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Sample records for enzymatic activities consistent

  1. Clustered mutations in hominid genome evolution are consistent with APOBEC3G enzymatic activity.

    PubMed

    Pinto, Yishay; Gabay, Orshay; Arbiza, Leonardo; Sams, Aaron J; Keinan, Alon; Levanon, Erez Y

    2016-05-01

    The gradual accumulation of mutations by any of a number of mutational processes is a major driving force of divergence and evolution. Here, we investigate a potentially novel mutational process that is based on the activity of members of the AID/APOBEC family of deaminases. This gene family has been recently shown to introduce-in multiple types of cancer-enzyme-induced clusters of co-occurring somatic mutations caused by cytosine deamination. Going beyond somatic mutations, we hypothesized that APOBEC3-following its rapid expansion in primates-can introduce unique germline mutation clusters that can play a role in primate evolution. In this study, we tested this hypothesis by performing a comprehensive comparative genomic screen for APOBEC3-induced mutagenesis patterns across different hominids. We detected thousands of mutation clusters introduced along primate evolution which exhibit features that strongly fit the known patterns of APOBEC3G mutagenesis. These results suggest that APOBEC3G-induced mutations have contributed to the evolution of all genomes we studied. This is the first indication of site-directed, enzyme-induced genome evolution, which played a role in the evolution of both modern and archaic humans. This novel mutational mechanism exhibits several unique features, such as its higher tendency to mutate transcribed regions and regulatory elements and its ability to generate clusters of concurrent point mutations that all occur in a single generation. Our discovery demonstrates the exaptation of an anti-viral mechanism as a new source of genomic variation in hominids with a strong potential for functional consequences. PMID:27056836

  2. Clustered mutations in hominid genome evolution are consistent with APOBEC3G enzymatic activity

    PubMed Central

    Pinto, Yishay; Gabay, Orshay; Arbiza, Leonardo; Sams, Aaron J.; Keinan, Alon

    2016-01-01

    The gradual accumulation of mutations by any of a number of mutational processes is a major driving force of divergence and evolution. Here, we investigate a potentially novel mutational process that is based on the activity of members of the AID/APOBEC family of deaminases. This gene family has been recently shown to introduce—in multiple types of cancer—enzyme-induced clusters of co-occurring somatic mutations caused by cytosine deamination. Going beyond somatic mutations, we hypothesized that APOBEC3—following its rapid expansion in primates—can introduce unique germline mutation clusters that can play a role in primate evolution. In this study, we tested this hypothesis by performing a comprehensive comparative genomic screen for APOBEC3-induced mutagenesis patterns across different hominids. We detected thousands of mutation clusters introduced along primate evolution which exhibit features that strongly fit the known patterns of APOBEC3G mutagenesis. These results suggest that APOBEC3G-induced mutations have contributed to the evolution of all genomes we studied. This is the first indication of site-directed, enzyme-induced genome evolution, which played a role in the evolution of both modern and archaic humans. This novel mutational mechanism exhibits several unique features, such as its higher tendency to mutate transcribed regions and regulatory elements and its ability to generate clusters of concurrent point mutations that all occur in a single generation. Our discovery demonstrates the exaptation of an anti-viral mechanism as a new source of genomic variation in hominids with a strong potential for functional consequences. PMID:27056836

  3. Enzymatically active ultrathin pepsin membranes.

    PubMed

    Raaijmakers, Michiel J T; Schmidt, Thomas; Barth, Monika; Tutus, Murat; Benes, Nieck E; Wessling, Matthias

    2015-05-11

    Enzymatically active proteins enable efficient and specific cleavage reactions of peptide bonds. Covalent coupling of the enzymes permits immobilization, which in turn reduces autolysis-induced deactivation. Ultrathin pepsin membranes were prepared by facile interfacial polycondensation of pepsin and trimesoyl chloride. The pepsin membrane allows for simultaneous enzymatic conversion and selective removal of digestion products. The large water fluxes through the membrane expedite the transport of large molecules through the pepsin layers. The presented method enables the large-scale production of ultrathin, cross-linked, enzymatically active membranes. PMID:25779668

  4. Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

    PubMed

    Cheng, Jonathan D; Valianou, Matthildi; Canutescu, Adrian A; Jaffe, Eileen K; Lee, Hyung-Ok; Wang, Hao; Lai, Jack H; Bachovchin, William W; Weiner, Louis M

    2005-03-01

    Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting

  5. High consistency enzymatic saccharification of sweet sorghum bagasse pretreated with liquid hot water.

    PubMed

    Wang, Wen; Zhuang, Xinshu; Yuan, Zhenhong; Yu, Qiang; Qi, Wei; Wang, Qiong; Tan, Xuesong

    2012-03-01

    A laboratory set-up was designed to carry out high consistency enzymatic saccharification of sweet sorghum bagasse (SSB) which was pretreated by liquid hot water (LHW). The effects of two impellers on enzymatic hydrolysis of SSB were investigated. Compared with the double-curved-blade impeller (DCBI), the plate-and-frame impeller (PFI) could improve glucose production by 10%. Tween80 and fed-batch hydrolysis method adopted in this study produced total sugar of 17.06 g/L more than batch hydrolysis and raised the substrate consistency to 30%. At the final substrate loading of 30%, the concentrations of cellobiose, glucose and xylose reached to 15.01 g/L, 88.95 g/L and 9.80 g/L, respectively, and the ethanol concentration reached to 43.36 g/L in the case of cellobiose and xylose were not fermented by Saccharomyces cerevisiae Y2034. This study is an attempt at improvement of enzyme hydrolyzing LHW-pretreated material at high consistency. PMID:22281144

  6. Enzymatic Activity of Xyloglucan Xylosyltransferase 51[OPEN

    PubMed Central

    Culbertson, Alan T.; Chou, Yi-Hsiang; Smith, Adrienne L.; Young, Zachary T.; Tietze, Alesia A.; Cottaz, Sylvain

    2016-01-01

    Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a β-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher Km and 9-fold lower kcat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone. PMID:27208276

  7. Enzymatic Activity of Xyloglucan Xylosyltransferase 5.

    PubMed

    Culbertson, Alan T; Chou, Yi-Hsiang; Smith, Adrienne L; Young, Zachary T; Tietze, Alesia A; Cottaz, Sylvain; Fauré, Régis; Zabotina, Olga A

    2016-07-01

    Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a β-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher Km and 9-fold lower kcat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone. PMID:27208276

  8. Nanoparticle Mediated Remote Control of Enzymatic Activity

    PubMed Central

    Knecht, Leslie D.; Ali, Nur; Wei, Yinan; Hilt, J. Zach; Daunert, Sylvia

    2012-01-01

    Nanomaterials have found numerous applications as tunable, remotely controlled platforms for drug delivery, hyperthermia cancer treatment, and various other biomedical applications. The basis for the interest lies in their unique properties achieved at the nanoscale that can be accessed via remote stimuli. These properties could then be exploited to simultaneously activate secondary systems that are not remotely actuatable. In this work, iron oxide nanoparticles are encapsulated in a bisacrylamide-crosslinked polyacrylamide hydrogel network along with a model dehalogenase enzyme, L-2-HADST. This thermophilic enzyme is activated at elevated temperatures and has been shown to have optimal activity at 70 °C. By exposing the Fe3O4 nanoparticles to a remote stimulus, an alternating magnetic field (AMF), enhanced system heating can be achieved, thus remotely activating the enzyme. The internal heating of the nanocomposite hydrogel network in the AMF results in a 2-fold increase in enzymatic activity as compared to the same hydrogel heated externally in a water bath, suggesting that the internal heating of the nanoparticles is more efficient than the diffusion limited heating of the water bath. This system may prove useful for remote actuation of biomedical and environmentally relevant enzymes and find applications in a variety of fields. PMID:22989219

  9. Artificial cytoskeletal structures within enzymatically active bio-inorganic protocells.

    PubMed

    Kumar, Ravinash Krishna; Li, Mei; Olof, Sam N; Patil, Avinash J; Mann, Stephen

    2013-02-11

    The fabrication of enzymatically active, semi-permeable bio-inorganic protocells capable of self-assembling a cytoskeletal-like interior and undergoing small-molecule dephosphorylation reactions is described. Reversible disassembly of an amino acid-derived supramolecular hydrogel within the internalized reaction space is used to tune the enzymatic activity of the nanoparticle-bounded inorganic compartments. PMID:23027575

  10. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    NASA Astrophysics Data System (ADS)

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  11. A General Framework for Thermodynamically Consistent Parameterization and Efficient Sampling of Enzymatic Reactions

    PubMed Central

    Saa, Pedro; Nielsen, Lars K.

    2015-01-01

    Kinetic models provide the means to understand and predict the dynamic behaviour of enzymes upon different perturbations. Despite their obvious advantages, classical parameterizations require large amounts of data to fit their parameters. Particularly, enzymes displaying complex reaction and regulatory (allosteric) mechanisms require a great number of parameters and are therefore often represented by approximate formulae, thereby facilitating the fitting but ignoring many real kinetic behaviours. Here, we show that full exploration of the plausible kinetic space for any enzyme can be achieved using sampling strategies provided a thermodynamically feasible parameterization is used. To this end, we developed a General Reaction Assembly and Sampling Platform (GRASP) capable of consistently parameterizing and sampling accurate kinetic models using minimal reference data. The former integrates the generalized MWC model and the elementary reaction formalism. By formulating the appropriate thermodynamic constraints, our framework enables parameterization of any oligomeric enzyme kinetics without sacrificing complexity or using simplifying assumptions. This thermodynamically safe parameterization relies on the definition of a reference state upon which feasible parameter sets can be efficiently sampled. Uniform sampling of the kinetics space enabled dissecting enzyme catalysis and revealing the impact of thermodynamics on reaction kinetics. Our analysis distinguished three reaction elasticity regions for common biochemical reactions: a steep linear region (0> ΔGr >-2 kJ/mol), a transition region (-2> ΔGr >-20 kJ/mol) and a constant elasticity region (ΔGr <-20 kJ/mol). We also applied this framework to model more complex kinetic behaviours such as the monomeric cooperativity of the mammalian glucokinase and the ultrasensitive response of the phosphoenolpyruvate carboxylase of Escherichia coli. In both cases, our approach described appropriately not only the kinetic

  12. Pesticide influence on soil enzymatic activities.

    PubMed

    Sannino, F; Gianfreda, L

    2001-11-01

    The influence of four pesticides, e.g. glyphosate, paraquat, atrazine, and carbaryl, on the activities of invertase, urease and phosphatase of twenty-two soils, numbered as 1-22, was investigated. Soils displayed a general variability of enzyme activities with invertase being more abundant than urease and phosphatase in the order listed. The addition of glyphosate and paraquat activated invertase and urease activities in several soils. Increments of invertase activity ranged from a very low increase (+4%) up to +204% in soils 11 and 14, respectively. Smaller increases were measured for urease. A general inhibitory effect (from 5% to 98%) was observed for phosphatase in the presence of glyphosate. The effects of atrazine and carbaryl on the three soil enzymes were evaluated against that exhibited by methanol, the solvent used for their solubilization. In almost all soils, atrazine further inhibited invertase activity with respect to the inhibitory effect shown by methanol. By contrast, consistent activation effects (from 61% to 10217%) were measured for urease with methanol alone and/or methanol-pesticide mixtures. Contradictory results were observed with phosphatase. Similarities found between the results obtained with enzymes in soils and those measured with synthetic enzyme complexes (e.g. free enzymes and/or clay-, organo-, and organo-clay-enzyme complexes) exposed to the same pesticides allowed some relationships between responses of soil enzymes to pesticides and soil properties to be hypothesized. PMID:11680737

  13. Enzymatic Dissolution of Biocomposite Solids Consisting of Phosphopeptides to Form Supramolecular Hydrogels.

    PubMed

    Shi, Junfeng; Yuan, Dan; Haburcak, Richard; Zhang, Qiang; Zhao, Chao; Zhang, Xixiang; Xu, Bing

    2015-12-01

    Enzyme-catalyzed dephosphorylation is essential for biomineralization and bone metabolism. Here we report the exploration of using enzymatic reaction to transform biocomposites of phosphopeptides and calcium (or strontium) ions to supramolecular hydrogels as a mimic of enzymatic dissolution of biominerals. (31) P NMR shows that strong affinity between the phosphopeptides and alkaline metal ions (e.g., Ca(2+) or Sr(2+) ) induces the formation of biocomposites as precipitates. Electron microscopy reveals that the enzymatic reaction regulates the morphological transition from particles to nanofibers. Rheology confirms the formation of a rigid hydrogel. As the first example of enzyme-instructed dissolution of a solid to form supramolecular nanofibers/hydrogels, this work provides an approach to generate soft materials with desired properties, expands the application of supramolecular hydrogelators, and offers insights to control the demineralization of calcified soft tissues. PMID:26462722

  14. Effects of organic carbon sequestration strategies on soil enzymatic activities

    NASA Astrophysics Data System (ADS)

    Puglisi, E.; Suciu, N.; Botteri, L.; Ferrari, T.; Coppolecchia, D.; Trevisan, M.; Piccolo, A.

    2009-04-01

    Greenhouse gases emissions can be counterbalanced with proper agronomical strategies aimed at sequestering carbon in soils. These strategies must be tested not only for their ability in reducing carbon dioxide emissions, but also for their impact on soil quality: enzymatic activities are related to main soil ecological quality, and can be used as early and sensitive indicators of alteration events. Three different strategies for soil carbon sequestration were studied: minimum tillage, protection of biodegradable organic fraction by compost amendment and oxidative polimerization of soil organic matter catalyzed by biometic porfirins. All strategies were compared with a traditional agricultural management based on tillage and mineral fertilization. Experiments were carried out in three Italian soils from different pedo-climatic regions located respectively in Piacenza, Turin and Naples and cultivated with maize or wheat. Soil samples were taken for three consecutive years after harvest and analyzed for their content in phosphates, ß-glucosidase, urease and invertase. An alteration index based on these enzymatic activities levels was applied as well. The biomimetic porfirin application didn't cause changes in enzymatic activities compared to the control at any treatment or location. Enzymatic activities were generally higher in the minimum tillage and compost treatment, while differences between location and date of samplings were limited. Application of the soil alteration index based on enzymatic activities showed that soils treated with compost or subjected to minimum tillage generally have a higher biological quality. The work confirms the environmental sustainability of the carbon sequestering agronomical practices studied.

  15. Study on beta-galactosidase enzymatic activity of herbal yogurt.

    PubMed

    Chowdhury, Banani Ray; Chakraborty, Runu; Raychaudhuri, Utpal

    2008-03-01

    Different types of herbal yogurts were developed by mixing standardized milk with pretreated herbs, namely tulsi leaf (Ocimum sanctum), pudina leaf (Mentha arvensis) and coriander leaf (Coriandrum sativum), with leaves separately and a 1:1 (v/v) mixture of the strains of lactic starter cultures---Lactobacillus acidophilus (NCIM 2903) and Lactobacillus plantarum (NCIM 2083)-followed by incubation at 40 degrees C for 6 h. The beta-galactosidase enzymatic activity of the abovementioned herbal yogurts was determined and interestingly noted to exhibit higher enzymatic activity compared with the control yogurt (without any herbs). Among all herbal yogurts, tulsi yogurt had the maximum beta-galactosidase activity. PMID:17852503

  16. Enzymatic activity of rodents acclimated to cold and long scotophase

    NASA Astrophysics Data System (ADS)

    Fourie, F. Le R.; Haim, A.

    1980-09-01

    Rodents representative of a diurnal species ( Rhabdomys pumilio) as well as a nocturnal species ( Praomys natalensis) were acclimated to cold (Ta = 8°C) at a photoperiod of LD 12:12 and a long scotophase (LD 8; 16) at a temperature of 25° C(Ta). Control groups were kept for both species at Ta = 25° C and LD 12:12 and winter acclimated individuals were obtained during July and August to serve as further reference. Blood samples obtained from the tail were analysed for enzymes representative of three major biochemical pathways. The enzymatic activity of LDH (glycolytic pathway), MDH (Krebs cycle) and G6PDH (hexose monophosphate shunt, as an indicator of gonadal activity) were monitored to represent metabolic activity of the respective cycles. Cold acclimated as well as winter acclimatized mice revealed similar enzymatic patterns for both species and significant increases in LDH and MDH were recorded with a concurrent decrease in G6PDH activity. Specimens exposed to long scotophase exhibited similar enzymatic patterns for both species studied, but enzymatic activity was higher than those of cold acclimated individuals. From these results it is concluded that cold as well as long scotophase induce metabolic adaptations through biochemical activity in the experimental animals. The effect of long scotophase is assumed to be an important factor in the induction of winter acclimatization.

  17. Macrophage Migration Inhibitory Factor (MIF) Enzymatic Activity and Lung Cancer

    PubMed Central

    Mawhinney, Leona; Armstrong, Michelle E; O’ Reilly, Ciaran; Bucala, Richard; Leng, Lin; Fingerle-Rowson, Gunter; Fayne, Darren; Keane, Michael P; Tynan, Aisling; Maher, Lewena; Cooke, Gordon; Lloyd, David; Conroy, Helen; Donnelly, Seamas C

    2014-01-01

    The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif P1G). Primary tumor growth was significantly attenuated in both Mif-KO and Mif P1G mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems. PMID:25826675

  18. Trisomy 21 consistently activates the interferon response

    PubMed Central

    Sullivan, Kelly D; Lewis, Hannah C; Hill, Amanda A; Pandey, Ahwan; Jackson, Leisa P; Cabral, Joseph M; Smith, Keith P; Liggett, L Alexander; Gomez, Eliana B; Galbraith, Matthew D; DeGregori, James; Espinosa, Joaquín M

    2016-01-01

    Although it is clear that trisomy 21 causes Down syndrome, the molecular events acting downstream of the trisomy remain ill defined. Using complementary genomics analyses, we identified the interferon pathway as the major signaling cascade consistently activated by trisomy 21 in human cells. Transcriptome analysis revealed that trisomy 21 activates the interferon transcriptional response in fibroblast and lymphoblastoid cell lines, as well as circulating monocytes and T cells. Trisomy 21 cells show increased induction of interferon-stimulated genes and decreased expression of ribosomal proteins and translation factors. An shRNA screen determined that the interferon-activated kinases JAK1 and TYK2 suppress proliferation of trisomy 21 fibroblasts, and this defect is rescued by pharmacological JAK inhibition. Therefore, we propose that interferon activation, likely via increased gene dosage of the four interferon receptors encoded on chromosome 21, contributes to many of the clinical impacts of trisomy 21, and that interferon antagonists could have therapeutic benefits. DOI: http://dx.doi.org/10.7554/eLife.16220.001 PMID:27472900

  19. Enzymatic activation of a matrix metalloproteinase inhibitor†

    PubMed Central

    Major Jourden, Jody L.; Cohen, Seth M.

    2010-01-01

    Matrix metalloproteinase inhibitors (MMPi) possessing a glucose protecting group on the zinc-binding group (ZBG) show a dramatic increase in inhibitory activity upon cleavage by β-glucosidase. PMID:20449263

  20. Bioorthogonal Enzymatic Activation of Caged Compounds.

    PubMed

    Ritter, Cornelia; Nett, Nathalie; Acevedo-Rocha, Carlos G; Lonsdale, Richard; Kräling, Katja; Dempwolff, Felix; Hoebenreich, Sabrina; Graumann, Peter L; Reetz, Manfred T; Meggers, Eric

    2015-11-01

    Engineered cytochrome P450 monooxygenase variants are reported as highly active and selective catalysts for the bioorthogonal uncaging of propargylic and benzylic ether protected substrates, including uncaging in living E. coli. observed selectivity is supported by induced-fit docking and molecular dynamics simulations. This proof-of-principle study points towards the utility of bioorthogonal enzyme/protecting group pairs for applications in the life sciences. PMID:26356324

  1. Members of the Chloride Intracellular Ion Channel Protein Family Demonstrate Glutaredoxin-Like Enzymatic Activity

    PubMed Central

    Al Khamici, Heba; Brown, Louise J.; Hossain, Khondker R.; Hudson, Amanda L.; Sinclair-Burton, Alxcia A.; Ng, Jane Phui Mun; Daniel, Elizabeth L.; Hare, Joanna E.; Cornell, Bruce A.; Curmi, Paul M. G.; Davey, Mary W.; Valenzuela, Stella M.

    2015-01-01

    The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function. PMID:25581026

  2. PARP1 Val762Ala polymorphism reduces enzymatic activity

    SciTech Connect

    Wang Xiaogan; Wang Zhaoqi; Tong Weimin . E-mail: tong@iarc.fr; Shen Yan

    2007-03-02

    Poly(ADP-ribose) polymerase 1 (PARP1) modifies a variety of nuclear proteins by poly(ADP-ribosyl)ation, and plays diverse roles in molecular and cellular processes. A common PARP1 single nucleotide polymorphism (SNP) at codon 762, resulting in the substitution of alanine (Ala) for valine (Val) in the catalytic domain has been implicated in susceptibility to cancer. To characterize the functional effect of this polymorphism on PARP1, we performed in vitro enzymatic analysis on PARP1-Ala762 and PARP1-Val762. We found that PARP1-Ala762 displayed 57.2% of the activity of PARP1-Val762 for auto-poly(ADP-ribosyl)ation and 61.9% of the activity of PARP1-Val762 for trans-poly(ADP-ribosyl)ation of histone H1. The kinetic characterization revealed that the K {sub m} of PARP1-Ala762 was increased to a 1.2-fold of the K {sub m} of PARP1-Val762 for trans-poly(ADP-ribosyl)ation. Thus, the PARP1 Val762Ala polymorphism reduces the enzymatic activity of PARP1 by increasing K {sub m}. This finding suggests that different levels of poly(ADP-ribosyl)ation by PARP1 might aid in understanding Cancer risk of carriers of the PARP1 Val762Ala polymorphism.

  3. Enzymatic activity preservation through entrapment within degradable hydrogel networks

    NASA Astrophysics Data System (ADS)

    Mariani, Angela Marie

    This dissertation aimed to design and develop a "biogel;" a reproducible, abiotic, and biocompatible polymer hydrogel matrix, that prolongs enzymatic stability allowing for rapid production of biomolecules. The researched entrapment method preserves enzyme activity within an amicable environment while resisting activity reduction in the presence of increased pH environmental challenges. These biogels can be used in a number of applications including repeated production of small molecules and in biosensors. Five main objectives were accomplished: 1) Biogels capable of maintaining enzymatic functionality post-entrapment procedures were fabricated; 2) Biogel activity dependence on crosslinker type and crosslink density was determined; 3) Biogel composition effects on sustained activity after storage were compared; 4) Biogel activity dependence on charged monomer moieties was evaluated, and 5) Combined optimization knowledge gained from the first four objectives was utilized to determine the protection of enzymes within hydrogels when challenged with an increased pH above 8. Biogels were fabricated by entrapping β-galactosidase (lactase) enzyme within acrylamide (ACR) gels crosslinked with poly(ethylene glycol) diacrylate (PEGDA, degradable through hydrolysis) or N,N'-methylenebisacrylamide (BIS, non-degradable). Initial hydrogel entrapment reduced activity to 40% in ACR/PEGDA gels, compared to a 75% reduction in initial activity of ACR/BIS biogels. Once entrapped, these enzymes resist activity reduction in the presence of environmental challenges, such as altering the pH from 7 to above 8. When biogels were challenged at a pH of 8, activity retention positively correlated to PEGDA crosslinker density; increasing from 48% to 91% retention in 30 to 40 mole % PEGDA biogels as compared to solution based control which retained only 23%. Retention of activity when perturbed from pH 7 is advantageous for biogel applications including the repeated production of desired small

  4. Controlling the enzymatic activity of a restriction enzyme by light

    PubMed Central

    Schierling, Benno; Noël, Ann-Josée; Wende, Wolfgang; Hien, Le Thi; Volkov, Eugeny; Kubareva, Elena; Oretskaya, Tatiana; Kokkinidis, Michael; Römpp, Andreas; Spengler, Bernhard; Pingoud, Alfred

    2010-01-01

    For many applications it would be desirable to be able to control the activity of proteins by using an external signal. In the present study, we have explored the possibility of modulating the activity of a restriction enzyme with light. By cross-linking two suitably located cysteine residues with a bifunctional azobenzene derivative, which can adopt a cis- or trans-configuration when illuminated by UV or blue light, respectively, enzymatic activity can be controlled in a reversible manner. To determine which residues when cross-linked show the largest “photoswitch effect,” i.e., difference in activity when illuminated with UV vs. blue light, > 30 variants of a single-chain version of the restriction endonuclease PvuII were produced, modified with azobenzene, and tested for DNA cleavage activity. In general, introducing single cross-links in the enzyme leads to only small effects, whereas with multiple cross-links and additional mutations larger effects are observed. Some of the modified variants, which carry the cross-links close to the catalytic center, can be modulated in their DNA cleavage activity by a factor of up to 16 by illumination with UV (azobenzene in cis) and blue light (azobenzene in trans), respectively. The change in activity is achieved in seconds, is fully reversible, and, in the case analyzed, is due to a change in V max rather than K m. PMID:20080559

  5. Physisorption of enzymatically active chymotrypsin on titania colloidal particles.

    PubMed

    Derr, Ludmilla; Dringen, Ralf; Treccani, Laura; Hildebrand, Nils; Ciacchi, Lucio Colombi; Rezwan, Kurosch

    2015-10-01

    In this study we use a straightforward experimental method to probe the presence and activity of the proteolytic enzyme α-chymotrypsin adsorbed on titania colloidal particles. We show that the adsorption of α-chymotrypsin on the particles is irreversible and pH-dependent. At pH 8 the amount of adsorbed chymotrypsin is threefold higher compared to the adsorption at pH 5. However, we observe that the adsorption is accompanied by a substantial loss of enzymatic activity, and only around 6-9% of the initial enzyme activity is retained. A Michaelis-Menten kinetics analysis of both unbound and TiO2-bound chymotrypsin shows that the K(M) value is increased from ∼10 μM for free chymotrypsin to ∼40 μM for the particle bound enzyme. Such activity decrease could be related by the hindered accessibility of substrate to the active site of adsorbed chymotrypsin, or by adsorption-induced structural changes. Our simple experimental method does not require any complex technical equipment, can be applied to a broad range of hydrolytic enzymes and to various types of colloidal materials. Our approach allows an easy, fast and reliable determination of particle surface-bound enzyme activity and has high potential for development of future enzyme-based biotechnological and industrial processes. PMID:26072448

  6. Activation of enzymatic chitin degradation by a lytic polysaccharide monooxygenase.

    PubMed

    Hamre, Anne Grethe; Eide, Kristine B; Wold, Hanne H; Sørlie, Morten

    2015-04-30

    For decades, the enzymatic conversion of recalcitrant polysaccharides such as cellulose and chitin was thought to solely rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. Here, we have examined the initial rate enhancement an LPMO (CBP21) has on the hydrolytic enzymes (ChiA, ChiB, and ChiC) of the chitinolytic machinery of Serratia marcescens through determinations of apparent k(cat) (k(cat)(app)) values on a β-chitin substrate. k(cat)(app) values were determined to be 1.7±0.1 s(-1) and 1.7±0.1 s(-1) for the exo-active ChiA and ChiB, respectively and 1.2±0.1 s(-1) for the endo-active ChiC. The addition of CBP21 boosted the k(cat)(app) values of ChiA and ChiB giving values of 11.1±1.5 s(-1) and 13.9±1.4 s(-1), while there was no effect on ChiC (0.9±0.1 s(-1)). PMID:25812992

  7. Cue Consistency Associated with Physical Activity Automaticity and Behavior.

    PubMed

    Pimm, Rosemary; Vandelanotte, Corneel; Rhodes, Ryan E; Short, Camille; Duncan, Mitch J; Rebar, Amanda L

    2016-01-01

    Physical activity is partly regulated by automatic processes such as habits (ie, well-learned responses to cues), but it remains unclear what cues trigger these processes. This study examined the relations of physical activity automaticity and behavior with the consistency of people, activity, routine, location, time, and mood cues present upon initiation of physical activity behavior. Australian adults (N = 1,244, 627 female, M age = 55 years) reported their physical activity automaticity, behavior, and the degree of consistency of these cues each time they start a physical activity behavior. Multiple regression models, which accounted for gender and age, revealed that more consistent routine and mood cues were linked to more physical activity automaticity; whereas more consistent time and people cues were linked to more physical activity behavior. Interventions may more effectively translate into long-lasting physical activity habits if they draw people's attention to the salient cues of time, people, routine, and mood. PMID:25864705

  8. Comparison of lab, pilot, and industrial scale low consistency mechanical refining for improvements in enzymatic digestibility of pretreated hardwood.

    PubMed

    Jones, Brandon W; Venditti, Richard; Park, Sunkyu; Jameel, Hasan

    2014-09-01

    Mechanical refining has been shown to improve biomass enzymatic digestibility. In this study industrial high-yield sodium carbonate hardwood pulp was subjected to lab, pilot and industrial refining to determine if the mechanical refining improves the enzymatic hydrolysis sugar conversion efficiency differently at different refining scales. Lab, pilot and industrial refining increased the biomass digestibility for lignocellulosic biomass relative to the unrefined material. The sugar conversion was increased from 36% to 65% at 5 FPU/g of biomass with industrial refining at 67.0 kWh/t, which was more energy efficient than lab and pilot scale refining. There is a maximum in the sugar conversion with respect to the amount of refining energy. Water retention value is a good predictor of improvements in sugar conversion for a given fiber source and composition. Improvements in biomass digestibility with refining due to lab, pilot plant and industrial refining were similar with respect to water retention value. PMID:25016156

  9. Chemical interaction of disulfiram with nitrosodimethylamine after in vitro enzymatic activation

    SciTech Connect

    Tacchi, A.M.; Bertram, B.; Wiessler, M.

    1984-02-01

    The in vitro reaction between disulfiram (DSF) and N-nitroso(/sup 14/C)dimethylamine ((/sup 14/C)NDMA) was studied. Incubations of DSF with (/sup 14/C)NDMA were carried out in the presence of rat liver microsomes, control 9000 g (S9) supernatant fraction and phenobarbital-induced S9 fraction. HPLC analysis and liquid scintillation measurement provided evidence for the formation of methyldiethyldithiocarbamate (MeDDTC) as a product of the reaction between diethyldithiocarbamate (DDTC), the main active metabolite of DSF and the 'methyl-cation' released by NDMA after enzymatic activation. The amount of MeDDTC found here was consistent with the rate of oxidation of NDMA to formaldehyde. Scintillation counting confirmed that other radioactive peaks, not due to MeDDTC, were unrelated to the methylation of L-cysteine by (/sup 14/C)NDMA.

  10. Enzymatic activity inside and outside of water-stable aggregates in soils under different land use

    NASA Astrophysics Data System (ADS)

    Garbuz, S. A.; Yaroslavtseva, N. V.; Kholodov, V. A.

    2016-03-01

    A method is presented for assessing the distribution of enzymatic activity inside and outside of water-stable aggregates. Two samples of water-stable aggregates >1 mm have been isolated from dry aggregates of 1-2 mm. To determine the enzymatic activity, a substrate has been added to one of the samples without disaggregation; the other sample has been preliminarily disaggregated. Enzymatic activity within waterstable aggregates has been assessed from the difference between the obtained results under the supposition that the penetration of substrate within the water-saturated aggregates is hampered, and enzymatic reactions occur only at the periphery. The levels and distributions of enzymatic (peroxidase, polyphenol oxidase, and catalase) activities in water-stable aggregates of soddy-podzolic soils under forest and plowland and typical chernozems of long-term field experiments have been studied. The peroxidase, polyphenol oxidase, and catalase activities of water-stable aggregates vary from 6 to 23, from 7 to 30, and from 5 to 7 mmol/(g h), respectively. The ratio between the enzymatic activities inside and outside of soil aggregates showed a higher dependence on soil type and land use, as well as on the input of organic matter and the structural state, than the general activity level in water-stable aggregates.

  11. Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces

    PubMed Central

    2015-01-01

    Ribonuclease A (RNase A) is a small globular enzyme that lyses RNA. The remarkable solution stability of its structure and enzymatic activity has led to its investigation to develop a new class of drugs for cancer chemotherapeutics. However, the successful clinical application of RNase A has been reported to be limited by insufficient stability and loss of enzymatic activity when it was coupled with a biomaterial carrier for drug delivery. The objective of this study was to characterize the structural stability and enzymatic activity of RNase A when it was adsorbed on different surface chemistries (represented by fused silica glass, high-density polyethylene, and poly(methyl-methacrylate)). Changes in protein structure were measured by circular dichroism, amino acid labeling with mass spectrometry, and in vitro assays of its enzymatic activity. Our results indicated that the process of adsorption caused RNase A to undergo a substantial degree of unfolding with significant differences in its adsorbed structure on each material surface. Adsorption caused RNase A to lose about 60% of its native-state enzymatic activity independent of the material on which it was adsorbed. These results indicate that the native-state structure of RNase A is greatly altered when it is adsorbed on a wide range of surface chemistries, especially at the catalytic site. Therefore, drug delivery systems must focus on retaining the native structure of RNase A in order to maintain a high level of enzymatic activity for applications such as antitumor chemotherapy. PMID:25420087

  12. Mimicking enzymatic active sites on surfaces for energy conversion chemistry.

    PubMed

    Gutzler, Rico; Stepanow, Sebastian; Grumelli, Doris; Lingenfelder, Magalí; Kern, Klaus

    2015-07-21

    Metal-organic supramolecular chemistry on surfaces has matured to a point where its underlying growth mechanisms are well understood and structures of defined coordination environments of metal atoms can be synthesized in a controlled and reproducible procedure. With surface-confined molecular self-assembly, scientists have a tool box at hand which can be used to prepare structures with desired properties, as for example a defined oxidation number and spin state of the transition metal atoms within the organic matrix. From a structural point of view, these coordination sites in the supramolecular structure resemble the catalytically active sites of metallo-enzymes, both characterized by metal centers coordinated to organic ligands. Several chemical reactions take place at these embedded metal ions in enzymes and the question arises whether these reactions also take place using metal-organic networks as catalysts. Mimicking the active site of metal atoms and organic ligands of enzymes in artificial systems is the key to understanding the selectivity and efficiency of enzymatic reactions. Their catalytic activity depends on various parameters including the charge and spin configuration in the metal ion, but also on the organic environment, which can stabilize intermediate reaction products, inhibits catalytic deactivation, and serves mostly as a transport channel for the reactants and products and therefore ensures the selectivity of the enzyme. Charge and spin on the transition metal in enzymes depend on the one hand on the specific metal element, and on the other hand on its organic coordination environment. These two parameters can carefully be adjusted in surface confined metal-organic networks, which can be synthesized by virtue of combinatorial mixing of building synthons. Different organic ligands with varying functional groups can be combined with several transition metals and spontaneously assemble into ordered networks. The catalytically active metal

  13. Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

    PubMed Central

    Li, Shan; Shi, Lianfa; Yang, Zhiyong; Zhang, Yongrong; Perez-Cordon, Gregorio; Huang, Tuxiong; Ramsey, Jeremy; Oezguen, Numan; Savidge, Tor C.

    2014-01-01

    TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (VHH) library raised against TcdB, we identified two VHH antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy. PMID:25404023

  14. GTP cyclohydrolase I expression and enzymatic activity are present in caveolae of endothelial cells

    PubMed Central

    Peterson, Timothy E.; d’Uscio, Livius V.; Cao, Sheng; Wang, Xiao-Li; Katusic, Zvonimir S.

    2009-01-01

    Tetrahydrobiopterin is an essential cofactor required for the synthesis of nitric oxide. GTP cyclohydrolase I (GTPCH I) is the rate limiting enzyme for tetrahydrobiopterin production in endothelial cells, yet little is known about the subcellular localization of this enzyme. In this study, we demonstrate that GTPCH I is localized to caveolar membrane microdomains along with caveolin-1 and endothelial nitric oxide synthase. GTPCH I activity was detected in isolated caveolar membranes from cultured endothelial cells. Confocal and electron microscopy analyses confirmed GTPCH I colocalization with caveolin-1. Consistent with in vitro studies, GTPCH I activity was evident in isolated caveolar microdomains from lung homogenates of wild-type mice. Importantly, a two-fold increase in GTPCH I activity was detected in the aortas of caveolin-1 deficient mice suggesting that caveolin-1 may be involved in the control of GTPCH I enzymatic activity. Indeed, overexpression of caveolin-1 inhibits GTPCH I activity, and tetrahydrobiopterin biosynthesis is activated by disruption of caveolae structure. These studies demonstrate that GTPCH I is targeted to caveolae microdomains in vascular endothelial cells and tetrahydrobiopterin production occurs in close proximity to endothelial nitric oxide synthase. Additionally, our findings provide new insights into the regulation of GTPCH I activity by the caveolar coat protein, caveolin-1. PMID:19104007

  15. Controlling enzymatic activity and kinetics in swollen mesophases by physical nano-confinement

    NASA Astrophysics Data System (ADS)

    Sun, Wenjie; Vallooran, Jijo J.; Zabara, Alexandru; Mezzenga, Raffaele

    2014-05-01

    Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them into a highly confined environment. We show that the enzymatic activity of a model enzyme, horseradish peroxidase (HRP), can be accurately controlled by relaxing its confinement within the cubic phases' water channels, when the aqueous channel diameters are systematically swollen with varying amount of hydration-enhancing sugar ester. The in-meso activity and kinetics of HRP are then systematically investigated by UV-vis spectroscopy, as a function of the size of the aqueous mesophase channels. The enzymatic activity of HRP increases with the swelling of the water channels. In swollen mesophases with water channel diameter larger than the HRP size, the enzymatic activity is more than double that measured in standard mesophases, approaching again the enzymatic activity of free HRP in bulk water. We also show that the physically-entrapped enzymes in the mesophases exhibit a restricted-diffusion-induced initial lag period and report the first observation of in-meso enzymatic kinetics significantly deviating from the normal Michaelis-Menten behaviour observed in free solutions, with deviations vanishing when enzyme confinement is released by swelling the mesophase.Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them

  16. A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

    PubMed Central

    Góra, Monika; Buckle, Ashley M.; Porebski, Benjamin T.; Kemp, E. Helen; Sutton, Brian J.; Czarnocka, Barbara; Banga, J. Paul

    2014-01-01

    simulations were consistent with these observations. Conclusions: Our results point to a redundant role for the propeptide sequence in TPO. The successful expression of TPOΔpro in a membrane-anchored, enzymatically active form that is insensitive to intramolecular proteolysis, and importantly is recognized by patients' autoantibodies, is a key advance for purification of substantial quantities of homogeneous preparation of TPO for crystallization, structural, and immunological studies. PMID:23668778

  17. Enzymatic assay for calmodulins based on plant NAD kinase activity

    SciTech Connect

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  18. Research of enzymatic activities of fresh juice and water infusions from dry herbs.

    PubMed

    Chudnicka, Alina; Matysik, Grazyna

    2005-06-01

    Research was done on the presence of enzymes in juice obtained from fresh plant material from Chamomilla recutita L. (Rauschel)-anthodium, Lamium album L.-flos, Calendula officinalis L.-flos, Plantaginis lanceolata L.-folium and Euphrasiae rostkoviana Hayne-herba, and in the prepared water infusion of these materials; the objective was to determine the activity of enzymes which beside biologically active substances may have an influence of the final therapeutic effect of the applied plant preparations. The research was conducted by means of the API ZYM system (bioMerieux). Higher enzymatic activities were found in fresh juices of the examined plant material than in prepared water infusions from dried plants. In both cases naphthol-AS-BI-phosphohydrolase should have highest activity. The second one in terms of activity out of 17 studied enzymes was acidic phosphatase. The highest enzymatic activity of fresh juice was found in Lamii albi flos and Calendulae officinalis flos. Water infusions showed the highest enzymatic activity in Lamii albi flos, Chamomille recutita anthodium and Plantaginis lanceolata folium. Drying the plant material resulted in decreased enzymatic activities but not in the case of naphthol-AS-BI-phosphohydrolase and acidic phosphatase which showed very low activities. The complex composition of plant materials in terms of content of biologically active substances may imply that the therapeutic effect might be directly related to the quantity and activity of plant enzymes present in preparations applied in therapeutics. PMID:15894139

  19. Biologically Active Oxylipins from Enzymatic and Nonenzymatic Routes in Macroalgae

    PubMed Central

    Barbosa, Mariana; Valentão, Patrícia; Andrade, Paula B.

    2016-01-01

    Marine algae are rich and heterogeneous sources of great chemical diversity, among which oxylipins are a well-recognized class of natural products. Algal oxylipins comprise an assortment of oxygenated, halogenated, and unsaturated functional groups and also several carbocycles, varying in ring size and position in lipid chain. Besides the discovery of structurally diverse oxylipins in macroalgae, research has recently deciphered the role of some of these metabolites in the defense and innate immunity of photosynthetic marine organisms. This review is an attempt to comprehensively cover the available literature on the chemistry, biosynthesis, ecology, and potential bioactivity of oxylipins from marine macroalgae. For a better understanding, enzymatic and nonenzymatic routes were separated; however, both processes often occur concomitantly and may influence each other, even producing structurally related molecules. PMID:26805855

  20. Different Quaternary Structures of Human RECQ1 Are Associated with Its Dual Enzymatic Activity

    PubMed Central

    Muzzolini, Laura; Beuron, Fabienne; Patwardhan, Ardan; Popuri, Venkateswarlu; Cui, Sheng; Niccolini, Benedetta; Rappas, Mathieu; Freemont, Paul S; Vindigni, Alessandro

    2007-01-01

    RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPγS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 Å × 130 Å with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding. PMID:17227144

  1. Enzymatic vitreolysis with recombinant tissue plasminogen activator for vitreomacular traction

    PubMed Central

    Raczyńska, Dorota; Lipowski, Paweł; Zorena, Katarzyna; Skorek, Andrzej; Glasner, Paulina

    2015-01-01

    Aims The aim of our research was to gain data about the efficacy of intravitreal injections of a recombinant tissue plasminogen activator (rTPA) in dissolving vitreoretinal tractions (VRTs). Materials and methods The study group consisted of patients of our Ophthalmology Clinic who had received an injection of rTPA (TPA Group) for an existent vitreomacular traction confirmed by optical coherence tomography and stereoscopic examinations. The control group consisted of patients who had declined treatment despite the existence of a vitreomacular traction confirmed by the same diagnostic methods. Each group consisted of 30 people (30 eyes). The observation period was 6 months. Conclusion In both groups some of the VRTs had dissolved. In the TPA group the traction dissolved in 10 patients (33.33%) and in the control group only in 5 (16.67%). It is also important to point out that the mean baseline membrane thickness was higher in the TPA group than in the control group. Observing patients in both groups we noticed that the dissolution of vitreoretinal membrane occurred most frequently in those cases where the membrane was thin. In the TPA group, the mean membrane thickness after 6 months decreased considerably. At the same time, no significant change in the membrane thickness could be observed in the control group. Observation of the retinal thickness allows us to draw the following conclusion: in the TPA group, the retinal thickness in the macular area (edema) had decreased over the study period, whereas in the control group it had increased. In those cases where the traction had dissolved, the edema of the retina decreased by the end of the 6-month period in both groups. In the TPA group, the dissolution of the membrane occurred most often within 3 months from the primary injection. Based on statistics, we can confirm that in the control group there was a decrease in visual acuity during the 6 months of the study period. At the same time, visual acuity in the TPA

  2. Thrombolytic efficacy and enzymatic activity of rt-PA-loaded echogenic liposomes.

    PubMed

    Bader, Kenneth B; Bouchoux, Guillaume; Peng, Tao; Klegerman, Melvin E; McPherson, David D; Holland, Christy K

    2015-08-01

    Echogenic liposomes (ELIP), that can encapsulate both recombinant tissue-type plasminogen activator (rt-PA) and microbubbles, are under development to improve the treatment of thrombo-occlusive disease. However, the enzymatic activity, thrombolytic efficacy, and stable cavitation activity generated by this agent has yet to be evaluated and compared to another established ultrasound-enhanced thrombolytic scheme. A spectrophotometric method was used to compare the enzymatic activity of the rt-PA incorporated into ELIP (t-ELIP) to that of rt-PA. An in vitro flow model was employed to measure the thrombolytic efficacy and dose of ultraharmonic emissions from stable cavitation for 120-kHz ultrasound exposure of three treatment schemes: rt-PA, rt-PA and the perfluorocarbon-filled microbubble Definity(®), and t-ELIP. The enzymatic activity of rt-PA incorporated into t-ELIP was 28 % that of rt-PA. Thrombolytic efficacy of t-ELIP or rt-PA and Definity(®) was equivalent when the dose of t-ELIP was adjusted to produce comparable enzymatic activity. Sustained bubble activity was nucleated from Definity but not from t-ELIP exposed to 120-kHz ultrasound. These results emphasize the advantages of encapsulating a thrombolytic and the importance of incorporating an insoluble gas required to promote sustained, stable cavitation activity. PMID:25829338

  3. Immobilization of Enzymes to Silver Island Films for Enhanced Enzymatic Activity

    PubMed Central

    Abel, Biebele; Aslan, Kadir

    2013-01-01

    Hypothesis The performance of the enzyme-based biosensors depends on the enzymatic activity and the use of an appropriate technique for immobilization of enzymes. The incorporation of silver island films (SIFs) into the enzyme-based biosensors is expected to enhance the enzymatic activity and to increase the detectability of analytes of interest. Experiments Two enzymes, β-galactosidase (β-Gal) and alkaline phosphatase (AP) were immobilized onto SIFs using the interactions of avidin-modified enzymes with (i) a monolayer of biotinylated bovine serum albumin (b-BSA) and/or (ii) a monolayer of biotinylated poly(ethylene-glycol)-amine (BEA molecular weight: 550 to 10000 Da). To confirm the effect of SIFs on enzymatic activity, two control surfaces (no silver) were also employed. Findings No enhancement in enzymatic activity for β-Gal on all SIFs was observed, which was attributed to the inhibition of β-Gal activity due to direct interactions of β-Gal with SIFs. The AP activity on SIFs with BEA was significantly larger than that observed on SIFs with b-BSA, where a 300% increase in AP activity was observed as compared to control surfaces. These observations suggest that SIFs can significantly enhance AP activity, which could help improve the detection limits of ELISAs and immunoassays that employ AP. PMID:24267340

  4. A Survey of Enzymatic Activity in Commercially Available Pool and Spa Products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many pool water treatment products currently available commercially claim that they work effectively by possessing enzyme activity (specifically lipase) that degrades common oil (lipid) contaminants found in pool water. Currently, there is no standard in measuring the enzymatic activity of these enz...

  5. A Survey of Enzymatic Activity in Commercially Available Pool and Spa Products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many pool water treatment products currently available commercially claim that they work effectively by possessing enzyme activity (specifically lipase) that degrades common oil (lipid) contaminants found in pool water. Currently, there is no standard in measuring the enzymatic activity of these en...

  6. Positive regulation of the enzymatic activity of gastric H(+),K(+)-ATPase by sialylation of its β-subunit.

    PubMed

    Fujii, Takuto; Watanabe, Midori; Shimizu, Takahiro; Takeshima, Hiroshi; Kushiro, Keiichiro; Takai, Madoka; Sakai, Hideki

    2016-06-01

    The gastric proton pump (H(+),K(+)-ATPase) consists of a catalytic α-subunit (αHK) and a glycosylated β-subunit (βHK). βHK glycosylation is essential for the apical trafficking and stability of αHK in gastric parietal cells. Here, we report the properties of sialic acids at the termini of the oligosaccharide chains of βHK. Sialylation of βHK was found in LLC-PK1 cells stably expressing αHK and βHK by staining of the cells with lectin-tagged fluorescent polymeric nanoparticles. This sialylation was also confirmed by biochemical studies using sialic acid-binding lectin beads and an anti-βHK antibody. The sialic acids of βHK are cleaved enzymatically by neuraminidase (sialidase) and nonenzymatically by an acidic solution (pH5). Interestingly, the enzymatic activity of H(+),K(+)-ATPase was significantly decreased by cleavage of the sialic acids of βHK. In contrast, βHK was not sialylated in the gastric tubulovesicles prepared from the stomach of fed hogs. The H(+),K(+)-ATPase activity in these tubulovesicles was not significantly altered by neuraminidase. Importantly, the sialylation of βHK was observed in the gastric samples prepared from the stomach of famotidine (a histamine H2 receptor antagonist)-treated rats, but not histamine (an acid secretagogue)-treated rats. The enzymatic activity of H(+),K(+)-ATPase in the samples of the famotidine-treated rats was significantly higher than in the histamine-treated rats. The effects of famotidine were weakened by neuraminidase. These results indicate that βHK is sialylated at neutral or weakly acidic pH, but not at acidic pH, suggesting that the sialic acids of βHK positively regulate the enzymatic activity of αHK. PMID:26922883

  7. Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities

    SciTech Connect

    Devash, Y.; Reichman, M.; Sela, I.; Reichenbach, N.L.; Suhadolnik, R.J.

    1985-01-29

    An enzyme that converts (/sup 3/H, /sup 32/P)ATP, with a /sup 3/H:/sup 32/P ratio of 1:1, to oligoadenylates with the same /sup 3/H:/sup 32/P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of /sup 3/H:/sup 32/P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.

  8. Enzymatic hydrolysis of rice protein with papain and antioxidation activity of hydrolysate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzymatic hydrolysis technology of rice protein and the antioxidant activity of the hydrolysate were studied. Substrate concentration,enzyme dose,pH value and temperature were selected as factors to optimize the hydrolysis parameters with single—factor and orthogonal tests. Results show the opti...

  9. Enzymatic hydrolysis of oleuropein from Olea europea (olive) leaf extract and antioxidant activities.

    PubMed

    Yuan, Jiao-Jiao; Wang, Cheng-Zhang; Ye, Jian-Zhong; Tao, Ran; Zhang, Yu-Si

    2015-01-01

    Oleuropein (OE), the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT) and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity) optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE) were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 μg/mL) was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods. PMID:25679050

  10. Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

    NASA Technical Reports Server (NTRS)

    Kerr, T. P.

    1985-01-01

    In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

  11. Effect of restricted motion in high temperature on enzymatic activity of the pancreas

    NASA Technical Reports Server (NTRS)

    Abdusattarov, A.; Smirnova, G. I.

    1980-01-01

    Effects of 30 day hypodynamia coupled with high temperature (35-36 C) on enzymatic activity of the pancreas of male adult rats were studied. The test animals were divided into four groups. Group one served as controls (freedom of movement and a temperature of 25-26 C, considered optimal). The remaining animals were divided into three additional groups: Group two freedom of movement but high temperature (35-36 C); group three hypodynamia but an optimal temperature; group four hypodynamia and 35-36 C. Considerable change in the enzymatic activity in the pancreas of the four groups is observed in three experimental groups (two, three, and four) as compared to the control (group one). The results indicate that adaption of the organism to the thermal factor and restricted movement is accompanied by a change in the enzymatic spectrum of the pancreas. With the combined effect of these two stresses under conditions of the adaption of the organism especially sharp shifts occur in the enzymatic activity.

  12. Analysis of rat cytosolic 9-cis-retinol dehydrogenase activity and enzymatic characterization of rat ADHII.

    PubMed

    Popescu, G; Napoli, J L

    2000-01-01

    We report the characterization of two enzymes that catalyze NAD(+)-dependent 9-cis-retinol dehydrogenase activity in rat liver cystol. Alcohol dehydrogenase class I (ADHI) contributes > 80% of the NA D+-dependent 9-cis-retinol dehydrogenase activity recovered, whereas alcohol dehydrogenase class II (ADHII), not identified previously at the protein level, nor characterized enzymatically in rat, accounts for approximately 2% of the activity. Rat ADHII exhibits properties different from those described for human ADHII. Moreover, rat ADHII-catalyzed rates of ethanol dehydrogenation are markedly lower than octanol or retinoid dehydrogenation rates. Neither ethanol nor 4-methylpyrazole inhibits the 9-cis-retinol dehydrogenase activity of rat ADHII. We propose that ADHII represents the previously observed additional retinoid oxidation activity of rat liver cytosol which occurred in the presence of either ethanol or 4-methylpyrazole. We also show that human and rat ADHII differ considerably in enzymatic properties. PMID:10606766

  13. Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance.

    PubMed

    Cui, Jiandong; Zhao, Yamin; Liu, Ronglin; Zhong, Cheng; Jia, Shiru

    2016-01-01

    Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to improve catalytic performance. However, activity of the most of the enzymes was declined after immobilization. Here, we develop a surfactant-activated lipase-inorganic flowerlike hybrid nanomaterials with rational design based on interfacial activation and self-assembly. The resulting surfactant-activated lipase-inorganic hybird nanoflower (activated hNF-lipase) exhibited 460% and 200% higher activity than native lipase and conventional lipase-inorganic hybird nanoflower (hNF-lipase). Furthermore, the activated hNF-lipase displayed good reusability due to its monodispersity and mechanical properties, and had excellent long-time stability. The superior catalytic performances were attributed to both the conformational modulation of surfactants and hierarchical structure of nanoflowers, which not only anchored lipases in an active form, but also decreased the enzyme-support negative interaction and mass-transfer limitations. This new biocatalytic system is promising to find widespread use in applications related to biomedicine, biosensor, and biodiesel. PMID:27297609

  14. Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance

    PubMed Central

    Cui, Jiandong; Zhao, Yamin; Liu, Ronglin; Zhong, Cheng; Jia, Shiru

    2016-01-01

    Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to improve catalytic performance. However, activity of the most of the enzymes was declined after immobilization. Here, we develop a surfactant-activated lipase-inorganic flowerlike hybrid nanomaterials with rational design based on interfacial activation and self-assembly. The resulting surfactant-activated lipase-inorganic hybird nanoflower (activated hNF-lipase) exhibited 460% and 200% higher activity than native lipase and conventional lipase-inorganic hybird nanoflower (hNF-lipase). Furthermore, the activated hNF-lipase displayed good reusability due to its monodispersity and mechanical properties, and had excellent long-time stability. The superior catalytic performances were attributed to both the conformational modulation of surfactants and hierarchical structure of nanoflowers, which not only anchored lipases in an active form, but also decreased the enzyme-support negative interaction and mass-transfer limitations. This new biocatalytic system is promising to find widespread use in applications related to biomedicine, biosensor, and biodiesel. PMID:27297609

  15. Directed enzymatic activation of 1-D DNA tiles.

    PubMed

    Garg, Sudhanshu; Chandran, Harish; Gopalkrishnan, Nikhil; LaBean, Thomas H; Reif, John

    2015-02-24

    The tile assembly model is a Turing universal model of self-assembly where a set of square shaped tiles with programmable sticky sides undergo coordinated self-assembly to form arbitrary shapes, thereby computing arbitrary functions. Activatable tiles are a theoretical extension to the Tile assembly model that enhances its robustness by protecting the sticky sides of tiles until a tile is partially incorporated into a growing assembly. In this article, we experimentally demonstrate a simplified version of the Activatable tile assembly model. In particular, we demonstrate the simultaneous assembly of protected DNA tiles where a set of inert tiles are activated via a DNA polymerase to undergo linear assembly. We then demonstrate stepwise activated assembly where a set of inert tiles are activated sequentially one after another as a result of attachment to a growing 1-D assembly. We hope that these results will pave the way for more sophisticated demonstrations of activated assemblies. PMID:25625898

  16. Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6.

    PubMed

    Sridar, Chitra; Kent, Ute M; Notley, Lisa M; Gillam, Elizabeth M J; Hollenberg, Paul F

    2002-06-01

    Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K(I) of 0.9 microM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity. PMID:12023523

  17. Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees.

    PubMed

    Molnárová, Jana; Vadkertiová, Renáta; Stratilová, Eva

    2014-07-01

    Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, β-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited β-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure. PMID:23744750

  18. Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii.

    PubMed

    Pedri, Z C; Lozano, L M S; Hermann, K L; Helm, C V; Peralta, R M; Tavares, L B B

    2015-11-01

    Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation. PMID:26675911

  19. Measuring In Vitro ATPase Activity for Enzymatic Characterization.

    PubMed

    Rule, Chelsea S; Patrick, Marcella; Sandkvist, Maria

    2016-01-01

    Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary. PMID:27584824

  20. The Influence of Encroaching Woodland on Grassland Enzymatic Activities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grass-dominated ecosystems around the world are experiencing woody plant invasion due to human land uses. Vast regions in southern Texas have been transformed from open grasslands to subtropical thorn woodlands during the past 150 yrs. The assumption is that the soil microbial activity in the remain...

  1. Soil disturbance increases soil microbial enzymatic activity in arid ecoregion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Functional diversity of the soil microbial community is commonly used in the assessment of soil health as it relates to the activity of soil microflora involved in carbon cycling. Soil microbes in different microenvironments will have varying responses to different substrates, thus catabolic fingerp...

  2. ENZYMATIC ACTIVITIES RELATED TO THE DECOMPOSITION OF CONIFEROUS LEAF LITTER

    EPA Science Inventory

    The rate of CO2 evolution at 23C and 75% moisture content for a diverse group of coniferous leaf litter samples from Oregon was measured. Significant correlations (1% level) were observed between the rate of CO2 evolution and the activity of amylase, cellulase, and xylanase (r=0....

  3. Evolution of robusta green coffee redox enzymatic activities with maturation.

    PubMed

    Montavon, Philippe; Bortlik, Karlheinz

    2004-06-01

    Oxidation reactions in coffee involve redox-sensitive polyphenols and appear to control the fragmentation of coffee storage proteins both in solution and during roasting. Coffee-specific nitrogenous flavor precursors may derive from this process. Accordingly, data converge to suggest that the redox status of the green bean before roasting might control the development of subsequent redox reactions during roasting. Consequently, we decided to identify biological events that may trigger or prevent oxidation during maturation of the coffee cherry and set the final redox status of the green bean. In a previous study, we observed that the sensitivity of green coffee to oxidative processes decreased along maturation. By using the very same samples originating from open-pollinated Robusta clones, we followed the activity of three essential redox enzymes: catalase (CAT), peroxidase (POD) and polyphenoloxidase (PPO). While CAT and POD activities increased with maturation, PPO activities decreased. Thanks to the identification of an atypical immature subclass, it appeared that CAT might be an essential factor in setting the final redox status of the green bean before the roasting event. PMID:15161235

  4. Activation of polymeric materials towards enzymatic postgrafting and cross-linking.

    PubMed

    Fatarella, E; Ciabatti, I; Cortez, J

    2012-10-10

    A methodology to activate inert polymeric materials to enzymatic functionalisation is described herein. Plasma irradiation can be used to graft compounds containing a moiety that is reactive towards an enzyme of interest. Subsequently, such enzyme can be used to either postgraft functional compounds or cross-link the polymeric materials. Argon plasma was utilised to graft 2-aminoethyl methacrylate onto cotton and wool fibres, introducing surface alkylamine groups to impart reactivity towards transglutaminase and tyrosinase. The efficiency of plasma grafting was verified by ATR-FTIR. Enzyme postgrafting of fluorescent peptides coupled with confocal microscopy was used to demonstrate transglutaminase activity towards cotton, a material typically inert to this enzyme. The grafting of alkylamines onto wool resulted in additional cross-linking by both enzymes, leading to significantly increased yarn breaking load and elongation at break. This technology permits the activation of inert materials towards enzymatic postgrafting, with applications in fields as diverse as textiles and biomaterials. PMID:22975121

  5. Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

    PubMed Central

    Guerra, Nelson P.; Pastrana Castro, Lorenzo

    2012-01-01

    The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, vmax decreased significantly (P < 0.05) and KM increased (although not always significantly) with the increase in t. The conformational changes produced in the starch chains as a consequence of the ageing seemed to affect negatively the diffusivity of the starch to the active site of the enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G]. PMID:22666116

  6. Immobilization of inorganic pyrophosphatase on nanodiamond particles retaining its high enzymatic activity.

    PubMed

    Rodina, Elena V; Valueva, Anastasiya V; Yakovlev, Ruslan Yu; Vorobyeva, Nataliya N; Kulakova, Inna I; Lisichkin, Georgy V; Leonidov, Nikolay B

    2015-01-01

    Nanodiamond (ND) particles are popular platforms for the immobilization of molecular species. In the present research, enzyme Escherichia coli inorganic pyrophosphatase (PPase) was immobilized on detonation ND through covalent or noncovalent bonding and its enzymatic activity was characterized. Factors affecting adsorption of PPase such as ND size and surface chemistry were studied. The obtained material is a submicron size association of ND particles and protein molecules in approximately equal amounts. Both covalently and noncovalently immobilized PPase retains a significant enzymatic activity (up to 95% of its soluble form) as well as thermostability. The obtained hybrid material has a very high enzyme loading capacity (∼1 mg mg(-1)) and may be considered as a promising delivery system of biologically active proteinaceous substances, particularly in the treatment of diseases such as calcium pyrophosphate crystal deposition disease and related pathologies. They can also be used as recoverable heterogeneous catalysts in the traditional uses of PPase. PMID:26489420

  7. Enzymatic activity in the presence of surfactants commonly used in dissolution media, Part 1: Pepsin.

    PubMed

    Guzman, Maria L; Marques, Margareth R; Olivera Me, Maria E; Stippler, Erika S

    2016-01-01

    The United States Pharmacopeia (USP) General Chapters Dissolution 〈711〉 and Disintegration and Dissolution of Dietary Supplements 〈2040〉 allows the use of enzymes in dissolution media when gelatin capsules do not conform to dissolution specifications due to cross linking. Possible interactions between enzymes and surfactants when used together in dissolution media could result in loss of the enzymatic activity. Pepsin is an enzyme commonly used in dissolution media, and in this work, the activity of pepsin was determined in the presence of different surfactants as usually found in case of dissolution tests of certain gelatin capsule formulations. Pepsin enzymatic activity was determined according to the Ninth Edition of the Food Chemicals Codex (FCC) 9 method, in dissolution conditions: simulated gastric fluid, 37 °C and 50 rpm. Sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), polysorbate 80 (Tween 80) and octoxynol 9 (Triton X100) in concentrations above and below their critical micellar concentrations were selected. Results showed a significant reduction in the activity of pepsin at all the concentrations of SDS assayed. On the contrary, CTAB, Tween 80, and Triton X100 did not alter the enzymatic activity at of pepsin any of the concentration assayed. This data demonstrates a rational selection of the surfactant to be used when pepsin is required in dissolution test. PMID:27047734

  8. Enzymatic activity in the presence of surfactants commonly used in dissolution media, Part 1: Pepsin

    PubMed Central

    Guzman, Maria L; Marques, Margareth R; Olivera ME, Maria E; Stippler, Erika S

    2016-01-01

    The United States Pharmacopeia (USP) General Chapters Dissolution 〈711〉 and Disintegration and Dissolution of Dietary Supplements 〈2040〉 allows the use of enzymes in dissolution media when gelatin capsules do not conform to dissolution specifications due to cross linking. Possible interactions between enzymes and surfactants when used together in dissolution media could result in loss of the enzymatic activity. Pepsin is an enzyme commonly used in dissolution media, and in this work, the activity of pepsin was determined in the presence of different surfactants as usually found in case of dissolution tests of certain gelatin capsule formulations. Pepsin enzymatic activity was determined according to the Ninth Edition of the Food Chemicals Codex (FCC) 9 method, in dissolution conditions: simulated gastric fluid, 37 °C and 50 rpm. Sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), polysorbate 80 (Tween 80) and octoxynol 9 (Triton X100) in concentrations above and below their critical micellar concentrations were selected. Results showed a significant reduction in the activity of pepsin at all the concentrations of SDS assayed. On the contrary, CTAB, Tween 80, and Triton X100 did not alter the enzymatic activity at of pepsin any of the concentration assayed. This data demonstrates a rational selection of the surfactant to be used when pepsin is required in dissolution test. PMID:27047734

  9. Enzymatically Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry

    PubMed Central

    2015-01-01

    Amyloid fibrils represent a generic class of protein structure associated with both pathological states and with naturally occurring functional materials. This class of protein nanostructure has recently also emerged as an excellent foundation for sophisticated functional biocompatible materials including scaffolds and carriers for biologically active molecules. Protein-based materials offer the potential advantage that additional functions can be directly incorporated via gene fusion producing a single chimeric polypeptide that will both self-assemble and display the desired activity. To succeed, a chimeric protein system must self-assemble without the need for harsh triggering conditions which would damage the appended functional protein molecule. However, the micrometer to nanoscale patterning and morphological control of protein-based nanomaterials has remained challenging. This study demonstrates a general approach for overcoming these limitations through the microfluidic generation of enzymatically active microgels that are stabilized by amyloid nanofibrils. The use of scaffolds formed from biomaterials that self-assemble under mild conditions enables the formation of catalytic microgels while maintaining the integrity of the encapsulated enzyme. The enzymatically active microgel particles show robust material properties and their porous architecture allows diffusion in and out of reactants and products. In combination with microfluidic droplet trapping approaches, enzymatically active microgels illustrate the potential of self-assembling materials for enzyme immobilization and recycling, and for biological flow-chemistry. These design principles can be adopted to create countless other bioactive amyloid-based materials with diverse functions. PMID:26030507

  10. Fungal cellulase is an elicitor but its enzymatic activity is not required for its elicitor activity.

    PubMed

    Ma, Yanan; Han, Chao; Chen, Jinyin; Li, Haiyun; He, Kun; Liu, Aixin; Li, Duochuan

    2015-01-01

    Plant-pathogenic fungi produce cellulases. However, little information is available on cellulase as an elicitor in plant-pathogen interactions. Here, an endocellulase (EG1) was isolated from Rhizoctonia solani. It contains a putative protein of 227 amino acids with a signal peptide and a family-45 glycosyl hydrolase domain. Its aspartic acid (Asp) residue at position 32 was changed to alanine (Ala), resulting in full loss of its catalytic activity. Wild-type and mutated forms of the endoglucanase were expressed in yeast and purified to homogeneity. The purified wild-type and mutant forms induced cell death in maize, tobacco and Arabidopsis leaves, and the transcription of three defence marker genes in maize and tobacco and 10 genes related to defence responses in maize. Moreover, they also induced the accumulation of reactive oxygen species (ROS), medium alkalinization, Ca(2+) accumulation and ethylene biosynthesis of suspension-cultured tobacco cells. Similarly, production of the EG1 wild-type and mutated forms in tobacco induced cell death using the Potato virus X (PVX) expression system. In vivo, expression of EG1 was also related to cell death during infection of maize by R. solani. These results provide direct evidence that the endoglucanase is an elicitor, but its enzymatic activity is not required for its elicitor activity. PMID:24844544

  11. Local modulation of steroid action: rapid control of enzymatic activity

    PubMed Central

    Charlier, Thierry D.; Cornil, Charlotte A.; Patte-Mensah, Christine; Meyer, Laurence; Mensah-Nyagan, A. Guy; Balthazart, Jacques

    2015-01-01

    Estrogens can induce rapid, short-lived physiological and behavioral responses, in addition to their slow, but long-term, effects at the transcriptional level. To be functionally relevant, these effects should be associated with rapid modulations of estrogens concentrations. 17β-estradiol is synthesized by the enzyme aromatase, using testosterone as a substrate, but can also be degraded into catechol-estrogens via hydroxylation by the same enzyme, leading to an increase or decrease in estrogens concentration, respectively. The first evidence that aromatase activity (AA) can be rapidly modulated came from experiments performed in Japanese quail hypothalamus homogenates. This rapid modulation is triggered by calcium-dependent phosphorylations and was confirmed in other tissues and species. The mechanisms controlling the phosphorylation status, the targeted amino acid residues and the reversibility seem to vary depending of the tissues and is discussed in this review. We currently do not know whether the phosphorylation of the same amino acid affects both aromatase and/or hydroxylase activities or whether these residues are different. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues. PMID:25852459

  12. Digestive enzymatic activity during ontogenetic development in zebrafish (Danio rerio).

    PubMed

    Guerrera, Maria Cristina; De Pasquale, Francesca; Muglia, Ugo; Caruso, Gabriella

    2015-12-01

    Despite the growing importance of zebrafish (Danio rerio) as an experimental model in biomedical research, some aspect of physiological and related morphological age dependent changes in digestive system during larval development are still unknown. In this paper, a biochemical and morphological study of the digestive tract of zebrafish was undertaken to record the functional changes occurring in this species during its ontogenetic development, particularly from 24 hr to 47 days post fertilization (dpf). Endo- and exo-proteases, as well as α-amylase enzymes, were quantified in zebrafish larvae before first feeding (7 dpf). The most morphologically significant events during the ontogenesis of the gut occurred between 3 dpf (mouth opening) and 7 dpf (end of exocrine pancreas differentiation). The presence of a wide range of digestive enzymes, already active at earlier zebrafish larval stages, closely related with the omnivorous diet of this species. Increasing enzyme activities were found with increasing age, probably in relation with intestinal mucosa folding and consequent absorption surface increase. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 699-706, 2015. © 2015 Wiley Periodicals, Inc. PMID:26477613

  13. Enzymatically active biomimetic micropropellers for the penetration of mucin gels

    PubMed Central

    Walker, Debora; Käsdorf, Benjamin T.; Jeong, Hyeon-Ho; Lieleg, Oliver; Fischer, Peer

    2015-01-01

    In the body, mucus provides an important defense mechanism by limiting the penetration of pathogens. It is therefore also a major obstacle for the efficient delivery of particle-based drug carriers. The acidic stomach lining in particular is difficult to overcome because mucin glycoproteins form viscoelastic gels under acidic conditions. The bacterium Helicobacter pylori has developed a strategy to overcome the mucus barrier by producing the enzyme urease, which locally raises the pH and consequently liquefies the mucus. This allows the bacteria to swim through mucus and to reach the epithelial surface. We present an artificial system of reactive magnetic micropropellers that mimic this strategy to move through gastric mucin gels by making use of surface-immobilized urease. The results demonstrate the validity of this biomimetic approach to penetrate biological gels, and show that externally propelled microstructures can actively and reversibly manipulate the physical state of their surroundings, suggesting that such particles could potentially penetrate native mucus. PMID:26824056

  14. Protein Conformational Gating of Enzymatic Activity in Xanthine Oxidoreductase

    SciTech Connect

    Ishikita, Hiroshi; Eger, Bryan T.; Okamoto, Ken; Nishino, Takeshi; Pai, Emil F.

