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Sample records for enzyme activity responses

  1. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  2. Magnetically responsive enzyme powders

    NASA Astrophysics Data System (ADS)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-04-01

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (-20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties.

  3. Boronic Acid-Appended Molecular Glues for ATP-Responsive Activity Modulation of Enzymes.

    PubMed

    Okuro, Kou; Sasaki, Mizuki; Aida, Takuzo

    2016-05-01

    Water-soluble linear polymers GumBAn (m/n = 18/6, 12/12, and 6/18) with multiple guanidinium ion (Gu(+)) and boronic acid (BA) pendants in their side chains were synthesized as ATP-responsive modulators for enzyme activity. GumBAn polymers strongly bind to the phosphate ion (PO4(-)) and 1,2-diol units of ATP via the Gu(+) and BA pendants, respectively. As only the Gu(+) pendants can be used for proteins, GumBAn is able to modulate the activity of enzymes in response to ATP. As a proof-of-concept study, we demonstrated that trypsin (Trp) can be deactivated by hybridization with GumBAn. However, upon addition of ATP, Trp was liberated to retrieve its hydrolytic activity due to a higher preference of GumBAn toward ATP than Trp. This event occurred in a much lower range of [ATP] than reported examples. Under cellular conditions, the hydrolytic activity of Trp was likewise modulated. PMID:27087468

  4. Response of oxidative enzyme activities to nitrogen deposition affects soil concentrations of dissolved organic carbon

    USGS Publications Warehouse

    Waldrop, M.P.; Zak, D.R.

    2006-01-01

    Recent evidence suggests that atmospheric nitrate (NO3- ) deposition can alter soil carbon (C) storage by directly affecting the activity of lignin-degrading soil fungi. In a laboratory experiment, we studied the direct influence of increasing soil NO 3- concentration on microbial C cycling in three different ecosystems: black oak-white oak (BOWO), sugar maple-red oak (SMRO), and sugar maple-basswood (SMBW). These ecosystems span a broad range of litter biochemistry and recalcitrance; the BOWO ecosystem contains the highest litter lignin content, SMRO had intermediate lignin content, and SMBW leaf litter has the lowest lignin content. We hypothesized that increasing soil solution NO 3- would reduce lignolytic activity in the BOWO ecosystem, due to a high abundance of white-rot fungi and lignin-rich leaf litter. Due to the low lignin content of litter in the SMBW, we further reasoned that the NO3- repression of lignolytic activity would be less dramatic due to a lower relative abundance of white-rot basidiomycetes; the response in the SMRO ecosystem should be intermediate. We increased soil solution NO3- concentrations in a 73-day laboratory incubation and measured microbial respiration and soil solution dissolved organic carbon (DOC) and phenolics concentrations. At the end of the incubation, we measured the activity of ??-glucosidase, N-acetyl-glucosaminidase, phenol oxidase, and peroxidase, which are extracellular enzymes involved with cellulose and lignin degradation. We quantified the fungal biomass, and we also used fungal ribosomal intergenic spacer analysis (RISA) to gain insight into fungal community composition. In the BOWO ecosystem, increasing NO 3- significantly decreased oxidative enzyme activities (-30% to -54%) and increased DOC (+32% upper limit) and phenolic (+77% upper limit) concentrations. In the SMRO ecosystem, we observed a significant decrease in phenol oxidase activity (-73% lower limit) and an increase in soluble phenolic concentrations

  5. Antioxidant enzymes activities of Burkholderia spp. strains-oxidative responses to Ni toxicity.

    PubMed

    Dourado, M N; Franco, M R; Peters, L P; Martins, P F; Souza, L A; Piotto, F A; Azevedo, R A

    2015-12-01

    Increased agriculture production associated with intense application of herbicides, pesticides, and fungicides leads to soil contamination worldwide. Nickel (Ni), due to its high mobility in soils and groundwater, constitutes one of the greatest problems in terms of environmental pollution. Metals, including Ni, in high concentrations are toxic to cells by imposing a condition of oxidative stress due to the induction of reactive oxygen species (ROS), which damage lipids, proteins, and DNA. This study aimed to characterize the Ni antioxidant response of two tolerant Burkholderia strains (one isolated from noncontaminated soil, SNMS32, and the other from contaminated soil, SCMS54), by measuring superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione S-transferase (GST) activities. Ni accumulation and bacterial growth in the presence of the metal were also analyzed. The results showed that both strains exhibited different trends of Ni accumulation and distinct antioxidant enzymes responses. The strain from contaminated soil (SCMS54) exhibited a higher Ni biosorption and exhibited an increase in SOD and GST activities after 5 and 12 h of Ni exposure. The analysis of SOD, CAT, and GR by nondenaturing PAGE revealed the appearance of an extra isoenzyme in strain SCMS54 for each enzyme. The results suggest that the strain SCMS54 isolated from contaminated soil present more plasticity with potential to be used in soil and water bioremediation. PMID:26289332

