequence were up to 82% homologous with those of several fungal subtilisins. One of the active site amino acids was detected and it occupied the same relative position as in the other subtilisins. Therefore, on the basis of these characteristics, the Proteinase is subtilisin-like. Purification of the enzyme was complicated by the fact that, when purified, it apparently underwent autolysis. The presence of extraneous protein stabilized the activity.
Anja I Pekkarinen - One of the best experts on this subject based on the ideXlab platform.
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purification and identification of barley hordeum vulgare l proteins that inhibit the Alkaline serine Proteinases of fusarium culmorum
Journal of Agricultural and Food Chemistry, 2003Co-Authors: Anja I Pekkarinen, Berne L JonesAbstract:: It has been proposed that microbial Proteinase inhibitors, which are present in abundance in cereal grains, protect the seed against plant pathogens. So far, however, very little is known about the interactions of those inhibitors with the Proteinases of phytopathogenic microbes. The increased Alkaline Proteinase activities of Fusarium head blight (FHB) diseased wheat and barley grain imply that the Fusarium fungi synthesize those enzymes during the colonization of the kernel. To study which barley proteins can inhibit Fusarium Proteinases, and hence, possibly protect the seed from FHB, the proteins of a grain extract have been separated and tested for their abilities to inhibit two Alkaline serine Proteinases that we previously isolated from F. culmorum. The proteins were separated by size exclusion, ion exchange, and reversed-phase-HPLC chromatographies. The purified inhibitors were identified by their molecular masses and N-terminal amino acid sequences. The proteins that inhibited the subtilisin-like Fusarium Proteinase were the chymotrypsin/subtilisin (CI) inhibitors 1A, 1B, and 2A and the barley alpha-amylase/subtilisin inhibitor (BASI). Only one of the purified proteins inhibited the trypsin-like Proteinase, the barley Bowman-Birk inhibitor (BBBI). No novel inhibitors were detected.
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purification and properties of an Alkaline Proteinase of fusarium culmorum
FEBS Journal, 2002Co-Authors: Anja I Pekkarinen, Berne L Jones, Marjaleena NikupaavolaAbstract:The disease Fusarium head blight (scab) causes severe problems for farmers and for the industries that use cereals. It is likely that the fungi that cause scab (Fusarium spp.) use various enzymes when they invade grains. We are studying enzymes that the fungi may use to hydrolyze grain proteins. To do this, Fusarium culmorum was grown in a gluten-containing medium from which an Alkaline serine Proteinase with a molecular mass of 28.7 kDa was purified by size-exclusion and cation exchange chromatographies. The enzyme was maximally active at pH 8.3–9.6 and 50 °C, but was unstable under these conditions. It hydrolyzed the synthetic substrates N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and, to a lesser extent, N-succinyl-Ala-Ala-Pro-Leu p-nitroanilide. It was inhibited by phenylmethanesulfonyl fluoride and chymostatin, but not by soybean trypsin or Bowman–Birk inhibitors. Parts of the amino-acid sequence were up to 82% homologous with those of several fungal subtilisins. One of the active site amino acids was detected and it occupied the same relative position as in the other subtilisins. Therefore, on the basis of these characteristics, the Proteinase is subtilisin-like. Purification of the enzyme was complicated by the fact that, when purified, it apparently underwent autolysis. The presence of extraneous protein stabilized the activity.
Marjaleena Nikupaavola - One of the best experts on this subject based on the ideXlab platform.
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purification and properties of an Alkaline Proteinase of fusarium culmorum
FEBS Journal, 2002Co-Authors: Anja I Pekkarinen, Berne L Jones, Marjaleena NikupaavolaAbstract:The disease Fusarium head blight (scab) causes severe problems for farmers and for the industries that use cereals. It is likely that the fungi that cause scab (Fusarium spp.) use various enzymes when they invade grains. We are studying enzymes that the fungi may use to hydrolyze grain proteins. To do this, Fusarium culmorum was grown in a gluten-containing medium from which an Alkaline serine Proteinase with a molecular mass of 28.7 kDa was purified by size-exclusion and cation exchange chromatographies. The enzyme was maximally active at pH 8.3–9.6 and 50 °C, but was unstable under these conditions. It hydrolyzed the synthetic substrates N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide and, to a lesser extent, N-succinyl-Ala-Ala-Pro-Leu p-nitroanilide. It was inhibited by phenylmethanesulfonyl fluoride and chymostatin, but not by soybean trypsin or Bowman–Birk inhibitors. Parts of the amino-acid sequence were up to 82% homologous with those of several fungal subtilisins. One of the active site amino acids was detected and it occupied the same relative position as in the other subtilisins. Therefore, on the basis of these characteristics, the Proteinase is subtilisin-like. Purification of the enzyme was complicated by the fact that, when purified, it apparently underwent autolysis. The presence of extraneous protein stabilized the activity.
J P Bouchara - One of the best experts on this subject based on the ideXlab platform.