    2012-05-24

    In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E{sub sq/hq}) is {approx}170 mV, a striking difference. The former greatly prefers NAD{sup +} as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD{sup +}), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 {angstrom} resolution crystal structures for XDH, XO, the NAD{sup +}- and NADH-complexed XDH, E{sub sq/hq} were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E{sub sq/hq} difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD{sup +} binding at XDH. Instead, the positive charge of the NAD{sup +} ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD{sup +} molecule all contribute to altering E{sub sq/hq} upon NAD{sup +} binding to XDH.

  15. Microbial and enzymatic activity of soil contaminated with azoxystrobin.

    PubMed

    Baćmaga, Małgorzata; Kucharski, Jan; Wyszkowska, Jadwiga

    2015-10-01

    The use of fungicides in crop protection still effectively eliminates fungal pathogens of plants. However, fungicides may dissipate to various elements of the environment and cause irreversible changes. Considering this problem, the aim of the presented study was to evaluate changes in soil biological activity in response to contamination with azoxystrobin. The study was carried out in the laboratory on samples of sandy loam with a pH of 7.0 in 1 Mol KCl dm(-3). Soil samples were treated with azoxystrobin in one of four doses: 0.075 (dose recommended by the manufacturer), 2.250, 11.25 and 22.50 mg kg(-1) soil DM (dry matter of soil). The control soil sample did not contain fungicide. Bacteria were identified based on 16S rRNA gene sequencing, and fungi were identified by internal transcribed spacer (ITS) region sequencing. The study revealed that increased doses of azoxystrobin inhibited the growth of organotrophic bacteria, actinomycetes and fungi. The fungicide also caused changes in microbial biodiversity. The lowest values of the colony development (CD) index were recorded for fungi and the ecophysiological (EP) index for organotrophic bacteria. Azoxystrobin had an inhibitory effect on the activity of dehydrogenases, catalase, urease, acid phosphatase and alkaline phosphatase. Dehydrogenases were found to be most resistant to the effects of the fungicide, while alkaline phosphatase in the soil recovered the balance in the shortest time. Four species of bacteria from the genus Bacillus and two species of fungi from the genus Aphanoascus were isolated from the soil contaminated with the highest dose of azoxystrobin (22.50 mg kg(-1)). PMID:26343782

  16. Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells

    PubMed Central

    Çağlayan, Melike; Horton, Julie K.; Wilson, Samuel H.

    2016-01-01

    We previously reported enzymatic activity assays for the base excision repair (BER) enzymes DNA polymerase β (pol β), aprataxin (APTX), and flap endonuclease 1 (FEN1) in cell extracts from Saccharomyces cerevisiae (Çağlayan and Wilson, 2014). Here, we describe a method to prepare cell extracts from vertebrate cells to investigate these enzymatic activities for the processing of the 5′-adenylated-sugar phosphate-containing BER intermediate. This new protocol complements our previous publication. The cell lines used are wild-type and APTX-deficient human lymphoblast cells from an Ataxia with Oculomotor Apraxia Type 1 (AOA1) disease patient, wild-type and APTX-null DT40 chicken B cells, and mouse embryonic fibroblast (MEF) cells. This protocol is a quick and efficient way to make vertebrate cell extracts without using commercial kits. PMID:27390764

  17. Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

    NASA Astrophysics Data System (ADS)

    Langer, Andreas; Schräml, Michael; Strasser, Ralf; Daub, Herwin; Myers, Thomas; Heindl, Dieter; Rant, Ulrich

    2015-07-01

    The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

  18. Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures.

    PubMed

    Souza, Débora Guerini; Bellaver, Bruna; Hansel, Gisele; Arús, Bernardo Assein; Bellaver, Gabriela; Longoni, Aline; Kolling, Janaina; Wyse, Angela T S; Souza, Diogo Onofre; Quincozes-Santos, André

    2016-07-01

    Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na(+)/K(+)-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate-glutamine cycle, as well as glutamate and D-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems. PMID:26915106

  19. The subunit composition of human extracellular superoxide dismutase (EC-SOD) regulate enzymatic activity

    PubMed Central

    Petersen, Steen V; Valnickova, Zuzana; Oury, Tim D; Crapo, James D; Chr Nielsen, Niels; Enghild, Jan J

    2007-01-01

    Background Human extracellular superoxide dismutase (EC-SOD) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. We have previously shown that the EC-SOD subunit exists in two distinct folding variants based on differences in the disulfide bridge pattern (Petersen SV, Oury TD, Valnickova Z, Thøgersen IB, Højrup P, Crapo JD, Enghild JJ. Proc Natl Acad Sci USA. 2003;100(24):13875–80). One variant is enzymatically active (aEC-SOD) while the other is inactive (iEC-SOD). The EC-SOD subunits are associated into covalently linked dimers through an inter-subunit disulfide bridge creating the theoretical possibility of 3 dimers (aa, ai or ii) with different antioxidant potentials. We have analyzed the quaternary structure of the endogenous EC-SOD disulfide-linked dimer to investigate if these dimers in fact exist. Results The analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (aa and ii) and a hetero-dimer (ai). Because EC-SOD is a tetramer the dimers may combine to generate 5 different mature EC-SOD molecules where the specific activity of each molecule is determined by the ratio of aEC-SOD and iEC-SOD subunits. Conclusion This finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity. This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity. PMID:17937792

  20. Acetylation of MnSOD directs enzymatic activity responding to cellular nutrient status or oxidative stress.

    PubMed

    Ozden, Ozkan; Park, Seong-Hoon; Kim, Hyun-Seok; Jiang, Haiyan; Coleman, Mitchell C; Spitz, Douglas R; Gius, David

    2011-02-01

    A fundamental observation in biology is that mitochondrial function, as measured by increased reactive oxygen species (ROS), changes significantly with age, suggesting a potential mechanistic link between the cellular processes governing longevity and mitochondrial metabolism homeostasis. In addition, it is well established that altered ROS levels are observed in multiple age-related illnesses including carcinogenesis, neurodegenerative, fatty liver, insulin resistance, and cardiac disease, to name just a few. Manganese superoxide dismutase (MnSOD) is the primary mitochondrial ROS scavenging enzyme that converts superoxide to hydrogen peroxide, which is subsequently converted to water by catalase and other peroxidases. It has recently been shown that MnSOD enzymatic activity is regulated by the reversible acetylation of specific, evolutionarily conserved lysine(s) in the protein. These results, suggest for the first time, that the mitochondria contain bidirectional post-translational signaling networks, similar to that observed in the cytoplasm and nucleus, and that changes in lysine acetylation alter MnSOD enzymatic activity. In addition, these new results demonstrate that the mitochondrial anti-aging or fidelity / sensing protein, SIRT3, responds to changes in mitochondrial nutrient and/or redox status to alter the enzymatic activity of specific downstream targets, including MnSOD that adjusts and/or maintains ROS levels as well as metabolic homeostatic poise. PMID:21386137

  1. Fusion-Triggered Switching of Enzymatic Activity on an Artificial Cell Membrane

    PubMed Central

    Mukai, Masaru; Sasaki, Yoshihiro; Kikuchi, Jun-ichi

    2012-01-01

    A nanosensory membrane device was constructed for detecting liposome fusion through changes in an enzymatic activity. Inspired by a biological signal transduction system, the device design involved functionalized liposomal membranes prepared by self-assembly of the following molecular components: a synthetic peptide lipid and a phospholipid as matrix membrane components, a Schiff's base of pyridoxal 5′-phosphate with phosphatidylethanolamine as a thermo-responsive artificial receptor, NADH-dependent L-lactate dehydrogenase as a signal amplifier, and Cu2+ ion as a signal mediator between the receptor and enzyme. The enzymatic activity of the membrane device was adjustable by changing the matrix lipid composition, reflecting the thermotropic phase transition behavior of the lipid membranes, which in turn controlled receptor binding affinity toward the enzyme-inhibiting mediator species. When an effective fusogen anionic polymer was added to these cationic liposomes, membrane fusion occurred, and the functionalized liposomal membranes responded with changes in enzymatic activity, thus serving as an effective nanosensory device for liposome fusion detection. PMID:22778625

  2. Rapid estimation of compost enzymatic activity by spectral analysis method combined with machine learning.

    PubMed

    Chakraborty, Somsubhra; Das, Bhabani S; Ali, Md Nasim; Li, Bin; Sarathjith, M C; Majumdar, K; Ray, D P

    2014-03-01

    The aim of this study was to investigate the feasibility of using visible near-infrared (VisNIR) diffuse reflectance spectroscopy (DRS) as an easy, inexpensive, and rapid method to predict compost enzymatic activity, which traditionally measured by fluorescein diacetate hydrolysis (FDA-HR) assay. Compost samples representative of five different compost facilities were scanned by DRS, and the raw reflectance spectra were preprocessed using seven spectral transformations for predicting compost FDA-HR with six multivariate algorithms. Although principal component analysis for all spectral pretreatments satisfactorily identified the clusters by compost types, it could not separate different FDA contents. Furthermore, the artificial neural network multilayer perceptron (residual prediction deviation=3.2, validation r(2)=0.91 and RMSE=13.38 μg g(-1) h(-1)) outperformed other multivariate models to capture the highly non-linear relationships between compost enzymatic activity and VisNIR reflectance spectra after Savitzky-Golay first derivative pretreatment. This work demonstrates the efficiency of VisNIR DRS for predicting compost enzymatic as well as microbial activity. PMID:24398221

  3. Enzymatic activation of autotaxin by divalent cations without EF-hand loop region involvement.

    PubMed

    Lee, J; Jung, I D; Nam, S W; Clair, T; Jeong, E M; Hong, S Y; Han, J W; Lee, H W; Stracke, M L; Lee, H Y

    2001-07-15

    Autotaxin (ATX) is a recently described member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family of proteins with potent tumor cell motility-stimulating activity. Like other NPPs, ATX is a glycoprotein with peptide sequences homologous to the catalytic site of bovine intestinal alkaline phosphodiesterase (PDE) and the loop region of an EF-hand motif. The PDE active site of ATX has been associated with the motility-stimulating activity of ATX. In this study, we examined the roles of the EF-hand loop region and of divalent cations on the enzymatic activities of ATX. Ca(2+) or Mg(2+) was each demonstrated to increase the PDE activity of ATX in a concentration-dependent manner, whereas incubation of ATX with chelating agents abolished this activity, indicating a requirement for divalent cations. Non-linear regression analysis of enzyme kinetic data indicated that addition of these divalent cations increases reaction velocity predominantly through an effect on V(max.) Three mutant proteins, Ala(740)-, Ala(742)-, and Ala(751)-ATX, in the EF-hand loop region of ATX had enzymatic activity comparable to that of the wild-type protein. A deletion mutation of the entire loop region resulted in slightly reduced PDE activity but normal motility-stimulating activity. However, the PDE activity of this same deletion mutant remained sensitive to augmentation by cations, strongly implying that cations exert their effect by interactions outside of the EF-hand loop region. PMID:11389881

  4. Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines

    PubMed Central

    Hanavan, Paul D.; Borges, Chad R.; Katchman, Benjamin A.; Faigel, Douglas O.; Ho, Thai H.; Ma, Chen-Ting; Sergienko, Eduard A.; Meurice, Nathalie; Petit, Joachim L.; Lake, Douglas F.

    2015-01-01

    Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a “proof-of-principle” that enzymatic inhibition of QSOX1 may have clinical relevancy. PMID:26158899

  5. DNA-fueled molecular machine for label-free and non-enzymatic ultrasensitive detection of telomerase activity.

    PubMed

    Sun, Panpan; Ran, Xiang; Liu, Chaoqun; Liu, Chaoying; Pu, Fang; Ren, Jinsong; Qu, Xiaogang

    2016-08-01

    Herein, a non-enzymatic and label-free strategy based on DNA-fueled molecular machine was developed for ultrasensitive detection of telomerase activity in cancer cell extracts even at the single-cell level. PMID:27405851

  6. Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

    PubMed

    Mertsalov, Ilya B; Novikov, Boris N; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M

    2016-07-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. PMID:27114558

  7. 15 CFR 930.36 - Consistency determinations for proposed activities.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND COASTAL RESOURCE MANAGEMENT FEDERAL CONSISTENCY WITH APPROVED COASTAL MANAGEMENT PROGRAMS... project (e.g., ongoing maintenance, waste disposal) which cumulatively has an effect upon any coastal use... coastal management issues and have similar enforceable policies, e.g., protection of a particular...

  8. 15 CFR 930.36 - Consistency determinations for proposed activities.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND COASTAL RESOURCE MANAGEMENT FEDERAL CONSISTENCY WITH APPROVED COASTAL MANAGEMENT PROGRAMS... project (e.g., ongoing maintenance, waste disposal) which cumulatively has an effect upon any coastal use... coastal management issues and have similar enforceable policies, e.g., protection of a particular...

  9. A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity.

    PubMed

    Guo, Shihui; Skala, Wolfgang; Magdolen, Viktor; Briza, Peter; Biniossek, Martin L; Schilling, Oliver; Kellermann, Josef; Brandstetter, Hans; Goettig, Peter

    2016-01-01

    Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology. PMID:26582203

  10. Aerobic and anaerobic enzymatic activity of orange roughy (Hoplostethus atlanticus) and alfonsino (Beryx splendens) from the Juan Fernandez seamounts area.

    PubMed

    Saavedra, L M; Quiñones, R A; Gonzalez-Saldía, R R; Niklitschek, E J

    2016-06-01

    The aerobic and anaerobic enzymatic activity of two important commercial bathypelagic species living in the Juan Fernández seamounts was analyzed: alfonsino (Beryx splendens) and orange roughy (Hoplostethus atlanticus). These seamounts are influenced by the presence of an oxygen minimum zone (OMZ) located between 160 and 250 m depth. Both species have vertical segregation; alfonsino is able to stay in the OMZ, while orange roughy remains at greater depths. In this study, we compare the aerobic and anaerobic capacity of these species, measuring the activity of key metabolic enzymes in different body tissues (muscle, heart, brain and liver). Alfonsino has higher anaerobic potential in its white muscle due to greater lactate dehydrogenase (LDH) activity (190.2 μmol NADH min(-1) g ww(-1)), which is related to its smaller body size, but it is also a feature shared with species that migrate through OMZs. This potential and the higher muscle citrate synthase and electron transport system activities indicate that alfonsino has greater swimming activity level than orange roughy. This species has also a high MDH/LDH ratio in its heart, brain and liver, revealing a potential capacity to conduct aerobic metabolism in these organs under prolonged periods of environmental low oxygen conditions, preventing lactic acid accumulation. With these metabolic characteristics, alfonsino may have increased swimming activity to migrate and also could stay for a period of time in the OMZ. The observed differences between alfonsino and orange roughy with respect to their aerobic and anaerobic enzymatic activity are consistent with their characteristic vertical distributions and feeding behaviors. PMID:26687132

  11. Yeasts from sub-Antarctic region: biodiversity, enzymatic activities and their potential as oleaginous microorganisms.

    PubMed

    Martinez, A; Cavello, I; Garmendia, G; Rufo, C; Cavalitto, S; Vero, S

    2016-09-01

    Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %). PMID:27469174

  12. Allocation of extracellular enzymatic activity in relation to litter composition, N deposition, and mass loss

    USGS Publications Warehouse

    Sinsabaugh, R. L.; Carreiro, M.M.; Repert, D.A.

    2002-01-01

    Decomposition of plant material is a complex process that requires interaction among a diversity of microorganisms whose presence and activity is subject to regulation by a wide range of environmental factors. Analysis of extracellular enzyme activity (EEA) provides a way to relate the functional organization of microdecomposer communities to environmental variables. In this study, we examined EEA in relation to litter composition and nitrogen deposition. Mesh bags containing senescent leaves of Quercus borealis (red oak), Acer rubrum (red maple) and Cornus florida (flowering dogwood) were placed on forest floor plots in southeastern New York. One-third of the plots were sprayed monthly with distilled water. The other plots were sprayed monthly with NH4NO3 solution at dose rates equivalent to 2 or 8 g N m-2 y-1. Mass loss, litter composition, fungal mass, and the activities of eight enzymes were measured on 13 dates for each litter type. Dogwood was followed for one year, maple for two, oak for three, For each litter type and treatment, enzymatic turnover activities were calculated from regressions of LN (%mass remaining) vs. cumulative activity. The decomposition of dogwood litter was more efficient than that of maple and oak. Maple litter had the lowest fungal mass and required the most enzymatic work to decompose, even though its mass loss rate was twice that of oak. Across litter types, N amendment reduced apparent enzymatic efficiencies and shifted EEA away from N acquisition and toward P acquisition, and away from polyphenol oxidation and toward polysaccharide hydrolysis. The effect of these shifts on decomposition rate varied with litter composition: dogwood was stimulated, oak was inhibited and maple showed mixed effects. The results show that relatively small shifts in the activity of one or two critical enzymes can significantly alter decomposition rates.

  13. Methods for determining enzymatic activity comprising heating and agitation of closed volumes

    DOEpatents

    Thompson, David Neil; Henriksen, Emily DeCrescenzo; Reed, David William; Jensen, Jill Renee

    2016-03-15

    Methods for determining thermophilic enzymatic activity include heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes, at least one enzyme is added, substantially simultaneously, to the closed volumes. At the predetermined reaction temperature, the closed volumes are agitated and then the activity of the at least one enzyme is determined. The methods are conducive for characterizing enzymes of high-temperature reactions, with insoluble substrates, with substrates and enzymes that do not readily intermix, and with low volumes of substrate and enzyme. Systems for characterizing the enzymes are also disclosed.

  14. Blocked Enzymatic Etching of Gold Nanorods: Application to Colorimetric Detection of Acetylcholinesterase Activity and Its Inhibitors.

    PubMed

    Saa, Laura; Grinyte, Ruta; Sánchez-Iglesias, Ana; Liz-Marzán, Luis M; Pavlov, Valeri

    2016-05-01

    The anisotropic morphology of gold nanorods (AuNRs) has been shown to lead to nonuniform ligand distribution and preferential etching through their tips. We have recently demonstrated that this effect can be achieved by biocatalytic oxidation with hydrogen peroxide, catalyzed by the enzyme horseradish peroxidase (HRP). We report here that modification of AuNRs with thiol-containing organic molecules such as glutathione and thiocholine hinders enzymatic AuNR etching. Higher concentrations of thiol-containing molecules in the reaction mixture gradually decrease the rate of enzymatic etching, which can be monitored by UV-vis spectroscopy through changes in the AuNR longitudinal plasmon band. This effect can be applied to develop novel optical assays for acetylcholinesterase (AChE) activity. The biocatalytic hydrolysis of acetylthiocholine by AChE yields thiocholine, which prevents enzymatic AuNR etching in the presence of HRP. Additionally, the same bioassay can be used for the detection of nanomolar concentrations of AChE inhibitors such as paraoxon and galanthamine. PMID:27070402

  15. Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

    NASA Astrophysics Data System (ADS)

    Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

    2014-07-01

    Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

  16. Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

    PubMed

    Maturano, Yolanda Paola; Assof, Mariela; Fabani, María Paula; Nally, María Cristina; Jofré, Viviana; Rodríguez Assaf, Leticia Anahí; Toro, María Eugenia; Castellanos de Figueroa, Lucía Inés; Vazquez, Fabio

    2015-11-01

    During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation. PMID:26386703

  17. Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity

    PubMed Central

    Kalb, Suzanne R.; Boyer, Anne E.; Barr, John R.

    2015-01-01

    Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A–G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin. PMID:26404376

  18. Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity.

    PubMed

    Kalb, Suzanne R; Boyer, Anne E; Barr, John R

    2015-09-01

    Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin. PMID:26404376

  19. Enzymatic improvement in the polyphenol extractability and antioxidant activity of green tea extracts.

    PubMed

    Hong, Yang-Hee; Jung, Eun Young; Park, Yooheon; Shin, Kwang-Soon; Kim, Tae Young; Yu, Kwang-Won; Chang, Un Jae; Suh, Hyung Joo

    2013-01-01

    This study describes increases in extraction efficiency and the bioconversion of catechins after treatment with several commercial enzymes. Tannase was also used to improve the anti-radical activities of green tea extracts. Enzymatic treatment with various commercial enzymes was introduced to improve the extraction efficiency of polyphenols. The total polyphenol, flavonoid, and catechin contents and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of the green tea extract treated with Viscozyme (VG) were significantly higher than those treated with other commercial enzymatic extractions (p<0.05). More than 95% of the epigallocatechingallate (EGCG) and of the epicatechingallate (ECG) was hydrolyzed to epigallocatechin (EGC) and to epicatechin (EC) in successive 20 min treatments with Viscozyme and tannase (TG). Due to its hydrolytic activity, treatment involving tannase resulted in a significant release of gallic acid (GA), EGC, and EC, leading to greater radical scavenging activities. Regarding the IC(50) values of the DPPH and 2,2-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, the green tea extract treated with TG showed values of 131.23 and 28.83 µg/mL, VG showed values of 224.70 and 32.54 µg/mL, and normal green tea extract (NG) showed values of 241.11 and 66.27 µg/mL, respectively. These results indicate that successive treatment with Viscozyme and tannase improves the extraction efficiency of polyphenols and increases radical scavenging activities. PMID:23291774

  20. Sensing Enzymatic Activity by Exposure and Selection of DNA-Encoded Probes.

    PubMed

    Jetson, Rachael R; Krusemark, Casey J

    2016-08-01

    A sensing approach is applied to encode quantitative enzymatic activity information into DNA sequence populations. The method utilizes DNA-linked peptide substrates as activity probes. Signal detection involves chemical manipulation of a probe population downstream of sample exposure and application of purifying, selective pressure for enzyme products. Selection-induced changes in DNA abundance indicate sample activity. The detection of protein kinase, protease, and farnesyltransferase activities is demonstrated. The assays were employed to measure enzyme inhibition by small molecules and activity in cell lysates using parallel DNA sequencing or quantitative PCR. This strategy will allow the extensive infrastructure for genetic analysis to be applied to proteomic assays, which has a number of advantages in throughput, sensitivity, and sample multiplexing. PMID:27355201

  1. Glycation of Ribonuclease A affects its enzymatic activity and DNA binding ability.

    PubMed

    Dinda, Amit Kumar; Tripathy, Debi Ranjan; Dasgupta, Swagata

    2015-11-01

    Prolonged non-enzymatic glycation of proteins results in the formation of advanced glycation end products (AGEs) that cause several diseases. The glycation of Ribonuclease A (RNase A) at pH 7.4 and 37 °C with ribose, glucose and fructose has been monitored by UV-vis, fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption ionization spectroscopy-time of flight (MALDI-TOF) methods. The enzymatic activity and DNA binding ability of glycated RNase A was also investigated by an agarose gel-based assay. A precipitation assay examined the ribonucleolytic activity of the glycated enzyme. An increase in incubation time resulted in the formation of high molecular weight AGEs with a decrease in ribonucleolytic activity. Ribose exhibits the highest potency as a glycating agent and showed the greatest reduction in the ribonucleolytic activity of the enzyme. Interestingly, glycated RNase A was unable to bind with the ribonuclease inhibitor (RI) and DNA. The glycated form of the protein was also found to be ineffective in DNA melting unlike native RNase A. PMID:26365067

  2. Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies

    PubMed Central

    Koch, Claudia; Eber, Fabian J; Azucena, Carlos; Förste, Alexander; Walheim, Stefan; Schimmel, Thomas; Bittner, Alexander M; Jeske, Holger; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania C

    2016-01-01

    Summary The rod-shaped nanoparticles of the widespread plant pathogen tobacco mosaic virus (TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus–host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for

  3. Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies.

    PubMed

    Koch, Claudia; Eber, Fabian J; Azucena, Carlos; Förste, Alexander; Walheim, Stefan; Schimmel, Thomas; Bittner, Alexander M; Jeske, Holger; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania C; Wege, Christina

    2016-01-01

    The rod-shaped nanoparticles of the widespread plant pathogen tobacco mosaic virus (TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus-host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for

  4. EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis.

    PubMed

    Galeano, E; Barroso, A A M; Vasconcelos, T S; López-Rubio, A; Albrecht, A J P; Victoria Filho, R; Carrer, H

    2016-01-01

    Weed resistance to herbicides is a natural phenomenon that exerts selection on individuals in a population. In Brazil, glyphosate resistance was recently detected in Digitaria insularis. The objective of this study was to elucidate mechanisms of weed resistance in this plant, including genetic variability, allelism, amino acid substitutions, gene expression, and enzymatic activity levels. Most of these have not previously been studied in this species. D. insularis DNA sequences were used to analyze genetic variability. cDNA from resistant and susceptible plants was used to identify mutations, alleles, and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) expression, using real-time quantitative reverse transcription-polymerase chain reaction. In addition, EPSPS activity was measured. We found a decrease in genetic variability between populations related to glyphosate application. Substitutions from proline to threonine and tyrosine to cysteine led to a decrease in EPSPS affinity for the glyphosate. In addition, the EPSPS enzymatic activity was slightly higher in resistant plants, whereas EPSPS gene expression was almost identical in both biotypes, suggesting feedback regulation at different levels. To conclude, our results suggest new molecular mechanisms used by D. insularis to increase glyphosate resistance. PMID:27525929

  5. Antioxidant activity and functional properties of enzymatic protein hydrolysates from common carp (Cyprinus carpio) roe (egg).

    PubMed

    Chalamaiah, M; Jyothirmayi, T; Diwan, Prakash V; Dinesh Kumar, B

    2015-09-01

    Previously, we have reported the composition, molecular mass distribution and in vivo immunomodulatory effects of common carp roe protein hydrolysates. In the current study, antioxidative activity and functional properties of common carp (Cyprinus carpio) roe (egg) protein hydrolysates, prepared by pepsin, trypsin and Alcalase, were evaluated. The three hydrolysates showed excellent antioxidant activities in a dose dependent manner in various in vitro models such as 2,2 diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2'-azino-bis(3-ethylbenzthiazoline-6)-sulfonic acid (ABTS(+)) radical scavenging activity, ferric reducing antioxidant power (FRAP) and ferrous ion (Fe(2+)) chelating ability. Enzymatic hydrolysis significantly increased protein solubility of the hydrolysates to above 62 % over a wide pH range (2-12). Carp roe hydrolysates exhibited good foaming and emulsification properties. The results suggest that bioactive carp roe protein hydrolysates (CRPHs) with good functional properties could be useful in health food/nutraceutical/pharmaceutical industry for various applications. PMID:26344996

  6. Novel, dually radiolabeled peptides for simultaneous monitoring of enzymatic activity and protein targets

    SciTech Connect

    Efrem Mebrahtu, Suzanne Lapi

    2012-12-13

    This application investigated a novel imaging approach to develop methods to incorporate multiple radionuclides into a single peptide at chemoselective sites for simultaneous monitoring of cell-bound protein targets as well as specific enzymatic activity, both of which are associated with enhanced tumor growth and metastasis. This imaging construct was synthesized in such a manner so that the PET radionuclide will remain associated with the tumor cells and the SPECT radionuclide was cleaved from the imaging agent. Measurement of the PET agent only will yield information about the tumor marker density while measurement of the amount of co-localization and mismatch of the two radionuclides will yield information about the enzymatic activity. This coincident measuring technique using both PET and SPECT agents allows us to draw correlations involving the interactions of enzymes (cathepsin, serine-protease urokinase (uPA) and matrix metalloproteases) and other cellular proteins which play a role in cancer growth and metastasis. This technique will allow for studies in xenograft or genetic models of cancer in the same animal at the same time, thus eliminating problems that may occur when trying to invoke comparisons across animals or timepoints. By using radionuclide imaging as opposed to other imaging modalities, this technique has the potential to be translatable and can exploit the high specific activity probes which can be generated with radiotracers. The proof of principle test of this system investigated simultaneous monitoring of matrix metalloprotease (MMP) activity in the extracellular matrix (ECM) as well as density of integrins on the cell surface, both of which can serve as tumor markers. The outcomes/deliverables of this project were as follows: 1. Peptides were synthesized dually labeled at chemospecific sites with PET and SPECT agents. 2. Stability (intrinsic and to radiolysis) and specific activity of these labeled compounds were determined. 3. The

  7. Isolation of Enzymatically Active Replication Complexes from Feline Calicivirus-Infected Cells

    PubMed Central

    Green, Kim Y.; Mory, Aaron; Fogg, Mark H.; Weisberg, Andrea; Belliot, Gaël; Wagner, Mariam; Mitra, Tanaji; Ehrenfeld, Ellie; Cameron, Craig E.; Sosnovtsev, Stanislav V.

    2002-01-01

    A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.0-kb) and subgenomic-length (2.5-kb) RNA molecules similar to those synthesized in FCV-infected cells. The identity of the viral proteins associated with the fraction was investigated. The 60-kDa VP1 major capsid protein was the most abundant viral protein detected. VP2, a minor structural protein encoded by open reading frame 3 (ORF3), was also present. Nonstructural proteins associated with the fraction included the precursor polypeptides Pro-Pol (76 kDa) and p30-VPg (43 kDa), as well as the mature nonstructural proteins p32 (derived from the N-terminal region of the ORF1 polyprotein), p30 (the putative “3A-like” protein), and p39 (the putative nucleoside triphosphatase). The isolation of enzymatically active RCs containing both viral and cellular proteins should facilitate efforts to dissect the contributions of the virus and the host to FCV RNA replication. PMID:12163578

  8. Enzymatically crosslinked dendritic polyglycerol nanogels for encapsulation of catalytically active proteins.

    PubMed

    Wu, Changzhu; Böttcher, Christoph; Haag, Rainer

    2015-02-01

    The enormous potential of nanogel scaffolds for protein encapsulation has been widely recognized. However, constructing stable polymeric nanoscale networks in a facile, mild, and controllable fashion still remains a technical challenge. Here, we present a novel nanogel formation strategy using horseradish peroxidase (HRP) catalyzed crosslinking on phenolic derivatized dendritic polyglycerol (dPG) in the presence of H2O2 in an inverse miniemulsion. This "enzymatic nanogelation" approach was efficient to produce stable 200 nm dPG nanogel particles, and was performed under physiological conditions, thus making it particularly beneficial for encapsulating biological proteins. Purification of the nanogels was easy to handle and practical because there was no need for a post-quenching step. Interestingly, the use of dPG resulted in higher HRP laden nanogels than for linear polyethylene glycol (PEG) analogs, which illustrates the benefits of dendritic backbones in nanogels for protein encapsulation. In addition, the mild immobilization contributed to the enhanced thermal stability and reusability of HRP. The nanogel preparation could be easily optimized to achieve the best HRP activity. Furthermore, a second enzyme, Candida antarctica lipase B (CalB), was successfully encapsulated and optimized for activity in dPG nanogels by the same enzymatic methodology, which shows the perspective applications of such techniques for encapsulation of diverse proteins. PMID:25519490

  9. Selection and validation of enzymatic activities as functional markers in wood biotechnology and fungal ecology.

    PubMed

    Mathieu, Yann; Gelhaye, Eric; Dumarçay, Stéphane; Gérardin, Philippe; Harvengt, Luc; Buée, Marc

    2013-02-15

    The dead wood and forest soils are sources of diversity and under-explored fungal strains with biotechnological potential, which require to be studied. Numerous enzymatic tests have been proposed to investigate the functional potential of the soil microbial communities or to test the functional abilities of fungal strains. Nevertheless, the diversity of these functional markers and their relevance in environmental studies or biotechnological screening does still have not been demonstrated. In this work, we assessed ten different extracellular enzymatic activities involved in the wood decaying process including β-etherase that specifically cleaves the β-aryl ether linkages in the lignin polymer. For this purpose, a collection of 26 fungal strains, distributed within three ecological groups (white, brown and soft rot fungi), has been used. Among the ten potential functional markers, the combinatorial use of only six of them allowed separation between the group of white and soft rot fungi from the brown rot fungi. Moreover, our results suggest that extracellular β-etherase is a rare and dispensable activity among the wood decay fungi. Finally, we propose that this set of markers could be useful for the analysis of fungal communities in functional and environmental studies, and for the selection of strains with biotechnological interests. PMID:23206919

  10. Enzymatic activity of Microsporum canis and Trichophyton mentagrophytes from breeding rabbits with and without skin lesions.

    PubMed

    Cafarchia, Claudia; Figueredo, Luciana A; Coccioli, Carmela; Camarda, Antonio; Otranto, Domenico

    2012-01-01

    Microsporum canis and Trichophyton mentagrophytes are zoophilic dermatophytes which can cause skin infections in animals and humans. The clinical expression of this infection strongly varies depending on host, fungal species as well as enzyme production. No comparative studies are available on the enzymatic activities of M. canis and T. mentagrophytes isolated from breeding rabbits. Thus, the aim of this work was to assess the capability of M. canis and T. mentagrophytes isolated from rabbits both with and without lesions in producing different enzymes. The relationship of dermatophyte enzymatic activities and presence/absence of skin lesions has also been investigated. A total of 260 isolates of T. mentagrophytes and 25 isolates of M. canis sampled both from healthy and lesioned skin of rabbits, as well as from air samples of positive farms were examined. The results showed that T. mentagrophytes and M. canis from rabbits produce different enzymes. However, only elastase and gelatinase were linked to the appearance of lesions in T. mentagrophytes infections, whereas lipase in those by M. canis. PMID:22175244

  11. Protein assembly onto patterned microfabricated devices through enzymatic activation of fusion pro-tag.

    PubMed

    Lewandowski, Angela T; Yi, Hyunmin; Luo, Xiaolong; Payne, Gregory F; Ghodssi, Reza; Rubloff, Gary W; Bentley, William E

    2008-02-15

    We report a versatile approach for covalent surface-assembly of proteins onto selected electrode patterns of pre-fabricated devices. Our approach is based on electro-assembly of the aminopolysaccharide chitosan scaffold as a stable thin film onto patterned conductive surfaces of the device, which is followed by covalent assembly of the target protein onto the scaffold surface upon enzymatic activation of the protein's "pro-tag." For our demonstration, the model target protein is green fluorescent protein (GFP) genetically fused with a pentatyrosine pro-tag at its C-terminus, which assembles onto both two-dimensional chips and within fully packaged microfluidic devices in situ and under flow. Our surface-assembly approach enables spatial selectivity and orientational control under mild experimental conditions. We believe that our integrated approach harnessing genetic manipulation, in situ enzymatic activation, and electro-assembly makes it advantageous for a wide variety of bioMEMS and biosensing applications that require facile "biofunctionalization" of microfabricated devices. PMID:17625789

  12. Enzymatic modification of chitosan by cinnamic acids: Antibacterial activity against Ralstonia solanacearum.

    PubMed

    Yang, Caifeng; Zhou, Yu; Zheng, Yu; Li, Changlong; Sheng, Sheng; Wang, Jun; Wu, Fuan

    2016-06-01

    This study aimed to identify chitosan polymers that have antibacterial activity against the bacterial wilt pathogen. The chitosan polymers were enzymatically synthesized using chitosan and five cinnamic acids (CADs): caffeic acid (CA), ferulic acid (FA), cinnamic acid (CIA), p-coumaric acid (COA) and chlorogenic acid (CHA), using laccase from Pleurotus ostreatus as a catalyst. The reaction was performed in a phosphate buffered solution under heterogenous reaction conditions. The chitosan derivatives (CTS-g-CADs) were characterized by FT-IR, XRD, TGA and SEM. FT-IR demonstrated that the reaction products bound covalently to the free amino groups or hydroxyl groups of chitosan via band of amide I or ester band. XRD showed a reduced packing density for grafted chitosan comparing to original chitosan. TGA demonstrated that CTS-g-CADs have a higher thermostability than chitosan. Additionally, chitosan and its derivatives showed similar antibacterial activity. However, the IC50 value of the chitosan-caffeic acid derivative (CTS-g-CA) against the mulberry bacterial wilt pathogen RS-5 was 0.23mg/mL, which was two-fifths of the IC50 value of chitosan. Therefore, the enzymatically synthesized chitosan polymers can be used to control plant diseases in biotechnological domains. PMID:26993531

  13. Improvement of antioxidant and moisture-preserving activities of Sargassum horneri polysaccharide enzymatic hydrolyzates.

    PubMed

    Shao, Ping; Chen, Xiaoxiao; Sun, Peilong

    2015-03-01

    In the previous study, we have found that polysaccharides isolated from Sargassum horneri exhibited bioactivities. The aim of this study was to investigate the antioxidant and moisture-preserving activities of molecular weight alteration of Sargassum horneri polysaccharide in vitro. For this purpose, the homogeneous active polysaccharide SHP was isolated from Sargassum horneri, and response surface methodology was employed to optimize the enzymatic degradation conditions to get SHP-derived fragments with different molecular weight. Results proved that the polysaccharide is capable of scavenging both ABTS and DPPH radicals in vitro. The study revealed that the polysaccharides had strong moisture-absorption and -retention capacities as compared to propanediol and glycerin. Furthermore, these data demonstrated that molecular weight had a certain effect on antioxidant activities and strong moisture-retention capacities of the polysaccharide from Sargassum horneri. PMID:25572719

  14. Integrated catalysis opens new arylation pathways via regiodivergent enzymatic C-H activation.

    PubMed

    Latham, Jonathan; Henry, Jean-Marc; Sharif, Humera H; Menon, Binuraj R K; Shepherd, Sarah A; Greaney, Michael F; Micklefield, Jason

    2016-01-01

    Despite major recent advances in C-H activation, discrimination between two similar, unactivated C-H positions is beyond the scope of current chemocatalytic methods. Here we demonstrate that integration of regioselective halogenase enzymes with Pd-catalysed cross-coupling chemistry, in one-pot reactions, successfully addresses this problem for the indole heterocycle. The resultant 'chemobio-transformation' delivers a range of functionally diverse arylated products that are impossible to access using separate enzymatic or chemocatalytic C-H activation, under mild, aqueous conditions. This use of different biocatalysts to select different C-H positions contrasts with the prevailing substrate-control approach to the area, and presents opportunities for new pathways in C-H activation chemistry. The issues of enzyme and transition metal compatibility are overcome through membrane compartmentalization, with the optimized process requiring no intermediate work-up or purification steps. PMID:27283121

  15. Integrated catalysis opens new arylation pathways via regiodivergent enzymatic C–H activation

    PubMed Central

    Latham, Jonathan; Henry, Jean-Marc; Sharif, Humera H.; Menon, Binuraj R. K.; Shepherd, Sarah A.; Greaney, Michael F.; Micklefield, Jason

    2016-01-01

    Despite major recent advances in C–H activation, discrimination between two similar, unactivated C–H positions is beyond the scope of current chemocatalytic methods. Here we demonstrate that integration of regioselective halogenase enzymes with Pd-catalysed cross-coupling chemistry, in one-pot reactions, successfully addresses this problem for the indole heterocycle. The resultant ‘chemobio-transformation' delivers a range of functionally diverse arylated products that are impossible to access using separate enzymatic or chemocatalytic C–H activation, under mild, aqueous conditions. This use of different biocatalysts to select different C–H positions contrasts with the prevailing substrate-control approach to the area, and presents opportunities for new pathways in C–H activation chemistry. The issues of enzyme and transition metal compatibility are overcome through membrane compartmentalization, with the optimized process requiring no intermediate work-up or purification steps. PMID:27283121

  16. Enzymatic browning and antioxidant activities in harvested litchi fruit as influenced by apple polyphenols.

    PubMed

    Zhang, Zhengke; Huber, Donald J; Qu, Hongxia; Yun, Ze; Wang, Hui; Huang, Zihui; Huang, Hua; Jiang, Yueming

    2015-03-15

    'Guiwei' litchi fruit were treated with 5 ga.i. L(-1) apple polyphenols (APP) and then stored at 25°C to investigate the effects on pericarp browning. APP treatment effectively reduced pericarp browning and retarded the loss of red colour. APP-treated fruit exhibited higher levels of anthocyanins and cyanidin-3-rutinoside, which correlated with suppressed anthocyanase activity. APP treatment also maintained membrane integrity and reduced oxidative damage, as indicated by a lower relative leakage rate, malondialdehyde content, and reactive oxygen species (ROS) generation. The data suggest that decompartmentalisation of peroxidase and polyphenoloxidase and respective browning substrates was reduced. In addition, APP treatment enhanced the activities of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), as well as non-enzymatic antioxidant capacity (DPPH radical-scavenging activity and reducing power), which might be beneficial in scavenging ROS. We propose that APP treatment is a promising safe strategy for controlling postharvest browning of litchi fruit. PMID:25308659

  17. Enzymatic digestive activity and absorption efficiency in Tagelus dombeii upon Alexandrium catenella exposure

    NASA Astrophysics Data System (ADS)

    Fernández-Reiriz, M. J.; Navarro, J. M.; Cisternas, B. A.; Babarro, J. M. F.; Labarta, U.

    2013-12-01

    We analyzed absorption efficiency (AE) and digestive enzyme activity (amylase, cellulase complex, and laminarinase) of the infaunal bivalve Tagelus dombeii originating from two geographic sites, Corral-Valdivia and Melinka-Aysén, which have different long-term paralytic shellfish poisoning (PSP) exposure rates. We report the effects of past feeding history (origin) on T. dombeii exposed to a mixed diet containing the toxic dinoflagellate Alexandrium catenella and another dinoflagellate-free control diet over a 12-day period in the laboratory. Absorption efficiency values of T. dombeii individuals that experienced PSP exposure in their habitat (Melinka-Aysén) remained unchanged during exposure to toxic food in the laboratory. In contrast, T. dombeii from a non-PSP exposure field site (Corral-Valdivia) showed a significant reduction in AE with toxic exposure time. This study established that the amylase and cellulase complexes were the most important enzymes in the digestive glands of Tagelus from both sites. The temporal evolution of enzymatic activity under toxic diet was fitted to exponential (amylase and cellulase) and to a logarithmic (laminarinase) models. In all fits, we found significant effect of origin in the model parameters. At the beginning of the experiment, higher enzymatic activity was observed for clams from Corral-Valdivia. The amylase activity decreased with time exposure for individuals from Corral and increased for individuals from Melinka. Cellulase activity did not vary over time for clams from Corral, but increased for individuals from Melinka and laminarinase activity decreased over time for individuals from Corral and remained unchanged over time for Melinka. A feeding history of exposure to the dinoflagellate A. catenella was reflected in the digestive responses of both T. dombeii populations.

  18. Enhancing cellular uptake of activable cell-penetrating peptide-doxorubicin conjugate by enzymatic cleavage.

    PubMed

    Shi, Nian-Qiu; Gao, Wei; Xiang, Bai; Qi, Xian-Rong

    2012-01-01

    The use of activable cell-penetrating peptides (ACPPs) as molecular imaging probes is a promising new approach for the visualization of enzymes. The cell-penetrating function of a polycationic cell-penetrating peptide (CPP) is efficiently blocked by intramolecular electrostatic interactions with a polyanionic peptide. Proteolysis of a proteinase-sensitive substrate present between the CPP and polyanionic peptide affords dissociation of both domains and enables the activated CPP to enter cells. This ACPP strategy could also be used to modify antitumor agents for tumor-targeting therapy. Here, we aimed to develop a conjugate of ACPP with antitumor drug doxorubicin (DOX) sensitive to matrix metalloproteinase-2 and -9 (MMP-2/9) for tumor-targeting therapy purposes. The ACPP-DOX conjugate was successfully synthesized. Enzymatic cleavage of ACPP-DOX conjugate by matrix metalloproteinase (MMP)-2/9 indicated that the activation of ACPP-DOX occurred in an enzyme concentration-dependent manner. Flow cytometry and laser confocal microscope studies revealed that the cellular uptake of ACPP-DOX was enhanced after enzymatic-triggered activation and was higher in HT-1080 cells (overexpressed MMPs) than in MCF-7 cells (under-expressed MMPs). The antiproliferative assay showed that ACPP had little toxicity and that ACPP-DOX effectively inhibited HT-1080 cell proliferation. These experiments revealed that the ACPP-DOX conjugate could be triggered by MMP-2/9, which enabled the activated CPP-DOX to enter cells. ACPP-DOX conjugate may be a potential prodrug delivery system used to carry antitumor drugs for MMP-related tumor therapy. PMID:22619516

  19. Ontogeny of nitric oxide synthase I and III protein expression and enzymatic activity in the guinea pig hippocampus.

    PubMed

    Kimura, K A; Reynolds, J N; Brien, J F

    1999-09-01

    60. NOS enzymatic activity increased throughout prenatal and postnatal life, and attained highest activity in the adult. The developmental profile of NOS III protein expression was similar to that for NOS enzymatic activity. There was differential expression of NOS I protein, which was low in the GD 50 fetus and increased rapidly during fetal development to attain adult level by GD 62. These data suggest that the guinea pig is a reliable animal model in which to investigate the roles of NO in normal hippocampal development and in mediating neuronal injury in this brain region. PMID:10521566

  20. Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity

    PubMed Central

    Washburn, Nathaniel; Schwab, Inessa; Ortiz, Daniel; Bhatnagar, Naveen; Lansing, Jonathan C.; Medeiros, Amy; Tyler, Steven; Mekala, Divya; Cochran, Edward; Sarvaiya, Hetal; Garofalo, Kevin; Meccariello, Robin; Meador, James W.; Rutitzky, Laura; Schultes, Birgit C.; Ling, Leona; Avery, William; Nimmerjahn, Falk; Manning, Anthony M.; Kaundinya, Ganesh V.; Bosques, Carlos J.

    2015-01-01

    Despite the beneficial therapeutic effects of intravenous immunoglobulin (IVIg) in inflammatory diseases, consistent therapeutic efficacy and potency remain major limitations for patients and physicians using IVIg. These limitations have stimulated a desire to generate therapeutic alternatives that could leverage the broad mechanisms of action of IVIg while improving therapeutic consistency and potency. The identification of the important anti-inflammatory role of fragment crystallizable domain (Fc) sialylation has presented an opportunity to develop more potent Ig therapies. However, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for clinical use. Therefore, we set out to develop the first, to our knowledge, robust and scalable process for generating a well-qualified sialylated IVIg drug candidate with maximum Fc sialylation devoid of unwanted alterations to the IVIg mixture. Here, we describe a controlled enzymatic, scalable process to produce a tetra-Fc–sialylated (s4-IVIg) IVIg drug candidate and its qualification across a wide panel of analytic assays, including physicochemical, pharmacokinetic, biodistribution, and in vivo animal models of inflammation. Our in vivo characterization of this drug candidate revealed consistent, enhanced anti-inflammatory activity up to 10-fold higher than IVIg across different animal models. To our knowledge, this candidate represents the first s4-IVIg suitable for clinical use; it is also a valuable therapeutic alternative with more consistent and potent anti-inflammatory activity. PMID:25733881

  1. Chitin extraction from shrimp shell using enzymatic treatment. Antitumor, antioxidant and antimicrobial activities of chitosan.

    PubMed

    Younes, Islem; Hajji, Sawssen; Frachet, Véronique; Rinaudo, Marguerite; Jellouli, Kemel; Nasri, Moncef

    2014-08-01

    Chitin was recovered through enzymatic deproteinization of the shrimp processing by-products. Different microbial and fish viscera proteases were tested for their deproteinization efficiency. High levels of protein removal of about 77±3% and 78±2% were recorded using Bacillus mojavensis A21 and Balistes capriscus proteases, respectively, after 3h of hydrolysis at 45°C using an enzyme/substrate ratio of 20U/mg. Therefore, these two crude proteases were used separately for chitin extraction and then chitosan preparation by N-deacetylation. Chitin and chitosan samples were then characterized by 13 Cross polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy and compared to samples prepared through chemical deproteinization. All chitins and chitosans showed identical spectra. Chitosans prepared through enzymatic deproteinization have practically the same acetylation degree but higher molecular weights compared to that obtained through chemical process. Antimicobial, antioxidant and antitumoral activitities of chitosan-M obtained by treatment with A21 proteases and chitosan-C obtained by alkaline treatment were investigated. Results showed that both chitosans inhibited the growth of most Gram-negative, Gram-positive bacteria and fungi tested. Furthermore, both chitosans exhibited antioxidant and antitumor activities which was dependent on the molecular weight. PMID:24950313

  2. Pertussis toxin analog with reduced enzymatic and biological activities is a protective immunogen.

    PubMed Central

    Kimura, A; Mountzouros, K T; Schad, P A; Cieplak, W; Cowell, J L

    1990-01-01

    Bordetella pertussis TOX3201 has a 12-base-pair insertion in the S1 subunit gene of pertussis toxin (PTX), which encodes for a 4-amino-acid insertion between residues 107 and 108 of the mature S1 subunit (Black et al., Science 240:656-659, 1988). This mutant strain has been shown to secrete a holotoxin analog of PTX, designated CRM3201, with reduced ADP-ribosyltransferase activity. In the present study, we evaluated the biochemical, biological, and immunoprotective activities of purified CRM3201. Assay of enzymatic activities showed that CRM3201 had 20 to 30% of the ADP-ribosyltransferase activity and 55 to 60% of the NAD glycohydrolase activity of native PTX. CRM3201, however, had only 2 to 6% of the activity of PTX in clustering CHO cells, promoting leukocytosis, inducing histamine sensitization, and potentiating an anaphylactic response to bovine serum albumin. In contrast, activities associated with the B oligomer (binding to fetuin, hemagglutination of goose erythrocytes, and lymphocyte mitogen activity) were comparable to those of native PTX. Injection of BALB/c mice with CRM3201 mixed with Al(OH)3 elicited high titers of antibody to PTX (as measured by enzyme-linked immunosorbent assay), which neutralized a leukocytosis-promoting dose of PTX in these mice and neutralized PTX in a CHO cell assay. Passive transfer of the anti-CRM3201 antibody protected 20-day-old Swiss-Webster mice against a lethal aerosol challenge with B. pertussis 18323. Active immunization with CRM3201 significantly reduced lung colonization in adult BALB/c mice with a B. pertussis respiratory infection. These results demonstrate (i) that the reduced ADP-ribosyltransferase activity of CRM3201 is associated with reductions in certain biological and toxic activities of PTX (the enzymatic and biological activities are not, however, totally concordant); (ii) that CRM3201 possesses a functional B oligomer; and (iii) that CRM3201 can induce toxin-neutralizing antibodies which protect mice

  3. Chemical and enzymatic reductive activation of acylfulvene to isomeric cytotoxic reactive intermediates

    PubMed Central

    Pietsch, Kathryn E.; Neels, James F.; Yu, Xiang; Gong, Jiachang; Sturla, Shana J.

    2011-01-01

    Acylfulvenes, a class of semisynthetic analogues of the sesquiterpene natural product illudin S, are cytotoxic towards cancer cells. The minor structural changes between illudin S and AFs translate to an improved therapeutic window in preclinical cell-based assays and xenograft models. AFs are, therefore, unique tools for addressing the chemical and biochemical basis of cytotoxic selectivity. AFs elicit cytotoxic responses by alkylation of biological targets, including DNA. While AFs are capable of direct alkylation, cytosolic reductive bioactivation to an electrophilic intermediate is correlated with enhanced cytotoxicity. Data obtained in this study illustrates chemical aspects of the process of AF activation. By tracking reaction mechanisms with stable isotope-labeled reagents, enzymatic versus chemical activation pathways for AF were compared for reactions involving the NADPH-dependent enzyme prostaglandin reductase 1 (PTGR1) or sodium borohydride, respectively. These two processes resulted in isomeric products that appear to give rise to similar patterns of DNA modification. The chemically activated isomer has been newly isolated and chemically characterized in this study, including an assessment of its relative stereochemistry, and stability at varying pH and under bioassay conditions. In mammalian cancer cells, this chemically activated analog was shown to not rely on further cellular activation to significantly enhance cytotoxic potency, in contrast to the requirements of AF. On the basis of this study, we anticipate that the chemically activated form of AF will serve as a useful chemical probe for evaluating biomolecular interactions independent of enzyme-mediated activation. PMID:21939268

  4. Mitochondrial Respiration Chain Enzymatic Activities in the Human Brain: Methodological Implications for Tissue Sampling and Storage.

    PubMed

    Ronsoni, Marcelo Fernando; Remor, Aline Pertile; Lopes, Mark William; Hohl, Alexandre; Troncoso, Iris H Z; Leal, Rodrigo Bainy; Boos, Gustavo Luchi; Kondageski, Charles; Nunes, Jean Costa; Linhares, Marcelo Neves; Lin, Kátia; Latini, Alexandra Susana; Walz, Roger

    2016-04-01

    Mitochondrial respiratory chain complexes enzymatic (MRCCE) activities were successfully evaluated in frozen brain samples. Epilepsy surgery offers an ethical opportunity to study human brain tissue surgically removed to treat drug resistant epilepsies. Epilepsy surgeries are done with hemodynamic and laboratory parameters to maintain physiology, but there are no studies analyzing the association among these parameters and MRCCE activities in the human brain tissue. We determined the intra-operative parameters independently associated with MRCCE activities in middle temporal neocortex (Cx), amygdala (AMY) and head of hippocampus (HIP) samples of patients (n = 23) who underwent temporal lobectomy using multiple linear regressions. MRCCE activities in Cx, AMY and HIP are differentially associated to trans-operative mean arterial blood pressure, O2 saturation, hemoglobin, and anesthesia duration to time of tissue sampling. The time-course between the last seizure occurrence and tissue sampling as well as the sample storage to biochemical assessments were also associated with enzyme activities. Linear regression models including these variables explain 13-17 % of MRCCE activities and show a moderate to strong effect (r = 0.37-0.82). Intraoperative hemodynamic and laboratory parameters as well as the time from last seizure to tissue sampling and storage time are associated with MRCCE activities in human samples from the Cx, AMYG and HIP. Careful control of these parameters is required to minimize confounding biases in studies using human brain samples collected from elective neurosurgery. PMID:26586405

  5. Antioxidative activities of hydrolysates from edible birds nest using enzymatic hydrolysis

    NASA Astrophysics Data System (ADS)

    Muhammad, Nurul Nadia; Babji, Abdul Salam; Ayub, Mohd Khan

    2015-09-01

    Edible bird's nest protein hydrolysates (EBN) were prepared via enzymatic hydrolysis to investigate its antioxidant activity. Two types of enzyme (alcalase and papain) were used in this study and EBN had been hydrolysed with different hydrolysis time (30, 60, 90 and 120 min). Antioxidant activities in EBN protein hydrolysate were measured using DPPH, ABTS+ and Reducing Power Assay. From this study, increased hydrolysis time from 30 min to 120 min contributed to higher DH, as shown by alcalase (40.59%) and papain (24.94%). For antioxidant assay, EBN hydrolysed with papain showed higher scavenging activity and reducing power ability compared to alcalase. The highest antioxidant activity for papain was at 120 min hydrolysis time with ABTS (54.245%), DPPH (49.78%) and Reducing Power (0.0680). Meanwhile for alcalase, the highest antioxidant activity was at 30 min hydrolysis time. Even though scavenging activity for EBN protein hydrolysates were high, the reducing power ability was quite low as compared to BHT and ascorbic Acid. This study showed that EBN protein hydrolysate with alcalase and papain treatments potentially exhibit high antioxidant activity which have not been reported before.

  6. Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration.

    PubMed

    Trapani, M R; Parisi, M G; Parrinello, D; Sanfratello, M A; Benenati, G; Palla, F; Cammarata, M

    2016-03-01

    The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self" have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different bacterial strains alters the expression of these enzymes and suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. Our study shows for the first time, a specific reaction and enzymatic responses following injection of bacteria in a cnidarian. PMID:26836977

  7. Exonic splicing signals impose constraints upon the evolution of enzymatic activity.

    PubMed

    Falanga, Alessia; Stojanović, Ozren; Kiffer-Moreira, Tina; Pinto, Sofia; Millán, José Luis; Vlahoviček, Kristian; Baralle, Marco

    2014-05-01

    Exon splicing enhancers (ESEs) overlap with amino acid coding sequences implying a dual evolutionary selective pressure. In this study, we map ESEs in the placental alkaline phosphatase gene (ALPP), absent in the corresponding exon of the ancestral tissue-non-specific alkaline phosphatase gene (ALPL). The ESEs are associated with amino acid differences between the transcripts in an area otherwise conserved. We switched out the ALPP ESEs sequences with the sequence from the related ALPL, introducing the associated amino acid changes. The resulting enzymes, produced by cDNA expression, showed different kinetic characteristics than ALPL and ALPP. In the organism, this enzyme will never be subjected to selection because gene splicing analysis shows exon skipping due to loss of the ESE. Our data prove that ESEs restrict the evolution of enzymatic activity. Thus, suboptimal proteins may exist in scenarios when coding nucleotide changes and consequent amino acid variation cannot be reconciled with the splicing function. PMID:24692663

  8. A Selective Glutathione Probe based on AIE Fluorogen and its Application in Enzymatic Activity Assay

    NASA Astrophysics Data System (ADS)

    Lou, Xiaoding; Hong, Yuning; Chen, Sijie; Leung, Chris Wai Tung; Zhao, Na; Situ, Bo; Lam, Jacky Wing Yip; Tang, Ben Zhong

    2014-03-01

    In this work, we design and synthesize a malonitrile-functionalized TPE derivative (TPE-DCV), which can react with thiol group through thiol-ene click reaction, leading to the fluorescence change of the system. Combined with the unique AIE property, TPE-DCV can selectively detect glutathione (GSH) but not cysteine or homocysteine. As the cleavage of GSSG with the aid of glutathione reductase produces GSH, which turns on the fluorescence of TPE-DCV, the ensemble of TPE-DCV and GSSG can thus serve as a label-free sensor for enzymatic activity assay of glutathione reductase. We also apply TPE-DCV for the detection of intracellular GSH in living cells.

  9. A Selective Glutathione Probe based on AIE Fluorogen and its Application in Enzymatic Activity Assay

    PubMed Central

    Lou, Xiaoding; Hong, Yuning; Chen, Sijie; Leung, Chris Wai Tung; Zhao, Na; Situ, Bo; Lam, Jacky Wing Yip; Tang, Ben Zhong

    2014-01-01

    In this work, we design and synthesize a malonitrile-functionalized TPE derivative (TPE-DCV), which can react with thiol group through thiol-ene click reaction, leading to the fluorescence change of the system. Combined with the unique AIE property, TPE-DCV can selectively detect glutathione (GSH) but not cysteine or homocysteine. As the cleavage of GSSG with the aid of glutathione reductase produces GSH, which turns on the fluorescence of TPE-DCV, the ensemble of TPE-DCV and GSSG can thus serve as a label-free sensor for enzymatic activity assay of glutathione reductase. We also apply TPE-DCV for the detection of intracellular GSH in living cells. PMID:24603274

  10. The Effects of Storage Conditions on the Preservation of Enzymatic Activity in Bone

    PubMed Central

    Cosby, Christi N.; Troiano, Nancy W.; Kacena, Melissa A.