  6. Response of Nodularia spumigena to pCO2 - Part 2: Exudation and extracellular enzyme activities

    NASA Astrophysics Data System (ADS)

    Endres, S.; Unger, J.; Wannicke, N.; Nausch, M.; Voss, M.; Engel, A.

    2012-04-01

    The filamentous and diazotrophic cyanobacterium Nodularia spumigena plays a major role in the productivity of the Baltic Sea as it forms extensive blooms regularly. Under phosphorus limiting conditions Nodularia spumigena has a high enzyme affinity for dissolved organic phosphorus (DOP) by production and release of alkaline phosphatase. Additionally, it is able to degrade proteinaceous compounds by expressing the extracellular enzyme leucine aminopeptidase. As atmospheric CO2 concentrations are increasing, we expect marine phytoplankton to experience changes in several environmental parameters including pH, temperature, and nutrient availability. The aim of this study was to investigate the combined effect of CO2-induced changes in seawater carbonate chemistry and of phosphate deficiency on the exudation of organic matter, and its subsequent recycling by extracellular enzymes in a Nodularia spumigena culture. Batch cultures of Nodularia spumigena were grown for 15 days aerated with three different pCO2 levels corresponding to values from glacial periods to future values projected for the year 2100. Extracellular enzyme activities as well as changes in organic and inorganic compound concentrations were monitored. CO2 treatment-related effects were identified for cyanobacterial growth, which in turn was influencing exudation and recycling of organic matter by extracellular enzymes. Biomass production was increased by 56.5% and 90.7% in the medium and high pCO2 treatment, respectively, compared to the low pCO2 treatment and simultaneously increasing exudation. During the growth phase significantly more mucinous substances accumulated in the high pCO2 treatment reaching 363 μg Gum Xanthan eq l-1 compared to 269 μg Gum Xanthan eq l-1 in the low pCO2 treatment. However, cell-specific rates did not change. After phosphate depletion, the acquisition of P from DOP by alkaline phosphatase was significantly enhanced. Alkaline phosphatase activities were increased by factor

  7. Tyrosinase inhibitory activity of cucumber compounds: enzymes responsible for browning in cucumber.

    PubMed

    Gandía-Herrero, Fernando; Jiménez, Mercedes; Cabanes, Juana; García-Carmona, Francisco; Escribano, Josefa

    2003-12-17

    The inhibition of mushroom tyrosinase by cucumber extracts was evaluated. The inhibitory effect was measured by both polarographic and spectrophotometric methods. The commercial aldehyde, trans,cis-2,6-nonadienal, described as a major volatile compound of cucumber, was characterized as a noncompetitive inhibitor against 4-tert-butylcatechol oxidation by mushroom tyrosinase. The K(I) obtained was 3.4 mM. Polyphenol oxidase (PPO) activity was not detected in cucumber skin extracts. However, the presence of PPO was revealed by Western blot; a single band was found with a M(r) of 53 kDa. These results support the assumption that the enzyme PPO is present in the cucumber skin, but its activity is inhibited. Peroxidase (PO) was also found in cucumber skin extracts. This enzyme was detected in the soluble fraction but not in the membrane fraction. The kinetic characterization of PO was carried out. Native isoelectric focusing revealed several acidic PO isoenzymes with a pI in the range between 5 and 6, a basic isoenzyme, and one principal neutral isoenzyme of pI = 7.2. PMID:14664542