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Role of PTX3 in Cystic Fibrosis-associated infections
2011Co-Authors: Y. Hamon, J P Bouchara, C. Person, J.l. Giniès, Y. DelnesteAbstract:PTX3, a soluble innate immunity receptor, binds to selected microbes and facilitates their clearance by phagocytes. PTX3 selectively binds to Pseudomonas aeruginosa and Aspergillus fumigatus, two microorganisms frequently colonizing the airways of patients with cystic fibrosis (CF), and sometimes causing true respiratory infections. PTX3 -/- mice are sensitive to A. fumigatus infection, highlighting the role of this protein in the protection against this pathogen. We thus hypothesized that PTX3 could be altered in CF patients and that this could be responsible, at least in part, to their susceptibility to some opportunistic pathogens. Serum and sputum samples from 30 CF patients (20 adults and 15 children) and 7 patients with chronic obstructive pulmonary disease (COPD) as the control group were analyzed for PTX3 expression and integrity by ELISA and Western-blotting, respectively. The role of endogenous or microbial proteases on recombinant human PTX3 was also analyzed. Results showed that PTX3 level was increased in CF and COPD serum, highlighting their infectious/inflammatory status, while, in contrast, PTX3 concentration was lower or undetectable in CF sputum than in COPD. Western-blotting showed that PTX3 is degraded in sputum samples from most of CF patients, but not in clinical specimens from COPD patients. The degradation of PTX3 was shown to be mediated by serine proteases. More precisely, both the neutrophil elastase and the Alkaline Proteinase from A. fumigatus have the ability to degrade in vitro PTX3. This study which shows that PTX3 is degraded in respiratory secretions from CF patients, provide new insights into the pathogenesis of microbial colonization of the airways and respiratory infections in CF patients, since degradation of PTX3 could be responsible, at least in part, for the sensitivity of CF patients to some opportunistic infections
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a 33 kda serine Proteinase from scedosporium apiospermum
Biochemical Journal, 1996Co-Authors: Gerald Larcher, B Cimon, F Symoens, G Tronchin, Dominique Chabasse, J P BoucharaAbstract:An extracellular Proteinase produced by the filamentous fungus Scedosporium apiospermum has been purified and characterized. Initially, in vitro conditions for enzyme synthesis were investigated. The highest yield of enzyme production was obtained when the fungus was cultivated in modified Czapek-Dox liquid medium supplemented with 0.1% bacteriological peptone and 1% (w/v) glucose as the nitrogen and carbon sources respectively. Purification to homogeneity of the Proteinase was accomplished by (NH4)2SO4 precipitation, followed by gel filtration through Sephadex G-75 and finally affinity chromatography through immobilized phenylalanine. Analysis of the purified enzyme by SDS/PAGE revealed a single polypeptide chain with an apparent molecular mass of 33 kDa. Further investigation of its physical and biochemical properties disclosed numerous similarities with those of the previously described serine Proteinase of Aspergillus fumigatus. The enzyme was not glycosylated and its pI was 9.3. Proteinase activity was optimum between 37 and 50 degrees C and at pH 9.0, but remained high within a large range of pH values between 7 and 11. The inhibition profile and N-terminal amino acid sequencing confirmed that this enzyme belongs to the subtilisin family of serine Proteinases. In agreement with this, the specific synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide proved to be an excellent substrate for the Proteinase with an estimated Km of 0.35 mM. Like the Alkaline Proteinase of A. fumigatus, this enzyme was able to degrade human fibrinogen, and thus may act as a mediator of the severe chronic bronchopulmonary inflammation from which cystic fibrosis patients suffer.
Hilton C. Deeth - One of the best experts on this subject based on the ideXlab platform.
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Diagnosing the cause of proteolysis in UHT milk
LWT - Food Science and Technology, 2003Co-Authors: Nivedita Datta, Hilton C. DeethAbstract:Proteolysis of UHT milk during storage at room temperature is a major factor limiting its shelf-life through changes in its flavour and texture. The latter is characterised by increases in viscosity leading in some cases to gel formation. The enzymes responsible for the proteolysis are the native milk Alkaline Proteinase, plasmin, and heat-stable, extracellular bacterial Proteinases produced by psychrotrophic bacterial contaminants in the milk prior to heat processing. These Proteinases react differently with the milk proteins and produce different peptides in the UHT milk. In order to differentiate these peptide products, reversed-phase HPLC and the fluorescamine method were used to analyse the peptides soluble in 12% trichloroacetic acid (TCA) and those soluble at pH 4.6. The TCA filtrate showed substantial peptide peaks only if the milk was contaminated by bacterial Proteinase, while the pH 4.6 filtrate showed peptide peaks when either or both bacterial and native milk Proteinases caused the proteolysis. Results from the fluorescamine test were in accordance with the HPLC results whereby the TCA filtrate exhibited significant proteolysis values only when bacterial Proteinases were present, but the pH 4.6 filtrates showed significant values when the milk contained either or both types of Proteinase. A procedure based on these analyses is proposed as a diagnostic test for determining which type of Proteinase - milk plasmin, bacterial Proteinase, or both - is responsible for proteolysis in UHT milk. © 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.