    2009-01-01

    Alkaline phosphatase and acid phosphatase are two major enzymatic measures of osteoblastic and osteoclastic activity, respectively. As a result, the preservation of the enzymes in bone specimens to near in vivo accuracy is essential. Despite standardization of the staining process, several factors related to the storage of blocks and slides before sectioning and staining impact the level of enzymes detected in the tissue. Block condition (intact, faced, or unstained) as well as environment (temperature and length of time in storage) affect alkaline phosphatase preservation while the acid phosphatase enzyme remains unaffected. We conclude that to optimally preserve alkaline phosphatase enzyme, methacrylate-embedded undecalcified murine bones should be stored as intact blocks. After sectioning, the faced blocks should be stored at 4°C for optimal enzyme staining of future sections. Furthermore, it is best to stain sections immediately after sectioning. PMID:20686670

  11. Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway

    PubMed Central

    Keller, Markus A.; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V.; Griffin, Julian L.; Ralser, Markus

    2016-01-01

    Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks. PMID:26824074

  12. An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity.

    PubMed Central

    Graves, M C; Lim, J J; Heimer, E P; Kramer, R A

    1988-01-01

    In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli. Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain. The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE. This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease. DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E. coli. This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E. coli. Extracts of E. coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro. These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation. Images PMID:3282230

  13. Linking Microbial Enzymatic Activities and Functional Diversity of Soil around Earthworm Burrows and Casts

    PubMed Central

    Lipiec, Jerzy; Frąc, Magdalena; Brzezińska, Małgorzata; Turski, Marcin; Oszust, Karolina

    2016-01-01

    The aim of this work was to evaluate the effect of earthworms (Lumbricidae) on the enzymatic activity and microbial functional diversity in the burrow system [burrow wall (BW) 0–3 mm, transitional zone (TZ) 3–7 mm, bulk soil (BS) > 20 mm from the BW] and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential) was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the BW or casts than in BS and TZ. Conversely, acid phosphomonoesterase had the largest value in the BS. Average Well Color Development in both the TZ and the BS (0.98–0.94 A590 nm) were more than eight times higher than in the BWs and casts. The lowest richness index in the BS (15 utilized substrates) increased by 86–113% in all the other compartments. The PC1 in principal component analysis mainly differentiated the BWs and the TZ. Utilization of all substrate categories was the lowest in the BS. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem. PMID:27625645

  14. Linking Microbial Enzymatic Activities and Functional Diversity of Soil around Earthworm Burrows and Casts.

    PubMed

    Lipiec, Jerzy; Frąc, Magdalena; Brzezińska, Małgorzata; Turski, Marcin; Oszust, Karolina

    2016-01-01

    The aim of this work was to evaluate the effect of earthworms (Lumbricidae) on the enzymatic activity and microbial functional diversity in the burrow system [burrow wall (BW) 0-3 mm, transitional zone (TZ) 3-7 mm, bulk soil (BS) > 20 mm from the BW] and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential) was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the BW or casts than in BS and TZ. Conversely, acid phosphomonoesterase had the largest value in the BS. Average Well Color Development in both the TZ and the BS (0.98-0.94 A590 nm) were more than eight times higher than in the BWs and casts. The lowest richness index in the BS (15 utilized substrates) increased by 86-113% in all the other compartments. The PC1 in principal component analysis mainly differentiated the BWs and the TZ. Utilization of all substrate categories was the lowest in the BS. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem. PMID:27625645

  15. Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions

    PubMed Central

    Yokogawa, Mariko; Tsushima, Takashi; Noda, Nobuo N.; Kumeta, Hiroyuki; Enokizono, Yoshiaki; Yamashita, Kazuo; Standley, Daron M.; Takeuchi, Osamu; Akira, Shizuo; Inagaki, Fuyuhiko

    2016-01-01

    Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions. PMID:26927947

  16. Optical Detection of Enzymatic Activity and Inhibitors on Non-Covalently Functionalized Fluorescent Graphene Oxide.

    PubMed

    Kang, Tae Woog; Jeon, Su-Ji; Kim, Hye-In; Park, Jung Hyun; Yim, DaBin; Lee, Hye-Rim; Ju, Jong-Min; Kim, Man-Jin; Kim, Jong-Ho

    2016-05-24

    It has been of great interest to measure the activity of acetylcholinesterase (AChE) and its inhibitor, as AChE is known to accelerate the aggregation of the amyloid beta peptides that underlie Alzheimer's disease. Herein, we report the development of graphene oxide (GO) fluorescence-based biosensors for the detection of AChE activity and AChE inhibitors. To this end, GO was non-covalently functionalized with phenoxy-modified dextran (PhO-dex-GO) through hydrophobic interaction; the resulting GO showed excellent colloidal stability and intense fluorescence in various aqueous solutions as compared to pristine GO and the GO covalently functionalized with dextran. The fluorescence of PhO-dex-GO remarkably increased as AChE catalyzed the hydrolysis of acetylthiocholine (ATCh) to give thiocholine and acetic acid. It was found that the turn-on fluorescence response of PhO-dex-GO to AChE activity was induced by protonation of carboxyl groups on it from the product of the enzymatic hydrolysis reaction, acetic acid. On the basis of its turn-on fluorescence response, PhO-dex-GO was able to report kinetic and thermodynamic parameters involving a maximum velocity, a Michaelis constant, and an inhibition dissociation constant for AChE activity and inhibition. These parameters enable us to determine the activity of AChE and the efficiency of the inhibitor. PMID:27136042

  17. Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions.

    PubMed

    Yokogawa, Mariko; Tsushima, Takashi; Noda, Nobuo N; Kumeta, Hiroyuki; Enokizono, Yoshiaki; Yamashita, Kazuo; Standley, Daron M; Takeuchi, Osamu; Akira, Shizuo; Inagaki, Fuyuhiko

    2016-01-01

    Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus-the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions. PMID:26927947

  18. Seasonal and spatial distribution of extracellular enzymatic activities and microbial incorporation of dissolved organic substrates in marine sediments

    SciTech Connect

    Meyer-Reil, L.

    1987-08-01

    Seasonal and spatial distributions of extracellular enzymatic activities and microbial incorporations of dissolved organic substrates were followed in sediments of the brackish water Kiel Bight (Baltic Sea). Enzymatic hydrolysis of polymeric organic compounds was determined by means of fluorogenic substrates; incorporation of dissolved organic substrates into microbial biomass was measured by using tritiated substances (acetate, leucine, and thymidine). Based on a recently developed core injection technique, substrates were injected in microliter portions into undisturbed sediment cores. Enzymatic and incorporation activities underwent strong seasonal variations related to the enrichment of organic material in the sediment surface following sedimentation events. The input of the phytoplankton bloom during autumn caused stimulation of both enzymatic hydrolysis of polymeric organic compounds and microbial incorporation of dissolved organic substrates. Following input by spring phytoplankton bloom, mainly incorporation activities were stimulated. In late spring the development of the benthic fauna obviously greatly influenced microbial activities. During summer individual periods of high microbial activities were observed which might be traced back to short-term sedimentation events.

  19. Enzymatic release of antitumor ether lipids by specific phospholipase A2 activation of liposome-forming prodrugs.

    PubMed

    Andresen, Thomas L; Davidsen, Jesper; Begtrup, Mikael; Mouritsen, Ole G; Jørgensen, Kent

    2004-03-25

    An enzymatically activated liposome-based drug-delivery concept involving masked antitumor ether lipids (AELs) has been investigated. This concept takes advantage of the cytotoxic properties of AEL drugs as well as the membrane permeability enhancing properties of these molecules, which can lead to enhanced drug diffusion into cells. Three prodrugs of AELs (proAELs) have been synthesized and four liposome systems, consisting of these proAELs, were investigated for enzymatic degradation by secretory phospholipase A(2) (sPLA(2)), resulting in the release of AELs. The three synthesized proAELs were (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphocholine (1-O-DPPC), (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)(350) (1-O-DPPE-PEG(350)), and 1-O-DPPE-PEG(2000) of which 1-O-DPPC was the main liposome component. All three phospholipids were synthesized from the versatile starting material (R)-O-benzyl glycidol. A phosphorylation method, employing methyl dichlorophosphate, was developed and applied in the synthesis of two analogues of (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol). Differential scanning calorimetry has been used to investigate the phase behavior of the lipid bilayers. A release study, employing calcein encapsulated in non-hydrolyzable 1,2-bis-O-octadecyl-sn-glycero-3-phosphocholine (D-O-SPC) liposomes, showed that proAELs, activated by sPLA(2), perturb membranes because of the detergent-like properties of the released hydrolysis products. A hemolysis investigation was conducted on human red blood cells, and the results demonstrate that proAEL liposomes display a very low hemotoxicity, which has been a major obstacle for using AELs in cancer therapy. The results suggest a possible way of combining a drug-delivery and prodrug concept in a single liposome system. Our investigation of the permeability-enhancing properties of the AEL molecules imply that by encapsulating conventional

  20. 15 CFR 930.38 - Consistency determinations for activities initiated prior to management program approval.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ..., DEPARTMENT OF COMMERCE OCEAN AND COASTAL RESOURCE MANAGEMENT FEDERAL CONSISTENCY WITH APPROVED COASTAL MANAGEMENT PROGRAMS Consistency for Federal Agency Activities § 930.38 Consistency determinations for... activities initiated prior to management program approval. 930.38 Section 930.38 Commerce and Foreign...

  1. Cell-free extracellular enzymatic activity is linked to seasonal temperature changes: a case study in the Baltic Sea

    NASA Astrophysics Data System (ADS)

    Baltar, Federico; Legrand, Catherine; Pinhassi, Jarone

    2016-05-01

    Extracellular enzymatic activities (EEAs) are a crucial step in the degradation of organic matter. Dissolved (cell-free) extracellular enzymes in seawater can make up a significant contribution of the bulk EEA. However, the factors controlling the proportion of dissolved EEA in the marine environment remain unknown. Here we studied the seasonal changes in the proportion of dissolved relative to total EEA (of alkaline phosphatase (APase), β-glucosidase (BGase), and leucine aminopeptidase (LAPase)), in the Baltic Sea for 18 months. The proportion of dissolved EEA ranged between 37 and 100, 0 and 100, and 34 and 100 % for APase, BGase, and LAPase, respectively. A consistent seasonal pattern in the proportion of dissolved EEA was found among all the studied enzymes, with values up to 100 % during winter and < 40 % during summer. A significant negative relation was found between the proportion of dissolved EEA and temperature, indicating that temperature might be a critical factor controlling the proportion of dissolved relative to total EEA in marine environments. Our results suggest a strong decoupling of hydrolysis rates from microbial dynamics in cold waters. This implies that under cold conditions, cell-free enzymes can contribute to substrate availability at large distances from the producing cell, increasing the dissociation between the hydrolysis of organic compounds and the actual microbes producing the enzymes. This might also suggest a potential effect of global warming on the hydrolysis of organic matter via a reduction of the contribution of cell-free enzymes to the bulk hydrolytic activity.

  2. Membrane Phospholipid Augments Cytochrome P4501a Enzymatic Activity by Modulating Structural Conformation during Detoxification of Xenobiotics

    PubMed Central

    Ghosh, Manik C.; Ray, Arun K.

    2013-01-01

    Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment. PMID:23469105

  3. Effect of compatible and noncompatible osmolytes on the enzymatic activity and thermal stability of bovine liver catalase.

    PubMed

    Sepasi Tehrani, H; Moosavi-Movahedi, A A; Ghourchian, H; Ahmad, F; Kiany, A; Atri, M S; Ariaeenejad, Sh; Kavousi, K; Saboury, A A

    2013-12-01

    Catalase is an important antioxidant enzyme that catalyzes the disproportionation of H2O2 into harmless water and molecular oxygen. Due to various applications of the enzyme in different sectors of industry as well as medicine, the enhancement of stability of the enzyme is important. Effect of various classes of compatible as well as noncompatible osmolytes on the enzymatic activity, disaggregation, and thermal stability of bovine liver catalase have been investigated. Compatible osmolytes, proline, xylitol, and valine destabilize the denatured form of the enzyme and, therefore, increase its disaggregation and thermal stability. The increase in the thermal stability is accompanied with a slight increase of activity in comparison to the native enzyme at 25 °C. On the other hand, histidine, a noncompatible osmolyte stabilizes the denatured form of the protein and hence causes an overall decrease in the thermal stability and enzymatic activity of the enzyme. Chemometric results have confirmed the experimental results and have provided insight into the distribution and number of mole fraction components for the intermediates. The increase in melting temperature (Tm) and enzymatic rate could be further amplified by the intrinsic effect of temperature enhancement on the enzymatic activity for the industrial purposes. PMID:23249140

  4. Enzymatic properties of immobilized Alcaligenes faecalis cells with cell-associated beta-glucosidase activity

    SciTech Connect

    Wheatly, M.A.; Phillips, C.R.

    1984-06-01

    Enzymatic properties of Alcaligenes faecalis cells immobilized in polyacrylamide were characterized and compared with those reported for the extracted enzyme, and with those measured for free cells. Many of the properties reflected those of the extracted enzyme rather than those measured in the free whole cells prior to immobilization, suggesting cell disruption during immobilization. These properties included the pH activity profile, a slightly broader pH stability profile, and the activation energy. Electron micrographs showed evidence of cell debris among the polymer matrix. The immobilized cells were not viable, and did not consume glucose. Thermal stability was less after immobilization with a half-line of 16 h at 45 degrees C, and 3.5 h at 50 degrees C. The immobilized preparation was more stable when stored lyophilized rather than in buffer, losing 23 and 52% activity, respectively, after six months. The enzyme was irreversibly inhibited by both acetate and citrate buffers. If the immobilized enzyme is to be used in conjunction with cellulases from Trichoderma reesei for cellulase saccharification, the optimal conditions would be pH 5.5 and 45 degrees C in a buffer containing no carboxylic acid groups.

  5. Topical formulations with superoxide dismutase: influence of formulation composition on physical stability and enzymatic activity.

    PubMed

    Di Mambro, Valéria M; Borin, Maria F; Fonseca, Maria J V

    2003-04-24

    Three different topical formulations were supplemented with superoxide dismutase (SOD) and evaluated concerning physical and chemical stabilities in order to determine the most stable formulation that would maintain SOD activity. Physical stability was evaluated by storing the formulation at room temperature, and at 37 and 45 degrees C for 28 days. Samples were collected at 7-day intervals for assessment of rheological behavior. Chemical stability was evaluated by the measurement of enzymatic activity in formulations stored at room temperature and at 45 degrees C for 75 days. The formulations showed a pseudoplastic behavior, with a flow index of less than 1. There was no significant difference in the initial values of flow index, hysteresis loop or minimum apparent viscosity. The simple emulsion and the one stabilized with hydroxyethylcellulose showed decreased viscosity by the 21st day and with higher temperature, but no significant changes concerning the presence of SOD. Although there were no significant changes concerning storage time or temperature, the formulation stabilized with hydroxyethylcellulose showed a marked loss of SOD activity. The addition of SOD to the formulations studied did not affect their physical stability. Simple emulsions or emulsions stabilized with carboxypolymethylene seem to be better bases for enzyme addition than emulsion stabilized with hydroxyethylcellulose. PMID:12852452

  6. Bioengineering of stainless steel surface by covalent immobilization of enzymes. Physical characterization and interfacial enzymatic activity.

    PubMed

    Caro, Anne; Humblot, Vincent; Méthivier, Christophe; Minier, Michel; Barbes, Lucica; Li, Joachim; Salmain, Michèle; Pradier, Claire-Marie

    2010-09-01

    Two hydrolytic enzymes, namely lysozyme and trypsin, were covalently immobilized onto stainless steel surfaces using wet chemistry processes. The immobilization strategy took advantage of the spontaneous physisorption of the polymer poly(ethylene imine) (PEI) onto stainless steel to yield a firmly attached, thin organic layer containing a high density of primary amine functions. Both enzymes were then covalently grafted to the surface via a glutaraldehyde cross-linker. Alternatively, a thicker underlayer of PEI was chemisorbed by cross-linking two PEI layers by glutaraldehyde. The effective presence of both enzymes on the stainless steel surfaces and their relative amount were assessed by immunochemical assays employing specific anti-enzyme antibodies. Eventually, the hydrolytic activity of the immobilized enzymes was evaluated by local enzymatic tests with suitable substrates. This work demonstrates that, although the amount of enzymes did not vary significantly with the underlayer thickness, their hydrolytic activity could be much improved by increasing the distance from the oxide surface and, likely, by favoring their accessibility. Our data suggest that the immobilization of enzymes on solid oxide surfaces is feasible and efficient, and that the enzymes retain catalytic activity. It may thus provide a promising route towards biofilm-resistant materials. PMID:20566201

  7. Downregulation of Rubisco Activity by Non-enzymatic Acetylation of RbcL.

    PubMed

    Gao, Xiang; Hong, Hui; Li, Wei-Chao; Yang, Lili; Huang, Jirong; Xiao, You-Li; Chen, Xiao-Ya; Chen, Gen-Yun

    2016-07-01

    Atmospheric carbon dioxide (CO2) is assimilated by the most abundant but sluggish enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Here we show that acetylation of lysine residues of the Rubisco large subunit (RbcL), including Lys201 and Lys334 in the active sites, may be an important mechanism in the regulation of Rubisco activities. It is well known that Lys201 reacts with CO2 for carbamylation, a prerequisite for both carboxylase and oxygenase activities of Rubisco, and Lys334 contacts with ribulose-1,5-bisphosphate (RuBP). The acetylation level of RbcL in plants is lower during the day and higher at night, inversely correlating with the Rubisco carboxylation activity. A search of the chloroplast proteome database did not reveal a canonical acetyltransferase; instead, we found that a plant-derived metabolite, 7-acetoxy-4-methylcoumarin (AMC), can non-enzymatically acetylate both native Rubisco and synthesized RbcL peptides spanning Lys334 or Lys201. Furthermore, lysine residues were modified by synthesized 4-methylumbelliferone esters with different electro- and stereo-substitutes, resulting in varied Rubisco activities. 1-Chloroethyl 4-methylcoumarin-7-yl carbonate (ClMC) could transfer the chloroethyl carbamate group to lysine residues of RbcL and completely inactivate Rubisco, whereas bis(4-methylcoumarin-7-yl) carbonate (BMC) improved Rubisco activity through increasing the level of Lys201 carbamylation. Our findings indicate that RbcL acetylation negatively regulates Rubisco activity, and metabolic derivatives can be designed to dissect and improve CO2 fixation efficiency of plants through lysine modification. PMID:27109602

  8. 29 CFR 779.205 - Enterprise must consist of “related activities.”

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 3 2014-07-01 2014-07-01 false Enterprise must consist of ârelated activities.â 779.205... STANDARDS ACT AS APPLIED TO RETAILERS OF GOODS OR SERVICES Employment to Which the Act May Apply; Enterprise Coverage Related Activities § 779.205 Enterprise must consist of “related activities.” The enterprise...

  9. 29 CFR 779.205 - Enterprise must consist of “related activities.”

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 3 2010-07-01 2010-07-01 false Enterprise must consist of ârelated activities.â 779.205... STANDARDS ACT AS APPLIED TO RETAILERS OF GOODS OR SERVICES Employment to Which the Act May Apply; Enterprise Coverage Related Activities § 779.205 Enterprise must consist of “related activities.” The enterprise...

  10. 29 CFR 779.205 - Enterprise must consist of “related activities.”

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 3 2011-07-01 2011-07-01 false Enterprise must consist of ârelated activities.â 779.205... STANDARDS ACT AS APPLIED TO RETAILERS OF GOODS OR SERVICES Employment to Which the Act May Apply; Enterprise Coverage Related Activities § 779.205 Enterprise must consist of “related activities.” The enterprise...

  11. 29 CFR 779.205 - Enterprise must consist of “related activities.”

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 3 2013-07-01 2013-07-01 false Enterprise must consist of ârelated activities.â 779.205... STANDARDS ACT AS APPLIED TO RETAILERS OF GOODS OR SERVICES Employment to Which the Act May Apply; Enterprise Coverage Related Activities § 779.205 Enterprise must consist of “related activities.” The enterprise...

  12. 29 CFR 779.205 - Enterprise must consist of “related activities.”

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 3 2012-07-01 2012-07-01 false Enterprise must consist of ârelated activities.â 779.205... STANDARDS ACT AS APPLIED TO RETAILERS OF GOODS OR SERVICES Employment to Which the Act May Apply; Enterprise Coverage Related Activities § 779.205 Enterprise must consist of “related activities.” The enterprise...

  13. Development of APE1 enzymatic DNA repair assays: low APE1 activity is associated with increase lung cancer risk.

    PubMed

    Sevilya, Ziv; Leitner-Dagan, Yael; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Lejbkowicz, Flavio; Rennert, Hedy S; Freedman, Laurence S; Rennert, Gad; Paz-Elizur, Tamar; Livneh, Zvi

    2015-09-01

    The key role of DNA repair in removing DNA damage and minimizing mutations makes it an attractive target for cancer risk assessment and prevention. Here we describe the development of a robust assay for apurinic/apyrimidinic (AP) endonuclease 1 (APE1; APEX1), an essential enzyme involved in the repair of oxidative DNA damage. APE1 DNA repair enzymatic activity was measured in peripheral blood mononuclear cell protein extracts using a radioactivity-based assay, and its association with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean APE1 enzyme activity in case patients was 691 [95% confidence interval (CI) = 655-727] units/ng protein, significantly lower than in control subjects (mean = 793, 95% CI = 751-834 units/ng protein, P = 0.0006). The adjusted odds ratio for lung cancer associated with 1 SD (211 units) decrease in APE1 activity was 2.0 (95% CI = 1.3-3.1; P = 0.002). Comparison of radioactivity- and fluorescence-based assays showed that the two are equivalent, indicating no interference by the fluorescent tag. The APE1Asp148Glu SNP was associated neither with APE1 enzyme activity nor with lung cancer risk. Taken together, our results indicate that low APE1 activity is associated with lung cancer risk, consistent with the hypothesis that 'bad DNA repair', rather than 'bad luck', is involved in cancer etiology. Such assays may be useful, along with additional DNA repair biomarkers, for risk assessment of lung cancer and perhaps other cancers, and for selecting individuals to undergo early detection techniques such as low-dose CT. PMID:26045303

  14. Formation of marine snow and enhanced enzymatic activities in oil-contaminated seawater

    NASA Astrophysics Data System (ADS)

    Ziervogel, K.; McKay, L.; Yang, T.; Rhodes, B.; Nigro, L.; Gutierrez, T.; Teske, A.; Arnosti, C.

    2010-12-01

    The fate of oil spilled into the ocean depends on its composition, as well as on biological, chemical, and physical characteristics of the spill site. We investigated the effects of oil addition from the Deepwater Horizon (DH) spill on otherwise uncontaminated water collected close to the spill site. Incubation on a roller table mimicked the physical dynamics of natural seawater, leading to the formation of marine snow-oil aggregates. We measured the enzymatic activities of heterotrophic microbes associated with the aggregates and in the surrounding water, and assessed microbial population and community composition as oil-marine snow aggregates formed and aged in the water. Surface seawater taken near the spill site in May 2010 that had no visible crude oil was incubated in 1-l glass bottles with (oil-bottles) and without (no-oil bottles) a seawater-oil mixture collected from the same site. In the oil-bottles formation of brownish, densely packed marine snow (2-3 cm diameter) was observed within the first hour of the roller table incubation. In contrast no-oil bottles showed aggregate formation only after 3 days, and aggregates were almost transparent, less abundant, and smaller in size (< 1cm diameter). Subsamples of the water surrounding the aggregates were taken throughout 21 days of the roller table incubation, and analyzed for bacterial abundance and community structure as well as the activities of hydrolytic enzymes that are used by heterotrophic bacteria to degrade organic matter. We monitored oil-degrading activities with MUF-stearate and -butyrate, and also measured b-glucosidase, alkaline phosphatase, aminopeptidase, and six different polysaccharide hydrolase activities. Enzymatic activities were up to one order of magnitude higher in the oil-bottles compared with the no-oil bottles throughout the entire incubation time. Butyrate hydrolysis was elevated throughout the time course of the incubation, and stearate hydrolysis was particularly high over the

  15. Single-molecule kinetics under force: probing protein folding and enzymatic activity with optical tweezers

    NASA Astrophysics Data System (ADS)

    Wong, Wesley

    2010-03-01

    Weak non-covalent bonds between and within single molecules govern many aspects of biological structure and function (e.g. DNA base-paring, receptor-ligand binding, protein folding, etc.) In living systems, these interactions are often subject to mechanical forces, which can greatly alter their kinetics and activity. My group develops and applies novel single-molecule manipulation techniques to explore and quantify these force-dependent kinetics. Using optical tweezers, we have quantified the force-dependent unfolding and refolding kinetics of different proteins, including the cytoskeletal protein spectrin in collaboration with E. Evans's group [1], and the A2 domain of the von Willebrand factor blood clotting protein in collaboration with T. Springer's group [2]. Furthermore, we have studied the kinetics of the ADAMTS13 enzyme acting on a single A2 domain, and have shown that physiolgical forces in the circulation can act as a cofactor for enzymatic cleavage, regulating hemostatic activity [2]. References: 1. E. Evans, K. Halvorsen, K. Kinoshita, and W.P. Wong, Handbook of Single Molecule Biophysics, P. Hinterdorfer, ed., Springer (2009). 2. X. Zhang, K. Halvorsen, C.-Z. Zhang, W.P. Wong, and T.A. Springer, Science 324 (5932), 1330-1334 (2009).

  16. α-Galactosidase-A Loaded-Nanoliposomes with Enhanced Enzymatic Activity and Intracellular Penetration.

    PubMed

    Cabrera, Ingrid; Abasolo, Ibane; Corchero, José L; Elizondo, Elisa; Gil, Pilar Rivera; Moreno, Evelyn; Faraudo, Jordi; Sala, Santi; Bueno, Dolores; González-Mira, Elisabet; Rivas, Merche; Melgarejo, Marta; Pulido, Daniel; Albericio, Fernando; Royo, Miriam; Villaverde, Antonio; García-Parajo, Maria F; Schwartz, Simó; Ventosa, Nora; Veciana, Jaume

    2016-04-01

    Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO2 -expanded liquid organic solution, shows the great potential of this CO2 -based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest. PMID:26890358

  17. Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine.

    PubMed

    Wang, Xiaohong; Schröder, Heinz C; Müller, Werner E G

    2014-01-01

    In recent years a paradigm shift in understanding of human bone formation has occurred that starts to change current concepts in tissue engineering of bone and cartilage. New discoveries revealed that fundamental steps in biomineralization are enzyme driven, not only during hydroxyapatite deposition, but also during initial bioseed formation, involving the transient deposition and subsequent transformation of calcium carbonate to calcium phosphate mineral. The principal enzymes mediating these reactions, carbonic anhydrase and alkaline phosphatase, open novel targets for pharmacological intervention of bone diseases like osteoporosis, by applying compounds acting as potential activators of these enzymes. It is expected that these new findings will give an innovation boost for the development of scaffolds for bone repair and reconstruction, which began with the use of bioinert materials, followed by bioactive materials and now leading to functional regenerative tissue units. These new developments have become possible with the discovery of the morphogenic activity of bioinorganic polymers, biocalcit, bio-polyphosphate and biosilica that are formed by a biogenic, enzymatic mechanism, a driving force along with the development of novel rapid-prototyping three-dimensional (3D) printing methods and bioprinting (3D cell printing) techniques that may allow a fabrication of customized implants for patients suffering in bone diseases in the future. PMID:25376489

  18. Phenylpropanoid Glycoside Analogues: Enzymatic Synthesis, Antioxidant Activity and Theoretical Study of Their Free Radical Scavenger Mechanism

    PubMed Central

    López-Munguía, Agustín; Hernández-Romero, Yanet; Pedraza-Chaverri, José; Miranda-Molina, Alfonso; Regla, Ignacio; Martínez, Ana; Castillo, Edmundo

    2011-01-01

    Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols. PMID:21674039

  19. Green Tea and Bone Marrow Transplantation: From Antioxidant Activity to Enzymatic and Multidrug-resistance Modulation.

    PubMed

    Peluso, Ilaria; Palmery, Maura; Vitalone, Annabella

    2016-10-25

    Epigallocatechin-3-gallate (EGCG), the main flavonoid of green tea (GT), could play an active role in the prevention of oxidative-stress-related diseases, such as hematologic malignancies. Some effects of EGCG are not imputable to antioxidant activity, but involve modulation of antioxidant enzymes and uric acid (UA) levels. The latter is the major factor responsible of the plasma non-enzymatic antioxidant capacity (NEAC). However, hyperuricemia is a frequent clinical feature caused by tumor lysis syndrome or cyclosporine side effects, both before and after bone marrow transplantation (BMT). Besides this, food-drug interactions could be associated with GT consumption and could have clinical implications. The molecular mechanisms involved in the redox and drug metabolizing/transporting pathways were discussed, with particular reference to the potential role of GT and EGCG in BMT. Moreover, on reviewing data on NEAC, isoprostanes, uric acid, and various enzymes from human studies on GT, its extract, or EGCG, an increase in NEAC, without effect on isoprostanes, and contrasting results on UA and enzymes were observed. Currently, few and contrasting available evidences suggest caution for GT consumption in BMT patients and more studies are needed to better understand the potential impact of EGCG on oxidative stress and metabolizing/transporting systems. PMID:26047551

  20. Molecular characterization and enzymatic activity of laccases in two Pleurotus spp. with different pathogenic behaviour.

    PubMed

    Punelli, Federico; Reverberi, Massimo; Porretta, Daniele; Nogarotto, Sara; Fabbri, Anna A; Fanelli, Corrado; Urbanelli, Sandra

    2009-03-01

    Pleurotus eryngii and P. ferulae, two species belonging to the P. eryngii complex, synthesize laccases, ligninolytic enzymes that play a role in the host-pathogen interaction in the first step of infection. Ecological studies have shown that although both fungi have been recognized as saprophytes, P. eryngii weakly pathogenic when colonizing the roots and stems of Eryngium campestre, whereas P. ferulae is mostly pathogenic to Ferula communis. The paper describes the genomic organization of four putative laccase genes (lac1, lac2, lac3, and lac5-like gene; gene names were assigned on the basis of sequence homologies) of P. eryngii and P. ferulae. The mRNA expression and enzymatic activity of the laccases were analysed under culture conditions where a source of lignin (wheat bran) or lyophilized roots of E. campestre or F. communis were present. These experiments indicated that the four lac-like genes were differentially regulated in the two mushrooms. Specifically, the addition of the lyophilized roots of the respective host plant to the culture media induced an advance in the mRNA expression of the four lac-like genes and a seven-fold higher total laccase activity in P. ferulae than in P. eryngii. The results obtained are discussed in relation to the possible role of laccases in the interaction of P. eryngii and P. ferulae with their respective host. PMID:19116166

  1. A 96-well electrochemical method for the screening of enzymatic activities.

    PubMed

    Abdellaoui, Sofiène; Noiriel, Alexandre; Henkens, Robert; Bonaventura, Celia; Blum, Loïc J; Doumèche, Bastien

    2013-04-01

    The rapid electrochemical screening of enzyme activities in bioelectronics is still a challenging issue. In order to solve this problem, we propose to use a 96-well electrochemical assay. This system is composed of 96 screen-printed electrodes on a printed circuit board adapted from a commercial system (carbon is used as the working electrode and silver chloride as the counter/reference electrode). The associated device allows for the measurements on the 96 electrodes to be performed within a few seconds. In this work, we demonstrate the validity of the screening method with the commercial laccase from the fungus Trametes versicolor. The signal-to-noise ratio (S/N) is found to be the best way to analyze the electrochemical signals. The S/N follows a saturation-like mechanism with a dynamic linear range of two decades ranging from 0.5 to 75 ng of laccase (corresponding to enzymatic activities from 62 × 10(-6) to 9.37 × 10(-3) μmol min(-1)) and a sensitivity of 3027 μg(-1) at +100 mV versus Ag/AgCl. Laccase inhibitors (azide and fluoride anions), pH optima, and interfering molecules could also be identified within a few minutes. PMID:23461701

  2. Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance.

    PubMed

    Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong

    2015-01-01

    Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities. PMID:26584633

  3. Three in one: Identification, expression and enzymatic activity of lysozymes in amphioxus.

    PubMed

    Xu, Na; Pan, Junli; Liu, Shousheng; Xue, Qinggang; Zhang, Shicui

    2014-10-01

    The lysozymes identified so far in animals belong to the g-type, c-type, and i-type. Vertebrate animals possess only the former two types, i.e., g- and c-types, while all the three types have been reported in invertebrates. Here we demonstrate that (1) three cDNAs that encode g-, c-, and i-type lysozymes, respectively, were identified in a single species of the amphioxus Branchiostoma japonicum; (2) all the 3-type genes displayed distinct tissue-specific expression pattern; (3) recombinant g-, c-, and i-type lysozymes all exhibited enzymatic activities; and (4) native g-, c-, and i-type lysozymes were identified in the different tissues of amphioxus. Collectively, these results suggest the presence of all the 3-type lysozymes in a single animal species, first such data ever reported. The presence of biologically active i-type lysozyme in amphioxus also suggests that i-type lysozyme gene is retained at least in Protochordata, contrasting to the previous proposal that i-type lysozyme gene has been lost in a common ancestor of all chordates. PMID:24968076

  4. Determination of myoglobin based on its enzymatic activity by stopped-flow spectrophotometry

    NASA Astrophysics Data System (ADS)

    Zheng, Qi; Liu, Zhihong; Cai, Ruxiu

    2005-04-01

    A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 °C was found to be proportional to the concentration of Mb in the range of 1.0 × 10 -6 to 4.0 × 10 -9 mol L -1. The detection limit of Mb was found to be 9.93 × 10 -10 mol L -1. The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin (BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.

  5. Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance

    PubMed Central

    Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong

    2015-01-01

    Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities. PMID:26584633

  6. Enzymatic activity in the rhizosphere of Spartina maritima: potential contribution for phytoremediation of metals.

    PubMed

    Reboreda, Rosa; Caçador, Isabel

    2008-02-01

    Extracellular enzymatic activity (EEA) of five enzymes (peroxidase, phenol oxidase, beta-glucosidase, beta-N-acetylglucosaminidase and acid phosphatase) was analysed in sediments colonised by Spartina maritima in two salt marshes (Rosário and Pancas) of the Tagus estuary (Portugal) with different characteristics such as sediment parameters and metal contaminant levels. Our aim was a better understanding of the influence of the halophyte on microbial activity in the rhizosphere under different site conditions, and its potential consequences for metal cycling and phytoremediation in salt marshes. Acid phosphatase and beta-N-acetylglucosaminidase presented significantly higher EEA in Rosário than in Pancas, whereas the opposite occurred for peroxidase. This was mainly attributed to differences in organic matter between the two sites. A positive correlation between root biomass and EEA of hydrolases (beta-glucosidase, beta-N-acetylglucosaminidase and acid phosphatase) was found, indicating a possible influence of the halophyte in sediment microbial function. This would potentially affect metal cycling in the rhizosphere through microbial reactions. PMID:17935772

  7. Angiotensin-converting enzyme inhibitory and antioxidant activities of enzymatically synthesized phenolic and vitamin glycosides.

    PubMed

    Charles, Rajachristu Einstein; Ponrasu, Thangavel; Sivakumar, Ramaiah; Divakar, Soundar

    2009-03-01

    Amyloglucosidase from Rhizopus mould and beta-glucosidase from sweet almond were employed for the preparation of phenolic and vitamin glycosides of vanillin, N-vanillylnonanamide, DL-dopa, dopamine, curcumin, alpha-tocopherol (vitamin E), pyridoxine (vitamin B(6)), ergocalciferol (vitamin D(2)), thiamin (vitamin B(1)) and riboflavin (vitamin B(2)). Approx. 20 enzymatically prepared phenolic and vitamin glycosides were subjected to ACE (angiotensin-converting enzyme) inhibition activity measurements, and 14 glycosides were tested for antioxidant activities. Both phenolic and vitamin glycosides exhibited IC(50) values for ACE inhibition in the 0.52+/-0.03-3.33+/-0.17 mM range and antioxidant activities ranging from 0.8+/-0.04 to 1.18+/-0.06 mM. Comparable ACE inhibition values were observed between free phenols and vitamin glycosides. However, antioxidant activities of glycosides were, in general, lesser than those of free phenols. Best IC(50) value for ACE inhibition were observed for 11-O-(D-fructofuranosyl)thiamin (0.52+/-0.03 mM), 3-hydroxy-4-O-(6-D-sorbitol)phenylalanine (0.56+/-0.03 mM), 4-O-(beta-D-glucopyranosyl)vanillin (0.61+/-0.03 mM), 4-O-(D-galactopyranosyl)vanillin (0.61+/-0.03 mM) and pyridoxine-D-glucoside (0.84+/-0.04 mM). Similarly, best IC(50) values for antioxidant activity were observed for 1,7-O-(bis-beta-D-glucopyranosyl)curcumin (0.8+/-0.04 mM), 4-O-(beta-D-glucopyranosyl)vanillin (0.9+/-0.05 mM), 3-hydroxy-4-O-(beta-D-galactopyranosyl-(1'-->4)beta-D-glucopyranosyl)phenylalanine (0.9+/-0.05 mM), 20-O-(D-glucopyranosyl)ergocalciferol (0.9+/-0.05 mM) and dopamine-D-galactoside (0.93+/-0.05 mM). PMID:18547170

  8. Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity.

    PubMed

    Lane, Michael D; Seelig, Burckhard

    2016-05-01

    Streptococcus pyogenes produces the cysteine protease streptopain (SpeB) as a critical virulence factor for pathogenesis. Despite having first been described seventy years ago, this protease still holds mysteries which are being investigated today. Streptopain can cleave a wide range of human proteins, including immunoglobulins, the complement activation system, chemokines, and structural proteins. Due to the broad activity of streptopain, it has been challenging to elucidate the functional results of its action and precise mechanisms for its contribution to S. pyogenes pathogenesis. To better study streptopain, several expression and purification schemes have been developed. These methods originally involved isolation from S. pyogenes culture but were more recently expanded to include recombinant Escherichia coli expression systems. While substantially easier to implement, the latter recombinant approach can prove challenging to reproduce, often resulting in mostly insoluble protein and poor purification yields. After extensive optimization of a wide range of expression and purification conditions, we applied the autoinduction method of protein expression and developed a two-step column purification scheme that reliably produces large amounts of purified soluble and highly active streptopain. This method reproducibly yielded 3 mg of streptopain from 50 mL of expression culture at >95% purity, with an activity of 5306 ± 315 U/mg, and no remaining affinity tags or artifacts from recombinant expression. This improved method therefore enables the facile production of the important virulence factor streptopain at higher yields, with no purification scars that might bias functional studies, and with an 8.1-fold increased enzymatic activity compared to previously described procedures. PMID:26773742

  9. Tissue-specific bioenergetic effects and increased enzymatic activities following acute sublethal peroral exposure to cyanide in the mallard duck.

    PubMed

    Ma, J; Pritsos, C A

    1997-02-01

    Protection of wildlife and in particular migratory birds, which are protected by the Migratory Bird Treaty Act, from cyanide waste in and around gold mining operations is an important environmental issue. We have investigated the bioenergetic effects of sublethal peroral cyanide exposure using the mallard duck (Anus platyrhynchos) as a model migratory bird. At cyanide concentrations well below levels considered safe by the mining industry and some regulatory agencies (50 ppm weak acid dissociable (WAD) cyanide) significant depletions of heart, liver, and brain tissue ATP levels were observed. Tissue ATP levels were restored to normal by 24 hr postexposure. Rhodanese and 3-mercaptopyruvate sulfurtransferase activities were determined in these tissues both for basal activity and post-cyanide exposure. Only brain tissue showed increased enzymatic activity following cyanide exposure, suggesting tissue-specific regulation of these enzymatic activities. These studies suggest that 50 ppm WAD cyanide is not a safe level of cyanide in water where avian wildlife exposure can occur. PMID:9070352

  10. The effects of mediator and granular activated carbon addition on degradation of trace organic contaminants by an enzymatic membrane reactor.

    PubMed

    Nguyen, Luong N; Hai, Faisal I; Price, William E; Leusch, Frederic D L; Roddick, Felicity; Ngo, Hao H; Guo, Wenshan; Magram, Saleh F; Nghiem, Long D

    2014-09-01

    The removal of four recalcitrant trace organic contaminants (TrOCs), namely carbamazepine, diclofenac, sulfamethoxazole and atrazine by laccase in an enzymatic membrane reactor (EMR) was studied. Laccases are not effective for degrading non-phenolic compounds; nevertheless, 22-55% removal of these four TrOCs was achieved by the laccase EMR. Addition of the redox-mediator syringaldehyde (SA) to the EMR resulted in a notable dose-dependent improvement (15-45%) of TrOC removal affected by inherent TrOC properties and loading rates. However, SA addition resulted in a concomitant increase in the toxicity of the treated effluent. A further 14-25% improvement in aqueous phase removal of the TrOCs was consistently observed following a one-off dosing of 3g/L granular activated carbon (GAC). Mass balance analysis reveals that this improvement was not due solely to adsorption but also enhanced biodegradation. GAC addition also reduced membrane fouling and the SA-induced toxicity of the effluent. PMID:24980029

  11. The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell

    PubMed Central

    Sumarheni; Gallet, Benoit; Fender, Pascal

    2016-01-01

    Penton-dodecahedron (Pt-Dd) derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP) has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP) with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents. PMID:27242929

  12. Changes in antioxidant and antiinflammatory activity of black bean (Phaseolus vulgaris L.) protein isolates due to germination and enzymatic digestion.

    PubMed

    López-Barrios, Lidia; Antunes-Ricardo, Marilena; Gutiérrez-Uribe, Janet A

    2016-07-15

    Germination is an inexpensive process to improve the nutritional properties of legumes. The effect of germinating black bean seeds on the production of cotyledon protein hydrolysates (CPH) with antioxidant and antiinflammatory activities was analyzed in this research. After simulated enzymatic digestion, the oxygen radical absorbance capacity (ORAC) of CPH obtained from germinated black beans was lower than that observed for raw cotyledons. There were no significant differences among CPH cellular antioxidant activities (CAA), except for the high CAA of the 120 min hydrolysate obtained from one day germinated black bean cotyledons. The most significant changes due to germination and enzymatic hydrolysis were observed for the inhibition of nitric oxide (NO) production in macrophages. The NO synthesis inhibition observed for raw CPH was reduced after simulated gastrointestinal digestion but for germinated samples the inhibition was doubled. Peptides derived from cell wall proteins produced during germination could be responsible of antiinflammatory activity. PMID:26948633

  13. Mutation of Asn28 Disrupts the Dimerization and Enzymatic Activity of SARS 3CL

    SciTech Connect

    Barrila, J.; Gabelli, S; Bacha, U; Amzel, M; Freire, E

    2010-01-01

    Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL{sup pro}, is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL{sup pro} and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 {angstrom} from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CLpro. Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K{sub d}. The crystallographic structure of the N28A mutant determined at 2.35 {angstrom} resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL{sup pro}.

  14. Activation and stabilization of the hydroperoxide lyase enzymatic extract from mint leaves (Mentha spicata) using selected chemical additives.

    PubMed

    Akacha, Najla B; Karboune, Salwa; Gargouri, Mohamed; Kermasha, Selim

    2010-03-01

    The effects of selected lyoprotecting excipients and chemical additives on the specific activity and the thermal stability of the hydroperoxide lyase (HPL) enzymatic extract from mint leaves were investigated. The addition of KCl (5%, w/w) and dextran (2.5%, w/w) to the enzymatic extract, prior to lyophilization, increased the HPL specific activity by 2.0- and 1.2-fold, respectively, compared to the control lyophilized extract. From half-life time (t (1/2)), it can be seen that KCl has enhanced the HPL stability by 1.3- to 2.3-fold, during long-period storage at -20 degrees Celsius and 4 degrees Celsius. Among the selected additives used throughout this study, glycine appeared to be the most effective one. In addition to the activation effect conferred by glycine, it also enhanced the HPL thermal stability. In contrast, polyhydroxyl-containing additives were not effective for stabilizing the HPL enzymatic extract. On the other hand, there was no signification increase in HPL activity and its thermal stability with the presence of Triton X-100. The results also showed that in the presence of glycine (10%), the catalytic efficiency of HPL was increased by 2.45-fold than that without additive. PMID:19430937

  15. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

    Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

    2015-02-24

    [FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

  16. Proteins and enzymatic activities in Erbaluce grape berries with different response to the withering process.

    PubMed

    Vincenzi, Simone; Tolin, Serena; Cocolin, Luca; Rantsiou, Kalliopi; Curioni, Andrea; Rolle, Luca

    2012-06-30

    During the off-vine natural withering process of Erbaluce (white) grapes to obtain "Erbaluce Caluso" Passito wine, some berries change in color from green-yellow to blue. This phenomenon appears at different extents in different years and might be related to several parameters, such as temperature and humidity during withering, grape composition and Botrytis cinerea loading. To better understand the mechanism involved in color variation, the metabolic changes corresponding to this event were studied. At the end of the withering process berries with different colors were separated using a reflectance spectrophotometer, obtaining three color classes identified as "green" (L*=40.3, a*=-0.56, b*=15.20), "gold" (L*=37.7, a*=5.01, b*=14.12) and "blue" (L*=28.6, a*=0.89, b*=-0.67). The three groups of berries had different water contents, the blue berries containing about 30% less water than the green ones. Samples were crushed and the juices were analyzed. The juice yield for blue berries was less than 50% of that of the other two classes, confirming their higher dehydration level. Protein extraction from de-seeded berries was carried out using two different protocols, the first involving a treatment with phenol (to remove polyphenolic substances) and the second based on an extraction with a mild detergent (to recover the proteins to be used for enzymatic analyses). No trace of laccase activity was found in any of the samples, although DNA analysis, by quantitative PCR, suggested the presence of B. cinerea infection in the blue grapes. Chitinase activity of the blue berries was only 30% of that of the other two samples, as confirmed also by zymographic analysis on electrophoretic gels. The same was found also for esterase activity, which was lower (of about 85%) in the blue berries, which, in contrast, showed the highest beta-glucosidase activity. The electrophoretic analysis of the protein extracts revealed strong differences among the samples. Compared to the green and

  17. Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

    PubMed

    Kalb, Suzanne R; Baudys, Jakub; Smith, Theresa J; Smith, Leonard A; Barr, John R

    2014-04-01

    Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum. PMID:24605815

  18. Comparison of enzymatic and non-enzymatic nitroethane anion formation: thermodynamics and contribution of tunneling.

    PubMed

    Valley, Michael P; Fitzpatrick, Paul F

    2004-05-26

    In the reaction of nitroalkane oxidase (NAO), the oxidation of nitroalkanes to the corresponding aldehydes or ketones is initiated by the deprotonation of the neutral nitroalkane. The energetics of nitroethane ionization for both the enzymatic and non-enzymatic reactions have been determined by measuring rate constants as a function of temperature. At 25 degrees C, the rate constant for the acetate-catalyzed reaction is a billionfold smaller than the kcat/Km value for NAO. This corresponds to a difference of 12.3 kcal/mol in the free energy of activation that is largely due to a difference in the activation enthalpy. Analysis of the temperature dependence of the deuterium kinetic isotope effects on the reactions yields similar DeltaEa and AH/AD values for the acetate, phosphate, and NAO-catalyzed reactions that fall within the semiclassical limits, consistent with similar contributions of tunneling to the enzymatic and non-enzymatic reactions. PMID:15149217

  19. Heavy metal concentrations and enzymatic activities in the functional zone sediments of Haizhou Bay, Lianyungang, Jiangsu, China.

    PubMed

    Li, Yu; Liu, Fu-cheng

    2015-11-01

    Surface sediments were collected at 31 sites covering five functional zones of Haizhou Bay, Lianyungang, Jiangsu, China. Heavy metal concentrations and enzymatic activity of phosphatase and urease were determined on a dry-weight basis of sediments. Metal concentrations in sediments were comparable to the Chinese National Standard of Marine Sediment Quality and were as follows: Cu, 8.60-55.8 mg kg(-1); Zn, 107-384 mg kg(-1); Pb, 33.6-200 mg kg(-1); Cd, 0.24-2.57 mg kg(-1); Cr, 30.3-92.1 mg kg(-1); As, 12.9-110 mg kg(-1); Ni, 15.8-49.6 mg kg(-1); Mn, 379-1272 mg kg(-1); and Fe, 13,790-38,240 mg kg(-1). A geoaccumulation index (I geo) was calculated to help researchers understand the status of pollutants in the sediments. I geo showed that Cd and As contamination existed in the study area. The mobility of the metals and the relationship between heavy metal concentrations of chemical fractions and enzymatic activities were also investigated. Results showed that Cd and Mn had higher mobility than other metals, and enzymatic activities may play an important role in controlling the bioavailability and transformation trend of heavy metals from one fraction to another in sediments. PMID:26431704

  20. Impaired enzymatic defensive activity, mitochondrial dysfunction and proteasome activation are involved in RTT cell oxidative damage.

    PubMed

    Cervellati, Carlo; Sticozzi, Claudia; Romani, Arianna; Belmonte, Giuseppe; De Rasmo, Domenico; Signorile, Anna; Cervellati, Franco; Milanese, Chiara; Mastroberardino, Pier Giorgio; Pecorelli, Alessandra; Savelli, Vinno; Forman, Henry J; Hayek, Joussef; Valacchi, Giuseppe

    2015-10-01

    A strong correlation between oxidative stress (OS) and Rett syndrome (RTT), a rare neurodevelopmental disorder affecting females in the 95% of the cases, has been well documented although the source of OS and the effect of a redox imbalance in this pathology has not been yet investigated. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have demonstrated in RTT cells high levels of H2O2 and HNE protein adducts. These findings correlated with the constitutive activation of NADPH-oxidase (NOX) and that was prevented by a NOX inhibitor and iron chelator pre-treatment, showing its direct involvement. In parallel, we demonstrated an increase in mitochondrial oxidant production, altered mitochondrial biogenesis and impaired proteasome activity in RTT samples. Further, we found that the key cellular defensive enzymes: glutathione peroxidase, superoxide dismutase and thioredoxin reductases activities were also significantly lower in RTT. Taken all together, our findings suggest that the systemic OS levels in RTT can be a consequence of both: increased endogenous oxidants as well as altered mitochondrial biogenesis with a decreased activity of defensive enzymes that leads to posttranslational oxidant protein modification and a proteasome activity impairment. PMID:26189585

  1. A Novel Red Clover Hydroxycinnamoyl Transferase Has Enzymatic Activities Consistent With a Role in Phaselic Acid Biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red clover (Trifolium pratense L.) leaves accumulate several micromol per g fresh weight of phaselic acid [2-O-(caffeoyl)-L-malate]. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forages like alfalfa (M...

  2. Influence of short-time imidacloprid and acetamiprid application on soil microbial metabolic activity and enzymatic activity.

    PubMed

    Wang, Fei; Yao, Jun; Chen, Huilun; Yi, Zhengji; Choi, Martin M F

    2014-09-01

    The influence of two neonicotinoids, i.e., imidacloprid (IMI) and acetamiprid (ACE), on soil microbial activities was investigated in a short period of time using a combination of the microcalorimetric approach and enzyme tests. Thermodynamic parameters such as Q T (J g(-1) soil), ∆H met (kJ mol(-1)), J Q/S (J g(-1) h(-1)), k (h(-1)), and soil enzymatic activities, dehydrogenase, phosphomonoesterase, arginine deaminase, and urease, were used to evaluate whole metabolic activity changes and acute toxicity following IMI and ACE treatment. Various profiles of thermogenic curves reflect different soil microbial activities. The microbial growth rate constant k, total heat evolution Q T (expect for IMI), and inhibitory ratio I show linear relationship with the doses of IMI and ACE. Q T for IMI increases at 0.0-20 μg g(-1) and then decreases at 20-80 μg g(-1), possibly attributing to the presence of tolerant microorganisms. The 50 % inhibitory ratios (IC50) of IMI and ACE are 95.7 and 77.2 μg g(-1), respectively. ACE displays slightly higher toxicity than IMI. Plots of k and Q T against microbial biomass-C indicate that the k and Q T are growth yield-dependent. IMI and ACE show 29.6; 40.4 and 23.0; and 23.3, 21.7, and 30.5 % inhibition of dehydrogenase, phosphomonoesterase, and urease activity, respectively. By contrast, the arginine deaminase activity is enhanced by 15.2 and 13.2 % with IMI and ACE, respectively. The parametric indices selected give a quantitative dose-response relationship of both insecticides and indicate that ACE is more toxic than IMI due to their difference in molecular structures. PMID:24819438

  3. Pretreatment and Enzymatic Hydrolysis

    SciTech Connect

    2006-06-01

    Activities in this project are aimed at overcoming barriers associated with high capital and operating costs and sub-optimal sugar yields resulting from pretreatment and subsequent enzymatic hydrolysis of biomass.

  4. Nickel hydroxide nanoparticle activated semi-metallic TiO(2) nanotube arrays for non-enzymatic glucose sensing.

    PubMed

    Gao, Zhi-Da; Guo, Jing; Shrestha, Nabeen K; Hahn, Robert; Song, Yan-Yan; Schmuki, Patrik

    2013-11-11

    Semi-metallic TiO2 nanotube arrays (TiOx Cy NTs) have been decorated uniformly with Ni(OH)2 nanoparticles without the aid of a polymer binder. The resulting hybrid nanotube arrays exhibit excellent catalytic activity towards non-enzymatic glucose electro-oxidation. The anodic current density of the glucose oxidation is significantly improved compared with traditional TiO2 nanotubes decorated with Ni(OH)2 . Moreover, the Ni(OH)2 /TiOx Cy NT-based electrode shows a fast response, high sensitivity, wide linear range, good selectivity and stability towards glucose electro-oxidation, and thus provides a promising and cost-effective sensing platform for non-enzymatic glucose detection. PMID:24115116

  5. Effect of commercial cellulases and refining on kraft pulp properties: correlations between treatment impacts and enzymatic activity components.

    PubMed

    Cui, Li; Meddeb-Mouelhi, Fatma; Laframboise, François; Beauregard, Marc

    2015-01-22

    The importance of enzymes as biotechnological catalysts for paper industry is now recognized. In this study, five cellulase formulations were used for fibre modification. The number of PFI revolutions decreased by about 50% while achieving the same freeness value (decrease in CSF by 200 mL) with the enzymatic pretreatment. The physical properties of handsheets were modified after enzymatic pretreatment followed by PFI refining. A slight decrease in tear strength was observed with enzymes C1 and C4 at pH 7 while the most decrease in tear was observed after C2, C3, C5 treatments. C1 and C4 which had xylanase activity improved paper properties, while other enzymes had a negative impact. Therefore, the intricate balance between cellulolytic and hemicellulolytic activity is the key to optimizing biorefining and paper properties. It was also observed that C1 impact was pH dependent, which supports the importance of pH in developing an enzymatic strategy for refining energy reduction. PMID:25439885

  6. New eutectic ionic liquids for lipase activation and enzymatic preparation of biodiesel†

    PubMed Central

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

    2012-01-01

    The enzymatic preparation of biodiesel has been hampered by the lack of suitable solvents with desirable properties such as high lipase compatibility, low cost, low viscosity, high biodegradability, and ease of product separation. Recent interest in using ionic liquids (ILs) as advanced reaction media has led to fast reaction rates and high yields in the enzymatic synthesis of biodiesel. However, conventional (i.e., cation–anion paired) ILs based on imidazolium and other quaternary ammonium salts remain too expensive for wide application at industrial scales. In this study, we report on newly-synthesized eutectic ILs derived from choline acetate or choline chloride coupled with biocompatible hydrogen-bond donors, such as glycerol. These eutectic solvents have favorable properties including low viscosity, high biodegradability, and excellent compatibility with Novozym® 435, a commercial immobilized Candida antarctica lipase B. Furthermore, in a model biodiesel synthesis system, we demonstrate high reaction rates for the enzymatic transesterification of Miglyol® oil 812 with methanol, catalyzed by Novozym® 435 in choline acetate/glycerol (1 : 1.5 molar ratio). The high conversion (97%) of the triglyceride obtained within 3 h, under optimal conditions, suggests that these novel eutectic solvents warrant further exploration as potential media in the enzymatic production of biodiesel. PMID:21283901

  7. Multi-compound polarization by DNP allows simultaneous assessment of multiple enzymatic activities in vivo

    NASA Astrophysics Data System (ADS)

    Wilson, David M.; Keshari, Kayvan R.; Larson, Peder E. Z.; Chen, Albert P.; Hu, Simon; Van Criekinge, Mark; Bok, Robert; Nelson, Sarah J.; Macdonald, Jeffrey M.; Vigneron, Daniel B.; Kurhanewicz, John

    2010-07-01

    Methods for the simultaneous polarization of multiple 13C-enriched metabolites were developed to probe several enzymatic pathways and other physiologic properties in vivo, using a single intravenous bolus. A new method for polarization of 13C sodium bicarbonate suitable for use in patients was developed, and the co-polarization of 13C sodium bicarbonate and [1- 13C] pyruvate in the same sample was achieved, resulting in high solution-state polarizations (15.7% and 17.6%, respectively) and long spin-lattice relaxation times ( T1) (46.7 s and 47.7 s respectively at 3 T). Consistent with chemical shift anisotropy dominating the T1 relaxation of carbonyls, T1 values for 13C bicarbonate and [1- 13C] pyruvate were even longer at 3 T (49.7 s and 67.3 s, respectively). Co-polarized 13C bicarbonate and [1- 13C] pyruvate were injected into normal mice and a murine prostate tumor model at 3 T. Rapid equilibration of injected hyperpolarized 13C sodium bicarbonate with 13C CO 2 allowed calculation of pH on a voxel by voxel basis, and simultaneous assessment of pyruvate metabolism with cellular uptake and conversion of [1- 13C] pyruvate to its metabolic products. Initial studies in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model demonstrated higher levels of hyperpolarized lactate and lower pH within tumor, relative to surrounding benign tissues and to the abdominal viscera of normal controls. There was no significant difference observed in the tumor lactate/pyruvate ratio obtained after the injection of co-polarized 13C bicarbonate and [1- 13C] pyruvate or polarized [1- 13C] pyruvate alone. The technique was extended to polarize four 13C labelled substrates potentially providing information on pH, metabolism, necrosis and perfusion, namely [1- 13C]pyruvic acid, 13C sodium bicarbonate, [1,4- 13C]fumaric acid, and 13C urea with high levels of solution polarization (17.5%, 10.3%, 15.6% and 11.6%, respectively) and spin-lattice relaxation values similar to those

  8. Priming effects and enzymatic activity in Israeli soils under treated wastewater and freshwater irrigation

    NASA Astrophysics Data System (ADS)

    Anissimova, Marina; Heinze, Stefanie; Chen, Yona; Tarchitzky, Jorge; Marschner, Bernd

    2014-05-01

    Irrigation of soils with treated wastewater (TWW) directly influences microbial processes of soil. TWW contains easily decomposable organic material, which can stimulate the activity of soil microorganisms and, as a result, lead to the excessive consumption of soil organic carbon pool. We investigated the effects of irrigation with TWW relative to those of irrigation with freshwater (FW) on the microbial parameters in soils with low (7%) and medium (13%) clay content in a lysimeter experiment. The objectives of our study were to (i) determine the impact of water quality on soil respiration and enzymatic activity influenced by clay content and depth, and (ii) work out the changes in the turnover of soil organic matter (PE, priming effects). Samples were taken from three soil depths (0-10, 10-20, and 40-60 cm). Soil respiration and PE were determined in a 21-days incubation experiment after addition of uniformly 14C-labeled fructose. Activity of 10 extracellular enzymes (EEA, from C-, N-, P-, and S-cycle), phenol oxidase and peroxidase activity (PO+PE), and dehydrogenase activity (DHA) were assayed. Microbial Community-Level Physiological Profiles (CLPP) using four substrates, and microbial biomass were determined. The results showed that the clay content acted as the main determinative factor. In the soil with low clay content the water quality had a greater impact: the highest PE (56%) was observed in the upper layer (0-10cm) under FW irrigation; EEA of C-, P-, and S-cycles was significantly higher in the upper soil layer under TWW irrigation. Microbial biomass was higher in the soil under TWW irrigation and decreased with increasing of depth (50 μg/g soil in the upper layer, 15 μg/g soil in the lowest layer). This tendency was also observed for DHA. Contrary to the low clay content, in the soil with medium clay content both irrigation types caused the highest PE in the lowest layer (65% under FW irrigation, 48% under TWW irrigation); the higher substrate

  9. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  10. Guinea pig phospholipase B, identification of the catalytic serine and the proregion involved in its processing and enzymatic activity.

    PubMed

    Nauze, Michel; Gonin, Lauriane; Chaminade, Brigitte; Perès, Christine; Hullin-Matsuda, Francoise; Perret, Bertrand; Chap, Hugues; Gassama-Diagne, Ama

    2002-11-15

    Guinea pig phospholipase B (GPPLB) is a glycosylated ectoenzyme of intestinal brush border membrane. It displays a broad substrate specificity and is activated by trypsin cleavage. The primary sequence contains four tandem repeat domains (I to IV) and several serines in lipase consensus sequences. We used site-directed mutagenesis to demonstrate that only the serine 399 present in repeat II is responsible for the various enzymatic activities of GPPLB. Furthermore, we characterized for the first time the retinyl esterase activity of the enzyme. We also constructed and expressed in COS-7 cells, an NH(2)-terminal repeat I deletion mutant which was detected at a very low level by immunoblot. However, confocal microscopy study showed a strong intracellular accumulation with a weak membrane expression of the mutated protein, indicating a role of the NH(2)-terminal repeat I in the processing of GPPLB. Nevertheless, the Western blot-detected protein presented a glycosylation and trypsin sensitivity patterns similar to wild type PLB. The mutant is also fully active without trypsin treatment, in contrast to native enzyme. Thus, we propose a structural model for GPPLB, in which the repeat I constitutes a lid covering the active site and impairing enzymatic activity, its removal by trypsin leading to an active protein. PMID:12194976

  11. The generalized active space concept in multiconfigurational self-consistent field methods.

    PubMed

    Ma, Dongxia; Li Manni, Giovanni; Gagliardi, Laura

    2011-07-28

    A multiconfigurational self-consistent field method based on the concept of generalized active space (GAS) is presented. GAS wave functions are obtained by defining an arbitrary number of active spaces with arbitrary occupation constraints. By a suitable choice of the GAS spaces, numerous ineffective configurations present in a large complete active space (CAS) can be removed, while keeping the important ones in the CI space. As a consequence, the GAS self-consistent field approach retains the accuracy of the CAS self-consistent field (CASSCF) ansatz and, at the same time, can deal with larger active spaces, which would be unaffordable at the CASSCF level. Test calculations on the Gd atom, Gd(2) molecule, and oxoMn(salen) complex are presented. They show that GAS wave functions achieve the same accuracy as CAS wave functions on systems that would be prohibitive at the CAS level. PMID:21806111

  12. Effects of different bulking agents on the maturity, enzymatic activity, and microbial community functional diversity of kitchen waste compost.