  8. Changes in the activities of starch metabolism enzymes in rice grains in response to elevated CO2 concentration.

    PubMed

    Xie, Li-Yong; Lin, Er-Da; Zhao, Hong-Liang; Feng, Yong-Xiang

    2016-05-01

    The global atmospheric CO(2) concentration is currently (2012) 393.1 μmol mol(-1), an increase of approximately 42 % over pre-industrial levels. In order to understand the responses of metabolic enzymes to elevated CO(2) concentrations, an experiment was conducted using the Free Air CO(2) Enrichment (FACE )system. Two conventional japonica rice varieties (Oryza sativa L. ssp. japonica) grown in North China, Songjing 9 and Daohuaxiang 2, were used in this study. The activities of ADPG pyrophosphorylase, soluble and granule-bound starch synthases, and soluble and granule-bound starch branching enzymes were measured in rice grains, and the effects of elevated CO(2) on the amylose and protein contents of the grains were analyzed. The results showed that elevated CO(2) levels significantly increased the activity of ADPG pyrophosphorylase at day 8, 24, and 40 after flower, with maximum increases of 56.67 % for Songjing 9 and 21.31 % for Daohuaxiang 2. Similarly, the activities of starch synthesis enzymes increased significantly from the day 24 after flower to the day 40 after flower, with maximum increases of 36.81 % for Songjing 9 and 66.67 % for Daohuaxiang 2 in soluble starch synthase (SSS), and 25.00 % for Songjing 9 and 36.44 % for Daohuaxiang 2 in granule-bound starch synthase (GBSS), respectively. The elevated CO(2) concentration significantly increased the activity of soluble starch branching enzyme (SSBE) at day 16, 32, and 40 after flower, and also significantly increased the activity of granule-bound starch branching enzyme (GBSBE) at day 8, 32, and 40 after flower. The elevated CO(2) concentration increased the peak values of enzyme activity, and the timing of the activity peaks for SSS and GBSBE were earlier in Songjing 9 than in Daohuaxiang 2. There were obvious differences in developmental stages between the two varieties of rice, which indicated that the elevated CO(2) concentration increased enzyme activity expression and starch synthesis, affecting the

  9. Changes in the activities of starch metabolism enzymes in rice grains in response to elevated CO2 concentration

    NASA Astrophysics Data System (ADS)

    Xie, Li-Yong; Lin, Er-Da; Zhao, Hong-Liang; Feng, Yong-Xiang

    2016-05-01

    The global atmospheric CO2 concentration is currently (2012) 393.1 μmol mol-1, an increase of approximately 42 % over pre-industrial levels. In order to understand the responses of metabolic enzymes to elevated CO2 concentrations, an experiment was conducted using the Free Air CO2 Enrichment (FACE )system. Two conventional japonica rice varieties ( Oryza sativa L. ssp. japonica) grown in North China, Songjing 9 and Daohuaxiang 2, were used in this study. The activities of ADPG pyrophosphorylase, soluble and granule-bound starch synthases, and soluble and granule-bound starch branching enzymes were measured in rice grains, and the effects of elevated CO2 on the amylose and protein contents of the grains were analyzed. The results showed that elevated CO2 levels significantly increased the activity of ADPG pyrophosphorylase at day 8, 24, and 40 after flower, with maximum increases of 56.67 % for Songjing 9 and 21.31 % for Daohuaxiang 2. Similarly, the activities of starch synthesis enzymes increased significantly from the day 24 after flower to the day 40 after flower, with maximum increases of 36.81 % for Songjing 9 and 66.67 % for Daohuaxiang 2 in soluble starch synthase (SSS), and 25.00 % for Songjing 9 and 36.44 % for Daohuaxiang 2 in granule-bound starch synthase (GBSS), respectively. The elevated CO2 concentration significantly increased the activity of soluble starch branching enzyme (SSBE) at day 16, 32, and 40 after flower, and also significantly increased the activity of granule-bound starch branching enzyme (GBSBE) at day 8, 32, and 40 after flower. The elevated CO2 concentration increased the peak values of enzyme activity, and the timing of the activity peaks for SSS and GBSBE were earlier in Songjing 9 than in Daohuaxiang 2. There were obvious differences in developmental stages between the two varieties of rice, which indicated that the elevated CO2 concentration increased enzyme activity expression and starch synthesis, affecting the final contents

  10. Photoperiodism and Enzyme Activity

    PubMed Central

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  11. Responses of soil microbial communities and enzyme activities to nitrogen and phosphorus additions in Chinese fir plantations of subtropical China

    NASA Astrophysics Data System (ADS)

    Dong, W. Y.; Zhang, X. Y.; Liu, X. Y.; Fu, X. L.; Chen, F. S.; Wang, H. M.; Sun, X. M.; Wen, X. F.