    PubMed

    Wang, Xiaojuan; Zhang, Wenwei; Gu, Jie; Gao, Hua; Qin, Qingjun

    2016-10-01

    Aerobic composting is an effective method for the disposal and utilization of kitchen waste. However, the addition of a bulking agent is necessary during kitchen waste composting because of its high moisture content and low C/N ratio. In order to select a suitable bulking agent, we investigated the influence of leaf litter (LL), sawdust (SD), and wheat straw (WS) on the enzymatic activity, microbial community functional diversity, and maturity indices during the kitchen waste composting process. The results showed that the addition of WS yielded the highest maturity (the C/N ratio decreased from 25 to 13, T value = 0.5, and germination index (GI) = 114.7%), whereas the compost containing SD as a bulking agent had the lowest maturity (GI = 32.4%). The maximum cellulase and urease activities were observed with the WS treatment on day 8, whereas the SD treatment had the lowest cellulase activity and the LL treatment had the lowest urease activity. The compost temperature and microbial activity (as the average well color development) showed that bulking the composts with SD prolonged the composting process. The diversity index based on the community-level physiological profile showed that the composts bulked with LL and WS had greater microbial community functional diversity compared with those bulked with SD. Thus, the maturity indexes and enzymatic activities suggest that WS is a suitable bulking agent for use in kitchen waste composting systems. PMID:26895274

  13. Enzymatic Activity of the Soybean Ecto-Apyrase GS52 Is Essential for Stimulation of Nodulation1[W][OA

    PubMed Central

    Tanaka, Kiwamu; Nguyen, Cuong T.; Libault, Marc; Cheng, Jianlin; Stacey, Gary

    2011-01-01

    Nitrogen is an essential nutrient for plant growth. In the Rhizobium-legume symbiosis, root nodules are the sites of bacterial nitrogen fixation, in which atmospheric nitrogen is converted into a form that plants can utilize. While recent studies suggested an important role for the soybean (Glycine max) ecto-apyrase GS52 in rhizobial root hair infection and root nodule formation, precisely how this protein impacts the nodulation process remains undetermined. In this study, the biochemical characteristics of the GS52 enzyme were investigated. Computer modeling of the GS52 apyrase structure identified key amino acid residues important for catalytic activity, which were subsequently mutagenized. Although the GS52 enzyme exhibited broad substrate specificity, its activity on pyrimidine nucleotides and diphosphate nucleotides was significantly higher than on ATP. This result was corroborated by structural modeling of GS52, which predicted a low specificity for the adenine base within the substrate-binding pocket of the enzyme. The wild-type enzyme and its inactive mutant forms were expressed in soybean roots in order to evaluate the importance of GS52 enzymatic activity for nodulation. The results indicated a clear correlation between GS52 enzymatic activity and nodule number. Altogether, our study indicates that the catalytic activity of the GS52 apyrase, likely acting on extracellular nucleotides, is critical for rhizobial infection and nodulation. PMID:21346172

  14. Predictive modelling of growth and measurement of enzymatic synthesis and activity by a cocktail of selected Enterobacteriaceae and Aeromonas hydrophila.

    PubMed

    Braun, P; Sutherland, J P

    2005-11-25

    The possibility was examined of developing a predictive model that would predict food spoilage by combining microbial growth (increase in cellular number) with extracellular enzymatic activity of a cocktail of five strains of Enterobacteriaceae: Escherichia coli, Enterobacter agglomerans, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus vulgaris and one Aeromonas hydrophila strain. Estimations of growth and enzyme activity were made within a three-dimensional matrix of conditions: temperature 2-20 degrees C, pH value 4.0-7.5 and water activity (a(w)) 0.95-0.995. A mathematical model was constructed which predicted growth based on increases in cell number. However, although notable effects of extracellular lipases and proteases were detected, it was not possible to model enzymatic activity and prepare a combined model because the data did not follow the characteristic profile that would allow curve-fitting. Nevertheless, the model for microbial growth and information relating to enzyme activity will be made freely available in a database on the internet. PMID:16154655

  15. Consistency of Moderate to Vigorous Physical Activity in Middle School Physical Education.

    PubMed

    Gill, Monique; Chan-Golston, Alec M; Rice, Lindsay N; Cole, Brian L; Koniak-Griffin, Deborah; Prelip, Michael L

    2016-01-01

    This study assessed the consistency of moderate to vigorous physical activity (MVPA) in a sample of middle school physical education lessons. Random intercept hierarchical linear regressions were employed to model the relationship between consistency of MVPA and independent variables, including lesson and teacher characteristics. Larger classes spent significantly more time in consistent MVPA in the absence of controlling for teacher characteristics. A significant interaction between class size and teacher experience suggests that experience may play a beneficial role in larger classes, and overall class size does not have to be a barrier to achieving high levels of MVPA. PMID:27536933

  16. Absorption of enzymatically active sup 125 I-labeled bovine milk xanthine oxidase fed to rabbits

    SciTech Connect

    Rzucidlo, S.J. ); Zikakis, J.P. )

    1990-05-01

    Rabbits fed a regular laboratory diet supplemented with a high-fat milk containing xanthine oxidase (XO) were studied to determine the presence of active XO in the blood. A pilot feeding study, where rabbits consumed a high-fat diet containing xanthine oxidase, showed a correlation between dairy food consumption and XO activity in the blood. Antibody to dietary XO was also found. In a second study, rabbits were fed ad libitum the high-fat milk and blood serum samples were tested weekly for XO activity. No elevation in serum XO activity was found. A third study showed that serum XO activity was increased when rabbits were force fed the high-fat milk. The final study consisted of force feeding {sup 125}I-labeled XO to one rabbit to ascertain whether the observed increase in serum XO was due to dietary or endogenous XO. Isoelectric focusing of sera collected from the test rabbit strongly suggested that at least a portion of the serum XO contained the radioactive label. This is the first direct evidence showing the uptake of dietary active XO from the gut.

  17. Inhibitory effect of pinostrobin from Renealmia alpinia, on the enzymatic and biological activities of a PLA2.

    PubMed

    Gómez-Betancur, Isabel; Pereañez, Jaime Andrés; Patiño, Arley Camilo; Benjumea, Dora

    2016-08-01

    Pinostrobin is a flavanone isolated from Renealmia alpinia, a plant used in folk medicine to treat snakebites. We tested the inhibitory ability of pinostrobin on the enzymatic, anticoagulant, myotoxic and edema-inducing activities of a PLA2 isolated from Crotalus durissus cumanensis venom. The compound displayed IC50 values of 1.76mM and 1.85mM (95% Confidence intervals: 1.34-2.18 and 1.21-2.45) on the PLA2 enzymatic activity, when either aggregated or monodispersed substrates were used, respectively. When mice were injected with PLA2 preincubated with 0.4, 2.0 and 4.0mM of pinostrobin, myotoxic activity induced by the PLA2 was inhibited up to 87%. Nevertheless, these values decreased up to 56% when the pinostrobin was injected into muscle after PLA2. Pinostrobin inhibited edema-forming and anticoagulant activities of the PLA2. In order to have insights on the mode of action of pinostrobin, intrinsic fluorescence and ultraviolet studies were performed. Results suggest that pinostrobin interacts directly with the PLA2. These findings were supported by molecular docking results, which suggested that pinostrobin forms hydrogen bonds with residues His48 and Asp49 of PLA2, besides, a π-π stacking interactions with those of residues Phe5 and Trp31, and rings C of flavanone and Tyr52 of the toxin. PMID:27109758

  18. Enzymatic Inhibitory Activity and Trypanocidal Effects of Extracts and Compounds from Siphoneugena densiflora O. Berg and Vitex polygama Cham

    PubMed Central

    Gallo, Margareth B. C.; Marques, Anna Sylvia F.; Vieira, Paulo C.; da Silva, Maria Fátima das G. F.; Fernandes, João B.; Silva, Márcio; Guido, Rafael V.; Oliva, Glaucius; Thiemann, Otávio H.; Albuquerque, Sérgio; Fairlamb, Alan H.

    2012-01-01

    Hexanic, methanolic, and hydroalcoholic extracts, and 34 isolated compounds from Vitex polygama Cham. (Lamiaceae, formely Verbenaceae) and Siphoneugena densiflora O. Berg (Myrtaceae) were screened for their trypanocidal effects on bloodstream forms of Trypanosoma cruzi and T. brucei, as well as for their enzymatic inhibitory activities on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) and trypanothione reductase (TR) enzymes from T. cruzi and adeninephosphoribosyl transferase (APRT) enzyme from Leishmania tarentolae. In general, polar extracts displayed strong effects and some of the tested compounds have shown good results in comparison to positive controls of the bioassays. PMID:18669023

  19. Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies

    PubMed Central

    Andjelković, Ana; Oliveira, Marcos T.; Cannino, Giuseppe; Yalgin, Cagri; Dhandapani, Praveen K.; Dufour, Eric; Rustin, Pierre; Szibor, Marten; Jacobs, Howard T.

    2015-01-01

    The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression. PMID:26672986

  20. Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity*

    PubMed Central

    Hughes, Siobhan; Jenkins, Victoria; Dar, Mohd Jamal; Engelman, Alan; Cherepanov, Peter

    2010-01-01

    Lens epithelium-derived growth factor (LEDGF) is an important co-factor of human immunodeficiency virus DNA integration; however, its cellular functions are poorly characterized. We now report identification of the Cdc7-activator of S-phase kinase (ASK) heterodimer as a novel interactor of LEDGF. Both kinase subunits co-immunoprecipitated with endogenous LEDGF from human cell extracts. Truncation analyses identified the integrase-binding domain of LEDGF as essential and minimally sufficient for the interaction with Cdc7-ASK. Reciprocally, the interaction required autophosphorylation of the kinase and the presence of 50 C-terminal residues of ASK. The kinase phosphorylated LEDGF in vitro, with Ser-206 being the major target, and LEDGF phosphorylated at this residue could be detected during S phase of the cell cycle. LEDGF potently stimulated the enzymatic activity of Cdc7-ASK, increasing phosphorylation of MCM2 in vitro by more than 10-fold. This enzymatic stimulation as well as phosphorylation of LEDGF depended on the protein-protein interaction. Intriguingly, removing the C-terminal region of ASK, involved in the interaction with LEDGF, resulted in a hyperactive kinase. Our results indicate that the interaction with LEDGF relieves autoinhibition of Cdc7-ASK kinase, imposed by the C terminus of ASK. PMID:19864417

  1. Enzymatic Activities of RNase H Domains of HIV-1 Reverse Transcriptase with Substrate Binding Domains of Bacterial RNases H1 and H2.

    PubMed

    Permanasari, Etin-Diah; Yasukawa, Kiyoshi; Kanaya, Shigenori

    2015-06-01

    Thermotoga maritima RNase H1 and Bacillus stearothermophilus RNase H2 have an N-terminal substrate binding domain, termed hybrid binding domain (TmaHBD), and N-terminal domain (BstNTD), respectively. HIV-1 reverse transcriptase (RT) is a heterodimer consisting of a P66 subunit and a P51 subunit. The P66 subunit contains a C-terminal RNase H domain, which exhibits RNase H activity either in the presence of Mg(2+) or Mn(2+) ions. The isolated RNase H domain of HIV-1 RT (RNH(HIV)) is inactive, possibly due to the lack of a substrate binding ability, disorder of a loop containing His539, and increased flexibility. To examine whether the activity of RNH(HIV) is restored by the attachment of TmaHBD or BstNTD to its N-terminus, two chimeric proteins, TmaHBD-RNH(HIV) and BstNTD-RNH(HIV), were constructed and characterized. Both chimeric proteins bound to RNA/DNA hybrid more strongly than RNH(HIV) and exhibited enzymatic activity in the presence of Mn(2+) ions. They did not exhibit activity or exhibited very weak activity in the presence of Mg(2+) ions. These results indicate that TmaHBD and BstNTD function as an RNA/DNA hybrid binding tag, and greatly increase the substrate binding affinity and Mn(2+)-dependent activity of RNH(HIV) but do not restore the Mg(2+)-dependent activity of RNH(HIV). PMID:25673083

  2. Enzymatically-Processed Wheat Bran Enhances Macrophage Activity and Has in Vivo Anti-Inflammatory Effects in Mice

    PubMed Central

    Kang, Hee; Lee, Mi-Gi; Lee, Jae-Kang; Choi, Yong-Hyun; Choi, Yong-Seok

    2016-01-01

    Wheat bran is a rich source of dietary fiber, of which arabinoxylan is the most abundant non-starch polysaccharide. Arabinoxylan has been known to exert in vivo immunological activities. Based on prior findings, we pretreated wheat bran with enzymatic hydrolysis to increase the release of soluble arabinoxylan and investigated whether oral administration of wheat bran altered macrophage activity in a mouse model. After four weeks of treatment, we isolated peritoneal macrophages for phagocytic receptor analysis and lipopolysaccharide (LPS)-induced inflammatory changes. In the second experiment, mice given wheat bran were intraperitoneally stimulated with LPS and serum levels of pro- and anti-inflammatory cytokines were determined. The expression of SRA and CD36, and phagocytic activity increased (p < 0.05, respectively). Ex vivo stimulation of macrophages by LPS resulted in reduced surface expression of CD40 (p < 0.05) and decreased production of nitric oxide (p < 0.005), tumor necrosis factor (TNF)-α (p < 0.005), interleukin (IL)-6 (p < 0.01), and IL-12 (p < 0.05). Mice treated with wheat bran showed decreased levels of serum TNF-α and IL-6 (p < 0.05, respectively) and an increased level of serum anti-inflammatory IL-10 (p < 0.05) in response to intraperitoneal LPS. Enzymatically-processed wheat bran boosts macrophage phagocytic capacity possibly through up-regulation of scavenger receptors and confers anti-inflammatory effects, indicating its potential as an immuno-enhancing functional food. PMID:27043618

  3. Ferredoxin:NADP+ oxidoreductase in junction with CdSe/ZnS quantum dots: characteristics of an enzymatically active nanohybrid.

    PubMed

    Szczepaniak, Krzysztof; Worch, Remigiusz; Grzyb, Joanna

    2013-05-15

    Ferredoxin:NADP(+) oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also found in non-photosynthetic tissues, where it is involved in redox reactions of biosynthetic pathways. In vivo it transfers electrons to nicotinamide adenine dinucleotide phosphate (NADP(+)), forming its reduced version, NADPH, while in vitro it can also use NADPH to reduce several substrates, such as ferricyanide, various quinones and nitriles. As an oxidoreductase catalyzing reaction of a broad range of substrates, FNR may be used in biotechnological processes. Quantum dots are semiconductor nanocrystals of a few to several nanometers diameter, having very useful luminescent properties. We present the spectroscopic and functional characteristics of a covalent conjugation of FNR and CdSe/ZnS quantum dots. Two types of quantum dots, of different diameter and emission maximum (550 and 650 nm), were used for comparison. Steady-state fluorescence and gel electrophoresis confirmed efficient conjugation, while fluorescence correlation spectroscopy (FCS) allowed for determination of the conjugates' radii. The nanohybrids sustained enzymatic activity; however, changes in maximal reaction rates and Michaelis constant were found. Detailed analysis of the kinetic parameters showed that the changes in the enzyme activity depend on the substrate used for activity measurement but also on the size of the quantum dots. The presented nanohybrids, as the first example using plant and photosynthetic enzyme as a protein partner, may became a tool to study photosynthesis as well as other biosynthetic and biotechnological processes, involving enzymatically catalyzed electron transfer. PMID:23611948

  4. Evaluation of oil removal efficiency and enzymatic activity in some fungal strains for bioremediation of petroleum-polluted soils

    PubMed Central

    2012-01-01

    Background Petroleum pollution is a global disaster and there are several soil cleaning methods including bioremediation. Methods In a field study, fugal strains were isolated from oil-contaminated sites of Arak refinery (Iran) and their growth ability was checked in potato dextrose agar (PDA) media containing 0-10% v/v crude oil, the activity of three enzymes (Catalase, Peroxidase and Phenol Oxidase) was evaluated in the fungal colonies and bioremediation ability of the fungi was checked in the experimental pots containing 3 kg sterilized soil and different concentrations of petroleum (0-10% w/w). Results Four fungal strains, Acromonium sp., Alternaria sp., Aspergillus terreus and Penicillium sp., were selected as the most resistant ones. They were able to growth in the subjected concentrations and Alternaria sp. showed the highest growth ability in the petroleum containing media. The enzyme assay showed that the enzymatic activity was increased in the oil-contaminated media. Bioremediation results showed that the studied fungi were able to decrease petroleum pollution. The highest petroleum removing efficiency of Aspergillus terreus, Penicillium sp., Alternaria sp. and Acromonium sp. was evaluated in the 10%, 8%, 8% and 2% petroleum pollution respectively. Conclusions Fungi are important microorganisms in decreasing of petroleum pollution. They have bioremediation potency that is related to their enzymatic activities. PMID:23369665

  5. Contrasting effects of untreated textile wastewater onto the soil available nitrogen-phosphorus and enzymatic activities in aridisol.

    PubMed

    Arif, Muhammad Saleem; Riaz, Muhammad; Shahzad, Sher Muhammad; Yasmeen, Tahira; Buttler, Alexandre; Garcıa-Gil, Juan Carlos; Roohi, Mahnaz; Rasool, Akhtar

    2016-02-01

    Water shortage and soil qualitative degradation are significant environmental problems in arid and semi-arid regions of the world. The increasing demand for water in agriculture and industry has resulted in the emergence of wastewater use as an alternative in these areas. Textile wastewater is produced in surplus amounts which poses threat to the environment as well as associated flora and fauna. A 60-day incubation study was performed to assess the effects of untreated textile wastewater at 0, 25, 50, 75, and 100% dilution levels on the physico-chemical and some microbial and enzymatic properties of an aridisol soil. The addition of textile wastewater provoked a significant change in soil pH and electrical conductivity and soil dehydrogenase and urease activities compared to the distilled-water treated control soil. Moreover, compared to the control treatment, soil phosphomonoesterase activity was significantly increased from 25 to 75% application rates, but decreased at 100% textile wastewater application rate. Total and available soil N contents increased significantly in response to application of textile wastewater. Despite significant increases in the soil total P contents after the addition of textile wastewater, soil available P content decreased with increasing concentration of wastewater. Changes in soil nutrient contents and related enzymatic activities suggested a dynamic match between substrate availability and soil N and P contents. Aridisols have high fixation and low P availability, application of textile wastewater to such soils should be considered only after careful assessment. PMID:26787271

  6. Enzymatic degradation of aromatic hydrocarbon intermediates using a recombinant dioxygenase immobilized onto surfactant-activated carbon nanotube.

    PubMed

    Suma, Yanasinee; Lim, Heejun; Kwean, Oh Sung; Cho, Suyeon; Yang, Junwon; Kim, Yohan; Kang, Christina S; Kim, Han S

    2016-06-01

    This study examined the enzymatic decomposition of aromatic hydrocarbon intermediates (catechol, 4-chlorocatechol, and 3-methylcatechol) using a dioxygenase immobilized onto single-walled carbon nanotube (SWCNT). The surfaces of SWCNTs were activated with surfactants. The dioxygenase was obtained by recombinant technique: the corresponding gene was cloned from Arthrobacter chlorophenolicus A6, and the enzyme was overexpressed and purified subsequently. The enzyme immobilization yield was 62%, and the high level of enzyme activity was preserved (60-79%) after enzyme immobilization. Kinetic analyses showed that the substrate utilization rates and the catalytic efficiencies of the immobilized enzyme for all substrates (target aromatic hydrocarbon intermediates) tested were similar to those of the free enzyme, indicating that the loss of enzyme activity was minimal during enzyme immobilization. The immobilized enzyme was more stable than the free enzyme against abrupt changes in pH, temperature, and ionic strength. Moreover, it retained high enzyme activity even after repetitive use. PMID:26810145

  7. Oxidant and enzymatic antioxidant status (gene expression and activity) in the brain of chickens with cold-induced pulmonary hypertension

    NASA Astrophysics Data System (ADS)

    Hassanpour, Hossein; Khalaji-Pirbalouty, Valiallah; Nasiri, Leila; Mohebbi, Abdonnaser; Bahadoran, Shahab

    2015-11-01

    To evaluate oxidant and antioxidant status of the brain (hindbrain, midbrain, and forebrain) in chickens with cold-induced pulmonary hypertension, the measurements of lipid peroxidation, protein oxidation, antioxidant capacity, enzymatic activity, and gene expression (for catalase, glutathione peroxidase, and superoxide dismutases) were done. There were high lipid peroxidation/protein oxidation and low antioxidant capacity in the hindbrain of cold-induced pulmonary hypertensive chickens compared to control ( P < 0.05). In the hypertensive chickens, superoxide dismutase activity was decreased (forebrain, midbrain, and hindbrain), while catalase activity was increased (forebrain and midbrain) ( P < 0.05). Glutathione peroxidase activity did not change. Relative gene expression of catalase and superoxide dismutases (1 and 2) was downregulated, while glutathione peroxidase was upregulated in the brain of the cold-induced pulmonary hypertensive chickens. Probably, these situations in the oxidant and antioxidant status of the brain especially hindbrain may change its function at cardiovascular center and sympathetic nervous system to exacerbate pulmonary hypertension.

  8. A Light-Activated Microheater for the Remote Control of Enzymatic Catalysis.

    PubMed

    Cao, Yuanyuan; Wang, Zhen; Liao, Shenglong; Wang, Jian; Wang, Yapei

    2016-01-18

    The remote control of enzymatic catalysis is of significant importance in disease treatment and industrial applications. Herein, we designed a microheater composed of a porous polylactic acid (PLA) matrix and polydopamine (PDA) with notable photothermal conversion capability. Starch hydrolysis, catalyzed by using α-amylase, was accelerated in the presence of the microheater under illumination with near-infrared light or natural sunlight at room temperature. Additionally, the methodology was extended to the preparation of microwave-absorbing materials with the deposition of polyaniline on porous PLA matrix. The porous morphology improves the energy-conversion efficiency. PMID:26603499

  9. Enhancement of activated sludge dewatering performance by combined composite enzymatic lysis and chemical re-flocculation with inorganic coagulants: Kinetics of enzymatic reaction and re-flocculation morphology.

    PubMed

    Chen, Zhan; Zhang, Weijun; Wang, Dongsheng; Ma, Teng; Bai, Runying

    2015-10-15

    The feasibility of combined process of composite enzymatic treatment and chemical flocculation with inorganic salt coagulants was investigated in this study. The evolution of extracellular polymeric substances (EPS) distribution, composition and morphological properties were analyzed to unravel the sludge conditioning mechanism. It was found that sludge filtration performance was deteriorated due to release of a large amount of biopolymers after enzymatic treatment. The change in EPS followed the pseudo-first-order kinetic equation well under enzymatic treatment. The feeding modes of enzymes had a significant influence on sludge lysis efficiency under compound enzymes treatment. Alpha amylase + protease was more effective in solubilization than other two addition modes (protease + α-amylase or simultaneous addition). The sludge floc re-formed and macromolecule biopolymers were effectively removed through coagulation process. At the same time, both of filtration rate and cake solid content of sludge treated with enzymes were improved with increasing dosage of coagulants, and ferric iron (FeCl3) had better performance in sludge dewaterability enhancement than polyaluminium chloride (PACl). In addition, sludge filtration property was slightly deteriorated, while the cake moisture reduction was favored at the optimal dosage of inorganic coagulants. PMID:26196306

  10. Amifostine Induces Antioxidant Enzymatic Activities in Normal Tissues and a Transplantable Tumor That Can Affect Radiation Response

    SciTech Connect

    Grdina, David J. Murley, Jeffrey S.; Kataoka, Yasushi; Baker, Kenneth L.; Kunnavakkam, Rangesh; Coleman, Mitchell C.; Spitz, Douglas R.

    2009-03-01

    Purpose: To determine whether amifostine can induce elevated manganese superoxide dismutase (SOD2) in murine tissues and a transplantable SA-NH tumor, resulting in a delayed tumor cell radioprotective effect. Methods and Materials: SA-NH tumor-bearing C3H mice were treated with a single 400 mg/kg or three daily 50 mg/kg doses of amifostine administered intraperitoneally. At selected time intervals after the last injection, the heart, liver, lung, pancreas, small intestine, spleen, and SA-NH tumor were removed and analyzed for SOD2, catalase, and glutathione peroxidase (GPx) enzymatic activity. The effect of elevated SOD2 enzymatic activity on the radiation response of SA-NH cells was determined. Results: SOD2 activity was significantly elevated in selected tissues and a tumor 24 h after amifostine treatment. Catalase and GPx activities remained unchanged except for significant elevations in the spleen. GPx was also elevated in the pancreas. SA-NH tumor cells exhibited a twofold elevation in SOD2 activity and a 27% elevation in radiation resistance. Amifostine administered in three daily fractions of 50 mg/kg each also resulted in significant elevations of these antioxidant enzymes. Conclusions: Amifostine can induce a delayed radioprotective effect that correlates with elevated levels of SOD2 activity in SA-NH tumor. If limited to normal tissues, this delayed radioprotective effect offers an additional potential for overall radiation protection. However, amifostine-induced elevation of SOD2 activity in tumors could have an unanticipated deleterious effect on tumor responses to fractionated radiation therapy, given that the radioprotector is administered daily just before each 2-Gy fractionated dose.

  11. fMRI activation patterns in an analytic reasoning task: consistency with EEG source localization

    NASA Astrophysics Data System (ADS)

    Li, Bian; Vasanta, Kalyana C.; O'Boyle, Michael; Baker, Mary C.; Nutter, Brian; Mitra, Sunanda

    2010-03-01

    Functional magnetic resonance imaging (fMRI) is used to model brain activation patterns associated with various perceptual and cognitive processes as reflected by the hemodynamic (BOLD) response. While many sensory and motor tasks are associated with relatively simple activation patterns in localized regions, higher-order cognitive tasks may produce activity in many different brain areas involving complex neural circuitry. We applied a recently proposed probabilistic independent component analysis technique (PICA) to determine the true dimensionality of the fMRI data and used EEG localization to identify the common activated patterns (mapped as Brodmann areas) associated with a complex cognitive task like analytic reasoning. Our preliminary study suggests that a hybrid GLM/PICA analysis may reveal additional regions of activation (beyond simple GLM) that are consistent with electroencephalography (EEG) source localization patterns.

  12. Effect of salinity tolerant PDH45 transgenic rice on physicochemical properties, enzymatic activities and microbial communities of rhizosphere soils

    PubMed Central

    Sahoo, Ranjan Kumar; Tuteja, Narendra

    2013-01-01

    The effect of genetically modified (GM) plants on environment is now major concern worldwide. The plant roots of rhizosphere soil interact with variety of bacteria which could be influenced by the transgene in GM plants. The antibiotic resistance genes in GM plants may be transferred to soil microbes. In this study we have examined the effect of overexpression of salinity tolerant pea DNA helicase 45 (PDH45) gene on microbes and enzymatic activities in the rhizosphere soil of transgenic rice IR64 in presence and absence of salt stress in two different rhizospheric soils (New Delhi and Odisha, India). The diversity of the microbial community and soil enzymes viz., dehydrogenase, alkaline phosphatase, urease and nitrate reductase was assessed. The results revealed that there was no significant effect of transgene expression on rhizosphere soil of the rice plants. The isolated bacteria were phenotyped both in absence and presence of salt and no significant changes were found in their phenotypic characters as well as in their population. Overall, the overexpression of PDH45 in rice did not cause detectable changes in the microbial population, soil enzymatic activities and functional diversity of the rhizosphere soil microbial community. PMID:23733066

  13. Modulated expression and enzymatic activities of Darkbarbel catfish, Pelteobagrus vachelli for oxidative stress induced by acute hypoxia and reoxygenation.

    PubMed

    Zhang, Guosong; Mao, Jianqiang; Liang, Fenfei; Chen, Jiawei; Zhao, Cheng; Yin, Shaowu; Wang, Li; Tang, Zhonglin; Chen, Shuqiao

    2016-05-01

    Large changes in oxygen availability in aquatic environments, ranging from anoxia through to hyperoxia, can lead to corresponding wide variation in the production of reactive oxygen species (ROS) by fish with aquatic respiration. In order to evaluate the effects of hypoxia and reoxygenation on oxidative stress in fish, the mRNA and protein expression of SODs (Cu/Zn-SOD and Mn-SOD) as well as indices (CP, LPO and MDA) and enzymatic activities (SOD, CAT, GPx, GR and GST) were analyzed in liver and brain tissues of Pelteobagrus vachelli. Predominant expression of PvSOD2 was detected in heart, brain, and liver. In contrast, PvSOD1 was highly expressed in liver. Based on the expression patterns of above parameters, we inferred that brain tissue of P. vachelli under 0.7 mg/L degree of acute hypoxia condition could experience hypometabolic states or no suffering stress, but brain tissue has effective mechanisms to minimize or prevent oxidative stress during the transition from hypoxia to reoxygenation. Our results also demonstrated an increased expression of SODs and enzymatic activities for oxidative stress in liver under hypoxic conditions, which supports the hypothesis that anticipatory preparation takes place in order to deal with the encountered oxidative stress during the recovery from hypoxia as proposed by M. Hermes-Lima. Therefore, this study will provide a clue to better understand the action mode of antioxidant genes and enzymes under oxidative stress in fish. PMID:26945243

  14. Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay

    SciTech Connect

    Jean, F.; Basak, A.; Chretien, M.; Lazure, C. , Quebec )

    1991-05-01

    A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human {alpha} 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.

  15. A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

    PubMed Central

    Matsunaga, Satoko; Masaoka, Takashi; Sawasaki, Tatsuya; Morishita, Ryo; Iwatani, Yasumasa; Tatsumi, Masashi; Endo, Yaeta; Yamamoto, Naoki; Sugiura, Wataru; Ryo, Akihide

    2015-01-01

    Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1. PMID:26583013

  16. Chromophoric dissolved organic matter and microbial enzymatic activity. A biophysical approach to understand the marine carbon cycle.

    PubMed

    Gonnelli, Margherita; Vestri, Stefano; Santinelli, Chiara

    2013-12-01

    This study reports the first information on extracellular enzymatic activity (EEA) combined with a study of DOM dynamics at the Arno River mouth. DOM dynamics was investigated from both a quantitative (dissolved organic carbon, DOC) and a qualitative (absorption and fluorescence of chromophoric DOM, CDOM) perspective. The data here reported highlight that the Arno River was an important source of both DOC and CDOM for this coastal area. CDOM optical properties suggested that terrestrial DOM did not undergo simple dilution at the river mouth but, other physical-chemical and biological processes were probably at work to change its molecular characteristics. This observation was further supported by the "potential" enzymatic activity of β-glucosidase (BG) and leucine aminopeptidase (LAP). Their Vmax values were markedly higher in the river water than in the seawater and their ratio suggested that most of the DOM used by microbes in the Arno River was polysaccharide-like, while in the seawater it was mainly protein-like. PMID:23850176

  17. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    SciTech Connect

    Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.; Juliano, Maria A.; Hayashi, Mirian A.F.; Oliveira, Vitor

    2010-01-01

    In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

  18. Antioxidant activities and functional properties of enzymatic protein hydrolysates from defatted Camellia oleifera seed cake.

    PubMed

    Li, Xu; Deng, Junlin; Shen, Shian; Li, Tian; Yuan, Ming; Yang, Ruiwu; Ding, Chunbang

    2015-09-01

    Seed cake protein (SCP) from Camellia oleifera was hydrolyzed by five commercial proteases (Flavorzyme, Trypsin, Neutrase, Papain, Alcalase). Amino acid composition, molecular weight distribution, antioxidant activity and functional property of the seed cake protein hydrolysates (SCPH) were investigated. Enzymatic hydrolysis improved protein solubility significantly but impaired the foaming and emulsifying property. Hydrolysate generated by alcalase had the highest hydrolysis degree (DH) and antioxidant activity, and displayed excellent protein solubility over wide range of pH, while hydrolysate prepared by flavorzyme showed better copper chelating capacity and emulsifying stability with low molecular weight distribution. Trypsin-treated SCPH showed better foaming property than original protein. The results indicated that enzyme type greatly influenced the molecular weight, functional property and antioxidant activity of SCPH. It was also found that electing appropriate protease and controlling the DH could be enhanced or reduced functional property according to actual applications. PMID:26344981

  19. Effects of cerium oxide nanoparticles on soil enzymatic activities and wheat grass nutrients uptake

    NASA Astrophysics Data System (ADS)

    Li, Biting; Chen, Yirui; Bai, Lingyun; Jacobson, Astrid; Darnault, Christophe

    2015-04-01

    The US National Science Foundation estimated that the use of nanomaterials and nanotechnology would reach a global market value of 1 million this year. Concomitant with the wide applications of nanoparticles is an increasing risk of adverse effects to the environment and human health. As a common nanomaterial used as a fuel catalyst and polish material, cerium (IV) oxide nanoparticles (CeO2 NP) were tested for their potential impact on soil health and plant growth. Through exposure by air, water, and solid deposition, nanoparticles may accumulate in soils and impact agricultural systems. The objectives of this research were to determine whether CeO2 NPs affect the growth of wheat grass and selected soil enzyme activities chose as indicators of soil health. Wheat grass was grown in plant boxes containing CeO2 NPs mixed with agricultural soil at different concentrations. Two control groups were included: one consisting of soil with plants but no CeO2 NPs, and one containing only soil, i.e., no NP or wheat plants added. The plants were grown for 10 weeks and harvested every two weeks in a laboratory under sodium growth lights. At the end of the each growing period, two weeks, soils were assayed for phosphatase, β-glucosidase, and urease activities, and NPK values. Spectrophotometer analyses were used to assess enzyme activities, and NPK values were tested by Clemson Agricultural Center. Wheat yields were estimated by shoot and root lengths and weights.

  20. Consistency in boldness, activity and exploration at different stages of life

    PubMed Central

    2013-01-01

    Background Animals show consistent individual behavioural patterns over time and over situations. This phenomenon has been referred to as animal personality or behavioural syndromes. Little is known about consistency of animal personalities over entire life times. We investigated the repeatability of behaviour in common voles (Microtus arvalis) at different life stages, with different time intervals, and in different situations. Animals were tested using four behavioural tests in three experimental groups: 1. before and after maturation over three months, 2. twice as adults during one week, and 3. twice as adult animals over three months, which resembles a substantial part of their entire adult life span of several months. Results Different behaviours were correlated within and between tests and a cluster analysis showed three possible behavioural syndrome-axes, which we name boldness, exploration and activity. Activity and exploration behaviour in all tests was highly repeatable in adult animals tested over one week. In animals tested over maturation, exploration behaviour was consistent whereas activity was not. Voles that were tested as adults with a three-month interval showed the opposite pattern with stable activity but unstable exploration behaviour. Conclusions The consistency in behaviour over time suggests that common voles do express stable personality over short time. Over longer periods however, behaviour is more flexible and depending on life stage (i.e. tested before/after maturation or as adults) of the tested individual. Level of boldness or activity does not differ between tested groups and maintenance of variation in behavioural traits can therefore not be explained by expected future assets as reported in other studies. PMID:24314274

  1. A consistent muscle activation strategy underlies crawling and swimming in Caenorhabditis elegans

    PubMed Central

    Butler, Victoria J.; Branicky, Robyn; Yemini, Eviatar; Liewald, Jana F.; Gottschalk, Alexander; Kerr, Rex A.; Chklovskii, Dmitri B.; Schafer, William R.

    2015-01-01

    Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force–posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control. PMID:25551155

  2. A consistent muscle activation strategy underlies crawling and swimming in Caenorhabditis elegans.

    PubMed

    Butler, Victoria J; Branicky, Robyn; Yemini, Eviatar; Liewald, Jana F; Gottschalk, Alexander; Kerr, Rex A; Chklovskii, Dmitri B; Schafer, William R

    2015-01-01

    Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force-posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control. PMID:25551155

  3. Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: comparison of wild-type protein and active-site mutant D27E.

    PubMed

    Ohmae, Eiji; Miyashita, Yurina; Tate, Shin-Ichi; Gekko, Kunihiko; Kitazawa, Soichiro; Kitahara, Ryo; Kuwajima, Kunihiro

    2013-12-01

    To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl2 concentration-dependence of the (1)H-(15)N HSQC spectra of the wild-type DHFR-folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased Km and Kd values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250MPa together with negative activation volumes of -4.0 or -4.8mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme. PMID:24140567

  4. Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

    PubMed Central

    Balasingham, Seetha V.; Zegeye, Ephrem Debebe; Homberset, Håvard; Rossi, Marie L.; Laerdahl, Jon K.; Bohr, Vilhelm A.; Tønjum, Tone

    2012-01-01

    XPB, also known as ERCC3 and RAD25, is a 3′→5′ DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3′→5′ DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg2+/Mn2+. Consistent with the 3′→5′ polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3′ overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3′ DNA tail, it was not active on substrates containing a 3′ RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB. PMID:22615856

  5. [Isolation of wood-decaying fungi and evaluation of their enzymatic activity (Quindío, Colombia)].

    PubMed

    Chaparro, Deisy Fernanda; Rosas, Diana Carolina; Varela, Amanda

    2009-12-31

    White rot fungi (Ascomycota and Basidiomycota) were collected on fallen trunks with different decay stages, in a subandean forest (La Montaña del Ocaso nature reserve), and it was evaluated their ligninolitic activity. They were cultured on malt extract agar. Then it was performed semiquantitative tests for laccase and cellobiose dehydrogenase (CDH) activity using ABTS and DCPIP as enzymatic inducers. Based on the results of these tests, the fungi with higher activities from trunks with different decay stages were selected: Cookeina sulcipes (for stage 1), a fungus from the family Corticiaceae (for stage 2), Xylaria polymorpha (for stage 3) and Earliella sp. (for stage 4). A fermentation was performed at 28 degrees C, during 11 days, in a rotatory shaker at 150 rpm. Biomass, glucose, proteins and enzyme activities measurements were performed daily. The fungi that were in the trunks with decay states from 1 to 3, showed higher laccase activity as the state of decay increased. A higher DCH activity was also associated with a higher. Also, there was a positive relationship between both enzymes' activities. Erliella was the fungus which presented the highest biomass production (1140,19 g/l), laccase activity (157 UL(-1)) and CDH activity (43,50 UL(-1)). This work is the first report of laccase and CDH activity for Cookeina sulcipes and Earliella sp. Moreover, it gives basis for the use of these native fungi in biotechnological applications and the acknowledgment of their function in the wood decay process in native forest. PMID:19796977

  6. Second-Order Perturbation Theory for Generalized Active Space Self-Consistent-Field Wave Functions.

    PubMed

    Ma, Dongxia; Li Manni, Giovanni; Olsen, Jeppe; Gagliardi, Laura

    2016-07-12

    A multireference second-order perturbation theory approach based on the generalized active space self-consistent-field (GASSCF) wave function is presented. Compared with the complete active space (CAS) and restricted active space (RAS) wave functions, GAS wave functions are more flexible and can employ larger active spaces and/or different truncations of the configuration interaction expansion. With GASSCF, one can explore chemical systems that are not affordable with either CASSCF or RASSCF. Perturbation theory to second order on top of GAS wave functions (GASPT2) has been implemented to recover the remaining electron correlation. The method has been benchmarked by computing the chromium dimer ground-state potential energy curve. These calculations show that GASPT2 gives results similar to CASPT2 even with a configuration interaction expansion much smaller than the corresponding CAS expansion. PMID:27276688

  7. Enzymatic and microbiological inhibitory activity in eggshell membranes as influenced by layer strains and age and storage variables.

    PubMed

    Ahlborn, G; Sheldon, B W

    2005-12-01

    Eggshell membranes (ESM) have been shown to exhibit antibacterial activity. The purpose of this study was to evaluate the enzymatic and biological [decimal reduction times (D-values)] activities of ESM as a function of bird breed, age, and ESM stabilization treatments. Younger White Leghorn (WL) hens produced ESM with 28% higher lysozyme activity than Rhode Island Red (RIR) layers. In contrast, older WL layers produced ESM with 17% less lysozyme activity than ESM from RIR layers. Similarly, beta-N-acetylglucosaminidase (beta-NAGase) ESM activities differed by hen age within breeds with younger hens yielding 14 to 16% more enzyme activity. D54 degrees C-values of Salmonella Typhimurium cells preexposed to WL ESM did not differ as a function of bird age (33, 50, and 81 wk). The ESM Lysozyme and beta-NAGase activities varied somewhat over a 6-mo storage study after treatment with 1 of 5 stabilization methods [i.e., storage at 4 degrees C, -20 degrees C, or ambient air storage after freeze drying, air drying (23 degrees C), or forced-air drying (50 degrees C)]. Both air and forced-air drying yielded significant reductions in beta-NAGase and lysozyme ESM activity (ca 12 to 30%) after the initial 24 h and then remained fairly stable during the extended storage. Freeze-dried samples retained the most enzymatic activity (95%) throughout the 6-mo trial, whereas refrigerated ESM lost 20 and 18% of the beta-NAGase and lysozyme activities, respectively. Frozen ESM lost 22% of the beta-NAGase activity, whereas lysozyme was nearly unaffected after 6 mo. The ESM biological activities against S. Typhimurium were not adversely impacted by layer breed or age. No significant loss in biological activity of ESM was detected 24 h after processing or after 6 mo of storage for refrigerated, frozen, and freeze-dried membranes, whereas significant reductions were observed for air- and heat-dried ESM. These findings demonstrate that ESM enzyme and biological activities are relatively

  8. Thiobarbituric acid reactive substances, Fe3+ reduction and enzymatic activities in cultures of Ganoderma australe growing on Drimys winteri wood.

    PubMed

    Elissetche, Juan-Pedro; Ferraz, André; Freer, Juanita; Mendonça, Régis; Rodríguez, Jaime

    2006-07-01

    Ganoderma australe is a basidiomycete responsible for a natural process of selective and extensive lignin degradation. Fatty acids, thiobarbituric acid reactive substances (TBARS), Fe3+-reduction and enzymatic activities were monitored in cultures of G. australe growing on Drimys winteri wood chips. Linoleic acid was de novo synthesized, and steadily increased during 12 weeks of cultivation. Part of the unsaturated fatty acids underwent peroxidation as TBARS accumulated with biodegradation time. TBARS accumulation was proportional to the wood weight and component losses. Manganese-dependent peroxidase and lignin peroxidase were not detected in the culture extracts, whereas laccase-induced oxidation of syringaldazine peaked after 2 weeks (104+/-9 micromol oxidized min(-1) kg(-1) of dry wood), subsequently decreasing. On the other hand, nonenzymatic Fe3+-reducing activity increased as a function of cultivation time and could be involved in the initiation of lipid peroxidation. PMID:16790026

  9. New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities.

    PubMed

    Moroz, Yurii S; Dunston, Tiffany T; Makhlynets, Olga V; Moroz, Olesia V; Wu, Yibing; Yoon, Jennifer H; Olsen, Alissa B; McLaughlin, Jaclyn M; Mack, Korrie L; Gosavi, Pallavi M; van Nuland, Nico A J; Korendovych, Ivan V

    2015-12-01

    Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution. PMID:26555770

  10. Enzymatic Methylation and Structure-Activity-Relationship Studies on Polycarcin V, a Gilvocarcin-Type Antitumor Agent

    PubMed Central

    Chen, Jhong-Min; Shepherd, Micah D.; Horn, Jamie; Leggas, Markos; Rohr, Jürgen

    2014-01-01

    Polycarcin V, a polyketide natural product of Streptomyces polyformus, was chosen to study structure-activity-relationships of the gilvocarcin group of antitumor antibiotics, because of a similar chemical structure and comparable bioactivity with gilvocarcin V, the principle compound of this group, and the feasibility of enzymatic modifications of its sugar moiety by auxiliary O-methyltransferases. Such enzymes were used to modify the interaction of the drug with histone H3, the biological target that interacts with the sugar moiety. Cytotoxicity assays revealed that a free 2’-OH group of the sugar moiety is essential to maintain the bioactivity of polycarcin V, apparently an important H-bond donor for the interaction with histone H3, while converting 3'-OH into an OCH3 group improved the bioactivity. Bis-methylated polycarcin derivatives revealed weaker activity than the parent compound, indicating that at least two H-bond donors in the sugar are necessary for optimal binding. PMID:25366963

  11. Monoclonal antibodies raised against 167-180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity.

    PubMed

    Dekaminaviciute, Dovile; Kairys, Visvaldas; Zilnyte, Milda; Petrikaite, Vilma; Jogaite, Vaida; Matuliene, Jurgita; Gudleviciene, Zivile; Vullo, Daniela; Supuran, Claudiu T; Zvirbliene, Aurelija

    2014-12-01

    Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application. PMID:24400872

  12. Childhood Sexual Violence and Consistent, Effective Contraception Use among Young, Sexually Active Urban Women

    PubMed Central

    Nelson, Deborah B.; Lepore, Stephen J.; Mastrogiannis, Dimitrios S.

    2015-01-01

    Unintended pregnancy (UP) is a significant public health problem. The consistent use of effective contraception is the primary method to prevent UP. We examined the role of childhood sexual and physical violence and current interpersonal violence on the risk of unintended pregnancy among young, urban, sexually active women. In particular, we were interested in examining the role of childhood violence and interpersonal violence while recognizing the psychological correlates of experiencing violence (i.e., high depressive symptoms and low self-esteem) and consistent use of contraception. For this assessment, 315 sexually active women living in Philadelphia PA were recruited from family planning clinics in 2013. A self-administered, computer-assisted interview was used to collect data on method of contraception use in the past month, consistency of use, experiences with violence, levels of depressive symptoms, self-esteem and sexual self-efficacy, substance use and health services utilization. Fifty percent of young sexually active women reported inconsistent or no contraception use in the past month. Inconsistent users were significantly more likely to report at least one prior episode of childhood sexual violence and were significantly less likely to have received a prescription for contraception from a health care provider. Inconsistent contraception users also reported significantly higher levels of depressive symptoms and significantly lower levels of self-esteem. The relation between childhood sexual violence and UP remained unchanged in the multivariate models adjusting for self-esteem or depressive symptoms. These findings highlight the long-term consequences of childhood sexual violence, independent of current depressive symptoms and low self-esteem, on consistent use of contraception. PMID:26010318

  13. Evolution of Enzymatic Activities in the Enolase Superfamily: D-Tartrate Dehydratase from Bradyrhizobium japonicum

    SciTech Connect

    Yew,W.; Fedorov, A.; Fedorov, E.; Wood, B.; Almo, S.; Gerlt, J.

    2006-01-01

    We focus on the assignment of function to and elucidation of structure-function relationships for a member of the mechanistically diverse enolase superfamily encoded by the Bradyrhizobium japonicum genome (bll6730; GI:27381841). As suggested by sequence alignments, the active site contains the same functional groups found in the active site of mandelate racemase (MR) that catalyzes a 1,1-proton transfer reaction: two acid/base catalysts, Lys 184 at the end of the second {beta}-strand, and a His 322-Asp 292 dyad at the ends of the seventh and sixth -strands, respectively, as well as ligands for an essential Mg{sup 2+}, Asp 213, Glu 239, and Glu 265 at the ends of the third, fourth, and fifth {beta}-strands, respectively. We screened a library of 46 acid sugars and discovered that only D-tartrate is dehydrated, yielding oxaloacetate as product. The kinetic constants (k{sub cat} = 7.3 s{sup -1}; k{sub cat}/K{sub M} = 8.5 x 10{sup 4} M{sup -1} s{sup -1}) are consistent with assignment of the D-tartrate dehydratase (TarD) function. The kinetic phenotypes of mutants as well as the structures of liganded complexes are consistent with a mechanism in which Lys 184 initiates the reaction by abstraction of the {alpha}-proton to generate a Mg{sup 2+}-stabilized enediolate intermediate, and the vinylogous -elimination of the 3-OH group is general acid-catalyzed by the His 322, accomplishing the anti-elimination of water. The replacement of the leaving group by solvent-derived hydrogen is stereorandom, suggesting that the enol tautomer of oxaloacetate is the product; this expectation was confirmed by its observation by {sup 1}H NMR spectroscopy. Thus, the TarD-catalyzed reaction is a 'simple' extension of the two-step reaction catalyzed by MR: base-catalyzed proton abstraction to generate a Mg{sup 2+}-stabilized enediolate intermediate followed by acid-catalyzed decomposition of that intermediate to yield the product.

  14. A two-parameter kinetic model based on a time-dependent activity coefficient accurately describes enzymatic cellulose digestion

    PubMed Central

    Kostylev, Maxim; Wilson, David

    2014-01-01

    Lignocellulosic biomass is a potential source of renewable, low-carbon-footprint liquid fuels. Biomass recalcitrance and enzyme cost are key challenges associated with the large-scale production of cellulosic fuel. Kinetic modeling of enzymatic cellulose digestion has been complicated by the heterogeneous nature of the substrate and by the fact that a true steady state cannot be attained. We present a two-parameter kinetic model based on the Michaelis-Menten scheme (Michaelis L and Menten ML. (1913) Biochem Z 49:333–369), but with a time-dependent activity coefficient analogous to fractal-like kinetics formulated by Kopelman (Kopelman R. (1988) Science 241:1620–1626). We provide a mathematical derivation and experimental support to show that one of the parameters is a total activity coefficient and the other is an intrinsic constant that reflects the ability of the cellulases to overcome substrate recalcitrance. The model is applicable to individual cellulases and their mixtures at low-to-medium enzyme loads. Using biomass degrading enzymes from a cellulolytic bacterium Thermobifida fusca we show that the model can be used for mechanistic studies of enzymatic cellulose digestion. We also demonstrate that it applies to the crude supernatant of the widely studied cellulolytic fungus Trichoderma reesei and can thus be used to compare cellulases from different organisms. The two parameters may serve a similar role to Vmax, KM, and kcat in classical kinetics. A similar approach may be applicable to other enzymes with heterogeneous substrates and where a steady state is not achievable. PMID:23837567

  15. States Lack Physical Activity Policies in Child Care That Are Consistent with National Recommendations

    PubMed Central

    Slining, Meghan M.; Benjamin Neelon, Sara E.

    2014-01-01

    Abstract Background: Child care facilities' policies can importantly impact health behaviors of toddlers and preschoolers. Our aim was to assess state regulations promoting physical activity (PA) in child care and compare regulations to national recommendations. Methods: We reviewed licensing and administrative regulations related to promoting PA for all states and territories for child care centers (centers) and family child care homes (homes). Three reviewers searched two sources (a publically available website and WestlawNext™) and compared regulations with 15 Institute of Medicine recommendations. We used Pearson's and Spearman's correlations to assess associations between geographic region, year of last update, and number of regulations consistent with the recommendations. Results: The average number and range of regulations in centers and homes was 4.1 (standard deviation [SD], 1.4; range, 0–8) and 3.8 (SD, 1.5; range, 0–7), respectively. Nearly all states had regulations consistent with providing an outdoor (centers, 98%; homes, 95%) and indoor (centers, 94%, homes, 92%) environment “with a variety of portable play equipment and adequate space.” No state had regulations for staff joining children, avoiding punishment for being physically active, yearly consultation from a PA expert, or providing training/education on PA for providers. Conclusions: There is room for improvement in child care regulations around PA for young children; PA promotion should be included with future updates to regulations. PMID:25354331

  16. Evidence consistent with the requirement of cresolase activity for suicide inactivation of tyrosinase.

    PubMed

    Land, Edward J; Ramsden, Christopher A; Riley, Patrick A; Stratford, Michael R L

    2008-11-01

    Tyrosinase is a mono-oxygenase with a dinuclear copper catalytic center which is able to catalyze both the ortho-hydroxylation of monophenols (cresolase activity) and the oxidation of catechols (catecholase activity) yielding ortho-quinone products. Tyrosinases appear to have arisen early in evolution and are widespread in living organisms where they are involved in several processes, including antibiosis, adhesion of molluscs, the hardening of the exoskeleton of insects, and pigmentation. Tyrosinase is the principal enzyme of melanin formation in vertebrates and is of clinical interest because of the possible utilization of its activity for targeted treatment of malignant melanoma. Tyrosinase is characterised by an irreversible inactivation that occurs during the oxidation of catechols. In a recent publication we proposed a mechanism to account for this feature based on the ortho-hydroxylation of catecholic substrates, during which process Cu(II) is reduced to Cu(0) which no longer binds to the enzyme and is eliminated (reductive elimination). Since this process is dependent on cresolase activity of tyrosinase, a strong prediction of the proposed inactivation mechanism is that it will not be exhibited by enzymes lacking cresolase activity. We show that the catechol oxidase readily extracted from bananas (Musa cavendishii) is devoid of cresolase activity and that the kinetics of catechol oxidation do not exhibit inactivation. We also show that a species with the molecular mass of the putative cresolase oxidation product is formed during tyrosinase oxidation of 4-methylcatechol. The results presented are entirely consistent with our proposed mechanism to account for suicide-inactivation of tyrosinase. PMID:18987457

  17. Lactogenic Activity of an Enzymatic Hydrolysate from Octopus vulgaris and Carica papaya in SD Rats.

    PubMed

    Cai, Bingna; Chen, Hua; Sun, Han; Sun, Huili; Wan, Peng; Chen, Deke; Pan, Jianyu

    2015-11-01

    The traditional Chinese medicine theory believes that octopus papaya soup can stimulate milk production in lactating women. The objective of this study was to determine whether dietary supplementation with an enzymatic hydrolysate of Octopus vulgaris and Carica papaya (EHOC) could increase milk production and nutritional indexes in Sprague Dawley (SD) rats. Female SD rats (n = 24) were fed a control diet (n = 8), EHOC-supplemented diet, or a positive control diet (Shengruzhi) from day 10 of pregnancy to day 10 of lactation. Maternal serum, mammary gland (day 10 of lactation), milk, and pup weight (daily) were collected for analysis. Results showed that the EHOC diet obviously elevated daily milk yield and pup weight compared to the control group (P < .05). The EHOC diet was found to increase the concentration of prolactin (PRL), progesterone (P), estradiol (E2), and growth hormone (GH) significantly in the circulation and mammary gland. Mammary glands of EHOC-treated dams showed clear lobuloalveolar development and proliferation of myoepithelial cells, but no striking variations were observed among the groups. Furthermore, the nutrition content and immune globulin concentration in the milk of EHOC-supplemented dams were higher than those of the control group, especially the cholesterol, glucose, and IgG were higher by 44.98% (P < .001), 42.76% (P < .01), and 42.23% (P < .01), respectively. In conclusion, this article demonstrates that EHOC administration has beneficial effects on milk production in the dams and on performance of the dam and pup. These results indicate that EHOC could be explored as a potentially lactogenic nutriment for lactating women. PMID:26270883

  18. Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats

    PubMed Central

    NISHIYAMA, Yoshihiro; NAKAYAMA, Shouta M.M.; WATANABE, Kensuke P.; KAWAI, Yusuke K.; OHNO, Marumi; IKENAKA, Yoshinori; ISHIZUKA, Mayumi

    2016-01-01

    Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies. PMID:26806536

  19. Identification of an Iron-Sulfur Cluster That Modulates the Enzymatic Activity in NarE, a Neisseria meningitidis ADP-ribosyltransferase*

    PubMed Central

    Del Vecchio, Mariangela; Pogni, Rebecca; Baratto, Maria Camilla; Nobbs, Angela; Rappuoli, Rino; Pizza, Mariagrazia; Balducci, Enrico

    2009-01-01

    In prokaryotes, mono-ADP-ribose transfer enzymes represent a family of exotoxins that display activity in a variety of bacterial pathogens responsible for causing disease in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report here that NarE, a putative ADP-ribosylating toxin previously identified from Neisseria meningitidis, which shares structural homologies with Escherichia coli heat labile enterotoxin and toxin from Vibrio cholerae, possesses an iron-sulfur center. The recombinant protein was expressed in E. coli, and when purified at high concentration, NarE is a distinctive golden brown in color. Evidence from UV-visible spectrophotometry and EPR spectroscopy revealed characteristics consistent of an iron-binding protein. The presence of iron was determined by colorimetric method and by an atomic absorption spectrophotometer. To identify the amino acids involved in binding iron, a combination of site-directed mutagenesis and UV-visible and enzymatic assays were performed. All four cysteine residues were individually replaced by serine. Substitution of Cys67 and Cys128 into serine caused a drastic reduction in the E420/E280 ratio, suggesting that these two residues are essential for the formation of a stable coordination. This modification led to a consistent loss in ADP-ribosyltransferase activity, while decrease in NAD-glycohydrolase activity was less dramatic in these mutants, indicating that the correct assembly of the iron-binding site is essential for transferase but not hydrolase activity. This is the first observation suggesting that a member of the ADP-ribosyltransferase family contains an Fe-S cluster implicated in catalysis. This observation may unravel novel functions exerted by this class of enzymes. PMID:19744927

  20. Identification of an iron-sulfur cluster that modulates the enzymatic activity in NarE, a Neisseria meningitidis ADP-ribosyltransferase.

    PubMed

    Del Vecchio, Mariangela; Pogni, Rebecca; Baratto, Maria Camilla; Nobbs, Angela; Rappuoli, Rino; Pizza, Mariagrazia; Balducci, Enrico

    2009-11-27

    In prokaryotes, mono-ADP-ribose transfer enzymes represent a family of exotoxins that display activity in a variety of bacterial pathogens responsible for causing disease in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report here that NarE, a putative ADP-ribosylating toxin previously identified from Neisseria meningitidis, which shares structural homologies with Escherichia coli heat labile enterotoxin and toxin from Vibrio cholerae, possesses an iron-sulfur center. The recombinant protein was expressed in E. coli, and when purified at high concentration, NarE is a distinctive golden brown in color. Evidence from UV-visible spectrophotometry and EPR spectroscopy revealed characteristics consistent of an iron-binding protein. The presence of iron was determined by colorimetric method and by an atomic absorption spectrophotometer. To identify the amino acids involved in binding iron, a combination of site-directed mutagenesis and UV-visible and enzymatic assays were performed. All four cysteine residues were individually replaced by serine. Substitution of Cys(67) and Cys(128) into serine caused a drastic reduction in the E(420)/E(280) ratio, suggesting that these two residues are essential for the formation of a stable coordination. This modification led to a consistent loss in ADP-ribosyltransferase activity, while decrease in NAD-glycohydrolase activity was less dramatic in these mutants, indicating that the correct assembly of the iron-binding site is essential for transferase but not hydrolase activity. This is the first observation suggesting that a member of the ADP-ribosyltransferase family contains an Fe-S cluster implicated in catalysis. This observation may unravel novel functions exerted by this class of enzymes. PMID:19744927

  1. Dynamics of microbiological parameters, enzymatic activities and worm biomass production during vermicomposting of effluent treatment plant sludge of bakery industry.

    PubMed

    Yadav, Anoop; Suthar, S; Garg, V K

    2015-10-01

    This paper reports the changes in microbial parameters and enzymatic activities during vermicomposting of effluent treatment plant sludge (ETPS) of bakery industry spiked with cow dung (CD) by Eisenia fetida. Six vermibins containing different ratios of ETPS and CD were maintained under controlled laboratory conditions for 15 weeks. Total bacterial and total fungal count increased upto 7th week and declined afterward in all the bins. Maximum bacterial and fungal count was 31.6 CFU × 10(6) g(-1) and 31 CFU × 10(4) g(-1) in 7th week. Maximum dehydrogenase activity was 1921 μg TPF g(-1) h(-1) in 9th week in 100 % CD containing vermibin, whereas maximum urease activity was 1208 μg NH4 (-)N g(-1) h(-1) in 3rd week in 100 % CD containing vermibin. The enzyme activity and microbial counts were lesser in ETPS containing vermibins than control (100 % CD). The growth and fecundity of the worms in different vermibins were also investigated. The results showed that initially biomass and fecundity of the worms increased but decreased at the later stages due to non-availability of the palatable feed. This showed that quality and palatability of food directly affect biological parameters of the system. PMID:25982984

  2. Regulation of MnSOD Enzymatic Activity by Sirt3 Connects the Mitochondrial Acetylome Signaling Networks to Aging and Carcinogenesis

    PubMed Central

    Tao, Randa; Vassilopoulos, Athanassios; Parisiadou, Loukia; Yan, Yufan

    2014-01-01

    Abstract Significance: It is a well-established scientific observation that mammalian cells contain fidelity or watchdog proteins that maintain the correct function of cellular organelles. Recent Advances: Over the past several years, the Sirtuin deacetylase family protein Sirt3 has emerged as a mitochondrial fidelity protein that directs energy generation and regulates reactive oxygen species (ROS) scavenging proteins. Loss of function or genetic mutation of these fidelity proteins has been shown to create a cellular environment that is permissive for the development of cellular damage associated with processes such as aging and carcinogenesis. Critical Issues: Mitochondria are the primary organelles that direct oxidative metabolism for the production of ATP; however, this is also a significant source of ROS. Thus, it is reasonable to propose that mitochondria should contain proteins that would signal downstream target molecules and/or ROS scavenger enzymes to maintain mitochondrial and cellular homeostatic poise. It is also reasonable to hypothesize that the mitochondria contain fidelity proteins similar to those found in the nucleus and cytoplasm. We discuss a new role of Sirt3 in the direction of the primary superoxide scavenger protein, manganese superoxide dismutase (MnSOD), and how the acetylation or deacetylation of several specific lysines appears to direct MnSOD enzymatic dismutase activity. Future Directions: Aberrant downstream regulation of MnSOD by Sirt3 may be a potential source of cellular damage that accumulates with aging to create a tumor-permissive phenotype. Future studies can explore the role of MnSOD in age-related illness using this new mechanism of enzymatic regulation. Antioxid. Redox Signal. 20, 1646–1654 PMID:23886445

  3. An ethoxylated alkyl phosphate (anionic surfactant) for the promotion of activities of proteases and its potential use in the enzymatic processing of wool.

    PubMed

    Zhang, Qinghua; Smith, Edward; Shen, Jinsong; Bishop, David

    2006-05-01

    Pretreatments of wool fabrics with cationic, anionic or non-ionic surfactants were investigated to reduce surface tension and improve the wettability of the fibres in order to promote protease activity on the fibres in subsequent processes. Results showed that an ethoxylated alkyl phosphate (specific anionic surfactant) as well as the widely used non-ionic surfactant was compatible with proteases in the enzymatic treatment of wool. There is therefore a potential for using specific anionic surfactants to achieve efficient enzymatic scouring processes. PMID:16791726

  4. Longitudinal changes in PON1 enzymatic activities in Mexican-American mothers and children with different genotypes and haplotypes

    SciTech Connect

    Huen, Karen; Harley, Kim; Bradman, Asa; Eskenazi, Brenda; Holland, Nina

    2010-04-15

    The paraoxonase 1 (PON1) enzyme prevents low-density lipoprotein oxidation and also detoxifies the oxon derivatives of certain neurotoxic organophosphate (OP) pesticides. PON1 activity in infants is low compared to adults, rendering them with lower metabolic and antioxidant capacities. We made a longitudinal comparison of the role of genetic variability on control of PON1 phenotypes in Mexican-American mothers and their children at the time of delivery (n = 388 and 338, respectively) and again 7 years later (n = 280 and 281, respectively) using generalized estimating equations models. At age 7, children's mean PON1 activities were still lower than those of mothers. This difference was larger in children with genotypes associated with low PON1 activities (PON1{sub -108TT}, PON1{sub 192QQ}, and PON1{sub -909CC}). In mothers, PON1 activities were elevated at delivery and during pregnancy compared to 7 years later when they were not pregnant (p < 0.001). In non-pregnant mothers, PON1 polymorphisms and haplotypes accounted for almost 2-fold more variation of arylesterase (AREase) and chlorpyrifos-oxonase (CPOase) activity than in mothers at delivery. In both mothers and children, the five PON1 polymorphisms (192, 55, -108, -909, -162) explained a noticeably larger proportion of variance of paraoxonase activity (62-78%) than AREase activity (12.3-26.6%). Genetic control of PON1 enzymatic activity varies in children compared to adults and is also affected by pregnancy status. In addition to known PON1 polymorphisms, unidentified environmental, genetic, or epigenetic factors may also influence variability of PON1 expression and therefore susceptibility to OPs and oxidative stress.

  5. Self-consistent simulation of CdTe solar cells with active defects

    SciTech Connect

    Brinkman, Daniel; Guo, Da; Akis, Richard; Ringhofer, Christian; Sankin, Igor; Fang, Tian; Vasileska, Dragica

    2015-07-21

    We demonstrate a self-consistent numerical scheme for simulating an electronic device which contains active defects. As a specific case, we consider copper defects in cadmium telluride solar cells. The presence of copper has been shown experimentally to play a crucial role in predicting device performance. The primary source of this copper is migration away from the back contact during annealing, which likely occurs predominantly along grain boundaries. We introduce a mathematical scheme for simulating this effect in 2D and explain the numerical implementation of the system. Lastly, we will give numerical results comparing our results to known 1D simulations to demonstrate the accuracy of the solver and then show results unique to the 2D case.

  6. Self-consistent simulation of CdTe solar cells with active defects

    SciTech Connect

    Brinkman, Daniel; Ringhofer, Christian; Guo, Da; Akis, Richard; Vasileska, Dragica; Sankin, Igor; Fang, Tian

    2015-07-21

    We demonstrate a self-consistent numerical scheme for simulating an electronic device which contains active defects. As a specific case, we consider copper defects in cadmium telluride solar cells. The presence of copper has been shown experimentally to play a crucial role in predicting device performance. The primary source of this copper is migration away from the back contact during annealing, which likely occurs predominantly along grain boundaries. We introduce a mathematical scheme for simulating this effect in 2D and explain the numerical implementation of the system. Finally, we will give numerical results comparing our results to known 1D simulations to demonstrate the accuracy of the solver and then show results unique to the 2D case.

  7. Self-consistent simulation of CdTe solar cells with active defects

    DOE PAGESBeta

    Brinkman, Daniel; Guo, Da; Akis, Richard; Ringhofer, Christian; Sankin, Igor; Fang, Tian; Vasileska, Dragica

    2015-07-21

    We demonstrate a self-consistent numerical scheme for simulating an electronic device which contains active defects. As a specific case, we consider copper defects in cadmium telluride solar cells. The presence of copper has been shown experimentally to play a crucial role in predicting device performance. The primary source of this copper is migration away from the back contact during annealing, which likely occurs predominantly along grain boundaries. We introduce a mathematical scheme for simulating this effect in 2D and explain the numerical implementation of the system. Lastly, we will give numerical results comparing our results to known 1D simulations tomore » demonstrate the accuracy of the solver and then show results unique to the 2D case.« less

  8. Conformational Changes in a Hyperthermostable Glycoside Hydrolase: Enzymatic Activity Is a Consequence of the Loop Dynamics and Protonation Balance

    PubMed Central

    de Oliveira, Leandro C.; da Silva, Viviam M.; Colussi, Francieli; Cabral, Aline D.; de Oliveira Neto, Mario; Squina, Fabio M.; Garcia, Wanius

    2015-01-01

    Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features. PMID:25723179

  9. Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance.

    PubMed

    de Oliveira, Leandro C; da Silva, Viviam M; Colussi, Francieli; Cabral, Aline D; de Oliveira Neto, Mario; Squina, Fabio M; Garcia, Wanius

    2015-01-01

    Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features. PMID:25723179

  10. A new generation of flowerlike horseradish peroxides as a nanobiocatalyst for superior enzymatic activity.

    PubMed

    Ocsoy, Ismail; Dogru, Esra; Usta, Seyda

    2015-01-01

    Although various supports including nanomaterials have been widely utilized as platforms for enzymes immobilization in order to enhance their catalytic activities, most of immobilized enzymes exhibited reduced activities compared to free enzymes. In this study, for the first time, we used iron ions (Fe(2+)) and horseradish peroxidase (HRP) enzyme together to synthesize flowerlike hybrid nanostructures with greatly enhanced activity and stability and reported an explanation of the enhancements in both catalytic activity and stability. We demonstrated that Fe(2+)-HRP hybrid nanoflower (HNF) showed catalytic activity of ∼ 512% and ∼ 710%, respectively when stored at +4 °C and room temperature (RT = 20 °C) compared to free HRP. In addition, the HNF stored at +4 °C lost only 2.9% of its original activity within 30 days while the HNF stored at RT lost approximately 10% of its original activity. However, under the same conditions, free HRP enzymes stored at +4 °C and RT lost 68% and 91% of their activities, respectively. We claim that the drastic increases in activities of HNF are associated with to high local HRP concentration in nanoscale dimension, appropriate HRP conformation, less mass transfer limitations, and role of Fe(2+) ion as an activator for HRP. Further biosensors studies based on enhanced activity and stability of HNF are currently underway. PMID:26047912

  11. Synthetic substrates specific to activated plasmin can monitor the enzymatic functional status in situ in breast cancer cells.

    PubMed

    Gohda, Keigo; Fujimori, Ko; Teno, Naoki; Wanaka, Keiko; Tsuda, Yuko

    2014-01-01

    We here strove to overcome the limitations of expression analyses such as PCR and IHC, based on molecular recognition between target and probe molecules, by designing synthetic substrates specific to the target molecules to directly estimate the enzymatic functionality in situ. The specific substrate contains a probing unit, which is an organic fragment for specific enzyme binding, and a reactive unit, which is a natural peptide subject to catalysis. In this study, the activation of plasminogen to plasmin was examined in MDA-MB231 breast cancer cells using the plasmin-specific synthetic substrates designed from their inhibitors. The localization and function of the activated plasmin were successfully visualized by fluorophore combined with the specific substrate concurrently. This would be the first time for activated plasmin at work in situ by direct observation. Our concept to directly monitor the functionality of target enzymes can be used straightforwardly for other proteases such as cathepsins or caspases. Also, this substrate concept as a 'tailor-made substrate' would be utilized as a novel functional molecular probe in vivo with appropriate detectable probes. PMID:24112688

  12. Plasmon-Enhanced Enzymatic Reactions 2:Optimization of Enzyme Activity by Surface Modification of Silver Island Films with Biotin-Poly (Ethylene-glycol)-Amine.

    PubMed

    Abel, Biebele; Aslan, Kadir

    2012-01-01

    Surface modification of silver island films (SIFs) was carried out with Biotin-Poly (Ethylene-glycol)-Amine (BEA), which acts as a cross-linker between the silver surface and horse radish peroxidase (HRP) enzyme for optimum plasmon-enhanced enzymatic activity. SIFs-deposited blank glass slides and SIFs-deposited 3-Aminopropyltriethoxysilane(APTES)-coated glass slides were used as our plasmonic surfaces.In this regard, three different extent of loading of SIFs were also prepared (low, medium and high) on APTES-coated glass slides. Streptavidin-linked HRP enzyme was attached to SIFs-deposited blank glass slides and SIFs-deposited APTES-coated glass slides through the well-known biotin-streptavidin interactions. The characterization of these surfaces was done using optical absorption spectroscopy. The loading of SIFs on glass slides was observed to have significant effect on the efficiency of plasmon-enhanced enzymatic activity, where an enhancement of 200% in the enzymatic activity was observed when compared to our previously used strategies for enzyme immobilization in our preceding work[1]. In addition, SIFs-deposited on APTES-coated glass slides were found to be re-usable for plasmon-enhanced enzymatic reactions unlike SIFs deposited on to blank glass slides. PMID:22485194

  13. Aldehyde PEGylation of laccase from Trametes versicolor in route to increase its stability: effect on enzymatic activity.

    PubMed

    Mayolo-Deloisa, Karla; González-González, Mirna; Simental-Martínez, Jesús; Rito-Palomares, Marco

    2015-03-01

    Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can be used in bioremediation, beverage (wine, fruit juice, and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking, and as a biosensor. Recently, the antiproliferative activity of laccase toward tumor cells has been reported. Because of the potential applications of this enzyme, the efforts for enhancing and stabilizing its activity have increased. Thus, the PEGylation of laccase can be an alternative. PEGylation is the covalent attachment of one or more molecules of methoxy poly(ethylene glycol) (mPEG) to a protein. Normally, during the PEGylation reaction, the activity is reduced but the stability increases; thus, it is important to minimize the loss of activity. In this work, the effects of molar ratio (1:4, 1:8, and 1:12), concentration of laccase (6 and 12 mg/ml), reaction time (4 and 17 h), molecular weight, and type of mPEG (20, 30, 40 kDa and 40 kDa-branched) were analyzed. The activity was measured using three substrates: ABTS, 2,6-dimethoxyphenol, and syringaldazine. The best conditions for laccase PEGylation were 12 mg/ml of laccase, molar ratio 1:4, and 4 h reaction time. Under these conditions, the enzyme was able to maintain nearly 100% of its enzymatic activity with ABTS. The PEGylation of laccase has not been extensively explored, so it is important to analyze the effects of this bioconjugation in route to produce a robust modified enzyme. PMID:25652594

  14. Seasonal Dynamics of Enzymatic Activities and Functional Diversity in Soils under Different Organic Management

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil microbial activity and diversity fluctuate seasonally under annual organic amendment for improving soil quality. We investigated the effects of municipal compost (MC), poultry litter (PL), and cover crops of spring oats and red clover (RC) on soil enzyme activities, and soil bacterial community...

  15. Biologically active peptides obtained by enzymatic hydrolysis of Adzuki bean seeds.

    PubMed

    Durak, Agata; Baraniak, Barbara; Jakubczyk, Anna; Świeca, Michał

    2013-12-01

    This study investigated the antioxidant and antihypertensive activities of peptides obtained from protein fractions of Adzuki bean seeds. Peptides were obtained by the use of hydrolytic enzymes in vitro under gastrointestinal conditions. A determination was made of the activity of the peptide inhibitors of the angiotensin I converting enzyme (ACE), and the antiradical and ion chelating activity of peptides from different protein fractions. The highest peptide levels after the absorption process (<7 kDa) were noted in the albumin fraction (50.69 μg/ml). Furthermore, it was found that peptides from the prolamin fraction were characterised by the highest antiradical activity and ACE inhibitory activity (IC50=0.17 mg/ml). Peptides obtained from the globulin fraction showed the highest ability to chelate iron ions, and peptides from the glutelin fraction were characterised as being the most effective in the chelation of copper ions. PMID:23870945

  16. Effect of prolonged and intermittent hypoxia on some cerebral enzymatic activities related to energy transduction.

    PubMed

    Dagani, F; Marzatico, F; Curti, D; Zanada, F; Benzi, G

    1984-12-01

    The adaptation to repeated, alternate normobaric hypoxic and normoxic exposures (12 h/day, for 5 days) and to pharmacological treatment was evaluated by studying the specific activities of some enzymes related to cerebral energy metabolism. Measurements were carried out on (a) the homogenate in toto, (b) the purified mitochondrial fraction, and (c) the crude synaptosomal fraction in different areas of rat brain--cerebral cortex, hippocampus, corpus striatum, hypothalamus, cerebellum, and medulla oblongata. The adaptation to intermittent normobaric hypoxic-normoxic exposures was characterized by significant modifications of some enzyme activities in synaptosomes (decrease of cytochrome oxidase activity in the hippocampus, corpus striatum, and cerebellum; decrease of malate dehydrogenase activity in the cerebellum) and in the purified mitochondrial fraction (increase of succinate dehydrogenase activity in the corpus striatum). Daily treatment with three doses of naftidrofuryl (10, 15, and 22.5 mg/kg i.m.) modified some enzyme activities affected or unaffected by intermittent hypoxia and, particularly, decreased acetylcholinesterase activity. PMID:6501447

  17. Enzymatic activation of cellulose acetate membrane for reducing of protein fouling.

    PubMed

    Koseoglu-Imer, Derya Y; Dizge, Nadir; Koyuncu, Ismail

    2012-04-01

    In this study, the surface of cellulose acetate (CA) ultrafiltration membrane was activated with serine protease (Savinase) enzyme to reduce protein fouling. Enzyme molecules were covalently immobilized with glutaraldehyde (cross-linking agent) onto the surface of CA membranes. The membrane activation was verified using filtration experiments and morphological analysis. Scanning electron microscopy (SEM) images and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy of the activated membrane when compared with raw membrane were confirmed that the enzyme was immobilized onto the membrane surface. The immobilization efficiencies changed from 13.2 to 41.2% according to the enzyme ratios from 2.5 to 10.0 mg/mL. However, the permeability values decreased from 232±6 to 121±4 L/m(2) h bar with increasing enzyme concentration from 2.5 to 10.0 mg/mL. In fouling experiments, bovine serum albumin (BSA) was used as the protein model solution and activated sludge was used as the model biological sludge. Enzyme-activated membranes exhibited good filtration performances and protein rejection efficiencies were compared with raw CA membrane. Also the relative flux reduction (RFR) ratios of membranes were calculated as 97% and 88% for raw CA and enzyme-activated membranes (5 mg/mL savinase), respectively. The membrane activated with Savinase enzyme could be proposed as a surface treatment method before filtration to mitigate protein fouling. PMID:22218336

  18. The Effect of Cadmium on COX-1 and COX-2 Gene, Protein Expression, and Enzymatic Activity in THP-1 Macrophages.

    PubMed

    Olszowski, Tomasz; Gutowska, Izabela; Baranowska-Bosiacka, Irena; Piotrowska, Katarzyna; Korbecki, Jan; Kurzawski, Mateusz; Chlubek, Dariusz

    2015-06-01

    The aim of this study was to examine the effects of cadmium in concentrations relevant to those detected in human serum on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression at mRNA, protein, and enzyme activity levels in THP-1 macrophages. Macrophages were incubated with various cadmium chloride (CdCl2) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM, and 2 μM CdCl2. The mRNA expression and protein levels of COXs were analyzed with RT-PCR and Western blotting, respectively. Prostaglandin E2 (PGE2) and stable metabolite of thromboxane B2 (TXB2) concentrations in culture media were determined using ELISA method. Our study demonstrates that cadmium at the highest tested concentrations modulates COX-1 and COX-2 at mRNA level in THP-1 macrophages; however, the lower tested cadmium concentrations appear to inhibit COX-1 protein expression. PGE2 and TXB2 production is not altered by all tested Cd concentrations; however, the significant stimulation of PGE2 and TXB2 production is observed when macrophages are exposed to both cadmium and COX-2 selective inhibitor, NS-398. The stimulatory effect of cadmium on COXs at mRNA level is not reflected at protein and enzymatic activity levels, suggesting the existence of some posttranscriptional, translational, and posttranslational events that result in silencing of those genes' expression. PMID:25645360

  19. Nanonets Derived from Turnip Mosaic Virus as Scaffolds for Increased Enzymatic Activity of Immobilized Candida antarctica Lipase B

    PubMed Central

    Cuenca, Sol; Mansilla, Carmen; Aguado, Marta; Yuste-Calvo, Carmen; Sánchez, Flora; Sánchez-Montero, Jose M.; Ponz, Fernando

    2016-01-01

    Elongated flexuous plant viral nanoparticles (VNPs) represent an interesting platform for developing different applications in nanobiotechnology. In the case of potyviruses, the virion external surface is made up of helically arrayed domains of the viral structural coat protein (CP), repeated over 2000 times, in which the N- and C-terminal domains of each CP are projected toward the exterior of the external virion surface. These characteristics provide a chemical environment rich in functional groups susceptible to chemical conjugations. We have conjugated Candida antarctica lipase B (CALB) onto amino groups of the external surface of the potyvirus turnip mosaic virus (TuMV) using glutaraldehyde as a conjugating agent. Using this approach, TuMV virions were transformed into scaffolds for CALB nanoimmobilization. Analysis of the resulting structures revealed the formation of TuMV nanonets onto which large CALB aggregates were deposited. The functional enzymatic characterization of the CALB-bearing TuMV nanonets showed that CALB continued to be active in the nanoimmobilized form, even gaining an increased relative specific activity, as compared to the non-immobilized form. These novel virus-based nanostructures may provide a useful new approach to enzyme nanoimmobilization susceptible to be industrially exploited. PMID:27148295

  20. Label-free electrochemical detection of botulinum neurotoxin type E based on its enzymatic activity using interdigitated electrodes

    NASA Astrophysics Data System (ADS)

    Hyun, Sang Hwa; Park, Dae Keun; Kang, Aeyeon; Kim, Soohyun; Kim, Daehee; Shin, Yu Mi; Song, Ji-Joon; Yun, Wan Soo

    2016-02-01

    We report a simple label-free electrochemical method of detecting low concentrations of botulinum neurotoxin type E light chain (BoNT/E LC) based on its peptide cleavage activity. Dual-mode cyclic voltammetry was employed to observe changes in the redox signal of ferri-/ferro-cyanide on interdigitated microelectrodes, whose surfaces were covered by peptides designed from synaptosomal-associated protein 25 to be cleaved by BoNT/E LC. With the introduction of BoNT/E LC, the redox signal showed a time-dependent increase due to cleavage of the immobilized peptide molecules. In addition to the increased redox signal intensity, its time-dependence can be considered as a strong evidence of BoNT/E sensing, since the time-dependent increase can only result from the enzymatic activity of BoNT/E LC. Using this method, BoNT/E LC, at concentrations as low as 5 pg/ml, was readily measurable with only an hour of incubation.

  1. Nanonets Derived from Turnip Mosaic Virus as Scaffolds for Increased Enzymatic Activity of Immobilized Candida antarctica Lipase B.

    PubMed

    Cuenca, Sol; Mansilla, Carmen; Aguado, Marta; Yuste-Calvo, Carmen; Sánchez, Flora; Sánchez-Montero, Jose M; Ponz, Fernando

    2016-01-01

    Elongated flexuous plant viral nanoparticles (VNPs) represent an interesting platform for developing different applications in nanobiotechnology. In the case of potyviruses, the virion external surface is made up of helically arrayed domains of the viral structural coat protein (CP), repeated over 2000 times, in which the N- and C-terminal domains of each CP are projected toward the exterior of the external virion surface. These characteristics provide a chemical environment rich in functional groups susceptible to chemical conjugations. We have conjugated Candida antarctica lipase B (CALB) onto amino groups of the external surface of the potyvirus turnip mosaic virus (TuMV) using glutaraldehyde as a conjugating agent. Using this approach, TuMV virions were transformed into scaffolds for CALB nanoimmobilization. Analysis of the resulting structures revealed the formation of TuMV nanonets onto which large CALB aggregates were deposited. The functional enzymatic characterization of the CALB-bearing TuMV nanonets showed that CALB continued to be active in the nanoimmobilized form, even gaining an increased relative specific activity, as compared to the non-immobilized form. These novel virus-based nanostructures may provide a useful new approach to enzyme nanoimmobilization susceptible to be industrially exploited. PMID:27148295

  2. Identification of a novel enzymatic activity from lactic acid bacteria able to degrade biogenic amines in wine.

    PubMed

    Callejón, S; Sendra, R; Ferrer, S; Pardo, I

    2014-01-01

    The main objectives of this study were the search for enzymatic activities responsible for biogenic amine (BA) degradation in lactic acid bacteria (LAB) strains isolated from wine, their identification, and the evaluation of their applicability for reducing BAs in wine. Fifty-three percent of the 76 LAB cell extracts showed activity against a mixture of histamine, tyramine, and putrescine when analyzed in-gel. The quantification of the degrading ability for each individual amine was tested in a synthetic medium and wine. Most of the bacteria analyzed were able to degrade the three amines in both conditions. The highest percentages of degradation in wine were those of putrescine: up to 41% diminution in 1 week. Enzymes responsible for amine degradation were isolated and purified from Lactobacillus plantarum J16 and Pediococcus acidilactici CECT 5930 strains and were identified as multicopper oxidases. This is the first report of an efficient BA reduction in wine by LAB. Furthermore, the identity of the enzymes involved has been revealed. PMID:23515835

  3. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid.

    PubMed

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  4. Marek's disease virus (MDV) ubiquitin-specific protease (USP) performs critical functions beyond its enzymatic activity during virus replication.

    PubMed

    Veiga, Inês B; Jarosinski, Keith W; Kaufer, Benedikt B; Osterrieder, Nikolaus

    2013-03-15

    Marek's disease virus (MDV) encodes an ubiquitin-specific protease (USP) within its UL36 gene. USP is highly conserved among herpesviruses and was shown to be important for MDV replication and pathogenesis in MDV's natural host, the chicken. To further investigate the role of MDV USP, several recombinant (r) MDVs were generated and their in vitro phenotypes were evaluated using plaque size and growth kinetics assays. We discovered that the N-terminus of pUL36 is essential for MDV replication and could not be complemented by ectopic expression of MDV USP. In addition, we demonstrated that the region located between the conserved glutamine (Q85) and leucine (L106) residues comprising the active site cysteine (C98) is also essential for MDV replication. Based on the analyses of the rMDVs generated here, we concluded that MDV USP likely contributes to the structure and/or stability of pUL36 and affects replication and oncogenesis of MDV beyond its enzymatic activity. PMID:23399034

  5. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

    PubMed Central

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  6. Methods, microfluidic devices, and systems for detection of an active enzymatic agent

    SciTech Connect

    Sommer, Gregory J; Hatch, Anson V; Singh, Anup K; Wang, Ying-Chih

    2014-10-28

    Embodiments of the present invention provide methods, microfluidic devices, and systems for the detection of an active target agent in a fluid sample. A substrate molecule is used that contains a sequence which may cleave in the presence of an active target agent. A SNAP25 sequence is described, for example, that may be cleaved in the presence of Botulinum Neurotoxin. The substrate molecule includes a reporter moiety. The substrate molecule is exposed to the sample, and resulting reaction products separated using electrophoretic separation. The elution time of the reporter moiety may be utilized to identify the presence or absence of the active target agent.

  7. Rescue of Enzymatic Function for Disease-associated RPE65 Proteins Containing Various Missense Mutations in Non-active Sites*

    PubMed Central

    Li, Songhua; Izumi, Tadahide; Hu, Jane; Jin, Heather H.; Siddiqui, Ahmed-Abdul A.; Jacobson, Samuel G.; Bok, Dean; Jin, Minghao

    2014-01-01

    Over 70 different missense mutations, including a dominant mutation, in RPE65 retinoid isomerase are associated with distinct forms of retinal degeneration; however, the disease mechanisms for most of these mutations have not been studied. Although some mutations have been shown to abolish enzyme activity, the molecular mechanisms leading to the loss of enzymatic function and retinal degeneration remain poorly understood. Here we show that the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, plays a critical role in regulating pathogenicity of three mutations (L22P, T101I, and L408P) by mediating rapid degradation of mutated RPE65s via a ubiquitination- and proteasome-dependent non-lysosomal pathway. These mutant RPE65s were misfolded and formed aggregates or high molecular complexes via disulfide bonds. Interaction of PSMD13 with mutant RPE65s promoted degradation of misfolded but not properly folded mutant RPE65s. Many mutations, including L22P, T101I, and L408P, were mapped on non-active sites. Although their activities were very low, these mutant RPE65s were catalytically active and could be significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. Sodium 4-phenylbutyrate and glycerol displayed a significant synergistic effect on the low temperature rescue of the mutant RPE65s by promoting proper folding, reducing aggregation, and increasing membrane association. Our results suggest that a low temperature eye mask and sodium 4-phenylbutyrate, a United States Food and Drug Administration-approved oral medicine, may provide a promising “protein repair therapy” that can enhance the efficacy of gene therapy by reducing the cytotoxic effect of misfolded mutant RPE65s. PMID:24849605

  8. Enzymatic activities and prokaryotic abundance in relation to organic matter along a West-East Mediterranean transect (TRANSMED cruise).

    PubMed

    Zaccone, R; Boldrin, A; Caruso, G; La Ferla, R; Maimone, G; Santinelli, C; Turchetto, M

    2012-07-01

    The distribution of extracellular enzymatic activities (EEA) [leucine aminopeptidase (LAP), ß-glucosidase (GLU), alkaline phosphatase (AP)], as well as that of prokaryotic abundance (PA) and biomass (PB), dissolved organic carbon (DOC), particulate organic carbon and particulate total nitrogen (POC, PTN), was determined in the epi-, meso-, and bathypelagic waters of the Mediterranean Sea along a West-East transect and at one Atlantic station located outside the Strait of Gibraltar. This study represents a synoptical evaluation of the microbial metabolism during early summer. Decreasing trends with depth were observed for most of the parameters (PA, PB, AP, DOC, POC, PTN). Significant differences between the western and eastern basins of the Mediterranean Sea were found, displaying higher rates of LAP and GLU and lower C/N ratios more in the eastern than in the western areas. Conversely, in the epipelagic layer, PA and PB were found to be higher in the western than in the eastern basins. PB was significantly related to DOC concentration (all data, n = 145, r = 0.53, P < 0.01), while significant correlations of EEA with POC and PTN were found in the epipelagic layer, indicating an active response of microbial metabolism to organic substrates. Specific enzyme activities normalized to cell abundance pointed out high values of LAP and GLU in the bathypelagic layer, especially in the eastern basin, while cell-specific AP was high in the epi- and bathypelagic zone of the eastern basin indicating a rapid regeneration of inorganic P for both prokaryotes and phytoplankton needs. Low activity and abundance characterized the Atlantic station, while opposite trends of these parameters were observed along the Mediterranean transect, showing the uncoupling between abundance and activity data. In the east Mediterranean Sea, decomposition processes increased probably in response to mesoscale structures which lead to organic matter downwelling. PMID:22349935

  9. Influence of intermittent hypoxia and pyrimidinic nucleosides on cerebral enzymatic activities related to energy transduction.

    PubMed

    Dagani, F; Marzatico, F; Curti, D; Taglietti, M; Zanada, F; Benzi, G

    1984-08-01

    The effect of intermittent normobaric hypoxia and of biological pyrimidines (uridine and cytidine) on the specific activities of some enzymes related to cerebral energy metabolism were studied. Measurement were carried out on the following: homogenate in toto; purified mitochondrial fraction; crude synaptosomal fraction, in different areas of rat brain: cerebral cortex, hippocampus, corpus striatum, hypothalamus, cerebellum, and medulla oblongata. Intermittent normobaric hypoxia (12 hours daily for 5 days) caused modifications of the enzyme activities in the homogenate in toto (decrease of hexokinase in cerebellum; increase of pyruvate kinase in medulla oblongata), in the purified mitochondrial fraction (increase of succinate dehydrogenase in the corpus striatum) and in the crude synaptosomal fraction (decrease of cytochrome oxidase activity in cerebral cortex, hippocampus, and cerebellum; decrease of malate dehydrogenase in hippocampus and cerebellum; decrease of lactate dehydrogenase in cerebellum). Daily treatment with cytidine or uridine altered some enzyme activities either affected or unaffected by intermittent hypoxia. PMID:6493441

  10. Integrin-mediated adhesion as self-sustained waves of enzymatic activation

    NASA Astrophysics Data System (ADS)

    Block, M. R.; Destaing, O.; Petropoulos, C.; Planus, E.; Albigès-Rizo, C.; Fourcade, B.

    2015-10-01

    Integrin receptors mediate interaction between the cellular actin-cytoskeleton and extracellular matrix. Based on their activation properties, we propose a reaction-diffusion model where the kinetics of the two-state receptors is modulated by their lipidic environment. This environment serves as an activator variable, while a second variable plays the role of a scaffold protein and controls the self-sustained activation of the receptors. Due to receptor diffusion which couples dynamically the activator and the inhibitor, our model connects major classes of reaction diffusion systems for excitable media. Spot and rosette solutions, characterized by receptor clustering into localized static or dynamic structures, are organized into a phase diagram. It is shown that diffusion and kinetics of receptors determines the dynamics and the stability of these structures. We discuss this model as a precursor model for cell signaling in the context of podosomes forming actoadhesive metastructures, and we study how generic signaling defects influence their organization.

  11. A methodology for preparing nanostructured biomolecular interfaces with high enzymatic activity

    NASA Astrophysics Data System (ADS)

    Wong, Lu Shin; Karthikeyan, Chinnan V.; Eichelsdoerfer, Daniel J.; Micklefield, Jason; Mirkin, Chad A.

    2012-01-01

    The development of a novel method for functionalizing nanopatterned surfaces with catalytically active proteins is reported. This method involves using dip-pen nanolithography (DPN) and polymer pen lithography (PPL) to generate nanoscale patterns of coenzyme A, followed by a phosphopantetheinyl transferase-mediated coupling between coenzyme A and proteins fused to the ybbR-tag. By exploiting the ability to generate protein features over large areas afforded by DPN and PPL, it was now possible to measure protein activity directly on these surfaces. It was found that proteins immobilized on the nanoscale features not only display higher activity per area with decreasing feature size, but are also robust and can be used for repeated catalytic cycles. The immobilization method is applicable to a variety of proteins and gives rise to superior activity compared to proteins attached in random orientations on the surface.

  12. Integrin-mediated adhesion as self-sustained waves of enzymatic activation.

    PubMed

    Block, M R; Destaing, O; Petropoulos, C; Planus, E; Albigès-Rizo, C; Fourcade, B

    2015-10-01

    Integrin receptors mediate interaction between the cellular actin-cytoskeleton and extracellular matrix. Based on their activation properties, we propose a reaction-diffusion model where the kinetics of the two-state receptors is modulated by their lipidic environment. This environment serves as an activator variable, while a second variable plays the role of a scaffold protein and controls the self-sustained activation of the receptors. Due to receptor diffusion which couples dynamically the activator and the inhibitor, our model connects major classes of reaction diffusion systems for excitable media. Spot and rosette solutions, characterized by receptor clustering into localized static or dynamic structures, are organized into a phase diagram. It is shown that diffusion and kinetics of receptors determines the dynamics and the stability of these structures. We discuss this model as a precursor model for cell signaling in the context of podosomes forming actoadhesive metastructures, and we study how generic signaling defects influence their organization. PMID:26565269

  13. Two-Photon Enzymatic Probes Visualizing Sub-cellular/Deep-brain Caspase Activities in Neurodegenerative Models

    PubMed Central

    Qian, Linghui; Zhang, Cheng-Wu; Mao, Yanli; Li, Lin; Gao, Nengyue; Lim, Kah-Leong; Xu, Qing-Hua; Yao, Shao Q.

    2016-01-01

    Caspases work as a double-edged sword in maintaining cell homeostasis. Highly regulated caspase activities are essential during animal development, but dysregulation might lead to different diseases, e.g. extreme caspase activation is known to promote neurodegeneration. At present, visualization of caspase activation has mostly remained at the cellular level, in part due to a lack of cell-permeable imaging probes capable of direct, real-time investigations of endogenous caspase activities in deep tissues. Herein, we report a suite of two-photon, small molecule/peptide probes which enable sensitive and dynamic imaging of individual caspase activities in neurodegenerative models under physiological conditions. With no apparent toxicity and the ability of imaging endogenous caspases both in different subcellular organelles of mammalian cells and in brain tissues, these probes serve as complementary tools to conventional histological analysis. They should facilitate future explorations of caspases at molecular, cellular and organism levels and inspire development of novel two-photon probes against other enzymes. PMID:27210613

  14. Two-Photon Enzymatic Probes Visualizing Sub-cellular/Deep-brain Caspase Activities in Neurodegenerative Models.

    PubMed

    Qian, Linghui; Zhang, Cheng-Wu; Mao, Yanli; Li, Lin; Gao, Nengyue; Lim, Kah-Leong; Xu, Qing-Hua; Yao, Shao Q

    2016-01-01

    Caspases work as a double-edged sword in maintaining cell homeostasis. Highly regulated caspase activities are essential during animal development, but dysregulation might lead to different diseases, e.g. extreme caspase activation is known to promote neurodegeneration. At present, visualization of caspase activation has mostly remained at the cellular level, in part due to a lack of cell-permeable imaging probes capable of direct, real-time investigations of endogenous caspase activities in deep tissues. Herein, we report a suite of two-photon, small molecule/peptide probes which enable sensitive and dynamic imaging of individual caspase activities in neurodegenerative models under physiological conditions. With no apparent toxicity and the ability of imaging endogenous caspases both in different subcellular organelles of mammalian cells and in brain tissues, these probes serve as complementary tools to conventional histological analysis. They should facilitate future explorations of caspases at molecular, cellular and organism levels and inspire development of novel two-photon probes against other enzymes. PMID:27210613

  15. ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes.

    PubMed

    Boschin, Giovanna; Scigliuolo, Graziana Maria; Resta, Donatella; Arnoldi, Anna

    2014-02-15

    The objective of this investigation was to compare the angiotensin converting enzyme (ACE)-inhibitory activity of the hydrolysates obtained by pepsin digestion of proteins of some legumes, such as chickpea, common bean, lentil, lupin, pea, and soybean, by using the same experimental procedure. The ACE-inhibitory activity was measured by using the tripeptide hippuryl-histidyl-leucine (HHL), as model peptide, and HPLC-DAD, as analytical method. The peptide mixtures of all legumes were active, with soybean and lupin the most efficient, with IC50 values of 224 and 226 μg/ml, respectively. Considering the promising results obtained with lupin, and aiming to identify the protein(s) that release(s) the peptides responsible for the activity, the peptides obtained from the pepsin digestion of some industrial lupin protein isolates and purified protein fractions were tested. The most active mixture, showing an IC50 value of 138 μg/ml, was obtained hydrolysing a mixture of lupin α+β conglutin. PMID:24128446

  16. Optimization of enzymatic hydrolysis of shrimp waste for recovery of antioxidant activity rich protein isolate.

    PubMed

    Sowmya, R; Ravikumar, T M; Vivek, R; Rathinaraj, K; Sachindra, N M

    2014-11-01

    Shrimp waste is an important source of astaxanthin, which occur as a complex with proteins, and protein isolates as well as carotenoids are known to possess antioxidant activity. Investigations were carried out to optimize hydrolysis of shrimp waste using a bacterial protease to obtain antioxidant activity rich protein isolate. The effect of three process variables namely enzyme concentration to waste, incubation temperature and time on carotenoid recovery, protein content, trichloro acetic acid (TCA) soluble peptide content and DiPhenyl Picryl Hydrazylchloride (DPPH) scavenging activity was evaluated using a fractionally factorial design. A high correlation coefficient (>0.90) between the observed and the predicted values indicated the appropriateness of the design employed. Maximum carotenoid recovery was obtained by hydrolysing the shrimp waste with 0.3 % enzyme for 4 h. DPPH radical scavenging activity of carotenoprotein isolate was markedly affected by enzyme concentration, temperature and time of hydrolysis. The study indicated that in order to obtain the carotenoprotein from shrimp waste with higher carotenoid content hydrolysing with an enzyme concentration of 0.2-0.4 %, at lower temperature of 25-30° upto 4 h is ideal. However, in order to obtain the protein isolate with increased antioxidant activity hydrolysing at higher temperature of 50 °C, with higher enzyme concentration of 0.5 % for shorter duration is more ideal. PMID:26396312

  17. Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Grant, C C; Messer, R J; Cieplak, W

    1994-01-01

    Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity. Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity. To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined. The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis. The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E. coli. A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells. The results indicate that trypsin-like cleavage of the A subunit of E. coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects. Images PMID:7927684

  18. Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

    PubMed Central

    Dolinska, Monika B.; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2014-01-01

    Background Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Methodology/Principal Findings The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Conclusions/Significance The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1. PMID:24392141

  19. Enzymatic activity induced by interactions with a nanofabricated hydrophobic Si surface

    NASA Astrophysics Data System (ADS)

    Nishiyama, Katsuhiko

    2013-07-01

    The binding of peptides of 2-10 glycine residues (2-10Gly) to papain on nanofabricated hydrophobic Si surfaces was investigated by molecular dynamics and docking simulations. 5Gly, 7Gly, 9Gly, and 10Gly were distributed on sites near the active center of papain on the Si surface, while 6-10Gly were distributed on sites near the active center of free papain. The Si surface changed the substrate specificity of papain, and modification of this surface should allow full control of substrate specificity. Molecular surgery of proteins in cells may be realized using papain on specially designed surfaces.

  20. Enzymatic Activity of α-L-Fucosidase and L-Fucokinase Across Vertebrate Animal Species‡

    PubMed Central

    Honas, Bradley J.; Glassman, Urlene M.; Wiese, Thomas J.

    2012-01-01

    The oligosaccharide portion of glycoproteins is known to modulate protein structure, function, and turnover. Our laboratory is interested in the metabolism of L-fucose, a normal constituent of eukaryotic glycoproteins. L-Fucose is unique in that it is the only levorotatory sugar utilized in mammalian systems. There is considerable interest in understanding the controls which determine the level of L-fucose attached to proteins, in order to generate stable and active glycoforms of protein for the treatment of disease. As part of a program to determine the controls on protein L-fucosylation, we have systematically determined the tissue distribution of the enzymes L-fucokinase and α-L-fucosidase in species across the vertebrate animal kingdom. In general, the level of α-L-fucosidase is higher than L-fucokinase level. The tissue with highest enzyme activity cannot be generalized, regardless of which enzyme is of interest. Furthermore, there is not a correlation between synthetic and catabolic enzyme activity within a tissue. L-Fucokinase can be detected in all tissues examined. Interestingly, we have also detected β-D-fucosidase activity, present in extraordinary levels in the liver and small intestine of snake. Whether this is due to a specific enzyme or whether it represents a broad specificity of the α-L-fucosidase is currently being investigated. PMID:19394435

  1. Arid soil microbial enzymatic activity profile as affected by geographical location and soil degradation status

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evaluating soil health is critical for any successful remediation effort. Arid lands, with their minimal carbon and water contents, low nutritional status and restricted, seasonal microbial activity pose specific challenges to soil health restoration and by extension, restoration of ecosystem repr...

  2. FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

    EPA Science Inventory

    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  3. Quantitation of CYP24A1 Enzymatic Activity With a Simple Two-Hybrid System

    PubMed Central

    Mugg, Amy; Legeza, Balazs; Tee, Meng Kian; Damm, Izabella; Long, Roger K.

    2015-01-01

    Context: Mutations of the CYP24A1 gene encoding the 24-hydroxylase (24OHase) that inactivates metabolites of vitamin D can cause hypercalcemia in infants and adults; in vitro assays of 24OHase activity have been difficult. Objective: We sought an alternative assay to characterize a CYP24A1 mutation in a young adult with bilateral nephrolithiasis and hypercalcemia associated with ingestion of excess vitamin D supplements and robust dairy intake for 5 years. Methods: CYP24A1 exons were sequenced from leukocyte DNA. Wild-type and mutant CYP24A1 cDNAs were expressed in JEG-3 cells, and 24OHase activity was assayed by a two-hybrid system. Results: The CYP24A1 missense mutation L409S was found on only one allele; no other mutation was found in exons or in at least 30 bp of each intron/exon junction. Based on assays of endogenous 24OHase activity and of activity from a transiently transfected CYP24A1 cDNA expression vector, JEG-3 cells were chosen over HepG2, Y1, MA10, and NCI-H295A cells for two-hybrid assays of 24OHase activity. The apparent Michaelis constant, Km(app), was 9.0 ± 2.0 nm for CYP24A1 and 8.6 ± 2.2 nm for its mutant; the apparent maximum velocity, Vmax(app), was 0.71 ± 0.055 d−1 for the wild type and 0.22 ± 0.026 d−1 for the mutant. As assessed by Vmax/Km, the L409S mutant has 32% of wild-type activity (P = .0012). Conclusions: The two-hybrid system in JEG-3 cells provides a simple, sensitive, quantitative assay of 24OHase activity. Heterozygous mutation of CYP24A1 may cause hypercalcemia in the setting of excessive vitamin D intake, but it is also possible that the patient had another, unidentified CYP24A1 mutation on the other allele. PMID:25375986

  4. The human hGSTA5 gene encodes an enzymatically active protein

    PubMed Central

    Singh, Sharda P.; Zimniak, Ludwika; Zimniak, Piotr

    2009-01-01

    Background Of the five human Alpha-class glutathione transferases, expression of hGSTA5 has not been experimentally documented, even though in silico the hGSTA5 sequence can be assembled into a mRNA and translated. The present work was undertaken to determine whether hGSTA5 is functional. Methods Human K562 cells were transfected with the hGSTA5 gene driven by the CMV promoter, and hGSTA5 cDNA was recovered from mature mRNA by reverse transcription. The cDNA was used in bacterial and eukaryotic protein expression systems. The resulting protein, after purification by glutathione affinity chromatography where appropriate, was tested for glutathione transferase activity. Results Human K562 cells transfected with the hGSTA5 gene under control of a CMV promoter produced a fully spliced mRNA which, after reverse transcription and expression in E. coli, yielded a protein that catalyzed the conjugation of the lipid peroxidation product 4-hydroxynonenal to glutathione. Similarly, transfection of human HEK-293 cells with the hGSTA5 gene driven by the CMV promoter led to an elevated 4-hydroxynonenal-conjugating activity in the cell lysate. In addition, translation of hGSTA5 cDNA in a cell-free eukaryotic system gave rise to a protein with 4-hydroxynonenal-conjugating activity. Conclusions hGSTA5 can be processed to a mature mRNA which is translation-competent, producing a catalytically active enzyme. General Significance Because a functional gene would not be maintained in the absence of selective pressure, we conclude that the native hGSTA5 promoter is active but has a spatially or temporally restricted expression pattern, and/or is expressed only under specific (patho)physiological conditions. PMID:19664689

  5. Periplasmic nitrate reductase and formate dehydrogenase: similar molecular architectures with very different enzymatic activities.

    PubMed

    Cerqueira, Nuno M F S A; Gonzalez, Pablo J; Fernandes, Pedro A; Moura, José J G; Ramos, Maria João

    2015-11-17

    It is remarkable how nature has been able to construct enzymes that, despite sharing many similarities, have simple but key differences that tune them for completely different functions in living cells. Periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) from the DMSOr family are representative examples of this. Both enzymes share almost identical three-dimensional protein foldings and active sites, in terms of coordination number, geometry and nature of the ligands. The substrates of both enzymes (nitrate and formate) are polyatomic anions that also share similar charge and stereochemistry. In terms of the catalytic mechanism, both enzymes have a common activation mechanism (the sulfur-shift mechanism) that ensures a constant coordination number around the metal ion during the catalytic cycle. In spite of these similarities, they catalyze very different reactions: Nap abstracts an oxygen atom from nitrate releasing nitrite, whereas FdH catalyzes a hydrogen atom transfer from formate and releases carbon dioxide. In this Account, a critical analysis of structure, function, and catalytic mechanism of the molybdenum enzymes periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) is presented. We conclude that the main structural driving force that dictates the type of reaction, catalyzed by each enzyme, is a key difference on one active site residue that is located in the top region of the active sites of both enzymes. In both enzymes, the active site is centered on the metal ion of the cofactor (Mo in Nap and Mo or W in Fdh) that is coordinated by four sulfur atoms from two pyranopterin guanosine dinucleotide (PGD) molecules and by a sulfido. However, while in Nap there is a Cys directly coordinated to the Mo ion, in FdH there is a SeCys instead. In Fdh there is also an important His that interacts very closely with the SeCys, whereas in Nap the same position is occupied by a Met. The role of Cys in Nap and SeCys in FdH is similar in both

  6. Intestinal morphology and enzymatic activity in newly weaned pigs fed contrasting fiber concentrations and fiber properties.

    PubMed

    Hedemann, M S; Eskildsen, M; Laerke, H N; Pedersen, C; Lindberg, J E; Laurinen, P; Knudsen, K E Bach

    2006-06-01

    The main objective of this study was to determine the effect of fiber source and concentration on morphological characteristics, mucin staining pattern, and mucosal enzyme activities in the gastrointestinal tract of pigs. The experiment included 50 pigs from 10 litters weaned at 4 wk of age (BW 8.6 +/- 1.4 kg) and divided into 5 treatment groups. Diets containing fiber of various physico-chemical properties and concentrations were formulated to contain 73, 104, or 145 g of dietary fiber/kg of DM. The diets were based on raw wheat and barley flours. Pectin and barley hulls, representing soluble and insoluble fiber sources, respectively, were used to increase the fiber concentration. The pigs were fed the experimental diets for 9 d, and then the pigs were euthanized and the entire gastrointestinal tract was removed. Tissue samples were taken from the mid and distal small intestine and from the mid colon. Inclusion of pectin in the diets significantly decreased (P < 0.001) ADFI and ADG compared with pigs fed no pectin. The villi and the crypts were shorter in pigs fed pectin-containing diets, but the villous height/crypt depth ratio was unaltered. Pectin significantly decreased the area of mucins in the crypts of the small intestine, indicating that the pigs fed the pectin-containing diet would probably be more susceptible to pathogenic bacteria, although this cannot be separated from the impact on ADFI. The lectin-binding pattern of the intestinal mucosa was unaffected by diet. The activity of lactase and maltase was increased in pigs fed diets with high fiber content, whereas sucrase activity was increased in pigs fed the pectin-containing diets. The activity of the peptidases, aminopeptidase N and dipeptidylpeptidase IV, was increased when feeding high fiber diets, whereas the activity of gamma-glutamyl transpeptidase remained unaffected by the experimental diets. In conclusion, the reduced feed intake observed with the pectin-containing diets could explain the

  7. High level expression and purification of the enzymatically active cytoplasmic region of human CD45 phosphatase from yeast.

    PubMed

    Pacitti, A; Stevis, P; Evans, M; Trowbridge, I; Higgins, T J

    1994-06-30

    The cytoplasmic region of human CD45 corresponding to residues 584-1281 was inserted downstream of the alcohol dehydrogenase promoter and transfected into a haploid strain of yeast. Expression of recombinant CD45 in yeast reached as high as 5% of the soluble protein. Following removal of cellular debris by centrifugation and an ammonium sulfate precipitation step, the enzyme was purified using phenyl-Sepharose chromatography, preparative gel filtration, Mono Q anion exchange chromatography and a final analytical gel filtration step. Enzymatically active material with a purity of > or = 98% was obtained with a yield approaching 50%. The final product gave a Km of 5.5 mM and a Vmax of 87.5 U/mg with p-nitrophenylphosphate and a Km and Vmax of 0.167 mM and 185 U/mg, respectively, with a phosphotyrosine peptide. The native enzyme purified from Jurkat cells showed comparable Kms with both substrates to the recombinant enzyme but displayed substantially lower Vmax values for both substrates. PMID:8031864

  8. Differential coral bleaching-Contrasting the activity and response of enzymatic antioxidants in symbiotic partners under thermal stress.

    PubMed

    Krueger, Thomas; Hawkins, Thomas D; Becker, Susanne; Pontasch, Stefanie; Dove, Sophie; Hoegh-Guldberg, Ove; Leggat, William; Fisher, Paul L; Davy, Simon K

    2015-12-01

    Mass coral bleaching due to thermal stress represents a major threat to the integrity and functioning of coral reefs. Thermal thresholds vary, however, between corals, partly as a result of the specific type of endosymbiotic dinoflagellate (Symbiodinium sp.) they harbour. The production of reactive oxygen species (ROS) in corals under thermal and light stress has been recognised as one mechanism that can lead to cellular damage and the loss of their symbiont population (Oxidative Theory of Coral Bleaching). Here, we compared the response of symbiont and host enzymatic antioxidants in the coral species Acropora millepora and Montipora digitata at 28°C and 33°C. A. millepora at 33°C showed a decrease in photochemical efficiency of photosystem II (PSII) and increase in maximum midday excitation pressure on PSII, with subsequent bleaching (declining photosynthetic pigment and symbiont density). M. digitata exhibited no bleaching response and photochemical changes in its symbionts were minor. The symbiont antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and catalase peroxidase showed no significant upregulation to elevated temperatures in either coral, while only catalase was significantly elevated in both coral hosts at 33°C. Increased host catalase activity in the susceptible coral after 5days at 33°C was independent of antioxidant responses in the symbiont and preceded significant declines in PSII photochemical efficiencies. This finding suggests a potential decoupling of host redox mechanisms from symbiont photophysiology and raises questions about the importance of symbiont-derived ROS in initiating coral bleaching. PMID:26310104

  9. Investigation of trypsin-CdSe quantum dot interactions via spectroscopic methods and effects on enzymatic activity

    NASA Astrophysics Data System (ADS)

    Kaur, Gurvir; Tripathi, S. K.

    2015-01-01

    The paper presents the interactions between trypsin and water soluble cadmium selenide (CdSe) quantum dots investigated by spectrophotometric methods. CdSe quantum dots have strong ability to quench the intrinsic fluorescence of trypsin by a static quenching mechanism. The quenching has been studied at three different temperatures where the results revealed that electrostatic interactions exist between CdSe quantum dots and trypsin and are responsible to stabilize the complex. The Scatchard plot from quenching revealed 1 binding site for quantum dots by trypsin, the same has been confirmed by making isothermal titrations of quantum dots against trypsin. The distance between donor and acceptor for trypsin-CdSe quantum dot complexes is calculated to be 2.8 nm by energy transfer mechanisms. The intrinsic fluorescence of CdSe quantum dots has also been enhanced by the trypsin, and is linear for concentration of trypsin ranging 1-80 μl. All the observations evidence the formation of trypsin-CdSe quantum dot conjugates, where trypsin retains the enzymatic activity which in turn is temperature and pH dependent.

  10. Divergence in the Enzymatic Activities of a Tomato and Solanum pennellii Alcohol Acyltransferase Impacts Fruit Volatile Ester Composition.

    PubMed

    Goulet, Charles; Kamiyoshihara, Yusuke; Lam, Nghi B; Richard, Théo; Taylor, Mark G; Tieman, Denise M; Klee, Harry J

    2014-10-29

    Tomato fruits accumulate a diverse set of volatiles including multiple esters. The content of ester volatiles is relatively low in tomato fruits (Solanum lycopersicum) and far more abundant in the closely related species S. pennellii. There are also qualitative variations in ester content between the two species. We have previously shown that high expression of a non-specific esterase is critical for the low overall ester content of S. lycopersicum fruit relative to S. pennellii fruit. Here, we show that qualitative differences in ester composition are the consequence of divergence in enzymatic activity of a ripening-related alcohol acyltransferase (AAT1). The S. pennellii AAT1 is more efficient than the tomato AAT1 for all the alcohols tested. The two enzymes have differences in their substrates preferences that explain variations observed in the volatiles. Together, the results illustrate how two related species have evolved to precisely adjust their volatile content by modulating the balance of synthesis and degradation of esters. PMID:25355057

  11. Divergence in the enzymatic activities of a tomato and Solanum pennellii alcohol acyltransferase impacts fruit volatile ester composition.

    PubMed

    Goulet, Charles; Kamiyoshihara, Yusuke; Lam, Nghi B; Richard, Théo; Taylor, Mark G; Tieman, Denise M; Klee, Harry J

    2015-01-01

    Tomato fruits accumulate a diverse set of volatiles including multiple esters. The content of ester volatiles is relatively low in tomato fruits (Solanum lycopersicum) and far more abundant in the closely related species Solanum pennellii. There are also qualitative variations in ester content between the two species. We have previously shown that high expression of a non-specific esterase is critical for the low overall ester content of S. lycopersicum fruit relative to S. pennellii fruit. Here, we show that qualitative differences in ester composition are the consequence of divergence in enzymatic activity of a ripening-related alcohol acyltransferase (AAT1). The S. pennellii AAT1 is more efficient than the tomato AAT1 for all the alcohols tested. The two enzymes have differences in their substrate preferences that explain the variations observed in the volatiles. The results illustrate how two related species have evolved to precisely adjust their volatile content by modulating the balance of the synthesis and degradation of esters. PMID:25578279

  12. [Study of the Sporothrix schenkii (yeast forms) extract. Electrophoretic and immunoelectrophoretic analyses: characterization of enzymatic activities].

    PubMed

    Walbaum, S; Duriez, T; Dujardin, L; Biguet, J

    1978-07-28

    An extract from living yeast forms of S. schenckii was prepared. The yeasts originated from a shake culture in B.H.I. broth (Difco) incubated for 3 days at 35 degrees C in darkness; they were harvested, washed and disrupted with glass beads in a model MSK Braun mechanical cell homogenizer; a freezing-thawing was added to improve the extract. After electrophoretic separation in agarose gel, the extract's components were characterized by their enzymic activity; with this technique, 30 bands were revealed. These enzymic activities were also investigated on the antigenic fractions of the extract revealed by a rabbit hyperimmunserum: 16 among 22 immunoprecipitates are identified by their catalytic properties. Study of the earliest precipitating antibodies (appearing-order and enzymic caracterization) in rabbits just immunized completes this work. How to ameliorate the quality of the extract by culture and extraction conditions is also specified. PMID:692628

  13. Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

    PubMed

    Zeymer, Cathleen; Werbeck, Nicolas D; Zimmermann, Sabine; Reinstein, Jochen; Hansen, D Flemming

    2016-09-12

    States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side-chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions. PMID:27534930

  14. Enzymatic activity of soluble and membrane tethered peptide pro-hormone convertase 1.

    PubMed

    Bruzzaniti, Angela; Mains, Richard E

    2002-05-01

    Pro-hormone convertases PC1 and PC2 perform endoproteolytic cleavages of precursors in peptide-containing secretory granules. PC1 and PC2 are soluble, secreted with bioactive peptides. Evolutionarily related PCs have membrane tethers, not secreted. We tethered PC1 to the transmembrane-cytoplasmic domains (CD) of a granule enzyme (peptidylglycine-alpha-amidating monooxygenase; PAM) and Golgi-localized PC8. The tethered PC1 is far more stable to elevated temperature and denaturants than soluble PC1, and more active. Both tethers allow PC1 to visit the cell surface transiently, cleaving soluble molecules outside the cell. Both membrane-bound PC1 chimeras cleave membrane PAM into soluble active fragments when PAM is expressed on adjacent cells. PMID:12084516

  15. Probing Mechanisms for Enzymatic Activity Enhancement of Organophosphorus Hydrolase in Functionalized Mesoporous Silica

    SciTech Connect

    Chen, Baowei; Lei, Chenghong; Shin, Yongsoon; Liu, Jun

    2009-12-25

    We have previously reported that organophosphorus hydrolase (OPH) can be spontaneously entrapped in functionalized mesoporous silica (FMS) with HOOC - as the functional groups and the entrapped OPH in HOOC-FMS showed enhanced enzyme specific activity. This work is to study the mechanisms that why OPH entrapped in FMS displayed the enhanced activity in views of OPH-FMS interactions using spectroscopic methods. The circular dichroism (CD) spectra show that, comparing to the secondary structure of OPH free in solution, OPH in HOOC-FMS displayed increased a-helix/b-strand transition of OPH with increased OPH loading density. The fluorescence emission spectra of Trp residues were used to assess the tertiary structural changes of the enzyme. There was a 42% increase in fluorescence. This is in agreement with the fact that the fluorescence intensity of OPH was increased accompanying with the increased OPH activity when decreasing urea concentrations in solution. The steady-state anisotropy was increased after OPH entrapping in HOOC-FMS comparing to the free OPH in solution, indicating that protein mobility was reduced upon entrapment. The solvent accessibility of Trp residues of OPH was probed by using acrylamide as a collisional quencher. Trp residues of OPH-FMS had less solvent exposure comparing with free OPH in solution due to its electrostatical binding to HOOC-FMS thereby displaying the increased fluorescence intensity. These results suggest the interactions of OPH with HOOC-FMS resulted in the protein immobilization and a favorable conformational change for OPH in the crowded confinement space and accordingly the enhanced activity.

  16. Effect of laser phototherapy on enzymatic activity of salivary glands of hamsters treated with 5-Fluorouracil.

    PubMed

    Campos, Luana; Nicolau, José; Arana-Chavez, Victor E; Simões, Alyne

    2014-01-01

    The chemotherapeutic agent 5-Fluorouracil (5-FU) can induce salivary gland hypofunction (SGH); however, previous studies did not reach final conclusions on the influence of this drug on glandular tissue. Thus, the aim of this study was to investigate the effect of 5-FU on submandibular (SMs) and sublingual glands (SLs), as well as, the effect of laser phototherapy (LPT) on SGH induced by 5-FU. Eighty-five hamsters were divided into three groups: control (C), chemotherapy (CT) and laser (L), and the SGH was induced by two injections of 5-FU in groups CT and L. The irradiation was performed using a diode (λ780 nm/20 mW/5 J cm(-2)/0.2 J and 10 s per point/spot size of 0.04 cm(2)) and applied daily. On the euthanasia day, SMs and SLs were removed and biochemical analyses were carried out. The lactate dehydrogenase activity was increased in group CT when compared with group C for SLs and SMs (P < 0.05). In addition, the peroxidase and catalase activities were increased and superoxide dismutase was decreased by 5-FU (P < 0.05). However, LPT appears to be a protective mechanism against oxidative stress, tending to alter the activity of these antioxidant enzymes, suggesting LPT as a promising therapy to modulate the 5-FU harmful effect. PMID:24172058

  17. Effect of high pressures on the enzymatic activity of commercial milk protein coagulants

    NASA Astrophysics Data System (ADS)

    Wiśniewska, Krystyna; Reps, Arnold; Jankowska, Agnieszka

    2014-04-01

    This study was aimed at determining the effect of high pressures in the range of 100-1000 MPa/15 min, applied in 100 MPa increments, on the coagulating and proteolytic activity of commercial coagulants produced with genetic engineering methods: Maxiren, Chymogen, Chymax and of a natural rennin preparation, Hala. The coagulating activity of Hala preparation differed compared with the other preparations, due to greater resistance to high pressures, especially in the range of 500-600 MPa. The preparations produced with genetic engineering methods lost their capability for milk protein coagulation by 500 MPa. Pressurization at 200 MPa contributed to their reduced capability for casein macroproteolysis. In contrast, an increase in Chymax, Chymogen, Maxiren and Hala preparations' hydrolytic capability for the macroproteolysis of isoelectric casein was observed upon pressure treatment at 100 and 400 MPa and for microproteolysis after pressure treatment at 200 MPa. Storage (48 h/5°C) of the pressurized preparations had an insignificant effect on their coagulating and proteolytic activities.

  18. Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II.

    PubMed

    Verma, Shikha; Mehta, Ranjit Kumar; Maiti, Prasanta; Röhm, Klaus-Heinrich; Sonawane, Avinash

    2014-07-01

    Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer-dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia. PMID:24721562

  19. Antimicrobial and antioxidant activities of clove essential oil and eugenyl acetate produced by enzymatic esterification.

    PubMed

    Vanin, Adriana B; Orlando, Tainara; Piazza, Suelen P; Puton, Bruna M S; Cansian, Rogério L; Oliveira, Debora; Paroul, Natalia

    2014-10-01

    This work reports the maximization of eugenyl acetate production by esterification of essential oil of clove in a solvent-free system using Novozym 435 as catalyst. The antimicrobial and antioxidant activities of clove essential oil and eugenyl acetate produced were determined. The conditions that maximized eugenyl acetate production were 60 °C, essential oil of clove to acetic anhydride ratio of 1:5, 150 rpm, and 10 wt% of enzyme, with a conversion of 99.87 %. A kinetic study was performed to assess the influence of substrates' molar ratio, enzyme concentration, and temperature on product yield. Results show that an excess of anhydride, enzyme concentration of 5.5 wt%, 50 °C, and essential oil of clove to acetic anhydride ratio of 1:5 afforded nearly a complete conversion after 2 h of reaction. Comparing the antibacterial activity of the essential oil of clove before and after esterification, we observed a decrease in the antimicrobial activity of eugenyl acetate, particularly with regard to minimum inhibitory concentration (MIC). Both eugenyl acetate and clove essential oil were most effective to the gram-negative than gram-positive bacteria group. The results showed a high antioxidant potential for essential oil before and particularly after the esterification reaction thus becoming an option for the formulation of new antioxidant products. PMID:25104002

  20. Initial characterization of a dually radiolabeled peptide for simultaneous monitoring of protein targets and enzymatic activity

    PubMed Central

    Mebrahtu, Efrem; Zheleznyak, Alexander; Hur, Minjun A.; Laforest, Richard; Lapi, Suzanne E.

    2016-01-01

    Objective The goal of this study was to develop dually radiolabeled peptides for simultaneous imaging of cancer cell localization by targeting the αvβ3 integrin and their pathophysiology by targeting the activity of the proteolytic enzyme MMP2, involved in the metastatic process. Methods A hybrid peptide c(RGDfE)K(DOTA)PLGVRY containing a RGD motif for binding to the αvβ3 integrin, a metal chelator (DOTA) for radiolabeling with [64Cu], and the MMP2 substrate cleavage sequence PLGVRY with terminal tyrosine for labeling with [123I] was synthesized, labeled with [64Cu] and [123I], and evaluated in vitro as a potential imaging agent. Results The peptide was synthesized and labeled with [64Cu] and [123I] with 300 and 40 μCi/μg (542 and 72.2 mCi/μmol) specific activities, respectively, and radiochemical purity of>98%.c(RGDfE)K(DOTA)PLGVRY demonstrated high affinity for αvβ3 integrins(Kd = 83.4 ± 13.2 nM) in both substrate competition and cell binding assays. c(RGDfE)K(DOTA)PLGVRY peptide, but not the scrambled version, c(RGDfE)K(DOTA)GRPLVY was specifically cleaved by MMP2. Conclusions These results demonstrate the feasibility of developing dually radiolabeled peptides for the simultaneous imaging of cancer cells and their pathophysiologic activity. PMID:23154178

  1. Effects of six selected antibiotics on plant growth and soil microbial and enzymatic activities.

    PubMed

    Liu, Feng; Ying, Guang-Guo; Tao, Ran; Zhao, Jian-Liang; Yang, Ji-Feng; Zhao, Lan-Feng

    2009-05-01

    The potential impact of six antibiotics (chlortetracycline, tetracycline and tylosin; sulfamethoxazole, sulfamethazine and trimethoprim) on plant growth and soil quality was studied by using seed germination test on filter paper and plant growth test in soil, soil respiration and phosphatase activity tests. The phytotoxic effects varied between the antibiotics and between plant species (sweet oat, rice and cucumber). Rice was most sensitive to sulfamethoxazole with the EC10 value of 0.1 mg/L. The antibiotics tested inhibited soil phosphatase activity during the 22 days' incubation. Significant effects on soil respiration were found for the two sulfonamides (sulfamethoxazole and sulfamethazine) and trimethoprim, whereas little effects were observed for the two tetracyclines and tylosin. The effective concentrations (EC10 values) for soil respiration in the first 2 days were 7 mg/kg for sulfamethoxazole, 13 mg/kg for sulfamethazine and 20 mg/kg for trimethoprim. Antibiotic residues in manure and soils may affect soil microbial and enzyme activities. PMID:19157661

  2. Screening for Genes Coding for Putative Antitumor Compounds, Antimicrobial and Enzymatic Activities from Haloalkalitolerant and Haloalkaliphilic Bacteria Strains of Algerian Sahara Soils

    PubMed Central

    Selama, Okba; Amos, Gregory C. A.; Djenane, Zahia; Borsetto, Chiara; Laidi, Rabah Forar; Porter, David; Nateche, Farida; Wellington, Elizabeth M. H.; Hacène, Hocine

    2014-01-01

    Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications. PMID:24977147

  3. Relationships between microbial extracellular enzymatic activity and suspended and sinking particulate organic matter: seasonal transformations in the North Water

    NASA Astrophysics Data System (ADS)

    Huston, A. L.; Deming, J. W.

    Despite the importance of hydrolytic activities by bacterial extracellular enzymes (EE) in the temperate ocean, little is known about the role of extracellular enzymatic activity (EEA) in determining the fate of particulate organic matter (POM) in polar seas. To explore the issue further, we measured various chemical and bacterial parameters in the near-0°C waters of the North Water during the months of May and July of 1998. Seawater (SW) samples were collected by Niskin bottle at the depth of the chlorophyll fluorescence maximum (8-90 m), while samples of sinking particles and aggregates were collected in short-term (0.5-1.2 d), unpoisoned, floating sediment traps deployed at depths typically below the mixed layer (50-136 m). Samples were analyzed for POC, PON, and abundance of total and actively respiring bacteria. They were also incubated with fluorescently tagged substrate analogs to measure potential maximal rates of three classes of EE (leucine-aminopeptidase, chitobiase, and β-glucosidase) at -1°C. The percentage of actively respiring bacteria was always higher in sediment trap samples than in SW (medians of 38% and 24% versus 10% and 12% in May and July, respectively). Cell-specific rates of EEA were also higher in the trap samples and, for both sample types, similar to published rates from temperate waters. Rates of EEA when scaled to the abundance of actively respiring bacteria, however, did not differ between sample types, suggesting that the elevated EEA associated with sinking material is due to the greater abundance of metabolically active cells supported by such material and not due to enhanced enzyme expression in general, as suggested by previous studies. In this study, leucine-aminopeptidase activity was always much higher than the other classes of EEA, becoming even more dominant later in the season; it always correlated positively with the abundance of both total and actively respiring bacteria. Enzyme ratios indicating protease dominance

  4. Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: characterization and application for enzymatic inhibition assays.