    2015-07-01

    Nitrogen (N) and phosphorus (P) additions to forest ecosystems are known to influence various above-ground properties, such as plant productivity and composition, and below-ground properties, such as soil nutrient cycling. However, our understanding of how soil microbial communities and their functions respond to nutrient additions in subtropical plantations is still not complete. In this study, we added N and P to Chinese fir plantations in subtropical China to examine how nutrient additions influenced soil microbial community composition and enzyme activities. The results showed that most soil microbial properties were responsive to N and/or P additions, but responses often varied depending on the nutrient added and the quantity added. For instance, there were more than 30 % greater increases in the activities of β-Glucosidase (βG) and N-acetyl-β-D-glucosaminidase (NAG) in the treatments that received nutrient additions compared to the control plot, whereas acid phosphatase (aP) activity was always higher (57 and 71 %, respectively) in the P treatment. N and P additions greatly enhanced the PLFA abundanceespecially in the N2P treatment, the bacterial PLFAs (bacPLFAs), fungal PLFAs (funPLFAs) and actinomycic PLFAs (actPLFAs) were about 2.5, 3 and 4 times higher, respectively, than in the CK. Soil enzyme activities were noticeably higher in November than in July, mainly due to seasonal differences in soil moisture content (SMC). βG or NAG activities were significantly and positively correlated with microbial PLFAs. There were also significant relationships between gram-positive (G+) bacteria and all three soil enzymes. These findings indicate that G+ bacteria is the most important microbial community in C, N, and P transformations in Chinese fir plantations, and that βG and NAG would be useful tools for assessing the biogeochemical transformation and metabolic activity of soil microbes. We recommend combined additions of N and P fertilizer to promote soil

  12. Effects of Pseudoalteromonas sp. BC228 on digestive enzyme activity and immune response of juvenile sea cucumber ( Apostichopus japonicus)

    NASA Astrophysics Data System (ADS)

    Ma, Yuexin; Sun, Feixue; Zhang, Congyao; Bao, Pengyun; Cao, Shuqing; Zhang, Meiyan

    2014-12-01

    A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus japonicus. Sea cucumber individuals were fed with the diets containing 0 (control), 105, 107 and 109 CFU g-1 diet of BC228 for 45 days. Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107 and 109 CFU g-1 diet of BC228 in comparison with control ( P < 0.01). The phagocytic activity in the coelomocytes of sea cucumber fed the diet supplemented with 107 CFU g-1 diet of BC228 was significantly higher than that of those fed control diet ( P < 0.05). In addition, 105 and 107 CFU g-1 diet of BC228 significantly enhanced lysozyme and phenoloxidase activities in the coelomic fluid of sea cucumber, respectively, in comparison with other diets ( P < 0.01). Sea cucumbers, 10 each diet, were challenged with Vibrio splendidus NB13 after 45 days of feeding. It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet. Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber, stimulated its immune response and enhanced its resistance to the infection of V. splendidus.

  13. Response of hydrolytic enzyme activities and nitrogen mineralization to fertilizer and organic matter application in subtropical paddy soils

    NASA Astrophysics Data System (ADS)

    Kader, Mohammed Abdul; Yeasmin, Sabina; Akter, Masuda; Sleutel, Steven

    2016-04-01

    Driving controllers of nitrogen (N) mineralization in paddy soils, especially under anaerobic soil conditions, remain elusive. The influence of exogenous organic matter (OM) and fertilizer application on the activities of five relevant enzymes (β-glucosaminidase, β-glucosidase, L-glutaminase, urease and arylamidase) was measured in two long-term field experiments. One 18-years field experiment was established on a weathered terrace soil with a rice-wheat crop rotation at the Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU) having five OM treatments combined with two mineral N fertilizer levels. Another 30-years experiment was established on a young floodplain soil with rice-rice crop rotation at the Bangladesh Agricultural University (BAU) having eight mineral fertilizer treatments combined with organic manure. At BSMRAU, N fertilizer and OM amendments significantly increased all enzyme activities, suggesting them to be primarily determined by substrate availability. At BAU, non-responsiveness of β-glucosidase activity suggested little effect of the studied fertilizer and OM amendments on general soil microbial activity. Notwithstanding probably equal microbial demand for N, β-glucosaminidase and L-glutaminase activities differed significantly among the treatments (P>0.05) and followed strikingly opposite trends and correlations with soil organic N mineralization. So enzymatic pathways to acquire N differed by treatment at BAU, indicating differences in soil N quality and bio-availability. L-glutaminase activity was significantly positively correlated to the aerobic and anaerobic N mineralization rates at both field experiments. Combined with negative correlations between β-glucosaminidase activity and N mineralization rates, it appears that terminal amino acid NH2 hydrolysis was a rate-limiting step for soil N mineralization at BAU. Future investigations with joint quantification of polyphenol accumulation and binding of N, alongside an

  14. Responses of soil microbial communities and enzyme activities to nitrogen and phosphorus additions in Chinese fir plantations of subtropical China

    NASA Astrophysics Data System (ADS)

    Dong, W. Y.; Zhang, X. Y.; Liu, X. Y.; Fu, X. L.; Chen, F. S.; Wang, H. M.; Sun, X. M.; Wen, X. F.

    2015-09-01

    Nitrogen (N) and phosphorus (P) additions to forest ecosystems are known to influence various above-ground properties, such as plant productivity and composition, and below-ground properties, such as soil nutrient cycling. However, our understanding of how soil microbial communities and their functions respond to nutrient additions in subtropical plantations is still not complete. In this study, we added N and P to Chinese fir plantations in subtropical China to examine how nutrient additions influenced soil microbial community composition and enzyme activities. The results showed that most soil microbial properties were responsive to N and/or P additions, but responses often varied depending on the nutrient added and the quantity added. For instance, there were more than 30 % greater increases in the activities of β-glucosidase (βG) and N-acetyl-β-D-glucosaminidase (NAG) in the treatments that received nutrient additions compared to the control plot, whereas acid phosphatase (aP) activity was always higher (57 and 71 %, respectively) in the P treatment. N and P additions greatly enhanced the phospholipid fatty acids (PLFAs) abundance especially in the N2P (100 kg ha-1 yr-1 of N +50 kg ha-1 yr-1 of P) treatment; the bacterial PLFAs (bacPLFAs), fungal PLFAs (funPLFAs) and actinomycic PLFAs (actPLFAs) were about 2.5, 3 and 4 times higher, respectively, than in the CK (control). Soil enzyme activities were noticeably higher in November than in July, mainly due to seasonal differences in soil moisture content (SMC). βG or NAG activities were significantly and positively correlated with microbial PLFAs. These findings indicate that βG and NAG would be useful tools for assessing the biogeochemical transformation and metabolic activity of soil microbes. We recommend combined additions of N and P fertilizer to promote soil fertility and microbial activity in this kind of plantation.

  15. Adsorption of enzymes to stimuli-responsive polymer brushes: Influence of brush conformation on adsorbed amount and biocatalytic activity.

    PubMed

    Koenig, Meike; Bittrich, Eva; König, Ulla; Rajeev, Bhadra Lakshmi; Müller, Martin; Eichhorn, Klaus-Jochen; Thomas, Sabu; Stamm, Manfred; Uhlmann, Petra

    2016-10-01

    Polyelectrolyte brushes can be utilized to immobilize enzymes on macroscopic surfaces. This report investigates the influence of the pH value of the surrounding medium on the amount and the activity of enzymes adsorbed to poly(2-vinylpyridine) and poly(acrylic acid) brushes, as well as the creation of thermoresponsive biocatalytically active coatings via the adsorption of enzymes onto a mixed brush consisting of a polyelectrolyte and temperature-sensitive poly(N-isopropylacryl amide). Spectroscopic ellipsometry and attenuated total reflection-Fourier transform infrared spectroscopy are used to monitor the adsorption process. Additionally, infrared spectra are evaluated in terms of the secondary structure of the enzymes. Glucose oxidase is used as a model enzyme, where the enzymatic activity is measured after different adsorption conditions. Poly(acrylic acid) brushes generally adsorb larger amounts of enzyme, while less glucose oxidase is found on poly(2-vinylpyridine), which however exhibits higher specific activity. This difference in activity could be attributed to a difference in secondary structure of the adsorbed enzyme. For glucose oxidase adsorbed to mixed brushes, switching of enzymatic activity between an active state at 20°C and a less active state at 40°C as compared to the free enzyme in solution is observed. However, this switching is strongly depending on pH in mixed brushes of poly(acrylic acid) and poly(N-isopropylacryl amide) due to interactions between the polymers. PMID:27447452