    PubMed

    Zhu, Yuan-Ting; Ren, Xiao-Yun; Liu, Yi-Ming; Wei, Ying; Qing, Lin-Sen; Liao, Xun

    2014-05-01

    Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe3O4-SiO2) possessed three dimensional core-shell structures with an average diameter of ~20 nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25 emu·g(-1). The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The Km and the Vmax values (0.02 mM, 6.40 U·mg(-1) enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29 mM, 3.16 U·mg(-1) enzyme). Furthermore, at an elevated temperature of 70 °C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds. PMID:24656379

  5. Protoporphyrins Enhance Oligomerization and Enzymatic Activity of HtrA1 Serine Protease

    PubMed Central

    Jo, Hakryul; Patterson, Victoria; Stoessel, Sean; Kuan, Chia-Yi; Hoh, Josephine

    2014-01-01

    High temperature requirement protein A1 (HtrA1), a secreted serine protease of the HtrA family, is associated with a multitude of human diseases. However, the exact functions of HtrA1 in these diseases remain poorly understood. We seek to unravel the mechanisms of HtrA1 by elucidating its interactions with chemical or biological modulators. To this end, we screened a small molecule library of 500 bioactive compounds to identify those that alter the formation of extracellular HtrA1 complexes in the cell culture medium. An initial characterization of two novel hits from this screen showed that protoporphyrin IX (PPP-IX), a precursor in the heme biosynthetic pathway, and its metalloporphyrin (MPP) derivatives fostered the oligomerization of HtrA1 by binding to the protease domain. As a result of the interaction with MPPs, the proteolytic activity of HtrA1 against Fibulin-5, a specific HtrA1 substrate in age-related macular degeneration (AMD), was increased. This physical interaction could be abolished by the missense mutations of HtrA1 found in patients with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Furthermore, knockdown of HtrA1 attenuated apoptosis induced by PPP-IX. These results suggest that PPP-IX, or its derivatives, and HtrA1 may function as co-factors whereby porphyrins enhance oligomerization and the protease activity of HtrA1, while active HtrA1 elevates the pro-apoptotic actions of porphyrin derivatives. Further analysis of this interplay may shed insights into the pathogenesis of diseases such as AMD, CARASIL and protoporphyria, as well as effective therapeutic development. PMID:25506911

  6. Antioxidant enzymatic activity is linked to waterlogging stress tolerance in citrus.

    PubMed

    Arbona, Vicent; Hossain, Zahed; López-Climent, María F; Pérez-Clemente, Rosa M; Gómez-Cadenas, Aurelio

    2008-04-01

    Soil flooding constitutes a seasonal factor that negatively affects plant performance and crop yields. In this work, the relationship between oxidative damage and flooding sensitivity was addressed in three citrus genotypes with different abilities to tolerate waterlogging. We examined leaf visible damage, oxidative damage in terms of malondialdehyde (MDA) concentration, leaf proline concentration, leaf and root ascorbate and glutathione contents and the antioxidant enzyme activities superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6) and glutathione reductase (EC 1.8.1.7). No differences in the extent of oxidative damage relative to controls were found among genotypes. However, a different ability to delay the apparition of oxidative damage was associated to a higher tolerance to waterlogging. This ability was linked to an enhanced activated oxygen species' scavenging capacity in terms of an increased antioxidant enzyme activity and higher content in polar antioxidant compounds. Therefore, the existence of a direct relationship between stress sensitivity and the early accumulation of MDA is proposed. In addition, data indicate that the protective role of proline has to be considered minimal as its accumulation was inversely correlated with tolerance to the stress. The positive antioxidant response in Carrizo citrange (Poncirus trifoliata L. Raf. x Citrus sinensis L. Osb.) and Citrumelo CPB 4475 (Poncirus trifoliata L. Raf. x Citrus paradisi L. Macf.) might be responsible for a higher tolerance to flooding stress, whereas in Cleopatra mandarin (Citrus reshni Hort. Ex Tan.), the early accumulation of MDA seems to be associated to an impaired ability for H2O2 scavenging. PMID:18333999

  7. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst)

    PubMed Central

    Martins, Julia M.; DeMarco, Ricardo; Jameson, David M.; Castro, Aline M.; Bossolan, Nelma R. S.; Wallace, B. A.; Araujo, Ana P. U.

    2016-01-01

    Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required. PMID:27351338

  8. Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes.

    PubMed

    Crofts, Naoko; Abe, Natsuko; Oitome, Naoko F; Matsushima, Ryo; Hayashi, Mari; Tetlow, Ian J; Emes, Michael J; Nakamura, Yasunori; Fujita, Naoko

    2015-08-01

    Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein-protein interactions in maize and wheat amyloplasts. This study investigated whether protein-protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200-400kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs-BEs, and, among BE isozymes, BEIIa-Pho1, and pullulanase-type DBE-BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch. PMID:25979995

  9. Hemolytic activity and solubilizing capacity of raffinose and melezitose fatty acid monoesters prepared by enzymatic synthesis.

    PubMed

    Carvalho, Luis; Morales, Juan C; Pérez-Victoria, José M; Pérez-Victoria, Ignacio

    2015-05-01

    The hemolytic activity and solubilizing capacity of two families of non-reducing trisaccharide fatty acid monoesters have been studied to assess their usefulness as surfactants for pharmaceutical applications. The carbohydrate-based surfactants investigated included homologous series of raffinose and melezitose monoesters bearing C10 to C18 acyl chains prepared by lipase-catalyzed synthesis in organic media. The hemolytic activity was determined in vitro using a static method based on the addition of the surfactants to an erythrocyte suspension and subsequent spectrophotometric determination of the released hemoglobin. The effect of the carbohydrate head group, the acyl chain length and the regioisomeric purity was investigated. In all cases, the carbohydrate monoester surfactants decreased their hemolytic activity (with respect to their critical micelle concentration) when increasing the length of the acyl chain. A very similar behaviour was observed either the carbohydrate head-group (raffinose and melezitose) or regardless of the regioisomeric purity. Interestingly, decanoyl (C10) and lauroyl (C12) monoesters were just marginally hemolytic at their critical micelle concentrations while the longer palmitoyl (C16) and (C18) stearoyl monoesters become hemolytic at concentrations much higher than their respective cmc. The palmitoyl and stearoyl monoesters also displayed higher solubilization capacity than the shorter acyl chain monoesters in a solubilization assay of a hydrophobic dye as a model drug mimic. These results suggest that raffinose and melezitose monoesters with long-chain fatty acids (C16 to C18) are promising surfactants for pharmaceutical applications and could be an alternative to the use of current commercial nonionic polyoxyethylene-based surfactants in parenteral formulations. PMID:25753196

  10. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).

    PubMed

    Lopes, Jose L S; Yoneda, Juliana S; Martins, Julia M; DeMarco, Ricardo; Jameson, David M; Castro, Aline M; Bossolan, Nelma R S; Wallace, B A; Araujo, Ana P U

    2016-01-01

    Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required. PMID:27351338

  11. Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes

    PubMed Central

    Crofts, Naoko; Abe, Natsuko; Oitome, Naoko F.; Matsushima, Ryo; Hayashi, Mari; Tetlow, Ian J.; Emes, Michael J.; Nakamura, Yasunori; Fujita, Naoko

    2015-01-01

    Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein–protein interactions in maize and wheat amyloplasts. This study investigated whether protein–protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200–400kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs–BEs, and, among BE isozymes, BEIIa–Pho1, and pullulanase-type DBE–BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch. PMID:25979995

  12. The enzymatic activity of lysyl oxidas-like-2 (LOXL2) is not required for LOXL2-induced inhibition of keratinocyte differentiation.

    PubMed

    Lugassy, Jennie; Zaffryar-Eilot, Shelly; Soueid, Sharon; Mordoviz, Amit; Smith, Victoria; Kessler, Ofra; Neufeld, Gera

    2012-01-27

    Lysyl oxidase-like-2 (LOXL2) induces tumor progression and fibrosis. It also inhibits the differentiation of keratinocytes promoting development of squamous cell carcinomas. Stimulation of HaCaT skin keratinocytes with exogenous LOXL2 or overexpression of LOXL2 in these cells inhibits their differentiation as manifested by inhibition of calcium or vitamin D-induced involucrin expression. The inhibition was abrogated by the LOXL2 function-blocking monoclonal antibody AB0023 as well as by an anti-LOXL2 polyclonal antibody. Surprisingly, a point-mutated form of LOXL2 (LOXL2(Y689F)) lacking enzymatic activity, as well as a LOXL2 deletion mutant lacking the entire catalytic domain, also inhibited calcium or vitamin D-induced up-regulation of involucrin expression, suggesting that the enzymatic activity of LOXL2 is not required for this activity. This conclusion was supported by experiments that showed that β-aminoproprionitrile, an irreversible competitive inhibitor of the enzymatic activity of all lysyl oxidases, is unable to abolish the LOXL2-induced inhibition of HaCaT cell differentiation. The activity of LOXL2(Y689F) required the presence of the fourth scavenger receptor-cysteine-rich (SRCR) domain of LOXL2, which is also the binding target of AB0023. Epitope-tagged LOXL2(Y689F) was internalized at 37 °C by HaCaT cells. The internalization was inhibited by AB0023 and by competition with unlabeled LOXL2, suggesting that these cells may express a LOXL2 receptor. Our results suggest that agents that inhibit the enzymatic activity of LOXL2 may not suffice to inhibit completely the effects of LOXL2 on complex processes that involve altered states of cellular differentiation. PMID:22157764

  13. Caspase-like enzymatic activity and the ascorbate-glutathione cycle participate in salt stress tolerance of maize conferred by exogenously applied nitric oxide

    PubMed Central

    Keyster, Marshall; Klein, Ashwil; Ludidi, Ndiko

    2012-01-01

    Salinity stress causes ionic stress (mainly from high Na+ and Cl- levels) and osmotic stress (as a result of inhibition of water uptake by roots and amplified water loss from plant tissue), resulting in cell death and inhibition of growth and ultimately adversely reducing crop productivity. In this report, changes in root nitric oxide content, shoot and root biomass, root H2O2 content, root lipid peroxidation, root cell death, root caspase-like enzymatic activity, root antioxidant enzymatic activity and root ascorbate and glutathione contents/redox states were investigated in maize (Zea mays L. cv Silverking) after long-term (21 d) salt stress (150 mM NaCl) with or without exogenously applied nitric oxide generated from the nitric oxide donor 2,2′-(Hydroxynitrosohydrazano)bis-ethane. In addition to reduced shoot and root biomass, salt stress increased the nitric oxide and H2O2 contents in the maize roots and resulted in elevated lipid peroxidation, caspase-like activity and cell death in the roots. Altered antioxidant enzymatic activities, along with changes in ascorbate and glutathione contents/redox status were observed in the roots in response to salt stress. The detrimental effects of salt stress in the roots were reversed by exogenously applied nitric oxide. These results demonstrate that exogenously applied nitric oxide confers salt stress tolerance in maize by reducing salt stress-induced oxidative stress and caspase-like activity through a process that limits accumulation of reactive oxygen species via enhanced antioxidant enzymatic activity. PMID:22476534

  14. Enhancing phytochemical levels, enzymatic and antioxidant activity of spinach leaves by chitosan treatment and an insight into the metabolic pathway using DART-MS technique.

    PubMed

    Singh, Shachi

    2016-05-15

    Phytochemicals are health promoting compounds, synthesized by the plants to protect them against biotic or abiotic stress. The metabolic pathways leading to the synthesis of these phytochemicals are highly inducible; therefore methods could be developed to enhance their production by the exogenous application of chemical inducers/elicitors. In the present experiment, chitosan was used as an elicitor molecule to improve the phytochemical content of spinach plant. When applied at a concentration of 0.01 mg/ml as a foliar spray, chitosan was able to cause an increase in the enzymatic (peroxidase, catalase and phenylalanine ammonium lyase (PAL)) and non enzymatic (total phenolics, flavonoids and proteins) defensive metabolites, as well as, in the total antioxidant activity of the spinach leaves. A 1.7-fold increase in the total phenolics, a 2-fold increase in total flavonoid and a 1.6-fold increase in total protein were achieved with the treatment. A higher level of enzymatic activity was observed with a 4-fold increase in peroxidase and approximately 3-fold increases in catalase and phenylalanine ammonium lyase activity. Antioxidant activity showed a positive correlation between phenolic compounds and the enzymatic activity. Direct analysis in real time mass spectrometry (DART-MS) was applied to generate the metabolite profile of control and treated leaves. DART analysis revealed the activation of phenylpropanoid pathway by chitosan molecule, targeting the synthesis of diverse classes of flavonoids and their glycosides. Important metabolites of stress response were also visible in the DART spectra, including proline and free sugars. PMID:26775959

  15. Biodegradation and extracellular enzymatic activities of Pseudomonas aeruginosa strain GF31 on β-cypermethrin.

    PubMed

    Tang, Aixing; Wang, Bowen; Liu, Youyan; Li, Qingyun; Tong, Zhangfa; Wei, Yingjun

    2015-09-01

    Pseudomonas aeruginosa strain GF31, isolated from a contaminated soil, can effectively degrade β-cypermethrin (β-CP), as well as fenpropathrin, fenvalerate, and cyhalothrin. The highest level of degradation (81.2 %) was achieved with the addition of peptone. Surprisingly, the enzyme responsible for degradation was mainly localized to the extracellular areas of the bacteria, in contrast to the other known pyrethroid-degrading enzymes, which are intracellular. Although intact bacterial cells function at about 30 °C for biodegradation, similar to other degrading strains, the crude extracellular extract of strain GF31 remained biologically active at 60 °C. Moreover, the extract fraction showed good storage stability, maintaining >50 % of its initial activity following storage at 25 °C for at least 20 days. Significant differences in the characteristics of the crude GF31 extracellular extract compared with the known pyrethroid-degrading enzymes indicate the presence of a novel pyrethroid-degrading enzyme. Furthermore, the identification of 3-phenoxybenzoic acid and 2,2-dimethylcyclopropanecarboxylate from the degradation products suggests the possibility that β-CP degradation by both the strain and the crude extracellular fraction is achieved through a hydrolysis pathway. Further degradation of these two metabolites may lead to the development of an efficient method for the mineralization of these types of pollutants. PMID:25921758

  16. Yersinia pestis lacZ expresses a beta-galactosidase with low enzymatic activity.

    PubMed

    Bobrov, Alexander G; Perry, Robert D

    2006-02-01

    Although very little, if any, beta-galactosidase activity is detected in Yersinia pestis by a standard Miller assay, we found that Y. pestis KIM6+ cells formed blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). Searches of the Y. pestis genome databases revealed the presence of noncontiguous sequences highly homologous to Escherichia coli lacZ, lacY, and lacI. Yersinia pestis lacZ is predicted to encode a 1060 amino-acid protein with 62% identity and 72% similarity to beta-galactosidase from E. coli. A deletion in the Y. pestis lacZ gene caused the formation of white colonies on X-gal-containing plates and beta-galactosidase activity was at background levels in the KIM6+lacZ mutant, while the complemented strain expressed about 190 Miller units. The Y. pestis lacZ promoter was not regulated by isopropylthiogalactoside or glucose. Finally, uptake of lactose by Y. pestis may be impaired. PMID:16436060

  17. A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions

    PubMed Central

    Schuster, Sabine; Roessler, Claudia; Meleshin, Marat; Zimmermann, Philipp; Simic, Zeljko; Kambach, Christian; Schiene-Fischer, Cordelia; Steegborn, Clemens; Hottiger, Michael O.; Schutkowski, Mike

    2016-01-01

    Sirtuins are NAD+ dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1–6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M−1s−1. These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM). PMID:26940860

  18. Surface energy modified chips for detection of conformational states and enzymatic activity in biomolecules.

    PubMed

    Asberg, Peter; Nilsson, K Peter R; Inganäs, Olle

    2006-02-28

    A novel patterning method for anchoring biomolecules and noncovalent assembled conjugated polyelectrolyte (CPE)/biomolecule complexes to a chip surface is presented. The surface energy of a hydrophilic substrate is modified using an elastomeric poly(dimethylsiloxane) (PDMS) stamp, containing a relief pattern. Modification takes place on the parts where the PDMS stamp is in conformal contact with the substrate and leaves low molecular weight PDMS residues on the surface resulting in a hydrophobic modification, and then biomolecules and CPE/biomolecule complexes are then adsorbed in a specific pattern. The method constitutes a discrimination system for different conformations in biomolecules using CPEs as reporters and the PDMS modified substrates as the discriminator. Detection of different conformations in two biomacromolecules, a synthetic peptide (JR2E) and a protein (calmodulin), reported by the CPE and resolved by fluorescence was demonstrated. Also, excellent enzyme activity in patterned CPE/horseradish peroxidase (HRP) enzyme was shown, demonstrating that this method can be used to pattern biomolecules with their activity retained. The method presented could be useful in various biochip applications, such as analyzing proteins and peptides in large-scale production, in making metabolic chips, and for making multi-microarrays. PMID:16489808

  19. An enzymatic activity isolated from Brassica oleracea specific for UV-irradiated DNA

    SciTech Connect

    Gallagher, P.E.; Lenhart, J.R.; Weiss, R.B. )

    1991-03-11

    As a consequence of a breakdown in the ozone layer, an increase in the amount of DNA damage caused by ultraviolet irradiation can be expected. Organisms have evolved mechanisms to repair numerous types of DNA damages. While these DNA repair systems have been well characterized in bacteria and to a lesser extent in mammalian cells, surprisingly little is known about repair of potentially harmful DNA lesions in plants. An enzyme that recognizes and incises UV irradiated DNA has been partially purified from the leaf tissue of Brassica oleracea. Glycosylase-produced base loss sites were detected by a nitrocellulose filter-binding assay using UV-irradiated PM2 viral DNA as the substrate. The optimal temperature for maximal enzyme activity is 47C with a pH optimum between 7.0 and 7.5. In addition, the endonuclease is active in both Tris and phosphate buffers, although it is stimulated by phosphate concentrations up to 25 mM. Currently, a number of synthetic polynucleotides as well as DNAs of defined sequence are being employed as substrates to determine the nature of the UV-induced lesion and the precise mechanism of action of the enzyme.

  20. Cathepsin S of Sciaenops ocellatus: Identification, transcriptional expression and enzymatic activity.

    PubMed

    Sun, Bo-Guang; Chi, Heng

    2016-01-01

    Cathepsin S is a member of cysteine cathepsins and belongs to the cathepsin L-like family. In mammals, it is known to participate in various physiological processes and host immune defense. In teleost fish, the function of cathepsin S is less investigated. In the present work, we characterized a cathepsin S homologue (SoCatS) from red drum (Sciaenops ocellatus), a commercially valuable fish in Chinese mariculture. Like all cathepsin S, SoCatS possesses a peptidase domain with four catalytically essential residues (Gln140, Cys146, His285, and Asn305) conserved in the cathepsin S of different organisms. SoCatS shares 60-90% overall sequence identities with known teleost cathepsin S. Phylogenetic profiling indicated that SoCatS is evolutionally close to the cathepsin S of other teleost fish, especially Miichthys miiuy, a member of Sciaenidae family like red drum. SoCatS expression was detected in various tissues and was enhanced by bacterial infection. Purified recombinant SoCatS exhibited apparent peptidase activity with maximum at 50°C and pH 7.5. This activity depended on the catalytic residue Cys146 and was severely reduced by the cathepsin inhibitor E-64. Our results suggest that SoCatS functions as a cysteine protease which is probably involved in the antibacterial immunity of red drum. PMID:26522244

  1. Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis.

    PubMed

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)(+) dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP(+) yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  2. Lignin binding to pancreatic lipase and its influence on enzymatic activity.

    PubMed

    Zhang, Juan; Xiao, Lin; Yang, Yucai; Wang, Zhaoxia; Li, Genxi

    2014-04-15

    In this paper, we find that the effect of lignin on pancreatic lipase (PL) is dependent on reaction medium and substrate used. Experimental results reveal that lignin can gradually bind to PL to form a PL-lignin complex, resulting in an increased activity of the enzyme. The binding process is spontaneous and the PL-lignin complex formation is an endothermic reaction induced by hydrophobic and electrostatic interaction. There is a non-radiation energy transfer from PL to lignin during the binding process, and the binding of lignin to PL conforms to a secondary exponential decay function. Moreover, the α-helix content of the enzyme will be changed and the rigidity of its side chain will be enhanced due to the formation of lignin-PL complex. This study has not only provided the activation effect of lignin on PL, but also given an insight into the interaction between lignin and the enzyme, which would benefit the application of lignin in the pharmacy and food industry, as well as other fields. PMID:24295682

  3. Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

    PubMed Central

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P.; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V.

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  4. A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions.

    PubMed

    Schuster, Sabine; Roessler, Claudia; Meleshin, Marat; Zimmermann, Philipp; Simic, Zeljko; Kambach, Christian; Schiene-Fischer, Cordelia; Steegborn, Clemens; Hottiger, Michael O; Schutkowski, Mike

    2016-01-01

    Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1-6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M(-1)s(-1). These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM). PMID:26940860

  5. Reversible Post-Translational Carboxylation Modulates The Enzymatic Activity Of N-Acetyl-L-Ornithine Transcarbamylase†

    PubMed Central

    Li, Yongdong; Yu, Xiaolin; Ho, Jeremy; Fushman, David; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang

    2010-01-01

    N-acetyl-L-ornithine transcarbamylase (AOTCase), rather than ornithine transcarbamylase (OTCase), is the essential carbamylase enzyme in the arginine biosynthesis of several plant and human pathogens. The specificity of this unique enzyme provides a potential target for controlling the spread of these pathogens. Recently, several crystal structures of AOTCase from Xanthomonas campestris (xc) have been determined. In these structures, an unexplained electron density at the tip of Lys302 side-chain was observed. Using 13C NMR spectroscopy, we show herein that Lys302 is post-translationally carboxylated. The structure of wild-type AOTCase complexed with the bisubstrate analogue, Nδ-(phosphonoacetyl)-Nα-acetyl-L-ornithine (PALAO), indicates that the carboxyl group on Lys302 forms a strong hydrogen bonding network with surrounding active site residues, Lys252, Ser253, His293, and Glu92 from the adjacent subunit either directly or via a water molecule. Furthermore, the carboxyl group is involved in binding N-acetyl-L-ornithine via a water molecule. Activity assays with the wild-type enzyme and several mutants demonstrate that the post translational modification of lysine 302 has an important role in catalysis. PMID:20695527

  6. Evolution of Enzymatic Activities in the Enolase Superfamily: Galactarate Dehydratase III from Agrobacterium tumefaciens C58

    PubMed Central

    2015-01-01

    The genome of Agrobacterium tumefaciens C58 encodes 12 members of the enolase superfamily (ENS), eight of which are members of the mandelate racemase (MR) subgroup and, therefore, likely to be acid sugar dehydratases. Using a library of 77 acid sugars for high-throughput screening, one protein (UniProt entry A9CG74; locus tag Atu4196) showed activity with both m-galactarate and d-galacturonate. Two families of galactarate dehydratases had been discovered previously in the ENS, GalrD/TalrD [Yew, W. S., et al. (2007) Biochemistry46, 9564–9577] and GalrD-II [Rakus, J. F., et al. (2009) Biochemistry48, 11546–11558]; these have different active site acid/base catalysis and have no activity with d-galacturonate. A9CG74 dehydrates m-galactarate to form 2-keto-3-deoxy-galactarate but does not dehydrate d-galacturonate as expected. Instead, when A9CG74 is incubated with d-galacturonate, 3-deoxy-d-xylo-hexarate or 3-deoxy-d-lyxo-hexarate is formed. In this reaction, instead of abstracting the C5 proton α to the carboxylate group, the expected reaction for a member of the ENS, the enzyme apparently abstracts the proton α to the aldehyde group to form 3-deoxy-d-threo-hexulosuronate that undergoes a 1,2-hydride shift similar to the benzylic acid rearrangement to form the observed product. A. tumefaciens C58 does not utilize m-galactarate as a carbon source under the conditions tested in this study, although it does utilize d-galacturonate, which is a likely precursor to m-galactarate. The gene encoding A9CG74 and several genome proximal genes were upregulated with d-galacturonate as the carbon source. One of these, a member of the dihydrodipicolinate synthase superfamily, catalyzes the dehydration and subsequent decarboxylation of 2-keto-3-deoxy-d-galactarate to α-ketoglutarate semialdehyde, thereby providing a pathway for the conversion of m-galactarate to α-ketoglutarate semialdehyde. PMID:24926996

  7. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Fuconate Dehydratase from Xanthomonas campestris

    SciTech Connect

    Yew,W.; Fedorov, A.; Fedorov, E.; Rakus, J.; Pierce, R.; Almo, S.; Gerlt, J.

    2006-01-01

    Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report the authors use both genome (operon) context and screening of a library of acid sugars to assign the L-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI: 21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of th third, fourth, and fifth-strands in the (/)7-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and sixth-strands (His 351 and Asp 324, respectively), and a Glue at the end of the eighth-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting 2-keto-3-deoxy-L-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to L-fuconate, FucD also catalyzes the dehydration of L-galactonate, D-arabinonate, D-altronate, L-talonate, and D-ribonate. The dehydrations of L-fuconate, L-galactonate, and D-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of L-talonate and D-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields L

  8. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Rhamnonate Dehydratase

    SciTech Connect

    Rakus,J.; Fedorov, A.; Fedorov, E.; Glaner, M.; Hubbard, B.; Delli, J.; Babbitt, P.; Almo, S.; Gerlt, J.

    2008-01-01

    The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy-l-mannonate) has the 'best' kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg2+; the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy-l-rhamnonate (obtained by reduction of the product with NaBH4). Like other members of the enolase superfamily, RhamD contains an N-terminal a + {beta} capping domain and a C-terminal ({beta}/a)7{beta}-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining '20s loop' in the capping domain is extended in length and the '50s loop' is truncated. The ligands for the Mg2+ are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth {beta}-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth {beta}-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg2+-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and delivers a proton to carbon-3

  9. Evolution of enzymatic activities in the enolase superfamily: galactarate dehydratase III from Agrobacterium tumefaciens C58.

    PubMed

    Groninger-Poe, Fiona P; Bouvier, Jason T; Vetting, Matthew W; Kalyanaraman, Chakrapani; Kumar, Ritesh; Almo, Steven C; Jacobson, Matthew P; Gerlt, John A

    2014-07-01

    The genome of Agrobacterium tumefaciens C58 encodes 12 members of the enolase superfamily (ENS), eight of which are members of the mandelate racemase (MR) subgroup and, therefore, likely to be acid sugar dehydratases. Using a library of 77 acid sugars for high-throughput screening, one protein (UniProt entry A9CG74; locus tag Atu4196) showed activity with both m-galactarate and d-galacturonate. Two families of galactarate dehydratases had been discovered previously in the ENS, GalrD/TalrD [Yew, W. S., et al. (2007) Biochemistry 46, 9564-9577] and GalrD-II [Rakus, J. F., et al. (2009) Biochemistry 48, 11546-11558]; these have different active site acid/base catalysis and have no activity with d-galacturonate. A9CG74 dehydrates m-galactarate to form 2-keto-3-deoxy-galactarate but does not dehydrate d-galacturonate as expected. Instead, when A9CG74 is incubated with d-galacturonate, 3-deoxy-d-xylo-hexarate or 3-deoxy-d-lyxo-hexarate is formed. In this reaction, instead of abstracting the C5 proton α to the carboxylate group, the expected reaction for a member of the ENS, the enzyme apparently abstracts the proton α to the aldehyde group to form 3-deoxy-d-threo-hexulosuronate that undergoes a 1,2-hydride shift similar to the benzylic acid rearrangement to form the observed product. A. tumefaciens C58 does not utilize m-galactarate as a carbon source under the conditions tested in this study, although it does utilize d-galacturonate, which is a likely precursor to m-galactarate. The gene encoding A9CG74 and several genome proximal genes were upregulated with d-galacturonate as the carbon source. One of these, a member of the dihydrodipicolinate synthase superfamily, catalyzes the dehydration and subsequent decarboxylation of 2-keto-3-deoxy-d-galactarate to α-ketoglutarate semialdehyde, thereby providing a pathway for the conversion of m-galactarate to α-ketoglutarate semialdehyde. PMID:24926996

  10. Rapid Bioorthogonal Chemistry Turn-on through Enzymatic or Long Wavelength Photocatalytic Activation of Tetrazine Ligation.

    PubMed

    Zhang, Han; Trout, William S; Liu, Shuang; Andrade, Gabriel A; Hudson, Devin A; Scinto, Samuel L; Dicker, Kevin T; Li, Yi; Lazouski, Nikifar; Rosenthal, Joel; Thorpe, Colin; Jia, Xinqiao; Fox, Joseph M

    2016-05-11

    Rapid bioorthogonal reactivity can be induced by controllable, catalytic stimuli using air as the oxidant. Methylene blue (4 μM) irradiated with red light (660 nm) catalyzes the rapid oxidation of a dihydrotetrazine to a tetrazine thereby turning on reactivity toward trans-cyclooctene dienophiles. Alternately, the aerial oxidation of dihydrotetrazines can be efficiently catalyzed by nanomolar levels of horseradish peroxidase under peroxide-free conditions. Selection of dihydrotetrazine/tetrazine pairs of sufficient kinetic stability in aerobic aqueous solutions is key to the success of these approaches. In this work, polymer fibers carrying latent dihydrotetrazines were catalytically activated and covalently modified by trans-cyclooctene conjugates of small molecules, peptides, and proteins. In addition to visualization with fluorophores, fibers conjugated to a cell adhesive peptide exhibited a dramatically increased ability to mediate contact guidance of cells. PMID:27078610

  11. Factors influencing the rate of non-enzymatic activation of carboxylic and amino acids by ATP

    NASA Technical Reports Server (NTRS)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1981-01-01

    The nonenzymatic formation of adenylate anhydrides of carboxylic and amino acids is discussed as a necessary step in the origin of the genetic code and protein biosynthesis. Results of studies are presented which have shown the rate of activation to depend on the pKa of the carboxyl group, the pH of the medium, temperature, the divalent metal ion catalyst, salt concentration, and the nature of the amino acid. In particular, it was found that of the various amino acids investigated, phenylalanine had the greatest affinity for the adenine derivatives adenosine and ATP. Results thus indicate that selective affinities between amino acids and nucleotides were important during prebiotic chemical evolution, and may have played a major role in the origin of protein synthesis and genetic coding.

  12. The antihyperlipidemic activities of enzymatic and acidic intracellular polysaccharides by Termitomyces albuminosus.

    PubMed

    Zhao, Huajie; Li, Shangshang; Zhang, Jianjun; Che, Gen; Zhou, Meng; Liu, Min; Zhang, Chen; Xu, Nuo; Lin, Lin; Liu, Yu; Jia, Le

    2016-10-20

    Two polysaccharides, EIPS and AIPS were obtained by the hydrolysis of IPS from Termitomyces albuminosus, and their pharmacological effects on blood lipid profiles metabolism and oxidative stress were investigated. The results demonstrated that EIPS was superior to IPS and AIPS on reducing hepatic lipid levels and preventing oxidative stress by improving serum enzyme activities (ALT, AST, and ALP), serum lipid levels (TC, TG, HDL-C, LDL-C and VLDL-C), hepatic lipid levels (TC and TG), and antioxidant status (SOD, GSH-Px, CAT, T-AOC, MDA, and LPO). These conclusions indicated that EIPS, AIPS and IPS might be suitable for functional foods and natural drugs on preventing the high-fat emulsion-induced hyperlipidemia. In addition, the monosaccharide compositions of IPS and its hydrolyzate were also processed. PMID:27474674

  13. Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

    PubMed Central

    2011-01-01

    Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

  14. Toxicity of perfluorooctanoic acid towards earthworm and enzymatic activities in soil.

    PubMed

    He, Wenxiang; Megharaj, Mallavarapu; Naidu, Ravi

    2016-07-01

    Perfluorooctanoic acid (PFOA) is a widespread persistent organic contaminant in the environment that has recently raised much of regulatory and public concern. Therefore, assessment of its ecological risk is a top priority research. Hence, this study investigated the toxicity of PFOA to beneficial microbial processes in the soil such as activities of dehydrogenase, urease and potential nitrification in addition to earthworm survival, weight loss and PFOA bioaccumulation in two contrasting soils. In general, PFOA caused inhibition of all the measured microbial processes in a dose-dependent manner and the inhibition was higher in Williamtown (WT) soil than Edinburgh (EB) soil. Thus, WT soil being sandy in nature with low clay content showed higher PFOA bioavailability and hence showed higher toxicity. There was no mortality in earthworms exposed up to 100 mg PFOA/kilogram soil in both the soils; however, there was a significant weight loss from 25 mg/kg onwards. This study clearly demonstrates that soil contamination of PFOA can lead to adverse effects on soil health. PMID:27329475

  15. Toward an Automatic Determination of Enzymatic Reaction Mechanisms and Their Activation Free Energies.

    PubMed

    Zinovjev, Kirill; Ruiz-Pernía, J Javier; Tuñón, Iñaki

    2013-08-13

    We present a combination of the string method and a path collective variable for the exploration of the free energy surface associated to a chemical reaction in condensed environments. The on-the-fly string method is employed to find the minimum free energy paths on a multidimensional free energy surface defined in terms of interatomic distances, which is a convenient selection to study bond forming/breaking processes. Once the paths have been determined, a reaction coordinate is defined as a measure of the advance of the system along these paths. This reaction coordinate can be then used to trace the reaction Potential of Mean Force from which the activation free energy can be obtained. This combination of methodologies has been here applied to the study, by means of Quantum Mechanics/Molecular Mechanics simulations, of the reaction catalyzed by guanidinoacetate methyltransferase. This enzyme catalyzes the methylation of guanidinoacetate by S-adenosyl-l-methionine, a reaction that involves a methyl transfer and a proton transfer and for which different reaction mechanisms have been proposed. PMID:26584125

  16. Vialinin A and thelephantin G, potent inhibitors of tumor necrosis factor-α production, inhibit sentrin/SUMO-specific protease 1 enzymatic activity.

    PubMed

    Yoshioka, Yasukiyo; Namiki, Daisuke; Makiuchi, Mao; Sugaya, Kouichi; Onose, Jun-Ichi; Ashida, Hitoshi; Abe, Naoki

    2016-09-01

    Several p-terphenyl compounds have been isolated from the edible Chinese mushroom Thelephora vialis. Vialinin A, a p-terphenyl compound, strongly inhibits tumor necrosis factor-α production and release. Vialinin A inhibits the enzymatic activity of ubiquitin-specific peptidase 5, one of the target molecules in RBL-2H3 cells. Here we examined the inhibitory effect of p-terphenyl compounds, including vialinin A, against sentrin/SUMO-specific protease 1 (SENP1) enzymatic activity. The half maximal inhibitory concentration values of vialinin A and thelephantin G against full-length SENP1 were 1.64±0.23μM and 2.48±0.02μM, respectively. These findings suggest that p-terphenyl compounds are potent SENP1 inhibitors. PMID:27491710

  17. Extraction and quantitation of coumarin from cinnamon and its effect on enzymatic browning in fresh apple juice: a bioinformatics approach to illuminate its antibrowning activity.

    PubMed

    Thada, Rajarajeshwari; Chockalingam, Shivashri; Dhandapani, Ramesh Kumar; Panchamoorthy, Rajasekar

    2013-06-01

    Enzymatic browning by polyphenoloxidase (PPO) affects food quality and taste in fruits and vegetables. Thus, the study was designed to reduce browning in apple juice by coumarin. The ethanolic extract of cinnamon was prepared and its coumarin content was quantitated by HPLC, using authentic coumarin (AC) as standard. The effect of cinnamon extract (CE) and AC on enzymatic browning, its time dependent effects, and the specific activity of PPO and peroxidase (POD) were studied in apple juice. The docking of coumarin with PPO and POD was also performed to elucidate its antibrowning mechanism. The CE (73%) and AC (82%) showed better reduction in browning, maintained its antibrowning effect at all time points, and significantly (p < 0.05) reduced the specific activity of PPO and POD when compared with controls. Coumarin showed strong interaction with binding pockets of PPO and POD, suggesting its potential use as inhibitor to enzyme mediated browning in apple juice. PMID:23683299

  18. Evolution of Enzymatic Activities in the Enolase Superfamily: D-Mannonate Dhydratase from Novosphingobium aromaticivorans

    SciTech Connect

    Rakus,J.; Fedorov, A.; Fedorov, E.; Glasner, M.; Vick, J.; Babbitt, P.; Almo, S.; Gerlt, J.

    2007-01-01

    The d-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of the wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-d-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the d-mannonate substrate. As previously observed in the structures of other members of the enolase superfamily, ManD contains two domains, an N-terminal a+{beta} capping domain and a ({beta}/a)7{beta}-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth {beta}-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second {beta}-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth {beta}-strands, respectively, that are found in many members of the superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second {beta}-strand and Arg 147 at the end of the second {beta}-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third {beta}-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-d-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg2+ are the only

  19. Genome-wide identification, 3D modeling, expression and enzymatic activity analysis of cell wall invertase gene family from cassava (Manihot esculenta Crantz).

    PubMed

    Yao, Yuan; Geng, Meng-Ting; Wu, Xiao-Hui; Liu, Jiao; Li, Rui-Mei; Hu, Xin-Wen; Guo, Jian-Chun

    2014-01-01

    The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6) were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a β-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(V)D), in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a β-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6), and Val residue from the WECVD (MeCWINV3 and 4) are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker. PMID:24786092

  20. Effects of low molecular weight sulfated galactan fragments from Botryocladia occidentalis on the pharmacological and enzymatic activity of sPLA2 from Crotalus durissus cascavella.

    PubMed

    Toyama, M H; Toyama, D O; Torres, V M; Pontes, G C; Farias, W R L; Melo, F R; Oliveira, S C B; Fagundes, F H R; Diz Filho, E B S; Cavada, B S

    2010-11-01

    Low molecular weight fragments of sulfated galactans (Boc-5 and Boc-10) from the red algae Botryocladia occidentalis significantly inhibited Crotalus durissus cascavella sPLA2 enzymatic activity. Equimolar ratios of sPLA2 to Boc-5 or Boc-10 resulted in allosteric inhibition of sPLA2. Under the conditions tested, we observed that both Boc-5 and Boc-10 strongly decreased edema, myonecrosis, and neurotoxicity induced by native sPLA2. PMID:21061146

  1. Improved enzymatic production of phenolated acylglycerols through alkyl phenolate intermediates.

    PubMed

    Yang, Zhiyong; Feddern, Vivian; Glasius, Marianne; Guo, Zheng; Xu, Xuebing

    2011-04-01

    A novel approach is reported for the synthesis of dihydrocaffoylated glycerols that consists of two steps: enzymatic synthesis of octyl dihydrocaffeate (as a synthetic intermediate) from octanol and dihydrocaffeic acid, and enzymatic interesterification of triacylglycerols with octyl dihydrocaffeate. Due to the good compatibility of the intermediate with triacylglycerols, an improved volumetric productivity [147 mol h(-1)(kg Novozym 435)(-1)] and high enzyme specific activity [up to 9.6 μmol(-1) min(-1)(g Novozym 435)(-1)] have been obtained. PMID:21120584

  2. A complete active space self-consistent field study of the photochemistry of nitrosamine

    SciTech Connect

    Pelaez, Daniel; Arenas, Juan F.; Otero, Juan C.; Soto, Juan

    2006-10-28

    Photodissociation mechanisms of nitrosamine (NH{sub 2}NO) have been studied at the complete active space self-consistent field level of theory in conjunction with atomic-natural-orbital-type basis sets. In addition, the energies of all the critical points and the potential energy curves connecting them have been recomputed with the multiconfigurational second-order perturbation method. Ground state minimum of nitrosamine has a C{sub 1} nonplanar structure with the hydrogen atoms of the amino moiety out of the plane defined by the N-N-O bonds. Electronic transitions to the three lowest states are allowed by selection rules: (i) S{sub 0}{yields}S{sub 3} (7.41 eV) has an oscillator strength of f=0.0006 and it is assigned as an (np{sub O}){sup 0}{yields}({pi}{sub NO}*){sup 2} transition, (ii) S{sub 0}{yields}S{sub 2} (5.86 eV) has an oscillator strength of f=0.14 and it is assigned as an np{sub N}{yields}{pi}{sub NO}* transition, and (iii) S{sub 0}{yields}S{sub 1} (2.98 eV) has an oscillator strength of f=0.002 and it is assigned as an np{sub O}{yields}{pi}{sub NO}* transition. It is found that N-N bond cleavage is the most likely process in all the photochemical relevant states, namely, S{sub 1} (1 {sup 1}A{sup ''}), S{sub 2} (2 {sup 1}A{sup '}), and T{sub 1} (1 {sup 3}A{sup ''}). While S{sub 1} and T{sub 1} yield exclusively homolytic dissociation: NH{sub 2}NO{yields}NH{sub 2} (1 {sup 2}B{sub 1})+NO(X {sup 2}{pi}), on S{sub 2} the latter process constitutes the major path, but two additional minor channels are also available: adiabatic homolytic dissociation: NH{sub 2}NO{yields}NH{sub 2} (1 {sup 2}A{sub 1})+NO(X {sup 2}{pi}), and adiabatic oxygen extrusion: NH{sub 2}NO{yields}NH{sub 2}N (1 {sup 3}A{sub 1})+O({sup 3}P). The excited species NH{sub 2} (1 {sup 2}A{sub 1}) experiences a subsequent ultrafast decay to the ground state, the final products in all cases the fragments being in their lowest electronic state. We have not found a unimolecular mechanism connecting

  3. Effect of self-alkalization on nitrite accumulation in a high-rate denitrification system: Performance, microflora and enzymatic activities.

    PubMed

    Li, Wei; Shan, Xiao-Yu; Wang, Zhi-Yao; Lin, Xiao-Yu; Li, Chen-Xu; Cai, Chao-Yang; Abbas, Ghulam; Zhang, Meng; Shen, Li-Dong; Hu, Zhi-Qiang; Zhao, He-Ping; Zheng, Ping

    2016-01-01

    The self-alkalization of denitrifying automatic circulation (DAC) reactor resulted in a large increase of pH up to 9.20 and caused a tremendous accumulation of nitrite up to 451.1 ± 49.0 mgN L(-1) at nitrate loading rate (NLR) from 35 kgN m(-3) d(-1) to 55 kgN m(-3) d(-1). The nitrite accumulation was greatly relieved even at the same NLR once the pH was maintained at 7.6 ± 0.2 in the system. Enzymatic assays indicated that the long-term bacterial exposure to high pH significantly inhibited the activity of copper type nitrite reductase (NirK) rather than the cytochrome cd1 type nitrite reductase (NirS). The terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the dominant denitrifying bacteria shifted from the NirS-containing Thauear sp. 27 to the NirK-containing Hyphomicrobium nitrativorans strain NL23 during the self-alkalization. The significant nitrite accumulation in the high-rate denitrification system could be therefore, due to the inhibition of Cu-containing NirK by high pH from the self-alkalization. The results suggest that the NirK-containing H. nitrativorans strain NL23 could be an ideal functional bacterium for the conversion of nitrate to nitrite, i.e. denitritation, which could be combined with anaerobic ammonium oxidation (Anammox) to develop a new process for nitrogen removal from wastewater. PMID:26595097

  4. Functional and Behavioral Product Information Representation and Consistency Validation for Collaboration in Product Lifecycle Activities

    ERIC Educational Resources Information Center

    Baysal, Mehmet Murat

    2012-01-01

    Information models that represent the function, assembly and behavior of artifacts are critical in the conceptual development of a product and its evaluation. Much research has been conducted in this area; however, existing models do not relate function, behavior and structure in a comprehensive and consistent way. In this work, NIST's Core…

  5. Studies on the effects of polyaspartate protease fertilizer enhancer in the absorptions of soil nutrition and the enzymatic activities of crops

    NASA Astrophysics Data System (ADS)

    Guoliang, Jiang; Dong, Yang; Yun, Liu; Guanghua, Zhang; Zhongjun, Li; Xinhua, Zhang

    2003-04-01

    The effects of polyaspartate protease fertilizer enhancer, made from oyster shell proteins, on the absorption of soil nutrition and the enzymatic activities of crops were studied. It has been found that the enhancer contributes 30%, 50% and 50% augmentation of nitrogen (N), phosphate (P) and potassium (K) absorption respectively and about 20% of nitrate reductase and peroxide enzyme activities of crops. These results show that polyaspartate protease fertilizer enhancer could improve significantly the absorption and utilization efficiencies of soil nutrition and the activities of nitrate reductase and peroxide enzyme of crops, thus elevating the utilization rates of chemical fertilizers to a certain extent.

  6. Enzymatic hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide by α-chymotrypsin in DMSO-water/AOT/n-heptane reverse micelles. A unique interfacial effect on the enzymatic activity.

    PubMed

    Moyano, Fernando; Setien, Evangelina; Silber, Juana J; Correa, N Mariano

    2013-07-01

    The reverse micelle (RM) media are very good as nanoreactors because they can create a unique microenvironment for carrying out a variety of chemical and biochemical reactions. The aim of the present work is to determine the influence of different water-dimethyl sulfoxide (DMSO) mixtures encapsulated in 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane RMs on the enzymatic hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide (Bz-Try-pNA) by α-chymotrypsin (α-CT). The reaction was first studied in homogeneous media at different DMSO-water mixture compositions and in DMSO-water/AOT/n-heptane RMs. The hydrolysis rates of Bz-Try-pNA catalyzed by α-CT were determined by UV-vis spectroscopy. The reaction follows the Michaelis-Menten mechanism and the kinetic parameters: kcat, KM, and kcat/KM were evaluated under different conditions. In this homogeneous media, DMSO plays an important role in the solubilization process of the peptide which is almost insoluble in water, but it has a tremendous impact on the inactivation of α-CT. It is shown that the enzyme dissolved in a 20% molar ratio of the DMSO-water mixture does not present enzymatic activity. Dynamic light scattering has been used to assess the formation of DMSO-water/AOT/heptane RMs at different DMSO compositions. The results also show that there is preferential solvation of the AOT RM interface by water molecules. To test the use of these RMs as nanoreactors, the kinetic parameters for the enzymatic reaction in these systems have been evaluated. The parameters were determined at fixed W(S) {W(S) = ([water] + [DMSO])/[AOT] = 20} at different DMSO-water compositions. The results show that the Michaelis-Menten mechanism is valid for α-CT in all the RM systems studied and that the reaction takes place at the RM interface. Surprisingly, it was observed that the enzyme encapsulated by the RMs show catalytic effects with similar kcat/KM values at any DMSO composition investigated, which evidence that DMSO molecules are

  7. Oil-containing waste water treating material consisting of modified active carbon

    SciTech Connect

    Sato, H.; Shigeta, S.; Takenaka, Y.

    1982-03-16

    An oil-containing waste water treating material comprises an active carbon upon whose surface is chemically bonded at least one nitrogenous compound which is an amine or a quaternarized derivative thereof.

  8. Biomechanics of the vibrissa motor plant in rat: rhythmic whisking consists of triphasic neuromuscular activity.

    PubMed

    Hill, Dan N; Bermejo, Roberto; Zeigler, H Philip; Kleinfeld, David

    2008-03-26

    The biomechanics of a motor plant constrain the behavioral strategies that an animal has available to extract information from its environment. We used the rat vibrissa system as a model for active sensing and determined the pattern of muscle activity that drives rhythmic exploratory whisking. Our approach made use of electromyography to measure the activation of all relevant muscles in both head-fixed and unrestrained rats and two-dimensional imaging to monitor the position of the vibrissae in head-fixed rats. Our essential finding is that the periodic motion of the vibrissae and mystacial pad during whisking results from three phases of muscle activity. First, the vibrissae are thrust forward as the rostral extrinsic muscle, musculus (m.) nasalis, contracts to pull the pad and initiate protraction. Second, late in protraction, the intrinsic muscles pivot the vibrissae farther forward. Third, retraction involves the cessation of m. nasalis and intrinsic muscle activity and the contraction of the caudal extrinsic muscles m. nasolabialis and m. maxillolabialis to pull the pad and the vibrissae backward. We developed a biomechanical model of the whisking motor plant that incorporates the measured muscular mechanics along with movement vectors observed from direct muscle stimulation in anesthetized rats. The results of simulations of the model quantify how the combination of extrinsic and intrinsic muscle activity leads to an enhanced range of vibrissa motion than would be available from the intrinsic muscles alone. PMID:18367610

  9. Deletion of a non-catalytic region increases the enzymatic activity of a β-agarase from Flammeovirga sp. MY04

    NASA Astrophysics Data System (ADS)

    Han, Wenjun; Gu, Jingyan; Liu, Huihui; Li, Fuchuan; Wu, Zhihong; Li, Yuezhong

    2015-10-01

    A Glycoside hydrolase (GH) typically contains one catalytic module and varied non-catalytic regions (NCRs). However, effects of the NCRs to the catalytic modules remain mostly unclear except the carbohydrate-binding modules (CBMs). AgaG4 is a GH16 endo- β-agarase of the agarolytic marine bacterium Flammeovirga sp. MY04. The enzyme consists of an extra sugar-binding peptide within the catalytic module, with no predictable CBMs but function-unknown sequences in the NCR, which is a new characteristic of agarase sequences. In this study, we deleted the NCR sequence, a 140-amino acid peptide at the C-terminus and expressed the truncated gene, agaG4-T140, in Escherichia coli. After purification and refolding, the truncated agarase rAgaG4-T140 retained the same catalytic temperature and pH value as rAgaG4. Using combined fluorescent labeling, HPLC and MS/MS techniques, we identified the end-products of agarose degradation by rAgaG4-T140 as neoagarotetraose and neoagarohexaose, with a final molar ratio of 1.53:1 and a conversion ratio of approximately 70%, which were similar to those of rAgaG4. However, the truncated agarase rAgaG4-T140 markedly decreased in protein solubility by 15 times and increased in enzymatic activities by 35 times. The oligosaccharide production of rAgaG4-T140 was approximately 25 times the weight of that produced by equimolar rAgaG4. This study provides some insights into the influences of NCR on the biochemical characteristics of agarase AgaG4 and implies some new strategies to improve the properties of a GH enzyme.

  10. pCO2 and enzymatic activity in a river floodplain system of the Danube under different hydrological settings.

    NASA Astrophysics Data System (ADS)

    Sieczko, Anna; Demeter, Katalin; Mayr, Magdalena; Meisterl, Karin; Peduzzi, Peter

    2014-05-01

    Surface waters may serve as either sinks or sources of CO2. In contrast to rivers, which are typically sources of CO2 to the atmosphere, the role of fringing floodplains in CO2 flux is largely understudied. This study was conducted in a river-floodplain system near Vienna (Austria). The sampling focused on changing hydrological situations, particularly on two distinct flood events: a typical 1-year flood in 2012 and an extraordinary 100-year flood in 2013. One objective was to determine partial pressure of CO2 (pCO2) in floodplain lakes with different degree of connectivity to the main channel, and compare the impact of these two types of floods. Another aim was to decipher which fraction of the dissolved organic matter (DOM) pool contributed to pCO2 by linking pCO2 with optical properties of DOM and extracellular enzymatic activity (EEA) of microbes. The EEA is a valuable tool, especially for assessing the non-chromophoric but rapidly utilized DOM-fraction during floods. In 2012 and 2013, the floodplain lakes were dominated by supersaturated pCO2 conditions, which indicates that they served as CO2 sources. Surprisingly, there were no significant differences in pCO2 between the two types of flood. Our findings imply that the extent of the flood had minor impact on pCO2, but the general occurrence of a flood appears to be important. During the flood in 2013 significantly more dissolved organic carbon (DOC) (p<0.05) was introduced into the floodplain. The optical measurements pointed towards more refractory DOM, with higher molecular weight and humic content during the flood in 2013 compared to 2012. However there were no significant differences in EEA between the two floods. Few days after beginning of the floods in 2012 and 2013, an increase in activity of carbon-acquiring enzymes (EEA-C) was observed. We also found positive correlations of pCO2with EEA-C both in 2012 (r=0.86, p<0.01) and in 2013 (r=0.73, p<0.05). The above findings imply that some fraction of DOM

  11. Antibacterial activity of hen egg white lysozyme modified by heat and enzymatic treatments against oenological lactic acid bacteria and acetic acid bacteria.

    PubMed

    Carrillo, W; García-Ruiz, A; Recio, I; Moreno-Arribas, M V

    2014-10-01

    The antimicrobial activity of heat-denatured and hydrolyzed hen egg white lysozyme against oenological lactic acid and acetic acid bacteria was investigated. The lysozyme was denatured by heating, and native and heat-denatured lysozymes were hydrolyzed by pepsin. The lytic activity against Micrococcus lysodeikticus of heat-denatured lysozyme decreased with the temperature of the heat treatment, whereas the hydrolyzed lysozyme had no enzymatic activity. Heat-denatured and hydrolyzed lysozyme preparations showed antimicrobial activity against acetic acid bacteria. Lysozyme heated at 90°C exerted potent activity against Acetobacter aceti CIAL-106 and Gluconobacter oxydans CIAL-107 with concentrations required to obtain 50% inhibition of growth (IC50) of 0.089 and 0.013 mg/ml, respectively. This preparation also demonstrated activity against Lactobacillus casei CIAL-52 and Oenococcus oeni CIAL-91 (IC50, 1.37 and 0.45 mg/ml, respectively). The two hydrolysates from native and heat-denatured lysozyme were active against O. oeni CIAL-96 (IC50, 2.77 and 0.3 mg/ml, respectively). The results obtained suggest that thermal and enzymatic treatments increase the antibacterial spectrum of hen egg white lysozyme in relation to oenological microorganisms. PMID:25285490

  12. Comparative study of enzymatic activities of new KatG mutants from low- and high-level isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

    PubMed

    Brossier, Florence; Boudinet, Marlène; Jarlier, Vincent; Petrella, Stéphanie; Sougakoff, Wladimir

    2016-09-01

    Resistance to isoniazid (INH-R) in Mycobacterium tuberculosis is mainly due to mutations at position 315 (S315T) of the catalase-peroxidase KatG. We identified 16 mutations (including 13 biochemically uncharacterized mutations) in KatG from INH-R clinical isolates of M. tuberculosis showing mutations other than S315T. The KatG enzymatic activities (catalase, peroxidase, free radical production and isonicotinoyl-NAD formation) of wild-type KatG and the 16 mutants were determined and correlated to their spatial location in a KatG model structure. Of all mutations studied, H270R, which conferred a high level of INH-R and results in the disruption of a coordination bond with the heme, caused complete loss of all enzymatic KatG activities. The mutants generally associated with a very high level of INH-R were all characterized by a drastic reduction in catalase activity and a marked decrease in INH activation activities. One mutant, A162E, displayed a behavior similar to S315T, i.e. a moderate decrease in catalase activity and a drastic decrease in the formation of the radical form of INH. Finally, the mutants associated with a low level of INH-R showed a moderate reduction in the four catalytic activities, likely stemming from an overall alteration of the folding and/or stability of the KatG protein. PMID:27553406

  13. Contrasted enzymatic cocktails reveal the importance of cellulases and hemicellulases activity ratios for the hydrolysis of cellulose in presence of xylans.

    PubMed

    Dondelinger, Eve; Aubry, Nathalie; Ben Chaabane, Fadhel; Cohen, Céline; Tayeb, Jean; Rémond, Caroline

    2016-03-01

    Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperproducer strain and a strain with β-glucosidase activity overexpression. By using various carbon sources (lactose, glucose, xylose, hemicellulosic hydrolysate) for strains growth, contrasted enzymatic activities were obtained. The enzymatic cocktails presented various levels of efficiency for the hydrolysis of cellulose Avicel into glucose, in presence of xylans, or not. These latter were also hydrolyzed with different extents according to cocktails. The most efficient cocktails (TR1 and TR3) on Avicel were richer in filter paper activity (FPU) and presented a low ratio FPU/β-glucosidase activity. Cocktails TR2 and TR5 which were produced on the higher amount of hemicellulosic hydrolysate, possess both high xylanase and β-xylosidase activities, and were the most efficient for xylans hydrolysis. When hydrolysis of Avicel was conducted in presence of xylans, a decrease of glucose release occurred for all cocktails compared to hydrolysis of Avicel alone. Mixing TR1 and TR5 cocktails with two different ratios of proteins (1/1 and 1/4) resulted in a gain of efficiency for glucose release during hydrolysis of Avicel in presence of xylans compared to TR5 alone. Our results demonstrate the importance of combining hemicellulase and cellulase activities to improve the yields of glucose release from Avicel in presence of xylans. In this context, strategies involving enzymes production with carbon sources comprising mixed C5 and C6 sugars or combining different cocktails produced on C5 or on C6 sugars are of interest for processes developed in the context of lignocellulosic biorefinery. PMID:27001439

  14. 15 CFR 930.154 - Listing activities subject to routine interstate consistency review.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... under subpart H of 15 CFR part 923. Each State submitting this program change shall include evidence of... this subpart and subpart H of 15 CFR part 923. States which have complied with paragraphs (a) through... are reasonably foreseeable. Listed activities may have different geographic location...

  15. 15 CFR 930.154 - Listing activities subject to routine interstate consistency review.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... under subpart H of 15 CFR part 923. Each State submitting this program change shall include evidence of... this subpart and subpart H of 15 CFR part 923. States which have complied with paragraphs (a) through... are reasonably foreseeable. Listed activities may have different geographic location...

  16. A hybrid attitude controller consisting of electromagnetic torque rods and an active fluid ring

    NASA Astrophysics Data System (ADS)

    Nobari, Nona A.; Misra, Arun K.

    2014-01-01

    In this paper, a novel hybrid actuation system for satellite attitude stabilization is proposed along with its feasibility analysis. The system considered consists of two magnetic torque rods and one fluid ring to produce the control torque required in the direction in which magnetic torque rods cannot produce torque. A mathematical model of the system dynamics is derived first. Then a controller is developed to stabilize the attitude angles of a satellite equipped with the abovementioned set of actuators. The effect of failure of the fluid ring or a magnetic torque rod is examined as well. It is noted that the case of failure of the magnetic torque rod whose torque is along the pitch axis is the most critical, since the coupling between the roll or yaw motion and the pitch motion is quite weak. The simulation results show that the control system proposed is quite fault tolerant.

  17. Screening of mammary carcinoma for hormone dependency in vitro. Enzymatic activity in short-term organotypic cultures of breast biopsies from 62 patients.

    PubMed

    Montessori, G A; Algard, F T; Van Netten, J P; Donald, J C

    1977-04-01

    Enzymatic activity in short-term organotypic cultures of breast biopsies from 62 patients. Am J Clin Pathol 67: 393-396, 1977. Mammary carcinomas from 62 patients were assessed for pentose shunt dehydrogenase activity initially and after 24-72 hours in organotypic cultures with or without exogenous hormones. Hormones tested were (1) estradiol, (2) testosterone, and (3) prolactin. Thirty-seven (60%) were judged hormone-independent, in vitro; 14 (23%) were judged hormone-dependent, in vitro; 11 (17%) were classed as "indeterminant." Clinical results of endocrine management of 13 cases and an appraisal of the usefulness of the method are presented. PMID:192068

  18. Nitric Oxide-Induced Calcium Release: Activation of Type 1 Ryanodine Receptor, a Calcium Release Channel, through Non-Enzymatic Post-Translational Modification by Nitric Oxide

    PubMed Central

    Kakizawa, Sho

    2013-01-01

    Nitric oxide (NO) is a typical gaseous messenger involved in a wide range of biological processes. In our classical knowledge, effects of NO are largely achieved by activation of soluble guanylyl cyclase to form cyclic guanosine-3′, 5′-monophosphate. However, emerging evidences have suggested another signaling mechanism mediated by NO: “S-nitrosylation” of target proteins. S-nitrosylation is a covalent addition of an NO group to a cysteine thiol/sulfhydryl (RSH), and categorized into non-enzymatic post-translational modification (PTM) of proteins, contrasted to enzymatic PTM of proteins, such as phosphorylation mediated by various protein kinases. Very recently, we found novel intracellular calcium (Ca2+) mobilizing mechanism, NO-induced Ca2+ release (NICR) in cerebellar Purkinje cells. NICR is mediated by type 1 ryanodine receptor (RyR1), a Ca2+ release channel expressed in endoplasmic-reticular membrane. Furthermore, NICR is indicated to be dependent on S-nitrosylation of RyR1, and involved in synaptic plasticity in the cerebellum. In this review, molecular mechanisms and functional significance of NICR, as well as non-enzymatic PTM of proteins by gaseous signals, are described. PMID:24130553

  19. Divergence in Enzymatic Activities in the Soybean GST Supergene Family Provides New Insight into the Evolutionary Dynamics of Whole-Genome Duplicates

    PubMed Central

    Liu, Hai-Jing; Tang, Zhen-Xin; Han, Xue-Min; Yang, Zhi-Ling; Zhang, Fu-Min; Yang, Hai-Ling; Liu, Yan-Jing; Zeng, Qing-Yin

    2015-01-01

    Whole-genome duplication (WGD), or polyploidy, is a major force in plant genome evolution. A duplicate of all genes is present in the genome immediately following a WGD event. However, the evolutionary mechanisms responsible for the loss of, or retention and subsequent functional divergence of polyploidy-derived duplicates remain largely unknown. In this study we reconstructed the evolutionary history of the glutathione S-transferase (GST) gene family from the soybean genome, and identified 72 GST duplicated gene pairs formed by a recent Glycine-specific WGD event occurring approximately 13 Ma. We found that 72% of duplicated GST gene pairs experienced gene losses or pseudogenization, whereas 28% of GST gene pairs have been retained in the soybean genome. The GST pseudogenes were under relaxed selective constraints, whereas functional GSTs were subject to strong purifying selection. Plant GST genes play important roles in stress tolerance and detoxification metabolism. By examining the gene expression responses to abiotic stresses and enzymatic properties of the ancestral and current proteins, we found that polyploidy-derived GST duplicates show the divergence in enzymatic activities. Through site-directed mutagenesis of ancestral proteins, this study revealed that nonsynonymous substitutions of key amino acid sites play an important role in the divergence of enzymatic functions of polyploidy-derived GST duplicates. These findings provide new insights into the evolutionary and functional dynamics of polyploidy-derived duplicate genes. PMID:26219583

  20. Consistent abnormalities in metabolic network activity in idiopathic rapid eye movement sleep behaviour disorder

    PubMed Central

    Wu, Ping; Yu, Huan; Peng, Shichun; Dauvilliers, Yves; Wang, Jian; Ge, Jingjie; Zhang, Huiwei; Eidelberg, David

    2014-01-01

    Rapid eye movement sleep behaviour disorder has been evaluated using Parkinson’s disease-related metabolic network. It is unknown whether this disorder is itself associated with a unique metabolic network. 18F-fluorodeoxyglucose positron emission tomography was performed in 21 patients (age 65.0 ± 5.6 years) with idiopathic rapid eye movement sleep behaviour disorder and 21 age/gender-matched healthy control subjects (age 62.5 ± 7.5 years) to identify a disease-related pattern and examine its evolution in 21 hemi-parkinsonian patients (age 62.6 ± 5.0 years) and 16 moderate parkinsonian patients (age 56.9 ± 12.2 years). We identified a rapid eye movement sleep behaviour disorder-related metabolic network characterized by increased activity in pons, thalamus, medial frontal and sensorimotor areas, hippocampus, supramarginal and inferior temporal gyri, and posterior cerebellum, with decreased activity in occipital and superior temporal regions. Compared to the healthy control subjects, network expressions were elevated (P < 0.0001) in the patients with this disorder and in the parkinsonian cohorts but decreased with disease progression. Parkinson’s disease-related network activity was also elevated (P < 0.0001) in the patients with rapid eye movement sleep behaviour disorder but lower than in the hemi-parkinsonian cohort. Abnormal metabolic networks may provide markers of idiopathic rapid eye movement sleep behaviour disorder to identify those at higher risk to develop neurodegenerative parkinsonism. PMID:25338949

  1. Enzymatic activities in different strains isolated from healthy and brittle leaf disease affected date palm leaves: study of amylase production conditions.

    PubMed

    Mouna, Jrad; Imen, Fendri; Choba Ines, Ben; Nourredine, Drira; Adel, Kadri; Néji, Gharsallah

    2015-02-01

    The present study aimed to investigate and compare the enzymatic production of endophytic bacteria isolated from healthy and brittle leaf disease affected date palm leaves (pectinase, cellulase, lipase, and amylase). The findings revealed that the enzymatic products from the bacterial isolates of healthy date palm leaves were primarily 33% amylolytic enzyme, 33 % cellulase, 25 % pectinase, and 25 % lipase. The isolates from brittle leaf disease date palm leaves, on the other hand, were noted to produce 16 % amylolytic enzyme, 20 % cellulose, 50 % pectinase, and 50 % lipase. The effects of temperature and pH on amylase, pectinase, and cellulose activities were investigated. The Bacillus subtilis JN934392 strain isolated from healthy date palm leaves produced higher levels of amylase activity at pH 7. A Box Behnken Design (BBD) was employed to optimize amylase extraction. Maximal activity was observed at pH and temperature ranges of pH 6-6.5 and 37-39 °C, respectively. Under those conditions, amylase activity was noted to be attained 9.37 U/ml. The results showed that the enzyme was able to maintain more than 50 % of its activity over a temperature range of 50-80 °C, with an optimum at 70 °C. This bacterial amylase showed high activity compared to other bacteria, which provides support for its promising candidacy for future industrial application. PMID:25432343

  2. Direct Measurements of Local Coupling between Myosin Molecules Are Consistent with a Model of Muscle Activation

    PubMed Central

    Walcott, Sam; Kad, Neil M.