  16. Active changes of lignification-related enzymes in pepper response to Glomus intraradices and/or Phytophthora capsici *

    PubMed Central

    Zheng, Hu-zhe; Cui, Chun-lan; Zhang, Yu-ting; Wang, Dan; Jing, Yu; Kim, Kil Yong

    2005-01-01

    The activities of enzymes responsible for lignification in pepper, pre-inoculation with arbuscular mycorrhizal (AM) fungus of Glomus intraradices and/or infection with pathogenic strain of Phytophthora capsici, and the biological control effect of G. intraradices on Phytophthora blight in pepper were investigated. The experiment was carried out with four treatments: (1) plants pre-inoculated with G. intraradices (Gi), (2) plants pre-inoculated with G. intraradices and then infected with P. capsici (Gi+Pc), (3) plants infected with P. capsici (Pc), and (4) plants without any of the two microorganisms (C). Mycorrhizal colonization rate was reduced by about 10% in pathogen challenged plants. Root mortality caused by infection of P. capsici was completely eliminated by pre-inoculation with antagonistic G. intraradices. On the ninth day after pathogen infection, Peroxidase (POD) activity increased by 116.9% in Pc-treated roots but by only 21.2% in Gi+Pc-treated roots, compared with the control, respectively. Polyphenol oxidase (PPO) and Phenylalanine ammonia-lyase (PAL) activities gradually increased during the first 3 d and dramatically decreased in Pc-treated roots but slightly decreased in Gi+Pc-treated roots, respectively. On the ninth day after pathogen infection, PPO and PAL decreased by 62.8% and 73.9% in Pc-treated roots but by only 19.8% and 19.5% in Gi+Pc-treated roots, compared with the control, respectively. Three major POD isozymes (45 000, 53 000 and 114 000) were present in Pc-treated roots, while two major bands (53 000 and 114 000) and one minor band (45 000) were present in spectra of Gi+Pc-treated roots, the 45 000 POD isozyme was significantly suppressed by G. intraradices, suggesting that the 45 000 POD isozyme was induced by the pathogen infection but not induced by the antagonistic G. intraradices. A 60 000 PPO isozyme was induced in Pc-treated roots but not induced in Gi+Pc-treated roots. All these results showed the inoculation of antagonistic G

  17. Enzyme activity determination using ultrasound

    NASA Astrophysics Data System (ADS)

    Holmes, M. J.; Southworth, T.; Watson, N. G.; Povey, M. J. W.

    2014-04-01

    Here are presented the results of a novel approach to the measurement of enzyme reaction rates in which ultrasound velocity measurement is used. Our results show enzyme activity is observable, in the acoustic context, and that furthermore this offers the potential to estimate the rate of reaction over different substrate concentrations and temperatures. Findings are corroborated with optical microscopy and rheological measurements. Ultrasound velocity measurement can be performed without the need for aliquot extraction and offers an efficient, non-invasive and dynamic method to monitor enzyme activity.

  18. Responses of absolute and specific soil enzyme activities to long term additions of organic and mineral fertilizer.

    PubMed

    Zhang, Xinyu; Dong, Wenyi; Dai, Xiaoqin; Schaeffer, Sean; Yang, Fengting; Radosevich, Mark; Xu, Lili; Liu, Xiyu; Sun, Xiaomin

    2015-12-01

    Long-term phosphorus (P) and nitrogen (N) applications may seriously affect soil microbial activity. A long-term field fertilizer application trial was established on reddish paddy soils in the subtropical region of southern China in 1998. We assessed the effects of swine manure and seven different rates or ratios of NPK fertilizer treatments on (1) the absolute and specific enzyme activities per unit of soil organic carbon (SOC) or microbial biomass carbon (MBC) involved in C, N, and P transformations and (2) their relationships with soil environmental factors and soil microbial community structures. The results showed that manure applications led to increases in the absolute and specific activities of soil β-1,4-glucosidase(βG), β-1,4-N-acetylglucosaminidase (NAG), and leucine aminopeptidase (LAP). The absolute and specific acid phosphatase (AP) activities decreased as mineral P fertilizer application rates and ratios increased. Redundancy analysis (RDA) showed that there were negative correlations between absolute and specific AP activities, pH, and total P contents, while there were positive correlations between soil absolute and specific βG, NAG, and LAP enzyme activities, and SOC and total N contents. RDA showed that the contents of actinomycete and Gram-positive bacterium PLFA biomarkers are more closely related to the absolute and specific enzyme activities than the other PLFA biomarkers (P<0.01). Our results suggest that both the absolute and specific enzyme activities could be used as sensitive soil quality indicators that provide useful linkages with the microbial community structures and environmental factors. To maintain microbial activity and to minimize environmental impacts, P should be applied as a combination of inorganic and organic forms, and total P fertilizer application rates to subtropical paddy soils should not exceed 44 kg P ha(-1) year(-1). PMID:26196069