    2015-01-01

    Muscle contracts due to ATP-dependent interactions of myosin motors with thin filaments composed of the proteins actin, troponin, and tropomyosin. Contraction is initiated when calcium binds to troponin, which changes conformation and displaces tropomyosin, a filamentous protein that wraps around the actin filament, thereby exposing myosin binding sites on actin. Myosin motors interact with each other indirectly via tropomyosin, since myosin binding to actin locally displaces tropomyosin and thereby facilitates binding of nearby myosin. Defining and modeling this local coupling between myosin motors is an open problem in muscle modeling and, more broadly, a requirement to understanding the connection between muscle contraction at the molecular and macro scale. It is challenging to directly observe this coupling, and such measurements have only recently been made. Analysis of these data suggests that two myosin heads are required to activate the thin filament. This result contrasts with a theoretical model, which reproduces several indirect measurements of coupling between myosin, that assumes a single myosin head can activate the thin filament. To understand this apparent discrepancy, we incorporated the model into stochastic simulations of the experiments, which generated simulated data that were then analyzed identically to the experimental measurements. By varying a single parameter, good agreement between simulation and experiment was established. The conclusion that two myosin molecules are required to activate the thin filament arises from an assumption, made during data analysis, that the intensity of the fluorescent tags attached to myosin varies depending on experimental condition. We provide an alternative explanation that reconciles theory and experiment without assuming that the intensity of the fluorescent tags varies. PMID:26536123

  3. The Root Apex of Arabidopsis thaliana Consists of Four Distinct Zones of Growth Activities

    PubMed Central

    De Cnodder, Tinne; Le, Jie

    2006-01-01

    In the growing apex of Arabidopsis thaliana primary roots, cells proceed through four distinct phases of cellular activities. These zones and their boundaries can be well defined based on their characteristic cellular activities. The meristematic zone comprises, and is limited to, all cells that undergo mitotic divisions. Detailed in vivo analysis of transgenic lines reveals that, in the Columbia-0 ecotype, the meristem stretches up to 200 µm away from the junction between root and root cap (RCJ). In the transition zone, 200 to about 520 µm away from the RCJ, cells undergo physiological changes as they prepare for their fast elongation. Upon entering the transition zone, they progressively develop a central vacuole, polarize the cytoskeleton and remodel their cell walls. Cells grow slowly during this transition: it takes ten hours to triplicate cell length from 8.5 to about 35 µm in the trichoblast cell files. In the fast elongation zone, which covers the zone from 520 to about 850 µm from the RCJ, cell length quadruplicates to about 140 µm in only two hours. This is accompanied by drastic and specific cell wall alterations. Finally, root hairs fully develop in the growth terminating zone, where root cells undergo a minor elongation to reach their mature lengths. PMID:19517000

  4. Consistent estimation of complete neuronal connectivity in large neuronal populations using sparse "shotgun" neuronal activity sampling.

    PubMed

    Mishchenko, Yuriy

    2016-10-01

    We investigate the properties of recently proposed "shotgun" sampling approach for the common inputs problem in the functional estimation of neuronal connectivity. We study the asymptotic correctness, the speed of convergence, and the data size requirements of such an approach. We show that the shotgun approach can be expected to allow the inference of complete connectivity matrix in large neuronal populations under some rather general conditions. However, we find that the posterior error of the shotgun connectivity estimator grows quickly with the size of unobserved neuronal populations, the square of average connectivity strength, and the square of observation sparseness. This implies that the shotgun connectivity estimation will require significantly larger amounts of neuronal activity data whenever the number of neurons in observed neuronal populations remains small. We present a numerical approach for solving the shotgun estimation problem in general settings and use it to demonstrate the shotgun connectivity inference in the examples of simulated synfire and weakly coupled cortical neuronal networks. PMID:27515518

  5. Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis.

    PubMed Central

    Kaufman, K D; Rapoport, B; Seto, P; Chazenbalk, G D; Magnusson, R P

    1989-01-01

    A full-length cDNA clone for human thyroid peroxidase (TPO) inserted into the mammalian cell expression vector pECE was stably transfected into Chinese hamster ovary (CHO) cells. Clones were assayed for human TPO mRNA, TPO protein, and TPO enzymatic activity. One subclone, expressing the highest TPO enzymatic activity, was used in further studies. FACS analysis of these cells preincubated in Hashimoto's serum revealed approximately 100-fold greater fluorescence compared with controls, indicating that recombinant TPO is expressed on the cell surface. Particulate antigen was extracted from these cells and studied by Western blot analysis using a panel of Hashimoto's sera of known antimicrosomal antibody (anti-MSA) titer. Under nonreducing conditions a broad, immunoreactive band of approximately 200 kD was observed, as well as a doublet of approximately 110 kD. All of the 36 Hashimoto's sera tested reacted with these bands, most in proportion to their anti-MSA titer. Six normal sera tested against this antigen(s) were nonreactive, as were the Hashimoto's sera tested against nontransfected CHO cells. Western blots under reducing conditions revealed a considerably diminished signal, with some of the sera of lower anti-MSA titer becoming negative, the loss of the 200-kD broad band, and the apparent conversion of the 110-kD doublet into a single band. Preincubation of cells in tunicamycin revealed no decrease in TPO immunoreactivity. In conclusion, we expressed enzymatically active human TPO in nonthyroidal eukaryotic cells. Our data prove that functionally active TPO is a major component of the thyroid microsomal antigen. Images PMID:2474568

  6. Discovery and analysis of consistent active sub-networks in cancers

    PubMed Central

    2013-01-01

    Gene expression profiles can show significant changes when genetically diseased cells are compared with non-diseased cells. Biological networks are often used to identify active subnetworks (ASNs) of the diseases from the expression profiles to understand the reason behind the observed changes. Current methodologies for discovering ASNs mostly use undirected PPI networks and node centric approaches. This can limit their ability to find the meaningful ASNs when using integrated networks having comprehensive information than the traditional protein-protein interaction networks. Using appropriate scoring functions to assess both genes and their interactions may allow the discovery of better ASNs. In this paper, we present CASNet, which aims to identify better ASNs using (i) integrated interaction networks (mixed graphs), (ii) directions of regulations of genes, and (iii) combined node and edge scores. We simplify and extend previous methodologies to incorporate edge evaluations and lessen their sensitivity to significance thresholds. We formulate our objective functions using mixed integer programming (MIP) and show that optimal solutions may be obtained. We compare the ASNs obtained by CASNet and similar other approaches to show that CASNet can often discover more meaningful and stable regulatory ASNs. Our analysis of a breast cancer dataset finds that the positive feedback loops across 7 genes, AR, ESR1, MYC, E2F2, PGR, BCL2 and CCND1 are conserved across the basal/triple negative subtypes in multiple datasets that could potentially explain the aggressive nature of this cancer subtype. Furthermore, comparison of the basal subtype of breast cancer and the mesenchymal subtype of glioblastoma ASNs shows that an ASN in the vicinity of IL6 is conserved across the two subtypes. This result suggests that subtypes of different cancers can show molecular similarities indicating that the therapeutic approaches in different types of cancers may be shared. PMID:23368093

  7. Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides.

    PubMed Central

    Augustyns, K; Godard, G; Hendrix, C; Van Aerschot, A; Rozenski, J; Saison-Behmoaras, T; Herdewijn, P

    1993-01-01

    Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA - DNA or RNA - DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not. Images PMID:7694231

  8. A sensitive radioisotopic method for the measurement of NAD(P)H: Its application to the assay of metabolites and enzymatic activities

    SciTech Connect

    Sener, A.; Malaisse, W.J. )

    1990-05-01

    A radioisotopic method for the assay of NADH or NADPH is presented, which is based on the conversion of 2-(U-{sup 14}C)ketoglutarate to {sup 14}C-labeled glutamate in the reaction catalyzed by glutamate dehydrogenase. The efficiency of the method is close to 75%, its precision (coefficient of variation) close to 5%, and its sensitivity close to 0.1 pmol/sample. This simple and rapid method can be applied to the measurement of several metabolites and enzymatic activities. In the present study, its application to the assay of sorbitol, 3-hydroxybutyrate, glutamate dehydrogenase, 3-hydroxybutyrate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase is documented.

  9. A Novel New Delhi Metallo-β-Lactamase Variant, NDM-14, Isolated in a Chinese Hospital Possesses Increased Enzymatic Activity against Carbapenems

    PubMed Central

    Zou, Dayang; Huang, Yong; Zhao, Xiangna; Liu, Wei; Dong, Derong; Li, Huan; Wang, Xuesong; Huang, Simo; Wei, Xiao; Yan, Xiabei; Yang, Zhan; Tong, Yigang

    2015-01-01

    A novel New Delhi metallo-β-lactamase (NDM) variant, NDM-14, was identified in clinical isolate Acinetobacter lwoffii JN49-1, which was recovered from an intensive care unit patient at a local hospital in China. NDM-14, which differs from other existing enzymes by an amino acid substitution at position 130 (Asp130Gly), possesses enzymatic activity toward carbapenems that is greater than that of NDM-1. Kinetic data indicate that NDM-14 has a higher affinity for imipenem and meropenem. PMID:25645836

  10. Oxygen isotope ratios of PO4: An inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life

    PubMed Central

    Blake, Ruth E.; Alt, Jeffrey C.; Martini, Anna M.

    2001-01-01

    The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (δ18Op) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO4–H2O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that δ18OP values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of δ18Op as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, δ18Op may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars. PMID:11226207

  11. A NOVEL RED CLOVER HYDROXYCINNAMOYL TRANSFERASE HAS ENZYMATIC ACTIVITIES CONSISTENT WITH A ROLE IN PHASALIC ACID [2-O-(CAFFEOYL)-L-MALATE] BIOSYNTHESIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phenylpropanoid o-diphenols accumulate in tissues of many plants functioning as defensive molecules and antioxidants. Red clover leaves accumulate high levels of two o-diphenols, phasalic acid [2-O-(caffeoyl)-L-malate] and clovamide [N-(caffeoyl)-L-DOPA]. In red clover, post-harvest oxidation of the...

  12. Enzymatic Regulation of Self-Assembling Peptide A9K2 Nanostructures and Hydrogelation with Highly Selective Antibacterial Activities.

    PubMed

    Bai, Jingkun; Chen, Cuixia; Wang, Jingxin; Zhang, Yu; Cox, Henry; Zhang, Jing; Wang, Yuming; Penny, Jeffrey; Waigh, Thomas; Lu, Jian R; Xu, Hai

    2016-06-22

    Hydrogels offer great potential for many biomedical and technological applications. For clinical uses, hydrogels that act as scaffold materials for cell culture, regenerative medicine, and drug delivery are required to have bactericidal properties. The amphiphilic peptide A9K2 was designed to effectively inhibit bacterial growth via a mechanism of membrane permeabilization. The present study demonstrated that addition of fetal bovine serum (FBS) or plasma amine oxidase (PAO) induced a sol-gel transition in A9K2 aqueous solutions. The transformation of A9K2 molecules catalyzed by lysyl oxidase (LO) in FBS or PAO accounted for the hydrogelation. Importantly, the enzymatic A9K2 hydrogel displayed high antibacterial ability against both Gram-negative and Gram-positive bacterial strains while showing extremely low mammalian cell cytotoxicity, thus demonstrating good biocompatibility. Under established coculture conditions, the peptide hydrogel showed excellent selectivity by favoring the adherence and spreading of mammalian cells, while killing pathogenic bacteria, thus avoiding bacterial contamination. These advantages endow the enzymatic A9K2 hydrogel with great potential for biomedical applications. PMID:27243270

  13. Humanized-Single Domain Antibodies (VH/VHH) that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

    PubMed Central

    Chavanayarn, Charnwit; Thanongsaksrikul, Jeeraphong; Thueng-in, Kanyarat; Bangphoomi, Kunan; Sookrung, Nitat; Chaicumpa, Wanpen

    2012-01-01

    Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA2). The PLA2 exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/VHH) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/VHH phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-VHH, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/VHH purified from the E. coli homogenates neutralized PLA2 enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/VHH covered the areas around the PLA2 catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/VHH would ameliorate/abrogate the principal toxicity of the venom PLA2 (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations. PMID:22852068

  14. Algal extracellular release in river-floodplain dissolved organic matter: response of extracellular enzymatic activity during a post-flood period

    PubMed Central

    Sieczko, Anna; Maschek, Maria; Peduzzi, Peter

    2015-01-01

    River-floodplain systems are susceptible to rapid hydrological events. Changing hydrological connectivity of the floodplain generates a broad range of conditions, from lentic to lotic. This creates a mixture of allochthonously and autochthonously derived dissolved organic matter (DOM). Autochthonous DOM, including photosynthetic extracellular release (PER), is an important source supporting bacterial secondary production (BSP). Nonetheless, no details are available regarding microbial extracellular enzymatic activity (EEA) as a response to PER under variable hydrological settings in river-floodplain systems. To investigate the relationship between bacterial and phytoplankton components, we therefore used EEA as a tool to track the microbial response to non-chromophoric, but reactive and ecologically important DOM. The study was conducted in three floodplain subsystems with distinct hydrological regimes (Danube Floodplain National Park, Austria). The focus was on the post-flood period. Enhanced %PER (up to 48% of primary production) in a hydrologically isolated subsystem was strongly correlated with β-glucosidase, which was related to BSP. This shows that—in disconnected floodplain backwaters with high terrestrial input—BSP can also be driven by autochthonous carbon sources (PER). In a semi-isolated section, in the presence of fresh labile material from primary producers, enhanced activity of phenol oxidase was observed. In frequently flooded river-floodplain systems, BSP was mainly driven by enzymatic degradation of particulate primary production. Our research demonstrates that EEA measurements are an excellent tool to describe the coupling between bacteria and phytoplankton, which cannot be deciphered when focusing solely on chromophoric DOM. PMID:25741326

  15. Algal extracellular release in river-floodplain dissolved organic matter: response of extracellular enzymatic activity during a post-flood period.

    PubMed

    Sieczko, Anna; Maschek, Maria; Peduzzi, Peter

    2015-01-01

    River-floodplain systems are susceptible to rapid hydrological events. Changing hydrological connectivity of the floodplain generates a broad range of conditions, from lentic to lotic. This creates a mixture of allochthonously and autochthonously derived dissolved organic matter (DOM). Autochthonous DOM, including photosynthetic extracellular release (PER), is an important source supporting bacterial secondary production (BSP). Nonetheless, no details are available regarding microbial extracellular enzymatic activity (EEA) as a response to PER under variable hydrological settings in river-floodplain systems. To investigate the relationship between bacterial and phytoplankton components, we therefore used EEA as a tool to track the microbial response to non-chromophoric, but reactive and ecologically important DOM. The study was conducted in three floodplain subsystems with distinct hydrological regimes (Danube Floodplain National Park, Austria). The focus was on the post-flood period. Enhanced %PER (up to 48% of primary production) in a hydrologically isolated subsystem was strongly correlated with β-glucosidase, which was related to BSP. This shows that-in disconnected floodplain backwaters with high terrestrial input-BSP can also be driven by autochthonous carbon sources (PER). In a semi-isolated section, in the presence of fresh labile material from primary producers, enhanced activity of phenol oxidase was observed. In frequently flooded river-floodplain systems, BSP was mainly driven by enzymatic degradation of particulate primary production. Our research demonstrates that EEA measurements are an excellent tool to describe the coupling between bacteria and phytoplankton, which cannot be deciphered when focusing solely on chromophoric DOM. PMID:25741326

  16. The stay-green phenotype of TaNAM-RNAi wheat plants is associated with maintenance of chloroplast structure and high enzymatic antioxidant activity.

    PubMed

    Checovich, Mariana L; Galatro, Andrea; Moriconi, Jorge I; Simontacchi, Marcela; Dubcovsky, Jorge; Santa-María, Guillermo E

    2016-07-01

    TaNAM transcription factors play an important role in controlling senescence, which in turn, influences the delivery of nitrogen, iron and other elements to the grain of wheat (Triticum aestivum) plants, thus contributing to grain nutritional value. While lack or diminished expression of TaNAMs determines a stay-green phenotype, the precise effect of these factors on chloroplast structure has not been studied. In this work we focused on the events undergone by chloroplasts in two wheat lines having either control or diminished TaNAM expression due to RNA interference (RNAi). It was found that in RNAi plants maintenance of chlorophyll levels and maximal photochemical efficiency of photosystem II were associated with lack of chloroplast dismantling. Flow cytometer studies and electron microscope analysis showed that RNAi plants conserved organelle ultrastructure and complexity. It was also found that senescence in control plants was accompanied by a low leaf enzymatic antioxidant activity. Lack of chloroplast dismantling in RNAi plants was associated with maintenance of protein and iron concentration in the flag leaf, the opposite being observed in control plants. These data provide a structural basis for the observation that down regulation of TaNAMs confers a functional stay-green phenotype and indicate that the low export of iron and nitrogen from the flag leaf of these plants is concomitant, within the developmental window studied, with lack of chloroplast degradation and high enzymatic antioxidant activity. PMID:27061370

  17. Evaluation of Biological and Enzymatic Activity of Soil in a Tropical Dry Forest: Desierto de la Tatacoa (Colombia) with Potential in Mars Terraforming and Other Similar Planets

    NASA Astrophysics Data System (ADS)

    Moreno Moreno, A. N.

    2009-12-01

    Desierto de la Tatacoa has been determined to be a tropical dry forest bioma, which is located at 3° 13" N 75° 13" W. It has a hot thermal floor with 440 msnm of altitude; it has a daily average of 28° C, and a maximum of 40° C, Its annual rainfall total can be upwards of 1250 mm. Its solar sheen has a daily average of 5.8 hours and its relative humidity is between 60% and 65%. Therefore, the life forms presents are very scant, and in certain places, almost void. It was realized a completely random sampling of soil from its surface down to 6 inches deep, of zones without vegetation and with soils highly loaded by oxides of iron in order to determine the number of microorganisms per gram and its subsequent identification. It was measured the soil basal respiration. Besides, it was determined enzymatic activity (catalase, dehydrogenase, phosphatase and urease). Starting with the obtained results, it is developes an alternative towards the study of soil genesis in Mars in particular, and recommendations for same process in other planets. Although the information found in the experiments already realized in Martian soil they demonstrate that doesnt exist any enzymatic activity, the knowledge of the same topic in the soil is proposed as an alternative to problems like carbonic fixing of the dense Martian atmosphere of CO2, the degradation of inorganic compounds amongst other in order to prepare the substratum for later colonization by some life form.

  18. Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

    NASA Astrophysics Data System (ADS)

    Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

    2014-03-01

    Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

  19. Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-08-22

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  20. Influence of crop rotation, intermediate crops, and organic fertilizers on the soil enzymatic activity and humus content in organic farming systems

    NASA Astrophysics Data System (ADS)

    Marcinkeviciene, A.; Boguzas, V.; Balnyte, S.; Pupaliene, R.; Velicka, R.

    2013-02-01

    The influence of crop rotation systems with different portions of nitrogen-fixing crops, intermediate crops, and organic fertilizers on the enzymatic activity and humus content of soils in organic farming was studied. The highest activity of the urease and invertase enzymes was determined in the soil under the crop rotation with 43% nitrogen-fixing crops and with perennial grasses applied twice per rotation. The application of manure and the growing of intermediate crops for green fertilizers did not provide any significant increase in the content of humus. The activity of urease slightly correlated with the humus content ( r = 0.30 at the significance level of 0.05 and r = 0.39 at the significance level of 0.01).

  1. Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro

    PubMed Central

    Li, Yan; Burclaff, Joseph; Anderson, James T.

    2016-01-01

    RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities. PMID:26820724

  2. Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro.

    PubMed

    Li, Yan; Burclaff, Joseph; Anderson, James T

    2016-01-01

    RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities. PMID:26820724

  3. Similar potential ATP-P production and enzymatic activities in the microplankton community off Concepción (Chile) under oxic and suboxic conditions

    NASA Astrophysics Data System (ADS)

    González, Rodrigo R.; Gutiérrez, Marcelo H.; Quiñones, Renato A.

    2007-11-01

    The effects of the oxygen minimum zone on the metabolism of the heterotrophic microplankton community (0.22-100 μm) in the Humboldt Current System, as well as the factors controlling its biomass production, remain unknown. Here we compare the effect of four sources of dissolved organic carbon (glucose, oxaloacetate, glycine, leucine) on microbial biomass production (such as ATP-P) and the potential enzymatic activities involved in catabolic pathways under oxic and suboxic conditions. Our results show significant differences ( p < 0.05) in the ATP-P production when induced by the different substrates that are used as dissolved organic carbon herein. The induction of ATP-P production is enhanced from glucose < oxaloacetate < glycine < leucine. Nevertheless, for individual substrates, no significant differences were found between incubation under oxic and suboxic conditions except in the case of leucine. For this amino acid, the induction of ATP-P synthesis was higher under suboxic than oxic conditions. The data sets of all the substrates used showed greater potential ATP-P production under suboxic than oxic conditions. The results of the potential enzymatic activities suggest that malate dehydrogenase has the highest signal of NADH oxidization activity in the microbial assemblage. Furthermore, for all experiments, the malate dehydrogenase activity data set had a significant relationship with ATP-P production. These findings suggest that the microbial community inhabiting the oxygen minimum zone has the same or greater potential growth than the community inhabiting more oxygenated strata of the water column and that malate dehydrogenase is the activity that best represents the metabolic potential of the community.

  4. Selection of Nanobodies that Block the Enzymatic and Cytotoxic Activities of the Binary Clostridium Difficile Toxin CDT

    PubMed Central

    Unger, Mandy; Eichhoff, Anna Marei; Schumacher, Lucas; Strysio, Moritz; Menzel, Stephan; Schwan, Carsten; Alzogaray, Vanina; Zylberman, Vanesa; Seman, Michel; Brandner, Johanna; Rohde, Holger; Zhu, Kai; Haag, Friedrich; Mittrücker, Hans-Willi; Goldbaum, Fernando; Aktories, Klaus; Koch-Nolte, Friedrich

    2015-01-01

    The spore-forming gut bacterium Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitalized patients. The major virulence factors are two large glucosylating cytotoxins. Hypervirulent strains (e.g. ribotype 027) with higher morbidity and mortality additionally produce the binary CDT toxin (Clostridium difficile transferase) that ADP-ribosylates actin and induces microtubule-based cell protrusions. Nanobodies are robust single domain antibodies derived from camelid heavy chain antibodies. Here we report the generation of functional nanobodies against the enzymatic CDTa and the heptameric receptor binding subunit CDTb. The nanobodies were obtained from a variable-domain repertoire library isolated from llamas immunized with recombinant CDTa or CDTb. Five CDTa-specific nanobodies blocked CDTa-mediated ADP-ribosylation of actin. Three CDTa-specific and two CDTb-specific nanobodies neutralized the cytotoxicity of CDTa+b. These nanobodies hold promise as new tools for research, diagnosis and therapy of C. difficile associated disease. PMID:25597743

  5. Large-Scale Variational Two-Electron Reduced-Density-Matrix-Driven Complete Active Space Self-Consistent Field Methods.

    PubMed

    Fosso-Tande, Jacob; Nguyen, Truong-Son; Gidofalvi, Gergely; DePrince, A Eugene

    2016-05-10

    A large-scale implementation of the complete active space self-consistent field (CASSCF) method is presented. The active space is described using the variational two-electron reduced-density-matrix (v2RDM) approach, and the algorithm is applicable to much larger active spaces than can be treated using configuration-interaction-driven methods. Density fitting or Cholesky decomposition approximations to the electron repulsion integral tensor allow for the simultaneous optimization of large numbers of external orbitals. We have tested the implementation by evaluating singlet-triplet energy gaps in the linear polyacene series and two dinitrene biradical compounds. For the acene series, we report computations that involve active spaces consisting of as many as 50 electrons in 50 orbitals and the simultaneous optimization of 1892 orbitals. For the dinitrene compounds, we find that the singlet-triplet gaps obtained from v2RDM-driven CASSCF with partial three-electron N-representability conditions agree with those obtained from configuration-interaction-driven approaches to within one-third of 1 kcal mol(-1). When enforcing only the two-electron N-representability conditions, v2RDM-driven CASSCF yields less accurate singlet-triplet energy gaps in these systems, but the quality of the results is still far superior to those obtained from standard single-reference approaches. PMID:27065086

  6. The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

    SciTech Connect

    Duong-Ly, Krisna C.; Gabelli, Sandra B.; Xu, WenLian; Dunn, Christopher A.; Schoeffield, Andrew J.; Bessman, Maurice J.; Amzel, L. Mario

    2011-09-06

    A Nudix enzyme from Bacillus cereus catalyzes the hydrolysis of CDP-choline to produce CMP and phosphocholine. Here, we show that in addition, the enzyme has a 3{prime} {yields} 5{prime} RNA exonuclease activity. The structure of the free enzyme, determined to a 1.8-{angstrom} resolution, shows that the enzyme is an asymmetric dimer. Each monomer consists of two domains, an N-terminal helical domain and a C-terminal Nudix domain. The N-terminal domain is placed relative to the C-terminal domain such as to result in an overall asymmetric arrangement with two distinct catalytic sites: one with an 'enclosed' Nudix pyrophosphatase site and the other with a more open, less-defined cavity. Residues that may be important for determining the asymmetry are conserved among a group of uncharacterized Nudix enzymes from Gram-positive bacteria. Our data support a model where CDP-choline hydrolysis is catalyzed by the enclosed Nudix site and RNA exonuclease activity is catalyzed by the open site. CDP-Chase is the first identified member of a novel Nudix family in which structural asymmetry has a profound effect on the recognition of substrates.

  7. Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant.

    PubMed Central

    Cieplak, W; Burnette, W N; Mar, V L; Kaljot, K T; Morris, C F; Chen, K K; Sato, H; Keith, J M

    1988-01-01

    The S1 subunit of pertussis toxin possesses two regions (homology boxes), each spanning 8 residues, that are nearly identical in sequence to similarly located regions in the enzymatically active A fragments of two other ADP-ribosylating toxins: cholera toxin and Escherichia coli heat-labile toxin. This observation suggests a functional role for one or both of these regions in enzymatic activity. We have examined the role of one of these regions, located near the amino terminus of the S1 subunit, by using a high-level recombinant expression system and progressive truncation of the gene sequence encoding the amino terminus of the molecule. A series of six truncated, recombinant proteins were produced at high levels in E. coli and examined for their enzymatic and antigenic properties. The three molecules that lacked most or all of the homology box delimited by amino acid residues 8 and 15 lacked detectable enzymatic activity. All of the three molecules in which the box was retained exhibited detectable activity. Only those recombinant molecules that possessed the homology box reacted with a neutralizing and passively protective monoclonal anti-S1 antibody. These findings identify the region of homology located near the amino terminus of S1 as an apparent enzymatic subsite and a potentially important antigenic determinant. Images PMID:2455296

  8. Global histone deacetylase enzymatic activity is an independent prognostic marker associated with a shorter overall survival in chronic lymphocytic leukemia patients

    PubMed Central

    Van Damme, Michaël; Crompot, Emerence; Meuleman, Nathalie; Mineur, Philippe; Dessars, Barbara; El Housni, Hakim; Bron, Dominique; Lagneaux, Laurence; Stamatopoulos, Basile

    2014-01-01

    Histone deacetylases (HDAC) play a crucial role in transcriptional regulation and are often deregulated in many cancers. However, global HDAC enzymatic activity has never been investigated in Chronic Lymphocytic Leukemia (CLL). We measured HDAC activity in protein extracts from CD19+ B-cells purified from 114 CLL patients with a median follow-up of 91 months (range: 11–376). HDAC activity was equivalent in CLL and normal B-cells but higher in patients who died during the study than in living patients (152.1 vs. 65.04 pmol; P = 0.0060). Furthermore, HDAC activity correlated with treatment-free survival (TFS; P = 0.0156) and overall survival (OS; P < 0.0001): patients with low HDAC activity (n = 75) had a median TFS and OS of 101 and >376 months, respectively, whereas patients with high HDAC activity (n = 39) had a median TFS and OS of 47 and 137 months, respectively. Multivariate analyses indicated that HDAC activity is an independent predictor of OS (hazard ratio = 7.68; P = 0.0017). Finally, HDAC activity increased after B-cell receptor stimulation using IgM, suggesting a role for microenvironment stimuli (n = 10; P = 0.0371). In conclusion, high HDAC activity in CLL B-cells is associated with shorter TFS and OS and is an independent marker of OS, refining the use of other prognostic factors. This work provides a biological base for the use of HDAC inhibitors in CLL treatment. PMID:25437053

  9. Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA.

    PubMed

    Specks, U; Fass, D N; Fautsch, M P; Hummel, A M; Viss, M A

    1996-07-29

    We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC-1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA. The enzymatic activity is inhibited by greater than 95% by alpha 1-PI. The rPR3 and the enzymatically inactive mutant rPR3-S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3's targeting to granules. This rPR3 is the first to be recognized by most c-ANCA from WG patients and all anti-PR3 ANCA that were detected by standard anti-PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure-function relationships and interaction with autoantibodies. PMID:8706874

  10. A Unified View of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gating: Combining the Allosterism of a Ligand-gated Channel with the Enzymatic Activity of an ATP-binding Cassette (ABC) Transporter*

    PubMed Central

    Kirk, Kevin L.; Wang, Wei

    2011-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ion channel in that its gating is coupled to an intrinsic enzymatic activity (ATP hydrolysis). This enzymatic activity derives from the evolutionary origin of CFTR as an ATP-binding cassette transporter. CFTR gating is distinct from that of a typical ligand-gated channel because its ligand (ATP) is usually consumed during the gating cycle. However, recent findings indicate that CFTR gating exhibits allosteric properties that are common to conventional ligand-gated channels (e.g. unliganded openings and constitutive mutations). Here, we provide a unified view of CFTR gating that combines the allosterism of a ligand-gated channel with its unique enzymatic activity. PMID:21296873

  11. Fully relativistic complete active space self-consistent field for large molecules: Quasi-second-order minimax optimization

    SciTech Connect

    Bates, Jefferson E.; Shiozaki, Toru

    2015-01-28

    We develop an efficient algorithm for four-component complete active space self-consistent field (CASSCF) methods on the basis of the Dirac equation that takes into account spin–orbit and other relativistic effects self-consistently. Orbitals are optimized using a trust-region quasi-Newton method with Hessian updates so that energies are minimized with respect to rotations among electronic orbitals and maximized with respect to rotations between electronic and positronic orbitals. Utilizing density fitting and parallel computation, we demonstrate that Dirac–Coulomb CASSCF calculations can be routinely performed on systems with 100 atoms and a few heavy-elements. The convergence behavior and wall times for octachloridodirhenate(III) and a tungsten methylidene complex are presented. In addition, the excitation energies of octachloridodirhenate(III) are reported using a state-averaged variant.

  12. An atomic orbital-based formulation of the complete active space self-consistent field method on graphical processing units

    SciTech Connect

    Hohenstein, Edward G.; Luehr, Nathan; Ufimtsev, Ivan S.; Martínez, Todd J.

    2015-06-14

    Despite its importance, state-of-the-art algorithms for performing complete active space self-consistent field (CASSCF) computations have lagged far behind those for single reference methods. We develop an algorithm for the CASSCF orbital optimization that uses sparsity in the atomic orbital (AO) basis set to increase the applicability of CASSCF. Our implementation of this algorithm uses graphical processing units (GPUs) and has allowed us to perform CASSCF computations on molecular systems containing more than one thousand atoms. Additionally, we have implemented analytic gradients of the CASSCF energy; the gradients also benefit from GPU acceleration as well as sparsity in the AO basis.

  13. Knockout of the p-Coumarate Decarboxylase Gene from Lactobacillus plantarum Reveals the Existence of Two Other Inducible Enzymatic Activities Involved in Phenolic Acid Metabolism

    PubMed Central

    Barthelmebs, Lise; Divies, Charles; Cavin, Jean-François

    2000-01-01

    Lactobacillus plantarum NC8 contains a pdc gene coding for p-coumaric acid decarboxylase activity (PDC). A food grade mutant, designated LPD1, in which the chromosomal pdc gene was replaced with the deleted pdc gene copy, was obtained by a two-step homologous recombination process using an unstable replicative vector. The LPD1 mutant strain remained able to weakly metabolize p-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids. We have shown that L. plantarum has a second acid phenol decarboxylase enzyme, better induced with ferulic acid than with p-coumaric acid, which also displays inducible acid phenol reductase activity that is mostly active when glucose is added. Those two enzymatic activities are in competition for p-coumaric and ferulic acid degradation, and the ratio of the corresponding derivatives depends on induction conditions. Moreover, PDC appeared to decarboxylate ferulic acid in vitro with a specific activity of about 10 nmol · min−1 · mg−1 in the presence of ammonium sulfate. Finally, PDC activity was shown to confer a selective advantage on LPNC8 grown in acidic media supplemented with p-coumaric acid, compared to the LPD1 mutant devoid of PDC activity. PMID:10919793

  14. Cytidylate cyclase activity in mouse tissues: the enzymatic conversion of cytidine 5'-triphosphate to cytidine 3',5'-cyclic monophosphate (cyclic CMP).

    PubMed

    Yamamoto, I; Takai, T; Mori, S

    1989-12-01

    Cytidylate cyclase activity, which enzymatically converts cytidine 5'-triphosphate (CTP) to cytidine 3',5'-cyclic monophosphate (cyclic CMP), has been demonstrated in mouse tissue homogenates by use of a highly sensitive enzyme immunoassay (EIA) specific for cyclic CMP. Cyclic CMP formation is dependent on the amount of homogenate and on the incubation time. Although the enzyme activity was detected at wide ranges of pH from 6.8 to 11.5, the maximal activity was observed at around pH 9.4. The optimal temperature was 37 degrees C. Cytidylate cyclase activity was almost completely lost if the homogenates were heated at 90 degrees C for 3 min prior to use. The enzyme reaction exhibited typical Michaelis-Menten kinetics with an apparent Km for CTP of approx. 0.31 mM. Cyclic CMP formation was greatly enhanced with 4 mM Mn2+, Mg2+, Co2+; Mn2+ was the most effective. Fe2+ and Ca2+ were without effect. Cu2+ and Zn2+ at a concentration of 0.1 to 0.5 mM were inhibitory to Mn2+-dependent activity. Moreover, the enzyme activity was inhibited by several nucleotides including ATP, ADP, 5'-AMP, and GTP. Cytidylate cyclase activity was found to be present in all homogenates from a variety of mouse tissues examined except heart, with the highest level found in brain, and the lowest in liver. PMID:2557087

  15. A Δ38 Deletion Variant of Human Transketolase as a Model of Transketolase-Like Protein 1 Exhibits No Enzymatic Activity

    PubMed Central

    Schneider, Stefan; Lüdtke, Stefan; Schröder-Tittmann, Kathrin; Wechsler, Cindy; Meyer, Danilo; Tittmann, Kai

    2012-01-01

    Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a “pseudo-TKTL1” Δ38 deletion variant of human TKT (TKTΔ38) as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism. PMID:23118983

  16. The Coupling of an Enzymatic Reaction to Transmembrane Flow of Electric Current in a Synthetic "Active Transport" System

    PubMed Central

    Blumenthal, R.; Caplan, S. R.; Kedem, O.

    1967-01-01

    If a chemical reaction is constrained to occur within an asymmetric structure, e.g. by the presence of bound or otherwise trapped enzyme, coupling of the reaction to the flow of one or more solutes, or to the flow of electric current, becomes possible. Such systems can serve as models in which transport is “driven” by chemical reaction. In this respect the processes involved are analogous to active transport, though the molecular mechanisms may be quite different from those in nature. A simple arrangement of this kind has been studied: a composite membrane consisting of two ion exchange membranes of opposite fixed charge, separated by an intermediate layer of solution containing papain. An uncharged substrate of low molecular weight acts as “fuel” for the system, N-acetyl-L-glutamic acid diamide. This material (not previously described) hydrolyzes in the presence of papain to ammonium N-acetyl-L-glutamine. The composite membrane gives rise to an electromotive force, ultimately reaching a stationary state, when clamped between two identical solutions in which the affinity of the reaction has been fixed. Onsager's reciprocity relation has not hitherto been tested in a case of coupling between chemical reaction and a vectorial flow (here electric current); its validity for this system, in which stationary-state coupling occurs, was established over the experimental range of affinities (up to 3 kcal/mole). PMID:19210996

  17. Enzymatic modification of schizophyllan.

    PubMed

    Leathers, Timothy D; Sutivisedsak, Nongnuch; Nunnally, Melinda S; Price, Neil P J; Stanley, April M

    2015-03-01

    An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of β-endoglucanase with specificity towards schizophyllan. Concentrated enzyme preparations from these strains showed specific activities of 1.7-4.3 U β-glucanase/mg protein. Using dilutions of these enzymes in time course digestions, schizophyllan was progressively modified to reduced molecular weight species. Glucose and oligosaccharides were found only in the more complete digestions, and thus modified schizophyllan can be produced quantitatively, without loss, to small molecules. Permethylation analysis confirmed that modified schizophyllan retains the fundamental linkage structure of native schizophyllan. Modified schizophyllan species showed progressively reduced viscosity profiles, and all exhibited pseudoplasticity in response to shear thinning. PMID:25335747

  18. Exogenous acetate ion reaches the type II copper centre in CueO through the water-excretion channel and potentially affects the enzymatic activity.

    PubMed

    Komori, Hirofumi; Kataoka, Kunishige; Tanaka, Sakiko; Matsuda, Nana; Higuchi, Yoshiki; Sakurai, Takeshi

    2016-07-01

    The acetate-bound form of the type II copper was found in the X-ray structure of the multicopper oxidase CueO crystallized in acetate buffer in addition to the conventional OH(-)-bound form as the major resting form. The acetate ion was retained bound to the type II copper even after prolonged exposure of a CueO crystal to X-ray radiation, which led to the stepwise reduction of the Cu centres. However, in this study, when CueO was crystallized in citrate buffer the OH(-)-bound form was present exclusively. This fact shows that an exogenous acetate ion reaches the type II Cu centre through the water channel constructed between domains 1 and 3 in the CueO molecule. It was also found that the enzymatic activity of CueO is enhanced in the presence of acetate ions in the solvent water. PMID:27380373

  19. Humanized-single domain antibodies (VH/VHH) that bound specifically to Naja kaouthia phospholipase A2 and neutralized the enzymatic activity.

    PubMed

    Chavanayarn, Charnwit; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Bangphoomi, Kunan; Sookrung, Nitat; Chaicumpa, Wanpen

    2012-07-01

    Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations. PMID:22852068

  20. Isotopic and enzymatic analyses of planktonic nitrogen utilisation in the vicinity of Cape Sines (Portugal) during weak upwelling activity

    NASA Astrophysics Data System (ADS)

    Slawyk, Gerd; Coste, Bernard; Collos, Yves; Rodier, Martine

    1997-01-01

    Using measurements of 15N uptake and activities of nitrate reductase and glutamine synthetase, the utilization of nitrogenous nutrients by microplankton in the Portuguese upwelling area was investigated. During this cruise the euphotic zone of coastal waters was in most cases bisected by a nitracline forming two layers. Total inorganic nitrogen uptake rates (NH 4+ + NO 3-) in the upper mixed and nitrate-impoverished layer ranged from 0.1 to 0.8 nM h -1 and were primarily supported by regenerated (ammonium) nitrogen (62-97%), whereas they varied between 0.9 and 10.4 nM h -1 in the deep nitrate-rich layer and were mainly driven by new (nitrate) nitrogen (52-82%). Depth profiles of Chl a-specific uptake rates for ammonium and nitrate paralleled those of absolute uptake rates, i.e. values of VNH 4+Chl were highest (up to 16.1 nmol μg -1 h -1) in nitrate-poor surface waters while values of VNO 3-Chl were maximum (up to 8.4 nmol μg -1 h -1)within the nitracline. This latter vertical ordering of planktonic nitrogen nutrition was consistent with an aged upwelling situation. However, applying several indices of cell metabolism and nutritional status, such as 15N uptake/enzyme activity, surge uptake internally controlled uptake, and V maxChl/K t ratios, we were able to demonstrate that the phytoplankton assemblages inhabiting the nutrient-impoverished upper layer still bore the signature of physically mediated nitrogen (nitrate) supply generated by active upwelling that had occurred during the week before our visit to the area. This signature was the most evident in samples from the station furthest inshore and faded with distance from shore as a result of the deepening of the nitrate isopleths (weakening of upwelling activity), which showed the same offshore trend. The appearance of nitrate-rich waters at the surface, after a strong pulse of upwelling favourable winds just before the end of the cruise, led to a five-fold increase in average (over the euphotic zone

  1. A sensitive electrochemical biosensor for detection of protein kinase A activity and inhibitors based on Phos-tag and enzymatic signal amplification.

    PubMed

    Yin, Huanshun; Wang, Mo; Li, Bingchen; Yang, Zhiqing; Zhou, Yunlei; Ai, Shiyun

    2015-01-15

    A simple, highly sensitive and selective electrochemical assay is developed for the detection of protein kinase A (PKA) activity based on the specific recognition utility of Phos-tag for kinase-induced phosphopeptides and enzymatic signal amplification. When the substrate peptide was phosphorylated by PKA reaction, they could specifically bind with Phos-tag-biotin in the presence of Zn(2+) through the formation of a specific noncovalent complex with the phosphomonoester dianion in phosphorylated peptides. Through the further specific interaction between biotin and avidin, avidin functionalized horseradish peroxidase (HRP) can be captured on the electrode surface. Under the catalytic effect of HRP, a sensitive electrochemical signal for benzoquinone was obtained, which was related to PKA activity. Under the optimal experiment conditions, the proposed electrochemical method presented dynamic range from 0.5 to 25 unit/mL with low detection limit of 0.15 unit/mL. This new detection strategy was also successfully applied to analyze the inhibition effect of inhibitors (ellagic acid and H-89) on PKA activity and monitored the PKA activity in cell lysates. Therefore, this Phos-tag-based electrochemical assay offers an alternative platform for PKA activity assay and inhibitor screening, and thus it might be a valuable tool for development of targeted therapy and clinical diagnosis. PMID:25048450

  2. Analysis of the link between the redox state and enzymatic activity of the HtrA (DegP) protein from Escherichia coli.

    PubMed

    Koper, Tomasz; Polit, Agnieszka; Sobiecka-Szkatula, Anna; Wegrzyn, Katarzyna; Scire, Andrea; Figaj, Donata; Kadzinski, Leszek; Zarzecka, Urszula; Zurawa-Janicka, Dorota; Banecki, Bogdan; Lesner, Adam; Tanfani, Fabio; Lipinska, Barbara; Skorko-Glonek, Joanna

    2015-01-01

    Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically. PMID:25710793

  3. Naturally occurring polyphenol, morin hydrate, inhibits enzymatic activity of N-methylpurine DNA glycosylase, a DNA repair enzyme with various roles in human disease

    PubMed Central

    Dixon, Monica; Woodrick, Jordan; Gupta, Suhani; Karmahapatra, Soumendra Krishna; Devito, Stephen; Vasudevan, Sona; Dakshanamurthy, Sivanesan; Adhikari, Sanjay; Yenugonda, Venkata M.; Roy, Rabindra

    2015-01-01

    Interest in the mechanisms of DNA repair pathways, including the base excision repair (BER) pathway specifically, has heightened since these pathways have been shown to modulate important aspects of human disease. Modulation of the expression or activity of a particular BER enzyme, N-methylpurine DNA glycosylase (MPG), has been demonstrated to play a role in carcinogenesis and resistance to chemotherapy as well as neurodegenerative diseases, which has intensified the focus on studying MPG-related mechanisms of repair. A specific small molecule inhibitor for MPG activity would be a valuable biochemical tool for understanding these repair mechanisms. By screening several small molecule chemical libraries, we identified a natural polyphenolic compound, morin hydrate, which inhibits MPG activity specifically (IC50 = 2.6 µM). Detailed mechanism analysis showed that morin hydrate inhibited substrate DNA binding of MPG, and eventually the enzymatic activity of MPG. Computational docking studies with an x-ray derived MPG structure as well as comparison studies with other structurally-related flavanoids offer a rationale for the inhibitory activity of morin hydrate observed. The results of this study suggest that the morin hydrate could be an effective tool for studying MPG function and it is possible that morin hydrate and its derivatives could be utilized in future studies focused on the role of MPG in human disease. PMID:25650313

  4. Phytase production by Rhizopus microsporus var. microsporus biofilm: characterization of enzymatic activity after spray drying in presence of carbohydrates and nonconventional adjuvants.

    PubMed

    Sato, Vanessa Sayuri; Jorge, João Atílio; Oliveira, Wanderley Pereira; Souza, Claudia Regina Fernandez; Guimarães, Luis Henrique Souza

    2014-02-28

    Microbial phytases are enzymes with biotechnological interest for the feed industry. In this article, the effect of spray-drying conditions on the stability and activity of extracellular phytase produced by R. microsporus var. microsporus biofilm is described. The phytase was spray-dried in the presence of starch, corn meal (>150 μm), soy bean meal (SB), corn meal (<150 μm) (CM), and maltodextrin as drying adjuvants. The residual enzyme activity after drying ranged from 10.7% to 60.4%, with SB and CM standing out as stabilizing agents. Water concentration and residual enzyme activity were determined in obtained powders as a function of the drying condition. When exposed to different pH values, the SB and CM products were stable, with residual activity above 50% in the pH range from 4.5 to 8.5 for 60 min. The use of CM as drying adjuvant promoted the best retention of enzymatic activity compared with SB. Spray drying of the R. microsporus var. microsporus phytase using different drying adjuvants showed interesting results, being quite feasible with regards their biotechnological applications, especially for poultry diets. PMID:24196167

  5. Analysis of the Link between the Redox State and Enzymatic Activity of the HtrA (DegP) Protein from Escherichia coli

    PubMed Central

    Koper, Tomasz; Polit, Agnieszka; Sobiecka-Szkatula, Anna; Wegrzyn, Katarzyna; Scire, Andrea; Figaj, Donata; Kadzinski, Leszek; Zarzecka, Urszula; Zurawa-Janicka, Dorota; Banecki, Bogdan; Lesner, Adam; Tanfani, Fabio; Lipinska, Barbara; Skorko-Glonek, Joanna

    2015-01-01

    Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically. PMID:25710793

  6. Communication: Smoothing out excited-state dynamics: Analytical gradients for dynamically weighted complete active space self-consistent field

    SciTech Connect

    Glover, W. J.

    2014-11-07

    State averaged complete active space self-consistent field (SA-CASSCF) is a workhorse for determining the excited-state electronic structure of molecules, particularly for states with multireference character; however, the method suffers from known issues that have prevented its wider adoption. One issue is the presence of discontinuities in potential energy surfaces when a state that is not included in the state averaging crosses with one that is. In this communication I introduce a new dynamical weight with spline (DWS) scheme that mimics SA-CASSCF while removing energy discontinuities due to unweighted state crossings. In addition, analytical gradients for DWS-CASSCF (and other dynamically weighted schemes) are derived for the first time, enabling energy-conserving excited-state ab initio molecular dynamics in instances where SA-CASSCF fails.

  7. Analytical gradients of the state-average complete active space self-consistent field method with density fitting

    NASA Astrophysics Data System (ADS)

    Delcey, Mickaël G.; Pedersen, Thomas Bondo; Aquilante, Francesco; Lindh, Roland

    2015-07-01

    An efficient implementation of the state-averaged complete active space self-consistent field (SA-CASSCF) gradients employing density fitting (DF) is presented. The DF allows a reduction both in scaling and prefactors of the different steps involved. The performance of the algorithm is demonstrated on a set of molecules ranging up to an iron-Heme b complex which with its 79 atoms and 811 basis functions is to our knowledge the largest SA-CASSCF gradient computed. For smaller systems where the conventional code could still be used as a reference, both the linear response calculation and the gradient formation showed a clear timing reduction and the overall cost of a geometry optimization is typically reduced by more than one order of magnitude while the accuracy loss is negligible.

  8. Analytical gradients of the state-average complete active space self-consistent field method with density fitting

    SciTech Connect

    Delcey, Mickaël G.; Pedersen, Thomas Bondo; Aquilante, Francesco; Lindh, Roland

    2015-07-28

    An efficient implementation of the state-averaged complete active space self-consistent field (SA-CASSCF) gradients employing density fitting (DF) is presented. The DF allows a reduction both in scaling and prefactors of the different steps involved. The performance of the algorithm is demonstrated on a set of molecules ranging up to an iron-Heme b complex which with its 79 atoms and 811 basis functions is to our knowledge the largest SA-CASSCF gradient computed. For smaller systems where the conventional code could still be used as a reference, both the linear response calculation and the gradient formation showed a clear timing reduction and the overall cost of a geometry optimization is typically reduced by more than one order of magnitude while the accuracy loss is negligible.

  9. Characteristics of maize biochar with different pyrolysis temperatures and its effects on organic carbon, nitrogen and enzymatic activities after addition to fluvo-aquic soil.

    PubMed

    Wang, Xiubin; Zhou, Wei; Liang, Guoqing; Song, Dali; Zhang, Xiaoya

    2015-12-15

    In this study, the characteristics of maize biochar produced at different pyrolysis temperatures (300, 450 and 600°C) and its effects on organic carbon, nitrogen and enzymatic activities after addition to fluvo-aquic soil were investigated. As pyrolysis temperature increased, ash content, pH, electrical conductivity, surface area, pore volume and aromatic carbon content of biochar increased while yield, ratios of oxygen:carbon and hydrogen: carbon and alkyl carbon content decreased. During incubation, SOC, total N, and ammonium-N contents increased in all biochar-amended treatments compared with the urea treatment; however, soil nitrate-N content first increased and then decreased with increasing pyrolysis temperature of the applied biochar. Extracellular enzyme activities associated with carbon transformation first increased and then decreased with biochars pyrolyzed at 450 and 600°C. Protease activity markedly increased with increased pyrolysis temperatures, whereas pyrolysis temperature had limited effect on soil urease activity. The results indicated that the responses of extracellular enzymes to biochar were dependent on the pyrolysis temperature, the enzyme itself and incubation time as well. PMID:26298256

  10. Neutral sphingomyelinase-2 is a redox sensitive enzyme: role of catalytic cysteine residues in regulation of enzymatic activity through changes in oligomeric state

    PubMed Central

    Dotson, P. Patrick; Karakashian, Alexander A.; Nikolova-Karakashian, Mariana N.

    2015-01-01

    Neutral sphingomyelinase-2 (nSMase-2) is the major sphingomyelinase activated in response to pro-inflammatory cytokines and during oxidative stress. It is a membrane-bound 655 amino acid protein containing 22 cysteine residues. In this study, we expressed recombinant mouse nSMase-2 protein in Escherichia coli, and investigated whether nSMase-2 is a redox sensitive enzyme. Our results demonstrate that nSMase-2 exists as both monomers and multimers that are associated with high and low enzymatic activity respectively. Mutational analysis of nSMase-2 identified within its C-terminal catalytic domain several oxidant-sensitive cysteine residues that were shown to be involved in enzyme oligomerization. Changing Cys617 to Ser for example is a gain-of-function mutation associated with a decreased propensity for oligomerization. Alternatively, nSMase-2 expression in a bacterial strain that lacks endogenous thioredoxin, Rosetta-gami2, results in increased oligomer formation and lower enzyme activity. Phenotypic rescue was accomplished by treating nSMase-2 lysates with recombinant human thioredoxin. This indicates that nSMase-2 may be a novel substrate for thioredoxin. FRET analysis confirmed the presence of nSMase-2 multimers in mammalian HEK cells and their localization to the plasma membrane. In conclusion, our results identify nSMase-2 as a redox-sensitive enzyme, whose basal activity is influenced by thioredoxin-mediated changes in its oligomeric state. PMID:25287744

  11. Kresoxim methyl dissipation kinetics and its residue effect on soil extra-cellular and intra-cellular enzymatic activity in four different soils of India.

    PubMed

    Sabale, Rupali P; Shabeer T P, Ahammed; Utture, Sagar C; Banerjee, Kaushik; Oulkar, Dasharath P; Adsule, Pandurang G; Deshmukh, Madhukar B

    2015-01-01

    The rate of degradation of kresoxim methyl and its effect on soil extra-cellular (acid phosphatase, alkaline phosphatase and β-glucosidase) and intra-cellular (dehydrogenase) enzymes were explored in four different soils of India. In all the tested soils, the degradation rate was faster at the beginning, which slowed down with time indicating a non-linear pattern of degradation. Rate of degradation in black soil was fastest followed by saline, brown and red soils, respectively and followed 1st or 1st + 1st order kinetics with half-life ranging between 1-6 days for natural soil and 1-19 days for sterile soils. The rate of degradation in natural against sterilized soils suggests that microbial degradation might be the major pathway of residue dissipation. Although small changes in enzyme activities were observed, kresoxim methyl did not have any significant deleterious effect on the enzymatic activity of the various test soils in long run. Simple correlation studies between degradation percentage and individual enzyme activities did not establish any significant relationships. The pattern and change of enzyme activity was primarily due to the effect of the incubation period rather than the effect of kresoxim methyl itself. PMID:25587778

  12. Neutral sphingomyelinase-2 is a redox sensitive enzyme: role of catalytic cysteine residues in regulation of enzymatic activity through changes in oligomeric state.

    PubMed

    Dotson, P Patrick; Karakashian, Alexander A; Nikolova-Karakashian, Mariana N

    2015-02-01

    Neutral sphingomyelinase-2 (nSMase-2) is the major sphingomyelinase activated in response to pro-inflammatory cytokines and during oxidative stress. It is a membrane-bound 655 amino acid protein containing 22 cysteine residues. In this study, we expressed recombinant mouse nSMase-2 protein in Escherichia coli, and investigated whether nSMase-2 is a redox sensitive enzyme. Our results demonstrate that nSMase-2 exists as both monomers and multimers that are associated with high and low enzymatic activity respectively. Mutational analysis of nSMase-2 identified within its C-terminal catalytic domain several oxidant-sensitive cysteine residues that were shown to be involved in enzyme oligomerization. Changing Cys(617) to Ser for example is a gain-of-function mutation associated with a decreased propensity for oligomerization. Alternatively, nSMase-2 expression in a bacterial strain that lacks endogenous thioredoxin, Rosetta-gami2, results in increased oligomer formation and lower enzyme activity. Phenotypic rescue was accomplished by treating nSMase-2 lysates with recombinant human thioredoxin. This indicates that nSMase-2 may be a novel substrate for thioredoxin. FRET analysis confirmed the presence of nSMase-2 multimers in mammalian HEK cells and their localization to the plasma membrane. In conclusion, our results identify nSMase-2 as a redox-sensitive enzyme, whose basal activity is influenced by thioredoxin-mediated changes in its oligomeric state. PMID:25287744

  13. Enzymatic conversion of carbon dioxide.

    PubMed

    Shi, Jiafu; Jiang, Yanjun; Jiang, Zhongyi; Wang, Xueyan; Wang, Xiaoli; Zhang, Shaohua; Han, Pingping; Yang, Chen

    2015-10-01

    With the continuous increase in fossil fuels consumption and the rapid growth of atmospheric CO2 concentration, the harmonious state between human and nature faces severe challenges. Exploring green and sustainable energy resources and devising efficient methods for CO2 capture, sequestration and utilization are urgently required. Converting CO2 into fuels/chemicals/materials as an indispensable element for CO2 capture, sequestration and utilization may offer a win-win strategy to both decrease the CO2 concentration and achieve the efficient exploitation of carbon resources. Among the current major methods (including chemical, photochemical, electrochemical and enzymatic methods), the enzymatic method, which is inspired by the CO2 metabolic process in cells, offers a green and potent alternative for efficient CO2 conversion due to its superior stereo-specificity and region/chemo-selectivity. Thus, in this tutorial review, we firstly provide a brief background about enzymatic conversion for CO2 capture, sequestration and utilization. Next, we depict six major routes of the CO2 metabolic process in cells, which are taken as the inspiration source for the construction of enzymatic systems in vitro. Next, we focus on the state-of-the-art routes for the catalytic conversion of CO2 by a single enzyme system and by a multienzyme system. Some emerging approaches and materials utilized for constructing single-enzyme/multienzyme systems to enhance the catalytic activity/stability will be highlighted. Finally, a summary about the current advances and the future perspectives of the enzymatic conversion of CO2 will be presented. PMID:26055659

  14. Ultrasonic-assisted enzymatic extraction of silymarin from the Silybum marianum seed shell and evaluation of its antioxidant activity in vitro

    PubMed Central

    Zhao, Fei; Li, XinHua

    2015-01-01

    This study revealed the optimal conditions for the Ultrasonic-Assisted Enzymatic Extraction (UAEE) of silymarin, and include: the concentration of ethanol, 50 %; enzyme concentration, 30 U/mg; liquid-solid ratio, 6:1; an extraction time of 120 min; and the ultrasonic power at 180 W. The extraction rate was 7.86 %, which is higher, by 74.67 %, than that of the silymarin extract from the Silybum marianum meal prepared by a distinct approach. SEM micrographs of the inner and outer surfaces of the Silybum marianum shell obtained by variant extractions demonstrated that the extraction of silymarin required the destruction of cell walls. The results suggest that UAEE is a promising alternative for the extraction of silymarin. The antioxidant activities of the silymarin were evaluated in vitro by its capabilities to scavenger the DPPH, hydroxyl and superoxide free radicals, as well as by its tyrosinase inhibitory activity. The results showed that silymarin has significant antioxidant activity, thus it can be used as a functional food material against oxidative stress. We believe that the knowledge gained from this study should contribute to the further development and application of this resource. PMID:27152106

  15. The role of the C-terminal region on the oligomeric state and enzymatic activity of Trypanosoma cruzi hypoxanthine phosphoribosyl transferase.

    PubMed

    Valsecchi, Wanda M; Cousido-Siah, Alexandra; Defelipe, Lucas A; Mitschler, André; Podjarny, Alberto; Santos, Javier; Delfino, José M

    2016-06-01

    Hypoxanthine phosphoribosyl transferase from Trypanosoma cruzi (TcHPRT) is a critical enzyme for the survival of the parasite. This work demonstrates that the full-length form in solution adopts a stable and enzymatically active tetrameric form, exhibiting large inter-subunit surfaces. Although this protein irreversibly aggregates during unfolding, oligomerization is reversible and can be modulated by low concentrations of urea. When the C-terminal region, which is predicted as a disordered stretch, is excised by proteolysis, TcHPRT adopts a dimeric state, suggesting that the C-terminal region acts as a main guide for the quaternary arrangement. These results are in agreement with X-ray crystallographic data presented in this work. On the other hand, the C-terminal region exhibits a modulatory role on the enzyme, as attested by the enhanced activity observed for the dimeric form. Bisphosphonates act as substrate-mimetics, uncovering long-range communications among the active sites. All in all, this work contributes to establish new ways applicable to the design of novel inhibitors that could eventually result in new drugs against parasitic diseases. PMID:26969784

  16. Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ-glutamyltranspeptidase activity.

    PubMed

    Zhang, Fei; Zheng, Qing-Zhong; Jiao, Qing-Cai; Liu, Jun-Zhong; Zhao, Gen-Hai

    2010-11-01

    Theanine (γ-glutamylethylamide) is the main amino acid component in green tea. The demand for theanine in the food and pharmaceutical industries continues to increase because of its special flavour and multiple physiological effects. In this research, an improved method for enzymatic theanine synthesis is reported. An economical substrate, glutamic acid γ-methyl ester, was used in the synthesis catalyzed by immobilized Escherichia coli cells with γ-glutamyltranspeptidase (GGT) activity. The results show that GGT activity with glutamic acid γ-methyl ester as substrate was about 1.2-folds higher than that with glutamine as substrate. Reaction conditions were optimized by using 300 mmol/l glutamic acid γ-methyl ester, 3,000 mmol/l ethylamine, and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C. Under these conditions, the immobilized cells were continuously used ten times, yielding an average glutamic acid γ-methyl ester to theanine conversion rate of 69.3%. Bead activity did not change significantly the first six times they were used, and the average conversion rate during the first six instances was 87.2%. The immobilized cells exhibited favourable operational stability. PMID:20238131

  17. Analysis of Non-Enzymatically Glycated Peptides: Neutral-Loss Triggered MS3 Versus Multi-Stage Activation Tandem Mass Spectrometry

    SciTech Connect

    Zhang, Qibin; Petyuk, Vladislav A.; Schepmoes, Athena A.; Orton, Daniel J.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Metz, Thomas O.

    2008-10-15

    Non-enzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. While electron transfer dissociation (ETD) has been shown to outperform collision-induced dissociation (CID) in sequencing glycated peptides by tandem mass spectrometry, ETD instrumentation is not yet available in all laboratories. In this study, we evaluated different advanced CID techniques (i.e., neutral-loss triggered MS3 and multi-stage activation) during LC-MSn analyses of Amadori-modified peptides enriched from human serum glycated in vitro. During neutral-loss triggered MS3 experiments, MS3 scans triggered by neutral-losses of 3 H2O or 3 H2O + HCHO produced similar results in terms of glycated peptide identifications. However, neutral losses of 3 H2O resulted in significantly more glycated peptide identifications during multi-stage activation experiments. Overall, the multi-stage activation approach produced more glycated peptide identifications, while the neutral-loss triggered MS3 approach resulted in much higher specificity. Both techniques offer a viable alternative to ETD for identifying glycated peptides when that method is unavailable.

  18. Degradation of di(2-ethyl hexyl) phthalate by Fusarium culmorum: Kinetics, enzymatic activities and biodegradation pathway based on quantum chemical modelingpathway based on quantum chemical modeling.

    PubMed

    Ahuactzin-Pérez, Miriam; Tlecuitl-Beristain, Saúl; García-Dávila, Jorge; González-Pérez, Manuel; Gutiérrez-Ruíz, María Concepción; Sánchez, Carmen

    2016-10-01

    Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer widely used in the manufacture of plastics, and it is an environmental contaminant. The specific growth rate (μ), maximum biomass (Xmax), biodegradation constant of DEHP (k), half-life (t1/2) of DEHP biodegradation and removal efficiency of DEHP, esterase and laccase specific activities, and enzymatic yield parameters were evaluated for Fusarium culmorum grown on media containing glucose and different concentrations of DEHP (0, 500 and 1000mg/L). The greatest μ and the largest Xmax occurred in media supplemented with 1000mg of DEHP/L. F. culmorum degraded 95% of the highest amount of DEHP tested (1000mg/L) within 60h of growth. The k and t1/2 were 0.024h(-1) and 28h, respectively, for both DEHP concentrations. The removal efficiency of DEHP was 99.8% and 99.9% for 1000 and 500mg/L, respectively. Much higher specific esterase activity than specific laccase activity was observed in all media tested. The compounds of biodegradation of DEHP were identified by GC-MS. A DEHP biodegradation pathway by F. culmorum was proposed on the basis of the intermolecular flow of electrons of the identified intermediate compounds using quantum chemical modeling. DEHP was fully metabolized by F. culmorum with butanediol as the final product. This fungus offers great potential in bioremediation of environments polluted with DEHP. PMID:27277206

  19. Effect of AOT on enzymatic activity of the organic solvent resistant tyrosinase from Streptomyces sp. REN-21 in aqueous solutions and water-in-oil microemulsions.

    PubMed

    Rodakiewicz-Nowak, Janina; Ito, Masaaki

    2005-04-15

    The effect of AOT (sodium-bis(2-ethylhexyl sulfosuccinate)) on enzymatic activity of the organic solvent resistant tyrosinase (OSRT) in aqueous phosphate buffer solutions and in water-in-oil microemulsions of the water/AOT/isooctane system has been investigated. In contrast to mushroom tyrosinase, AOT does not activate OSRT in aqueous solutions, altering its activity very little at concentrations lower than 2 mM. Increasing contents of AOT in isooctane reduce the observed initial reaction rates of oxidation of t-butylcatechol (tBC) and 4-methylcatechol (4-MC). Similarly to mushroom tyrosinase, the effect has been described using an equation based on preferential binding of the substrates by surfactant interface layers. The apparent Michaelis-Menten substrate binding constants increase linearly with AOT concentration (with slopes of 0.12+/-0.02 and 0.051+/-0.006 for tBC and 4-MC, respectively), and the effective enzyme turnover number in the microemulsions remains practically constant. PMID:15780309

  20. Modulation of Enzymatic Activity and Biological Function of Listeria monocytogenes Broad-Range Phospholipase C by Amino Acid Substitutions and by Replacement with the Bacillus cereus Ortholog

    PubMed Central

    Zückert, Wolfram R.; Marquis, Hélène; Goldfine, Howard

    1998-01-01

    The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium’s ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens α-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells. PMID:9746585

  1. Rational design of functional and tunable oscillating enzymatic networks.

    PubMed

    Semenov, Sergey N; Wong, Albert S Y; van der Made, R Martijn; Postma, Sjoerd G J; Groen, Joost; van Roekel, Hendrik W H; de Greef, Tom F A; Huck, Wilhelm T S

    2015-02-01

    Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks. PMID:25615670

  2. Rational design of functional and tunable oscillating enzymatic networks

    NASA Astrophysics Data System (ADS)

    Semenov, Sergey N.; Wong, Albert S. Y.; van der Made, R. Martijn; Postma, Sjoerd G. J.; Groen, Joost; van Roekel, Hendrik W. H.; de Greef, Tom F. A.; Huck, Wilhelm T. S.

    2015-02-01

    Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks.

  3. Heterotrophic bacterial production and extracellular enzymatic activity in sinking particulate matter in the western North Pacific Ocean

    PubMed Central

    Yamada, Namiha; Fukuda, Hideki; Ogawa, Hiroshi; Saito, Hiroaki; Suzumura, Masahiro

    2012-01-01

    Heterotrophic activities on sinking particulate matter (SPM) play an important role in SPM fluxes in the ocean. To demonstrate regional differences in heterotrophic activities on SPM, we measured heterotrophic bacterial production (HBP) in seawater (HBPSW) and SPM (HBPSPM) as well as potential extracellular enzyme activity (EEA) in SPM on a transect along 155°E in the western North Pacific Ocean in the subarctic (44°N), the Kuroshio Extension area (35°N), and the subtropical gyre (20°N). Depth-integrated HBPSW from the surface to 500 m was comparable between the locations, whereas HBPSPM at 44°N was substantially lower than at the other sites. We found the highest particulate organic carbon (POC) export flux and export efficiency to bathypelagic depths, and the lowest water temperatures, at 44°N. We found significant correlations between leucine aminopeptidase (LAPase) activity, β-glucosidase (BGase) activity, POC flux and particulate organic nitrogen flux. LAPase activity was two orders of magnitude higher than BGase activity, with a BGase:LAPase activity ratio of 0.027. There were no significant correlations between HBP and EEA in SPM except for lipase, and lipase activity was significantly correlated with temperature. We propose that hydrographic conditions are an important factor controlling heterotrophic bacterial activity and export efficiency of organic carbon to the deep ocean, as are the sources and abundance of SPM produced in the euphotic zone via primary production. PMID:23109933

  4. Engineering Protein Allostery: 1.05 Å Resolution Structure and Enzymatic Properties of a Na[superscript +]-activated Trypsin

    SciTech Connect

    Page, Michael J.; Carrell, Christopher J.; Di Cera, Enrico

    2008-05-28

    Some trypsin-like proteases are endowed with Na{sup +}-dependent allosteric enhancement of catalytic activity, but this important mechanism has been difficult to engineer in other members of the family. Replacement of 19 amino acids in Streptomyces griseus trypsin targeting the active site and the Na{sup +}-binding site were found necessary to generate efficient Na{sup +} activation. Remarkably, this property was linked to the acquisition of a new substrate selectivity profile similar to that of factor Xa, a Na{sup -} activated protease involved in blood coagulation. The X-ray crystal structure of the mutant trypsin solved to 1.05 {angstrom} resolution defines the engineered Na{sup +} site and active site loops in unprecedented detail. The results demonstrate that trypsin can be engineered into an efficient allosteric protease, and that Na+ activation is interwoven with substrate selectivity in the trypsin scaffold.

  5. [Enhanced enzymatic hydrolysis of excess sludge by surfactant].

    PubMed

    Yu, Jing; Luo, Kun; Yang, Qi; Li, Xiao-Ming; Xie, Bing-Xin; Yang, Guo-Jing; Mo, Chuang-Rong

    2011-08-01

    In order to enhance the efficiency of enzymatic hydrolysis of excess sludge, sodium dodecyl sulfate (SDS) was added to the system to explore the feasibility of promotion the enzyme hydrolysis. The results showed that the enzymatic hydrolysis of excess sludge could be greatly improved by SDS, and the mixed enzymes system was more effective than that by single enzyme system. SCOD releasing increased linearly with the increase of SDS dosage at the mixed enzymes concentration of 0.06 g/g. SCOD/TCOD increased from 1.3% to 54.3% and VSS reduction achieved to 43.2% at the SDS dosage of 0.20 g/g. Further studies indicated that SDS could improve the activity of external enzymes. At SDS dosage of 0.10 g/g, the protease activity of SDS + protease showed a 2. 3-time increase and the amylase activity of SDS + amylase showed a 1.2-time increase compared with enzymatic treatment. After 4 h hydrolysis, the concentration of protein, NH4+ -N and soluble sugar in SDS + mixed enzymes system were improved by 85.4%, 92.5% and 64.0%, respectively. Correspondingly, sludge hydrolysis within prior 4 h was consistent with first-order reaction dynamics. The reaction rate constant (K) of soluble sugar increased from 0.23 to 0.41, which indicated that the reaction rate of hydrolysis increased significantly. PMID:22619958

  6. Single-Unit Activity during Natural Vision: Diversity, Consistency, and Spatial Sensitivity among AF Face Patch Neurons

    PubMed Central

    Russ, Brian E.; Elnaiem, Heba D.; Kurnikova, Anastasia I.; Leopold, David A.

    2015-01-01

    Several visual areas within the STS of the macaque brain respond strongly to faces and other biological stimuli. Determining the principles that govern neural responses in this region has proven challenging, due in part to the inherently complex stimulus domain of dynamic biological stimuli that are not captured by an easily parameterized stimulus set. Here we investigated neural responses in one fMRI-defined face patch in the anterior fundus (AF) of the STS while macaques freely view complex videos rich with natural social content. Longitudinal single-unit recordings allowed for the accumulation of each neuron's responses to repeated video presentations across sessions. We found that individual neurons, while diverse in their response patterns, were consistently and deterministically driven by the video content. We used principal component analysis to compute a family of eigenneurons, which summarized 24% of the shared population activity in the first two components. We found that the most prominent component of AF activity reflected an interaction between visible body region and scene layout. Close-up shots of faces elicited the strongest neural responses, whereas far away shots of faces or close-up shots of hindquarters elicited weak or inhibitory responses. Sensitivity to the apparent proximity of faces was also observed in gamma band local field potential. This category-selective sensitivity to spatial scale, together with the known exchange of anatomical projections of this area with regions involved in visuospatial analysis, suggests that the AF face patch may be specialized in aspects of face perception that pertain to the layout of a social scene. PMID:25855170

  7. Posttranslational Modification of Maize Chloroplast Pyruvate Orthophosphate Dikinase Reveals the Precise Regulatory Mechanism of Its Enzymatic Activity.

    PubMed

    Chen, Yi-Bo; Lu, Tian-Cong; Wang, Hong-Xia; Shen, Jie; Bu, Tian-Tian; Chao, Qing; Gao, Zhi-Fang; Zhu, Xin-Guang; Wang, Yue-Feng; Wang, Bai-Chen

    2014-04-01

    In C4 plants, pyruvate orthophosphate dikinase (PPDK) activity is tightly dark/light regulated by reversible phosphorylation of an active-site threonine (Thr) residue; this process is catalyzed by PPDK regulatory protein (PDRP). Phosphorylation and dephosphorylation of PPDK lead to its inactivation and activation, respectively. Here, we show that light intensity rather than the light/dark transition regulates PPDK activity by modulating the reversible phosphorylation at Thr-527 (previously termed Thr-456) of PPDK in maize (Zea mays). The amount of PPDK (unphosphorylated) involved in C4 photosynthesis is indeed strictly controlled by light intensity, despite the high levels of PPDK protein that accumulate in mesophyll chloroplasts. In addition, we identified a transit peptide cleavage site, uncovered partial amino-terminal acetylation, and detected phosphorylation at four serine (Ser)/Thr residues, two of which were previously unknown in maize. In vitro experiments indicated that Thr-527 and Ser-528, but not Thr-309 and Ser-506, are targets of PDRP. Modeling suggests that the two hydrogen bonds between the highly conserved residues Ser-528 and glycine-525 are required for PDRP-mediated phosphorylation of the active-site Thr-527 of PPDK. Taken together, our results suggest that the regulation of maize plastid PPDK isoform (C4PPDK) activity is much more complex than previously reported. These diverse regulatory pathways may work alone or in combination to fine-tune C4PPDK activity in response to changes in lighting. PMID:24710069

  8. Chemo-Enzymatic Synthesis of Optically Active γ- and δ-Decalactones and Their Effect on Aphid Probing, Feeding and Settling Behavior.

    PubMed

    Boratyński, Filip; Dancewicz, Katarzyna; Paprocka, Marlena; Gabryś, Beata; Wawrzeńczyk, Czesław

    2016-01-01

    The enantiomerically enriched γ- and δ-decalactones (4a and 4b) were prepared from corresponding racemic primary-secondary 1,4- and 1,5-diols (1a and 1b), as products of enzymatic oxidation catalyzed by different alcohol dehydrogenases. The results of biotransformations indicated that the oxidation processes catalyzed by alcohol dehydrogenase (HLADH), both isolated from horse liver and recombinant in Escherichia coli, were characterized by the highest degree of conversion with moderate enantioselectivity of the reaction. Useful, environmentally friendly extraction procedure of decalactones (4a and 4b) based on hydrodistillation using a Deryng apparatus was developed. Both racemic lactones (4a and 4b), as well as their enantiomerically enriched isomers, were tested for feeding deterrent activity against Myzus persicae. The effect of these compounds on probing, feeding and settling behavior of M. persicae was studied in vivo. The deterrent activity of decalactones (4a and 4b) against aphids depended on the size of the lactone ring and the enantiomeric purity of the compounds. δ-Decalactone (4b) appeared inactive against M. persicae while γ-decalactone (4a) restrained aphid probing at ingestional phase. Only (-)-(S)-γ-decalactone (4a) had strong and durable (i.e. lasting for at least 24 hours) limiting effect, expressed at phloem level. PMID:26741824

  9. Chemo-Enzymatic Synthesis of Optically Active γ- and δ-Decalactones and Their Effect on Aphid Probing, Feeding and Settling Behavior

    PubMed Central

    Boratyński, Filip; Dancewicz, Katarzyna; Paprocka, Marlena; Gabryś, Beata; Wawrzeńczyk, Czesław

    2016-01-01

    The enantiomerically enriched γ- and δ-decalactones (4a and 4b) were prepared from corresponding racemic primary-secondary 1,4- and 1,5-diols (1a and 1b), as products of enzymatic oxidation catalyzed by different alcohol dehydrogenases. The results of biotransformations indicated that the oxidation processes catalyzed by alcohol dehydrogenase (HLADH), both isolated from horse liver and recombinant in Escherichia coli, were characterized by the highest degree of conversion with moderate enantioselectivity of the reaction. Useful, environmentally friendly extraction procedure of decalactones (4a and 4b) based on hydrodistillation using a Deryng apparatus was developed. Both racemic lactones (4a and 4b), as well as their enantiomerically enriched isomers, were tested for feeding deterrent activity against Myzus persicae. The effect of these compounds on probing, feeding and settling behavior of M. persicae was studied in vivo. The deterrent activity of decalactones (4a and 4b) against aphids depended on the size of the lactone ring and the enantiomeric purity of the compounds. δ-Decalactone (4b) appeared inactive against M. persicae while γ-decalactone (4a) restrained aphid probing at ingestional phase. Only (–)-(S)-γ-decalactone (4a) had strong and durable (i.e. lasting for at least 24 hours) limiting effect, expressed at phloem level. PMID:26741824

  10. Effect of permethrin, anthracene and mixture exposure on shell components, enzymatic activities and proteins status in the Mediterranean clam Venerupis decussata.

    PubMed

    Sellami, Badreddine; Khazri, Abdelhafidh; Mezni, Amine; Louati, Héla; Dellali, Mohamed; Aissa, Patricia; Mahmoudi, Ezzeddine; Beyrem, Hamouda; Sheehan, David

    2015-01-01

    Anthracene (ANT) and permethrin (PER) are two of the more toxic compounds reaching the marine environment. This study aimed to determine the impact of these molecules on Venerupis decussata, an economically important species cultured on the Tunisian coast. Shell structure and its possible transformation upon exposure to the two contaminants were studied by X-ray diffraction and gravimetric analyses. Results revealed a phase transition in shell composition from aragonite to calcite after PER exposure, to a mixture of PER and ANT (Mix) but not for ANT alone. Catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GST) activities were determined in digestive gland and gills after exposure to ANT, PER and Mix to assess the impact of the contamination on the oxidative status of V. decussata. Enzyme activities increased in the digestive gland after PER treatment and in the gills after ANT treatment. PER exposure significantly reduced the levels of free thiols and increased levels of carbonylated proteins in the digestive gland, as compared to controls. In contrast, ANT exposure significantly reduced free thiols and increased the number of carbonylated proteins in the gills. Mix induced additive effects as measured by both enzymatic and proteomic approaches. The present study suggests that PER has a strong effect on shell structure; that PER and ANT exposure generate compound-dependent oxidative stress in the tissues of V. decussata and that a mixture of the two compounds has synergistic effects on biochemical response. PMID:25461742

  11. Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities

    SciTech Connect

    Kajikawa, Takao; Kataoka, Kunishige; Sakurai, Takeshi

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Proton transfer pathway to dioxygen in CueO was identified. Black-Right-Pointing-Pointer Glu506 is the key amino acid to transport proton. Black-Right-Pointing-Pointer The Ala mutation at Glu506 formed a compensatory proton transfer pathway. Black-Right-Pointing-Pointer The Ile mutation at Glu506 shut down the hydrogen bond network. -- Abstract: CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase.

  12. Time-dependent restricted-active-space self-consistent-field singles method for many-electron dynamics

    SciTech Connect

    Miyagi, Haruhide; Bojer Madsen, Lars

    2014-04-28

    The time-dependent restricted-active-space self-consistent-field singles (TD-RASSCF-S) method is presented for investigating TD many-electron dynamics in atoms and molecules. Adopting the SCF notion from the muticonfigurational TD Hartree-Fock (MCTDHF) method and the RAS scheme (single-orbital excitation concept) from the TD configuration-interaction singles (TDCIS) method, the TD-RASSCF-S method can be regarded as a hybrid of them. We prove that, for closed-shell N{sub e}-electron systems, the TD-RASSCF-S wave function can be fully converged using only N{sub e}/2 + 1 ⩽ M ⩽ N{sub e} spatial orbitals. Importantly, based on the TD variational principle, the converged TD-RASSCF-S wave function with M = N{sub e} is more accurate than the TDCIS wave function. The accuracy of the TD-RASSCF-S approach over the TDCIS is illustrated by the calculation of high-order harmonic generation spectra for one-dimensional models of atomic helium, beryllium, and carbon in an intense laser pulse. The electronic dynamics during the process is investigated by analyzing the behavior of electron density and orbitals. The TD-RASSCF-S method is accurate, numerically tractable, and applicable for large systems beyond the capability of the MCTDHF method.

  13. Effect of cholecystokinin-pancreozymin and secretin on the volume, composition, and enzymatic activity of hepatic bile in rabbits

    PubMed Central

    Kirchmayer, S.; Tarnawski, A.; Drożdż, H.; Cichecka, K.

    1972-01-01

    In rabbits given cholecystokinin-pancreozymin intravenously a statistically significant increase in bile volume and alkaline phosphatase (AP) activity was observed. There was also an increase in the total amount of protein, cholesterol, bicarbonates Na, K, Ca, Mg, and the total activity of alkaline phosphatase and leucineaminopeptidase (LAP) secreted in bile within 90 minutes after pancreozymin injection. Pancreozymin had no effect on the concentration and output of taurocholates and the activity of gammaglutamyltranspeptidase (GGTP) and alanineaminotransferase (ALAT) in bile. Secretin neither changes the volume nor the concentration of any of the investigated components in rabbit bile. PMID:18668839

  14. Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

    PubMed

    Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

    2015-11-15

    Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. PMID:26057733

  15. Gonadotropin-releasing hormone 1 directly affects corpora lutea lifespan in Mediterranean buffalo (Bubalus bubalis) during diestrus: presence and in vitro effects on enzymatic and hormonal activities.

    PubMed

    Zerani, Massimo; Catone, Giuseppe; Maranesi, Margherita; Gobbetti, Anna; Boiti, Cristiano; Parillo, Francesco

    2012-08-01

    The expression of gonadotropin-releasing hormone (GNRH) receptor (GNRHR) and the direct role of GNRH1 on corpora lutea function were studied in Mediterranean buffalo during diestrus. Immunohistochemistry evidenced at early, mid, and late luteal stages the presence of GNRHR only in large luteal cells and GNRH1 in both small and large luteal cells. Real-time PCR revealed GNRHR and GNRH1 mRNA at the three luteal stages, with lowest values in late corpora lutea. In vitro corpora lutea progesterone production was greater in mid stages and lesser in late luteal phases, whereas prostaglandin F2 alpha (PGF2alpha) increased from early to late stages, and PGE2 was greater in the earlier-luteal phase. Cyclooxygenase 1 (prostaglandin-endoperoxide synthase 1; PTGS1) activity did not change during diestrus, whereas PTGS2 increased from early to late stages, and PGE2-9-ketoreductase (PGE2-9-K) was greater in late corpora lutea. PTGS1 activity was greater than PTGS2 in early corpora lutea and lesser in late luteal phase. In corpora lutea cultured in vitro, the GNRH1 analog (buserelin) reduced progesterone secretion and increased PGF2alpha secretion as well as PTGS2 and PGE2-9-K activities at mid and late stages. PGE2 release and PTGS1 activity were increased by buserelin only in late corpora lutea. These results suggest that GNRH is expressed in all luteal cells of buffalo, whereas GNRHR is only expressed in large luteal phase. Additionally, GNRH directly down-regulates corpora lutea progesterone release, with the concomitant increases of PGF2alpha production and PTGS2 and PGE2-9-K enzymatic activities. PMID:22592497

  16. Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2.

    PubMed

    Shetty, Jagathpala; Sinville, Rondedrick; Shumilin, Igor A; Minor, Wladek; Zhang, Jianhai; Hawkinson, Jon E; Georg, Gunda I; Flickinger, Charles J; Herr, John C

    2016-05-01

    The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents. PMID:26777341

  17. Effect of ferrous chloride on biogas production and enzymatic activities during anaerobic fermentation of cow dung and Phragmites straw.

    PubMed

    Zhang, Huayong; Tian, Yonglan; Wang, Lijun; Mi, Xueyue; Chai, Yang

    2016-06-01

    The effect of ferrous (added as FeCl2) on the anaerobic co-digestion of Phragmites straw and cow dung was studied by investigating the biogas properties, pH values, organic matter degradation (COD) and enzyme activities (cellulase, protease and dehydrogenase) at different stages of mesophilic fermentation. The results showed that Fe(2+) addition increased the cumulative biogas yields by 18.1 % by extending the peak period with high daily biogas yields. Meanwhile, the methane (CH4) contents in the Fe(2+) added groups were generally higher than the control group before the 15th day. The pH values were not significantly impacted by Fe(2+) concentrations during the fermentation process. The COD concentrations, cellulase, protease and dehydrogenase activities varied with the added Fe(2+) concentrations and the stages of the fermentation process. At the beginning stage of fermentation (4th day), Fe(2+) addition increased the biogas production by improving the cellulase and dehydrogenase activities which caused a decline in COD. At the peak stage of fermentation (8th day), Fe(2+) addition enhanced the cellulase and protease activities, and resulted in lower COD contents than the control group. When the biogas yields decreased again (13th day), the COD contents varied similar with the protease and dehydrogenase activities, whilst cellulase activities were not sensitive to Fe(2+) concentrations. At the end of fermentation (26th day), Fe(2+) addition decreased the cellulase activities, led to lower COD contents and finally resulted the lower biogas yields than the control group. Taking the whole fermentation process into account, the promoting effect of Fe(2+) addition on biogas yields was mainly attributed to the extension of the gas production peak stage and the improvement of cellulase activities. PMID:26862032

  18. Enzymatic modification of schizophyllan

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of ß-endoglucanase with specif...

  19. Enzymatically Regulated Peptide Pairing and Catalysis for the Bioanalysis of Extracellular Prometastatic Activities of Functionally Linked Enzymes

    PubMed Central

    Li, Hao; Huang, Yue; Yu, Yue; Li, Tianqi; Li, Genxi; Anzai, Jun-ichi

    2016-01-01

    Diseases such as cancer arise from systematical reconfiguration of interactions of exceedingly large numbers of proteins in cell signaling. The study of such complicated molecular mechanisms requires multiplexed detection of the inter-connected activities of several proteins in a disease-associated context. However, the existing methods are generally not well-equipped for this kind of application. Here a method for analyzing functionally linked protein activities is developed based on enzyme controlled pairing between complementary peptide helix strands, which simultaneously enables elaborate regulation of catalytic activity of the paired peptides. This method has been used to detect three different types of protein modification enzymes that participate in the modification of extracellular matrix and the formation of invasion front in tumour. In detecting breast cancer tissue samples using this method, up-regulated activity can be observed for two of the assessed enzymes, while the third enzyme is found to have a subtle fluctuation of activity. These results may point to the application of this method in evaluating prometastatic activities of proteins in tumour. PMID:27140831

  20. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals.

    PubMed Central

    Casadaban, M J; Chou, J; Cohen, S N

    1980-01-01

    We report the construction and use of a series of plasmid vectors suitable for the detection and cloning of translational control signals and 5' coding sequences of exogenously derived genes. In these plasmids, the first eight codons of the amino-terminal end of the lactose operon beta-galactosidase gene, lacZ, were removed, and unique BamHI, EcoRI, and SmaI (XmaI) endonuclease cleavage sites were incorporated adjacent to the eighth codon of lacZ. Introduction of deoxyribonucleic acid fragments containing appropriate regulatory signals and 5' coding sequences into such lac fusion plasmids led to the production of hybrid proteins consisting of the carboxyl-terminal segment of a beta-galactosidase remnant plus a peptide fragment that contained the amino-terminal amino acids encoded by the exogenous deoxyribonucleic acid sequence. These hybrid peptides retained beta-galactosidase enzymatic activity and yielded a Lac+ phenotype. Such hybrid proteins are useful for purifying peptide sequences encoded by exogenous deoxyribonucleic acid fragments and for studies relating the structure and function of specific peptide segments. Images PMID:6162838

  1. Enzymatic desulfurization of coal

    SciTech Connect

    Marquis, J.K. . School of Medicine); Kitchell, J.P. )

    1988-10-07

    Our current efforts to develop clean coal technology, emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  2. Enzymatic desulfurization of coal

    SciTech Connect

    Marquis, J.K. . School of Medicine); Kitchell, J.P. )

    1988-12-15

    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  3. Enzymatic desulfurization of coal

    SciTech Connect

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V. ); Marquis, J.K. . School of Medicine)

    1989-06-16

    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes as well as commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  4. Effect of processing on physicochemical composition, bioactive compounds and enzymatic activity of yellow mombin (Spondias mombin L.) tropical juice.

    PubMed

    de Carvalho, Joelia Marques; Maia, Geraldo Arraes; da Fonseca, Ana Valquíria V; de Sousa, Paulo Henrique M; Rodrigues, Sueli

    2015-02-01

    Yellow mombin (Spondias mombin, L.) is a tropical fruit that presents exotic taste and aroma, being source of carotenoids and phenolics compounds. It presents a good potential for processing, despite some restriction related with the presence of high amounts of peroxidase (POD) and pectinmethylesterase (PME) which can cause sensory changes in the product. This work addresses the evaluation of changes in POD and PME enzyme activity during the traditional industrial processing used to produce tropical juices in Brazil. The enzyme activity was determined after the main steps of the processing: fruit pulping, homogenization and pasteurization. Although both enzymes presented significant activity loss during processing, the final product showed residual activity for PME (25 %) and POD (2.5 %). PME showed to be more thermal resistant than POD in yellow mombin juice. Considering the compounds with antioxidant activity, yellow mombin presented high amounts of carotenoids and phenolics when compared to other tropical fruits such as passion fruit and pineapple. Although the processing of the fruit resulted in significative phenolic loss, the carotenoids content was not affected significantly by the processing. PMID:25694737

  5. Structural and Enzymatic Characterization of ABgp46, a Novel Phage Endolysin with Broad Anti-Gram-Negative Bacterial Activity

    PubMed Central

    Oliveira, Hugo; Vilas Boas, Diana; Mesnage, Stéphane; Kluskens, Leon D.; Lavigne, Rob; Sillankorva, Sanna; Secundo, Francesco; Azeredo, Joana

    2016-01-01

    The present study demonstrates the antibacterial potential of a phage endolysin against Gram-negative pathogens, particularly against multidrug resistant strains of Acinetobacter baumannii. We have cloned, heterologously expressed and characterized a novel endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1 and tested its antibacterial activity against several multidrug-resistant A. baumannii strains. LC-MS revealed that ABgp46 is an N-acetylmuramidase, that is also active over a broad pH range (4.0–10.0) and temperatures up to 50°C. Interestingly, ABgp46 has intrinsic and specific anti-A. baumannii activity, reducing multidrug resistant strains by up to 2 logs within 2 h. By combining ABgp46 with several organic acids that act as outer membrane permeabilizing agents, it is possible to increase and broaden antibacterial activity to include other Gram-negative bacterial pathogens. In the presence of citric and malic acid, ABgp46 reduces A. baumannii below the detection limit (>5 log) and more than 4 logs Pseudomonas aeruginosa and Salmonella typhimurium strains. Overall, this globular endolysin exhibits a broad and high activity against Gram-negative pathogens, that can be enhanced in presence of citric and malic acid, and be used in human and veterinary medicine. PMID:26955368

  6. Propeptide of carboxypeptidase Y provides a chaperone-like function as well as inhibition of the enzymatic activity.

    PubMed Central

    Winther, J R; Sørensen, P

    1991-01-01

    The zymogen of the vacuolar carboxypeptidase Y from Saccharomyces cerevisiae was purified and characterized with respect to activation as well as refolding in vitro. The purified procarboxypeptidase Y has no detectable activity but can be efficiently activated by proteinase K from Tritirachium album. We used this method of activation as a tool for the investigation of refolding procarboxypeptidase Y in vitro. The proenzyme, denatured in 6 M guanidinium chloride, is renatured efficiently after dilution of the denaturant, whereas the mature enzyme regains little activity in the same procedure. Changes in intrinsic fluorescence reveal the mature enzyme to be considerably more stable than the proenzyme toward denaturation with guanidinium chloride. This suggests that the propeptide induces a metastable structure important for overcoming energy barriers that might otherwise obstruct a productive folding pathway. The relatively large number of charged amino acid residues and a high theoretical potential for alpha-helix formation in the carboxypeptidase Y propeptide suggest a structural similarity to a number of other propeptides and heat shock proteins. PMID:1924396

  7. Detection of antibacterial activity of an enzymatic hydrolysate generated by processing rainbow trout by-products with trout pepsin.

    PubMed

    Wald, Maleen; Schwarz, Karin; Rehbein, Hartmut; Bußmann, Bettina; Beermann, Christopher

    2016-08-15

    Trout by-product hydrolysates, generated using trout pepsin, were characterized and studied in terms of their antibacterial effects against food contaminants and fish farming pathogens. After a hydrolysis time of 25 min, the hydrolysates demonstrated inhibitory activity against several gram-positive and gram-negative bacteria. The degree of hydrolysis (DH) was found to exert a considerable influence on antibacterial activity, with a significant increase in the observed inhibitory effect at the beginning of hydrolysis. The highest antibacterial activity was obtained at a DH of 30% (enzyme/protein ratio 0.04 U/mg of protein, enzyme activity 6.5 U/mg protein, hydrolysis conditions 37°C, pH 3.0). The highest antibacterial activity detected was against the fish farming bacteria Flavobacterium psychrophilum and Renibacterium salmoninarum, with minimal inhibition concentrations of 2mg/ml and 5mg/ml, respectively. The amino acid determination of the hydrolysate (DH 30%) revealed that lysine, leucine, alanine, arginine, glycine, aspartic acid and glutamic acid residues represented the major amino acids. PMID:27006234

  8. Enzymatic Synthesis of Structured Lipids using a Novel Cold-Active Lipase from Pichia lynferdii NRRL Y-7723

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Structured lipids (SL) were synthesized by the acidolysis of borage oil with caprylic acid using lipases. Six commercial lipases from different sources and a novel lipase from Pichia lynferdii NRRL Y-7723 were screened for their acidolysis activities and Lipozyme RM IM and NRRL Y-7723 lipase were s...

  9. Heat treatment of curdlan enhances the enzymatic production of biologically active β-(1,3)-glucan oligosaccharides.

    PubMed

    Kumagai, Yuya; Okuyama, Masayuki; Kimura, Atsuo

    2016-08-01

    Biologically active β-(1,3)-glucan oligosaccharides were prepared from curdlan using GH64 enzyme (KfGH64). KfGH64 showed low activity toward native curdlan; thereby pretreatment conditions of curdlan were evaluated. KfGH64 showed the highest activity toward curdlan with heat treatment. The most efficient pretreatment (90°C for 0.5h) converted approximately 60% of curdlan into soluble saccharides under the optimized enzyme reaction conditions (pH 5.5, 37°C, 100rpm mixing speed, 24h, and 10μg of KfGH64/1g of curdlan). The resulting products were predominantly laminaripentaose and a small amount of β-(1,3)-glucans with an average degree of polymerization (DP) of 13 and 130. The products did not contain small oligosaccharides (DP<5), indicating that the hydrolysis of heat-treated curdlan by KfGH64 is a suitable method for the production of biologically active β-(1,3)-glucan oligosaccharides. PMID:27112889

  10. Influence of Tableting on Enzymatic Activity of Papain along with Determination of Its Percolation Threshold with Microcrystalline Cellulose

    PubMed Central

    Sharma, Manu; Sharma, Vinay; Majumdar, Dipak K.

    2014-01-01

    The binary mixture tablets of papain and microcrystalline cellulose (MCC), dicalcium phosphate dihydrate (DCP), carrageenan, tragacanth, and agar were prepared by direct compression. Carrageenan, tragacanth, and agar provided maximum protection to enzyme activity compared to MCC and DCP. However, stability studies indicated highest loss of enzyme activity with carrageenan, tragacanth, and agar. Therefore, compression behaviour of different binary mixtures of papain with MCC at different compaction pressures, that is, 40–280 MPa, was studied according to Heckel equation. The compressibility studies of binary mixtures indicated brittle behavior of papain. The application of percolation theory on the relationship between critical density as a function of enzyme activity and mixture composition revealed the presence of percolation threshold for binary mixture. Papain-MCC mixture composition showed significant percolation threshold at 18.48% (w/w) papain loading. Microcrystalline cellulose provided higher protection during stability study. However, higher concentrations of microcrystalline cellulose, probably as dominant particles, do not protect the enzyme with their plastic deformation. Below the percolation threshold, that is, 18.48% (w/w) papain amount in mixture with plastic excipient, activity loss increases strongly because of higher shearing forces during compaction due to system dominance of plastic particles. This mixture range should therefore be avoided to get robust formulation of papain. PMID:27350972

  11. The effect of drinking milk containing conjugated linoleic acid on fecal microbiological profile, enzymatic activity, and fecal characteristics in humans

    PubMed Central

    Farnworth, Edward R; Chouinard, Yvan P; Jacques, Helene; Venkatramanan, Sudha; Maf, Akier A; Defnoun, Sabrina; Jones, Peter JH

    2007-01-01

    Background The primary objective was to determine whether consumption of conjugated linoleic acids (CLAs) affected the fecal microbiota composition, fecal enzyme activity or fecal composition. Methods Human subjects consumed (1 L/day) cows' milk (4% fat) containing (5 mg/g fat) cis-9, trans-11 CLA (CONT), (32 mg/g fat) cis-9, trans-11 CLA (NAT) and (32 mg/g fat) trans-10, cis-12 CLA and cis-9, trans-11 CLA (SYN) for 8 weeks, in addition to their normal diet. Milk feeding periods were separated by 4 week washout periods. Fecal samples were obtained at the beginning (day 0) and the end (day 56) of each milk feeding period. Fecal samples were analysed for microbiological profile, enzyme activity, pH and short chain fatty acid content. Results Samples taken at day 0 and day 56 indicated that the numbers of lactobacilli and bifidobacteria significantly decreased after consumption of all experimental milks; total aerobes, total anaerobes, enterobacteria, and enterococci + streptococci did not change. At day 56, the activities of β-glucosidase, nitroreductase, and urease enzymes had decreased compared to samples taken on day 0 for all treatments. β-glucuronidase activity did not change. Fecal pH and ammonia content did not change. Conclusion It was concluded that observed changes could have been attributed to increased milk intake; no differences could be attributed to consumption of the different CLAs. PMID:17620127

  12. Characterization of an extracellular biofunctional alginate lyase from marine Microbulbifer sp. ALW1 and antioxidant activity of enzymatic hydrolysates.

    PubMed

    Zhu, Yanbing; Wu, Liyun; Chen, Yanhong; Ni, Hui; Xiao, Anfeng; Cai, Huinong

    2016-01-01

    A novel alginate-degrading marine bacterium Microbulbifer sp. ALW1 was isolated from rotten brown alga. An extracellular alginate lyase was purified to electrophoretic homogeneity and had a molecular mass of about 26.0 kDa determined by SDS-PAGE and size exclusion chromatography. This enzyme showed activities towards both polyguluronate and polymannuronate indicating its bifunctionality while with preference for the former substrate. Using sodium alginate as a substrate, strain ALW1 alginate lyase was optimally active at 45 °C and pH 7.0. It was stable at 25 °C, 30 °C, 35 °C and 40 °C, but not stable at 50 °C. This alginate lyase showed good stability over a broad pH range (5.0-9.0). The enzyme activity was increased to 5.1 times by adding NaCl to a final concentration of 0.5M. Strain ALW1 alginate lyase produced disaccharide (majority) and trisaccharide from alginate indicating that this enzyme could be a good tool for preparation of alginate oligosaccharides with low degree of polymerization (DP). The alginate oligosaccharides displayed the scavenging abilities towards radicals (DPPH, ABTS(+) and hydroxyl) and the reducing power. Therefore, the hydrolysates exhibited the antioxidant activity and had potential as a natural antioxidant. PMID:26686613

  13. Towards a Self-Consistent Physical Framework for Modeling Coupled Human and Physical Activities during the Anthropocene

    NASA Astrophysics Data System (ADS)

    Garrett, T. J.

    2014-12-01

    Studies of the response of global climate to anthropogenic activities rely upon scenarios for future human activity to provide a range of possible trajectories for greenhouse gases emissions over the coming century. Sophisticated integrated models are used to explore not only what will happen, but what should happen in order to optimize societal well-being. Hundreds of equations might be used to account for the interplay between human decisions, technological change, and macroeconomic priniciples. In contrast, the model equations used to describe geophysical phenomena look very different because they are a) purely deterministic and b) consistent with basic thermodynamic laws. This inconsistency between macroeconomics and physics suggests a rather unhappy marriage. During the Anthropocene the evolution of humanity and our environment will become increasingly intertwined. Representing such a coupling suggests a need for a common theoretical basis. To this end, the approach that is described here is to treat civilization like any other physical process, that is as an open, non-equilibrium thermodynamic system that dissipates energy and diffuses matter in order to sustain existing circulations and to further its material growth. Theoretical arguments and over 40 years of measurements show that a very general representation of global economic wealth (not GDP) has been tied to rates of global primary energy consumption through a constant 7.1 ± 0.1 mW per year 2005 USD. This link between physics and economics leads to very simple expressions for how fast civilization and its rate of energy consumption grow. These are expressible as a function of rates of energy and material resource discovery and depletion, and of the magnitude of externally imposed decay. The equations are validated through hindcasts that show, for example, that economic conditions in the 1950s can be invoked to make remarkably accurate forecasts of present rates of global GDP growth and primary energy

  14. Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antarctic region

    PubMed Central

    2012-01-01

    Background Antarctica has been successfully colonized by microorganisms despite presenting adverse conditions for life such as low temperatures, high solar radiation, low nutrient availability and dryness. Although these “cold-loving” microorganisms are recognized as primarily responsible for nutrient and organic matter recycling/mineralization, the yeasts, in particular, remain poorly characterized and understood. The aim of this work was to study the yeast microbiota in soil and water samples collected on King George Island. Results A high number of yeast isolates was obtained from 34 soil and 14 water samples. Molecular analyses based on rDNA sequences revealed 22 yeast species belonging to 12 genera, with Mrakia and Cryptococcus genera containing the highest species diversity. The species Sporidiobolus salmonicolor was by far the most ubiquitous, being identified in 24 isolates from 13 different samples. Most of the yeasts were psychrotolerant and ranged widely in their ability to assimilate carbon sources (consuming from 1 to 27 of the 29 carbon sources tested). All species displayed at least 1 of the 8 extracellular enzyme activities tested. Lipase, amylase and esterase activity dominated, while chitinase and xylanase were less common. Two yeasts identified as Leuconeurospora sp. and Dioszegia fristingensis displayed 6 enzyme activities. Conclusions A high diversity of yeasts was isolated in this work including undescribed species and species not previously isolated from the Antarctic region, including Wickerhamomyces anomalus, which has not been isolated from cold regions in general. The diversity of extracellular enzyme activities, and hence the variety of compounds that the yeasts may degrade or transform, suggests an important nutrient recycling role of microorganisms in this region. These yeasts are of potential use in industrial applications requiring high enzyme activities at low temperatures. PMID:23131126

  15. Data of enzymatic activities of the electron transport chain and ATP synthase complexes in mouse hepatoma cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

    PubMed

    Hwang, Hye Jin; Steidemann, Michelle; Dunivin, Taylor K; Rizzo, Mike; LaPres, John J

    2016-09-01

    2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most widely studied ligand of the aryl hydrocarbon receptor (AHR). The AHR-dependent TCDD-induced mitochondrial hyperpolarization (Tappenden et al., 2011) [1] and reduced oxygen consumption rate of intact mouse hepatoma cells (Huang et al., in press) [2] in the previous studies suggest that these alterations can be related to enzymatic activities of the electron transport chain (ETC) and ATP synthase in oxidative phosphorylation (OXPHOS) system. Here, we evaluated the activity of each complex in the OXPHOS system using in vitro enzymatic assays. The calculated enzymatic activity of each complex was normalized against the activity of citrate synthase. To combine each value from an independent experiment, normalized enzyme activities from cells exposed to TCDD were converted to fold changes via comparison to the activity relative to time-matched vehicle control. The averaged fold change for each treatment suggests more replicates are needed in order to clearly evaluate a difference between treatments. PMID:27284569

  16. Enzyme orientation for direct electron transfer in an enzymatic fuel cell with alcohol oxidase and laccase electrodes.

    PubMed

    Arrocha, Andrés A; Cano-Castillo, Ulises; Aguila, Sergio A; Vazquez-Duhalt, Rafael

    2014-11-15

    A new full enzymatic fuel cell was built and characterized. Both enzymatic electrodes were molecularly oriented to enhance the direct electron transfer between the enzyme active site and the electrode surface. The anode consisted in immobilized alcohol oxidase on functionalized carbon nanotubes with 4-azidoaniline, which acts as active-site ligand to orientate the enzyme molecule. The cathode consisted of immobilized laccase on functionalized graphite electrode with 4-(2-aminoethyl) benzoic acid. The enzymatic fuel cell reaches 0.5 V at open circuit voltage with both, ethanol and methanol, while in short circuit the highest current intensity of 250 μA cm(-2) was obtained with methanol. Concerning the power density, the methanol was the best substrate reaching 60 μW cm(-2), while with ethanol 40 μW cm(-2) was obtained. PMID:24953844

  17. Comparative study of human intestinal and hepatic esterases as related to enzymatic properties and hydrolizing activity for ester-type drugs.

    PubMed

    Inoue, M; Morikawa, M; Tsuboi, M; Ito, Y; Sugiura, M

    1980-08-01

    In attempts to determine the exact role of intestinal esterase in the body, we purified esterases from human intestinal mucosa and liver, and compared the enzymatic properties and substrate specificities with those of purified esterases. Esterase from human liver was purified 58-fold, by treatment with butanol, DE-52 and DEAE Sephadex A-50 column chromatographies, Sephadex G-200 gel filtration, and isoelectric focusing. The purified preparation showed a single band by polyacylamide gel electrophoresis. The molecular weights of intestinal and hepatic esterases were determined to be 53,000-55,000 and 180,000, respectively, by gel filtration on Sephadex G-200. The activity of the purified intestinal and hepatic esterases was strongly inhibited by diethyl-p-nitrophenyl phosphate and diisopropyl fluorophosphate, and was not inhibited by eserine sulfate and p-chloromercuribenzoate. Moreover, the purified esterases hydrolyzed ester-type drugs such as aspirin, clofibrate, indanyl carbenicillin and procaine. Hepatic esterase had properties similar to those of intestinal esterase with respect to the sensitivity to organophosphate and the substrate specificity. However, the two purified esterases differed in properties such as molecular weight, isoelectric point, thermostability and optimal pH. PMID:7206363

  18. The effects of a copper-based antifouling paint on mortality and enzymatic activity of a non-target marine organism.

    PubMed

    Katranitsas, A; Castritsi-Catharios, J; Persoone, G

    2003-11-01

    Antifouling paints are used on a wide range of underwater structures in order to protect them from the development of fouling organisms. The leaching of the toxic substances from the matrix of the paint causes toxic effects not only to the fouling organisms but also on other "non-target" biota. The present study addresses the impact of the antifouling paint Flexgard VI-II on brine shrimp nauplii selected as convenient test organisms. The surface to volume (S/V) concept developed by Persoone and Castritsi-Catharios (1989) was used to determine S/V-LC50s for the test biota exposed to PVC test panels of 400-1000 mm2 surface coated with the antifouling paint in test vessels containing 20 ml seawater. Total ATPase and Mg2+-ATPase were also analyzed for coated surface areas inducing less than 50% mortality in the brine shrimp nauplii. The calculated S/V-LC50 (24 h) was 24.6 mm2/ml, which shows the high toxic character of the antifouling paint. Decreased enzymatic activities were noted in the brine shrimp nauplii exposed to test panels of 50 and 100 mm2 in 20 ml seawater. The present study indicates that the "surface to volume" concept is an interesting methodology that can be applied with both lethal and sublethal effect criteria for the determination of toxic stress from leaches of painted surfaces. PMID:14607547

  19. A conserved arginine-containing motif crucial for the assembly and enzymatic activity of the mixed lineage leukemia protein-1 core complex.

    PubMed

    Patel, Anamika; Vought, Valarie E; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S

    2008-11-21

    The mixed lineage leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5), and the absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the molecular details of these interactions are unknown. To gain insight into the interactions among these proteins, we have determined the biophysical basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine-containing motif, called the "Win" or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5's recognition of arginine 3765 of MLL1 is essential for the assembly and enzymatic activity of the MLL1 core complex in vitro. PMID:18829457

  20. Microwave-assisted aqueous enzymatic extraction of oil from pumpkin seeds and evaluation of its physicochemical properties, fatty acid compositions and antioxidant activities.

    PubMed

    Jiao, Jiao; Li, Zhu-Gang; Gai, Qing-Yan; Li, Xiao-Juan; Wei, Fu-Yao; Fu, Yu-Jie; Ma, Wei

    2014-03-15

    Microwave-assisted aqueous enzymatic extraction (MAAEE) of pumpkin seed oil was performed in this study. An enzyme cocktail comprised of cellulase, pectinase and proteinase (w/w/w) was found to be the most effective in releasing oils. The highest oil recovery of 64.17% was achieved under optimal conditions of enzyme concentration (1.4%, w/w), temperature (44°C), time (66 min) and irradiation power (419W). Moreover, there were no significant variations in physicochemical properties of MAAEE-extracted oil (MAAEEO) and Soxhlet-extracted oil (SEO), but MAAEEO exhibited better oxidation stability. Additionally, MAAEEO had a higher content of linoleic acid (57.33%) than SEO (53.72%), and it showed stronger antioxidant activities with the IC50 values 123.93 and 152.84, mg/mL, according to DPPH radical scavenging assay and β-carotene/linoleic acid bleaching test. SEM results illustrated the destruction of cell walls and membranes by MAAEE. MAAEE is, therefore, a promising and environmental-friendly technique for oil extraction in the food industry. PMID:24206680

  1. D-2-Hydroxyglutarate producing neo-enzymatic activity inversely correlates with frequency of the type of isocitrate dehydrogenase 1 mutations found in glioma

    PubMed Central

    2014-01-01

    Background IDH mutations frequently occur in diffuse gliomas and result in a neo-enzymatic activity that results in reduction of α-ketoglutarate to D-2-hydroxyglutarate. In gliomas, the frequency of IDH1 mutations in codon 132 increases in the order R132L-R132S-R132G-R132C-R132H with R132H constituting more than 90% of all IDH1 mutations. Results We determined the levels of D-2-hydroxyglutarate in glioma tissues with IDH1 mutations. D-2-hydroxyglutarate levels increased in the order of R132H-R132C-R132S/R132G/R132L. We expressed and purified IDH1 wild type and mutant protein for biochemical characterization. Enzyme kinetics of mutant IDH protein correlated well with D-2-hydroxyglutarate production in cells with R132H exhibiting the highest and R132L the lowest KM for α-ketoglutarate. Addition of D-2-hydroxyglutarate to the medium of cell lines revealed an inhibitory effect at higher concentrations. Migration of LN229 increased at lower D-2-hydroxyglutarate concentrations while higher concentrations showed no effect. Conclusion These findings may suggest natural selection against the rare IDH1R132 mutations in human glioma due to toxicity caused by high levels of D-2-hydroxyglutarate. PMID:24529257

  2. Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay

    PubMed Central

    Prevato, Marua; Ferlenghi, Ilaria; Bonci, Alessandra; Uematsu, Yasushi; Anselmi, Giulia; Giusti, Fabiola; Bertholet, Sylvie; Legay, Francois; Telford, John Laird; Settembre, Ethan C.; Maione, Domenico; Cozzi, Roberta

    2015-01-01

    Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments. The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA). PMID:26280677

  3. Functional significance of Asn-linked glycosylation of proteinase 3 for enzymatic activity, processing, targeting, and recognition by anti-neutrophil cytoplasmic antibodies.

    PubMed

    Specks, Ulrich; Fass, David N; Finkielman, Javier D; Hummel, Amber M; Viss, Margaret A; Litwiller, Robert D; McDonald, Cari J

    2007-01-01

    Proteinase 3 (PR3) is a neutral serine protease stored in neutrophil granules. It has substantial sequence homology with elastase, cathepsin G and azurocidin. PR3 is the target antigen for autoantibodies (ANCA) in Wegener's granulomatosis, a necrotizing vasculitis syndrome. ANCA have been implicated in the pathogenesis of this disease. PR3 has two potential Asn-linked glycosylation sites. This study was designed to determine the occupancy of these glycosylation sites, and to evaluate their effect on enzymatic function, intracellular processing, targeting to granules and recognition by ANCA. We found that glycosylation occurs at both sites in native neutrophil PR3 and in wild type recombinant PR3 (rPR3) expressed in HMC-1 cells. Using glycosylation deficient rPR3 mutants we found that glycosylation at Asn-147, but not at Asn-102, is critical for thermal stability, and for optimal hydrolytic activity of PR3. Efficient amino-terminal proteolytic processing of rPR3 is dependent on glycosylation at Asn-102. Targeting to granules is not dependent on glycosylation, but unglycosylated rPR3 gets secreted preferentially into media supernatants. Finally, a capture ELISA for ANCA detection, using rPR3 glycosylation variants as target antigens, reveals that in about 20% of patients, epitope recognition by ANCA is affected by the glycosylation status of PR3. PMID:17158864

  4. Structural characterization of the novel aminoglycoside phosphotransferase AphVIII from Streptomyces rimosus with enzymatic activity modulated by phosphorylation.

    PubMed

    Boyko, Konstantin M; Gorbacheva, Marina A; Korzhenevskiy, Dmitry A; Alekseeva, Maria G; Mavletova, Dilara A; Zakharevich, Natalia V; Elizarov, Sergey M; Rudakova, Natalia N; Danilenko, Valery N; Popov, Vladimir O

    2016-09-01

    Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3'-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 Å. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop with unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding. PMID:27338640

  5. An enzymatic deconjugation method for the analysis of small molecule active drugs on antibody-drug conjugates.

    PubMed

    Li, Yi; Gu, Christine; Gruenhagen, Jason; Yehl, Peter; Chetwyn, Nik P; Medley, Colin D

    2016-01-01

    Antibody-drug conjugates (ADCs) are complex therapeutic agents that use the specific targeting properties of antibodies and the highly potent cytotoxicity of small molecule drugs to selectively eliminate tumor cells while limiting the toxicity to normal healthy tissues. Two critical quality attributes of ADCs are the purity and stability of the active small molecule drug linked to the ADC, but these are difficult to assess once the drug is conjugated to the antibody. In this study, we report a enzyme deconjugation approach to cleave small molecule drugs from ADCs, which allows the drugs to be subsequently characterized by reversed-phase high performance liquid chromatography. The model ADC we used in this study utilizes a valine-citrulline linker that is designed to be sensitive to endoproteases after internalization by tumor cells. We screened several proteases to determine the most effective enzyme. Among the 3 cysteine proteases evaluated, papain had the best efficiency in cleaving the small molecule drug from the model ADC. The deconjugation conditions were further optimized to achieve complete cleavage of the small molecule drug. This papain deconjugation approach demonstrated excellent specificity and precision. The purity and stability of the active drug on an ADC drug product was evaluated and the major degradation products of the active drug were identified. The papain deconjugation method was also applied to several other ADCs, with the results suggesting it could be applied generally to ADCs containing a valine-citrulline linker. Our results indicate that the papain deconjugation method is a powerful tool for characterizing the active small molecule drug conjugated to an ADC, and may be useful in ensuring the product quality, efficacy and the safety of ADCs. PMID:26891281

  6. Characterization of the Enzymatic Activity of SETDB1 and Its 1:1 Complex with ATF7IP.

    PubMed

    Basavapathruni, Aravind; Gureasko, Jodi; Porter Scott, Margaret; Hermans, William; Godbole, Adarsh; Leland, Peter A; Boriack-Sjodin, P Ann; Wigle, Tim J; Copeland, Robert A; Riera, Thomas V

    2016-03-22

    The protein methyltransferase (PMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing S-adenosylmethionine (SAM) as the methyl donor and its catalytic activity, has been reported to be regulated by a partner protein ATF7IP. Here, we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed and 1:1 stoichiometric complexes were purified for comparison against SETDB1 enzyme alone. We employed both radiometric flashplate-based and SAMDI mass spectrometry assays to follow methylation on histone H3 15-mer peptides, where lysine 9 was either unmodified, monomethylated, or dimethylated. Results show that SETDB1 and the SETDB1:ATF7IP complex efficiently catalyze both monomethylation and dimethylation of H3K9 peptide substrates. The activity of the binary complex was 4-fold lower than SETDB1 alone. This difference was due to a decrease in the value of kcat as the substrate KM values were comparable between SETDB1 and the SETDB1:ATF7IP complex. H3K9 methylation by SETDB1 occurred in a distributive manner, and this too was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9. Our results indicate that ATF7IP does not directly modulate SETDB1 catalytic activity, suggesting alternate roles, such as affecting cellular localization or mediating interaction with additional binding partners. PMID:26813693

  7. Synthesis and enzymatic photo-activity of an O2 tolerant hydrogenase-CdSe@CdS quantum rod bioconjugate.

    PubMed

    Hamon, C; Ciaccafava, A; Infossi, P; Puppo, R; Even-Hernandez, P; Lojou, E; Marchi, V

    2014-05-21

    This communication reports on the preparation of stable and photo-active nano-heterostructures composed of O2 tolerant [NiFe] hydrogenase extracted from the Aquifex aeolicus bacterium grafted onto hydrophilic CdSe/CdS quantum rods in view of the development of H2/O2 biofuel cells. The resulting complex is efficient towards H2 oxidation, displays good stability and new photosensitive properties. PMID:24468861

  8. Ureases display biological effects independent of enzymatic activity: is there a connection to diseases caused by urease-producing bacteria?

    PubMed

    Olivera-Severo, D; Wassermann, G E; Carlini, C R

    2006-07-01

    Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori. PMID:16862275

  9. Curcumin Blocks Naproxen-Induced Gastric Antral Ulcerations through Inhibition of Lipid Peroxidation and Activation of Enzymatic Scavengers in Rats.

    PubMed

    Kim, Jeong-Hwan; Jin, Soojung; Kwon, Hyun Ju; Kim, Byung Woo

    2016-08-28

    Curcumin is a polyphenol derived from the plant Curcuma longa, which is used for the treatment of diseases associated with oxidative stress and inflammation. The present study was undertaken to determine the protective effect of curcumin against naproxen-induced gastric antral ulcerations in rats. Different doses (10, 50, and 100 mg/kg) of curcumin or vehicle (curcumin, 0 mg/kg) were pretreated for 3 days by oral gavage, and then gastric mucosal lesions were caused by 80 mg/kg naproxen applied for 3 days. Curcumin significantly inhibited the naproxen-induced gastric antral ulcer area and lipid peroxidation in a dose-dependent manner. In addition, curcumin markedly increased activities of radical scavenging enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase in a dose-dependent manner. Specifically, 100 mg/kg curcumin completely protected the gastric mucosa against the loss in the enzyme, resulting in a drastic increase of activities of radical scavenging enzymes up to more than the level of untreated normal rats. Histological examination obviously showed that curcumin prevents naproxen-induced gastric antral ulceration as a result of direct protection of the gastric mucosa. These results suggest that curcumin blocks naproxen-induced gastric antral ulcerations through prevention of lipid peroxidation and activation of radical scavenging enzymes, and it may offer a potential remedy of gastric antral ulcerations. PMID:27197667

  10. Comparative Composition and Antioxidant Activity of Peptide Fractions Obtained by Ultrafiltration of Egg Yolk Protein Enzymatic Hydrolysates

    PubMed Central

    Chay Pak Ting, Bertrand P.; Mine, Yoshinori; Juneja, Lekh R.; Okubo, Tsutomu; Gauthier, Sylvie F.; Pouliot, Yves

    2011-01-01

    The objective of the study was to compare the antioxidant activity of two distinct hydrolysates and their peptide fractions prepared by ultrafiltration (UF) using membranes with molecular weight cut-off of 5 and 1 kDa. The hydrolysates were a delipidated egg yolk protein concentrate (EYP) intensively hydrolyzed with a combination of two bacterial proteases, and a phosphoproteins (PPP) extract partially hydrolyzed with trypsin. Antioxidant activity, as determined by the oxygen radical absorbance capacity (ORAC) assay, was low for EYP and PPP hydrolysates with values of 613.1 and 489.2 μM TE·g−1 protein, respectively. UF-fractionation of EYP hydrolysate increased slightly the antioxidant activity in permeate fractions (720.5–867.8 μM TE·g−1 protein). However, ORAC values were increased by more than 3-fold in UF-fractions prepared from PPP hydrolysate, which were enriched in peptides with molecular weight lower than 5 kDa. These UF-fractions were characterized by their lower N/P atomic ratio and higher phosphorus content compared to the same UF-fractions obtained from EYP-TH. They also contained high amounts of His, Met, Leu, and Phe, which are recognized as antioxidant amino acids, but also high content in Lys and Arg which both represent target amino acids of trypsin used for the hydrolysis of PPP. PMID:24957729

  11. Enzymatic Synthesis of l-Ascorbyl Fatty Acid Esters Under Ultrasonic Irradiation and Comparison of Their Antioxidant Activity and Stability.

    PubMed

    Jiang, Chen; Lu, Yuyun; Li, Zhuo; Li, Cunzhi; Yan, Rian

    2016-06-01

    A series of novel l-ascorbyl fatty acid esters were synthesized by catalization of Novozym(®) 435 under ultrasonic irradiation and characterized by infrared spectroscopy, electrospray ionization mass spectra, and nuclear magnetic resonance. Their properties especially antioxidant activity and stability were investigated. The results showed that the reducing power, the scavenging activity of hydroxyl radical and 2,2-diphenyl-1-picrylhydrazyl radical were decreased with the increase of the number of carbon atoms in fatty acid. The hydroxyl radical scavenging activity and reducing power of l-ascorbyl saturated fatty acid esters were better than that of tert-butylhydroquinone. The induction period in lipid oxidation of l-ascorbyl saturated fatty acid esters and tert-butylhydroquinone were longer than that of l-ascorbyl unsaturated fatty acid esters and l-ascorbic acid both in soybean oil and lard. Besides, the l-ascorbyl fatty acid esters showed different stabilities in different conditions by comparing with l-ascorbic acid, and the l-ascorbyl saturated fatty acid esters were more stable than l-ascorbyl unsaturated fatty acid esters in ethanol solution. PMID:27100741

  12. Structural Basis of Enzymatic Activity for the Ferulic Acid Decarboxylase (FADase) from Enterobacter sp. Px6-4

    PubMed Central

    Liang, Lianming; Sun, Yuna; Huang, Jingwen; Li, Xuemei; Cao, Yi; Meng, Zhaohui; Zhang, Ke-Qin

    2011-01-01

    Microbial ferulic acid decarboxylase (FADase) catalyzes the transformation of ferulic acid to 4-hydroxy-3-methoxystyrene (4-vinylguaiacol) via non-oxidative decarboxylation. Here we report the crystal structures of the Enterobacter sp. Px6-4 FADase and the enzyme in complex with substrate analogues. Our analyses revealed that FADase possessed a half-opened bottom β-barrel with the catalytic pocket located between the middle of the core β-barrel and the helical bottom. Its structure shared a high degree of similarity with members of the phenolic acid decarboxylase (PAD) superfamily. Structural analysis revealed that FADase catalyzed reactions by an “open-closed” mechanism involving a pocket of 8×8×15 Å dimension on the surface of the enzyme. The active pocket could directly contact the solvent and allow the substrate to enter when induced by substrate analogues. Site-directed mutagenesis showed that the E134A mutation decreased the enzyme activity by more than 60%, and Y21A and Y27A mutations abolished the enzyme activity completely. The combined structural and mutagenesis results suggest that during decarboxylation of ferulic acid by FADase, Trp25 and Tyr27 are required for the entering and proper orientation of the substrate while Glu134 and Asn23 participate in proton transfer. PMID:21283705

  13. Role of enzymatic activity in muscle damage and cytotoxicity induced by Bothrops asper Asp49 phospholipase A2 myotoxins: are there additional effector mechanisms involved?

    PubMed Central

    Mora-Obando, Diana; Díaz, Cecilia; Angulo, Yamileth; Gutiérrez, José María

    2014-01-01

    Viperid venoms often contain mixtures of Asp49 and Lys49 PLA2 myotoxin isoforms, relevant to development of myonecrosis. Given their difference in catalytic activity, mechanistic studies on each type require highly purified samples. Studies on Asp49 PLA2s have shown that enzyme inactivation using p-bromophenacyl bromide (p-BPB) drastically affects toxicity. However, based on the variable levels of residual toxicity observed in some studies, it has been suggested that effector mechanisms independent of catalysis may additionally be involved in the toxicity of these enzymes, possibly resembling those of the enzymatically inactive Lys49 myotoxins. A possibility that Lys49 isoforms could be present in Asp49 PLA2 preparations exists and, if undetected in previous studies, could explain the variable residual toxicity. This question is here addressed by using an enzyme preparation ascertained to be free of Lys49 myotoxins. In agreement with previous reports, inactivation of the catalytic activity of an Asp49 myotoxin preparation led to major inhibition of toxic effects in vitro and in vivo. The very low residual levels of myotoxicity (7%) and cytotoxicity (4%) observed can be attributed to the low, although detectable, enzyme remaining active after p-BPB treatment (2.7%), and would be difficult to reconcile with the proposed existence of additional catalytic-independent toxic mechanisms. These findings favor the concept that the effector mechanism of toxicity of Asp49 PLA2 myotoxins from viperids fundamentally relies on their ability to hydrolyze phospholipids, arguing against the proposal that membrane disruption may also be caused by additional mechanisms that are independent of catalysis. PMID:25276503

  14. In Vitro Evaluation of Enzymatic and Antifungal Activities of Soil-Actinomycetes Isolates and Their Molecular Identification by PCR

    PubMed Central

    Keikha, Nasser; Ayatollahi Mousavi, Seyyed Amin; Nakhaei, Ali Reza; Yadegari, Mohammad Hossein; Shahidi Bonjar, Gholam Hossein; Amiri, Somayyeh

    2015-01-01

    Background: Human cutaneous infection caused by a homogeneous group of keratinophilic fungi called dermatophytes. These fungi are the most common infectious agents in humans that are free of any population and geographic area. Microsporum canis is a cause of dermatophytosis (Tinea) in recent years in Iran and atypical strain has been isolated in Iran. Its cases occur sporadically due to M. canis transmission from puppies and cats to humans. Since this pathogenic dermatophyte is eukaryotes, chemical treatment with antifungal drugs may also affect host tissue cells. Objectives: The aim of the current study was to find a new antifungal agent of soil-Actinomycetes from Kerman province against M. canis and Actinomycete isolates were identified by PCR. Materials and Methods: A number of hundred Actinomycete isolated strains were evaluated from soil of Kerman province, for their antagonistic activity against the M. canis. M. canis of the Persian Type Culture Collection (PTCC) was obtained from the Iranian Research Organization for Science and Technology (IROST). Electron microscope studies of these isolates were performed based on the physiological properties of these antagonists including lipase, amylase, protease and chitinase activities according to the relevant protocols and were identified using gene 16SrDNA. Results: In this study the most antagonist of Actinomycete isolates with antifungal activity against M. canis isolates of L1, D5, Ks1m, Km2, Kn1, Ks8 and Ks1 were shown in vitro. Electron microscopic studies showed that some fungal strains form spores, mycelia and spore chain. Nucleotide analysis showed that Ks8 had maximum homology (98%) to Streptomyces zaomyceticus strain xsd08149 and L1 displayed 100% homology to Streptomyces sp. HVG6 using 16SrDNA studies. Conclusions: Our findings showed that Streptomyces has antifungal effects against M. canis. PMID:26060560

  15. Synthesis of enzymatically active human alpha-L-iduronidase in Arabidopsis cgl (complex glycan-deficient) seeds.

    PubMed

    Downing, Willa L; Galpin, Jason D; Clemens, Sabine; Lauzon, Shauna M; Samuels, A Lacey; Pidkowich, Mark S; Clarke, Lorne A; Kermode, Allison R

    2006-03-01

    As an initial step to develop plants as systems to produce enzymes for the treatment of lysosomal storage disorders, Arabidopsis thaliana wild-type (Col-0) plants were transformed with a construct to express human alpha-l-iduronidase (IDUA; EC 3.2.1.76) in seeds using the promoter and other regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene. IDUA protein was easily detected on Western blots of extracts from the T(2) seeds, and extracts contained IDUA activity as high as 2.9 nmol 4-methylumbelliferone (4 MU)/min/mg total soluble protein (TSP), corresponding to approximately 0.06 microg IDUA/mg TSP. The purified protein reacted with an antibody specific for xylose-containing plant complex glycans, indicating its transit through the Golgi complex. In an attempt to avoid maturation of the N-linked glycans of IDUA, the same IDUA transgene was introduced into the Arabidopsis cgl background, which is deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of complex glycan biosynthesis. IDUA activity and protein levels were significantly higher in transgenic cgl vs. wild-type seeds (e.g. maximum levels were 820 nmol 4 MU/min/mg TSP, or 18 microg IDUA/mg TSP). Affinity-purified IDUA derived from cgl mutant seeds showed a markedly reduced reaction with the antibody specific for plant complex glycans, despite transit of the protein to the apoplast. Furthermore, gel mobility changes indicated that a greater proportion of its N-linked glycans were susceptible to digestion by Streptomyces endoglycosidase H, as compared to IDUA derived from seeds of wild-type Arabidopsis plants. The combined results indicate that IDUA produced in cgl mutant seeds contains glycans primarily in the high-mannose form. This work clearly supports the viability of using plants for the production of human therapeutics with high-mannose glycans. PMID:17177794

  16. Exposure to phenanthrene and depuration: Changes on gene transcription, enzymatic activity and lipid peroxidation in gill of scallops Nodipecten nodosus.

    PubMed

    Piazza, Rômi S; Trevisan, Rafael; Flores-Nunes, Fabrício; Toledo-Silva, Guilherme; Wendt, Nestor; Mattos, Jacó J; Lima, Daína; Taniguchi, Satie; Sasaki, Silvio Tarou; Mello, Álvaro C P; Zacchi, Flávia L; Serrano, Miguel A S; Gomes, Carlos H A M; Bícego, Márcia C; Almeida, Eduardo A de; Bainy, Afonso C D

    2016-08-01

    Understanding the mechanism of phenanthrene (PHE) biotransformation and related cellular responses in bivalves can be an important tool to elucidate the risks of polycyclic aromatic hydrocarbons (PAHs) to aquatic organisms. In the present study it was analyzed the transcriptional l