  19. The temperature response of fungal enzyme kinetics

    NASA Astrophysics Data System (ADS)

    Curran, M.; Lu, Y.; Taylor, J.; Allison, S. D.

    2013-12-01

    Extracellular enzymes produced and excreted by microbes mediate the decomposition of carbon (C), nitrogen (N), and phosphorus (P) -containing compounds in their environment. Climate change has the potential to alter the rate of decomposition especially in high latitude regions where stocks of recalcitrant, or long-lived, C are abundant. This project compares extracellular enzyme activity (EEA) across ten fungi strains within the model family Neurospora in order to assess the range of variation in temperature sensitivities of fungal enzyme Vmax and Km. Vmax values of most enzymes tested increased exponentially,which was hypothesized and consistant with thermodynamic principles. We also hypothesized that Neurospora strains would exhibit different EEA temperature sensitivities based on their native climate. We observed strain-dependent variation in enzyme temperature responses consistent with strain-specific adaptation to local conditions. Since fungi are the major decomposers of organic carbon in high-latitude ecosystems, an increase in EEA in-situ would result in higher carbon dioxide emissions. These findings suggest a shift in fungal processing of soil organic carbon and nutrients in response to changing climate.

  20. Toxic responses and antioxidative enzymes activity of Scenedesmus obliquus exposed to fenhexamid and atrazine, alone and in mixture.

    PubMed

    Mofeed, Jelan; Mosleh, Yahia Y

    2013-09-01

    Laboratory studies were conducted to determine the effects of different concentrations of fenhexamid and atrazine (25, 50 and 100 µg L(-1)) on growth and oxidative stress on Scenedesmus obliquus (microalgae) after exposure for 24, 48, and 96 h. In addition, residues of fenhexamid and atrazine were determined in the culture medium after 96 h; 52%, 44% and 43% of fenhexamid remained in the medium for the lowest, middle and highest concentrations, respectively. Atrazine concentration decreased significantly in the medium with time. The reduction was faster with the lowest concentration (-53%), than in the highest concentration (-46%), while it was intermediate with 50 µg L(-1) (-47%). The antioxidative enzyme activities were used as biomarkers to evaluate the toxic effects of fenhexamid and atrazine on the microalgae. Enzymatic activities were measured in the presence of each compound alone after 24, 48 and 96 h and also in mixture after 24h exposure. The results showed that fenhexamid and atrazine induced antioxidative enzyme activities (GST, CAT and GR) at different concentrations. Catalase activities (CAT) in both pesticides treated-algae were significantly increased. Additionally, an increase in gulathione-S-transferase (GST) was observed in algae after 24, 48 and 96 h of exposure to both fenhexamid and atrazine. Antioxidative enzymes in fenhexamid and atrazine mixture treatment showed an antagonistic interaction after 24h of exposure in algae. PMID:23796667

  1. Mutagenesis of the novel Hericium erinaceus ribonuclease, RNase He1, reveals critical responsible residues for enzyme stability and activity.

    PubMed

    Kobayashi, Hiroko; Motoyoshi, Naomi; Itagaki, Tadashi; Inokuchi, Norio

    2014-01-01

    Here, we determined the sequence of a cDNA encoding a guanylic acid-specific ribonuclease (RNase He1) from Hericium erinaceus that exhibits high sequence identity (59%) with RNase Po1, an enzyme with anti-cancer activity and which is found in Pleurotus ostreatus. RNase He1 and RNase Po1 have similar structures and heat stabilities; hence, RNase He1 may also have potential as an anti-cancer agent. Therefore, we initiated structure-function studies to further characterize the enzyme. Based on the RNase Po1 structure, RNase He1 is predicted to form 3 disulfide bonds involving Cys7-Cys98, Cys5-Cys83, and Cys47-Cys81 linkages. The Cys5Ala mutant exhibited no RNase activity, whereas the Cys81Ala mutant retained RNase activity, but had reduced heat stability. Therefore, the Cys5-Cys83 bond in RNase He1 is essential for the structure of the RNase active site region. Similarly, the Cys47-Cys81 bond helps maintain the conformational stability of the active site region, and may contribute to the greater heat stability of RNase He1. PMID:25366489

  2. Enzyme-responsive nanomaterials for controlled drug delivery

    NASA Astrophysics Data System (ADS)

    Hu, Quanyin; Katti, Prateek S.; Gu, Zhen

    2014-10-01

    Enzymes underpin physiological function and exhibit dysregulation in many disease-associated microenvironments and aberrant cell processes. Exploiting altered enzyme activity and expression for diagnostics, drug targeting, and drug release is tremendously promising. When combined with booming research in nanobiotechnology, enzyme-responsive nanomaterials used for controlled drug release have achieved significant development and have been studied as an important class of drug delivery strategies in nanomedicine. In this review, we describe enzymes such as proteases, phospholipases and oxidoreductases that serve as delivery triggers. Subsequently, we explore recently developed enzyme-responsive nanomaterials with versatile applications for extracellular and intracellular drug delivery. We conclude by discussing future opportunities and challenges in this area.

  3. Enzyme-Responsive Nanomaterials for Controlled Drug Delivery

    PubMed Central

    Hu, Quanyin; Katti, Prateek S.; Gu, Zhen

    2015-01-01

    Enzymes underpin physiological function and exhibit dysregulation in many disease-associated microenvironments and aberrant cell processes. Exploiting altered enzyme activity and expression for diagnostics, drug targeting, and drug release is tremendously promising. When combined with booming research in nanobiotechnology, enzyme-responsive nanomaterials for controlled drug release have achieved significant development and been studied as an important class of drug delivery devices in nanomedicine. In this review, we describe enzymes such as proteases, phospholipase and oxidoreductases that serve as delivery triggers. Subsequently, we explore recently developed enzyme-responsive nanomaterials with versatile applications for extracellular and intracellular drug delivery. We conclude by discussing future opportunities and challenges in this area. PMID:25251024

  4. Activity and Transcriptional Responses of Hepatopancreatic Biotransformation and Antioxidant Enzymes in the Oriental River Prawn Macrobrachium nipponense Exposed to Microcystin-LR.

    PubMed

    Yuan, Julin; Wang, Xueqin; Gu, Zhiming; Zhang, Yingying; Wang, Zaizhao

    2015-10-01

    Microcystins (MCs) are a major group of cyanotoxins with side effects in many organisms; thus, compounds in this group are recognized as potent stressors and health hazards in aquatic ecosystems. In order to assess the toxicity of MCs and detoxification mechanism of freshwater shrimp Macrobrachium nipponense, the full-length cDNAs of the glutathione S-transferase (gst) and catalase (cat) genes were isolated from the hepatopancreas. The transcription level and activity changes in the biotransformation enzyme (glutathione S-transferase (GST)) and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)) in the hepatopancreas of M. nipponense exposed to MC-LR (0.2, 1, 5, and 25 μg/L) for 12, 24, 72 and 96 h were analyzed. The results showed that the isolated full-length cDNAs of cat and gst genes from M. nipponense displayed a high similarity to other crustaceans, and their mRNAs were mainly expressed in the hepatopancreas. MC-LR caused significant increase of GST activity following 48-96 h (p < 0.05) and an increase in SOD activity especially in 24- and 48-h exposures. CAT activity was activated when exposed to MC-LR in 12-, 24- and 48-h exposures and then it was inhibited at 96-h exposure. There was no significant effect on GPx activity after the 12- and 24-h exposures, whereas it was significantly stimulated after the 72- and 96-h exposures (p < 0.05). The transcription was altered similarly to enzyme activity, but the transcriptional response was generally more immediate and had greater amplitude than enzymatic response, particularly for GST. All of the results suggested that MC-LR can induce antioxidative modulation variations in M. nipponense hepatopancreas in order to eliminate oxidative damage. PMID:26457718

  5. Activity and Transcriptional Responses of Hepatopancreatic Biotransformation and Antioxidant Enzymes in the Oriental River Prawn Macrobrachium nipponense Exposed to Microcystin-LR

    PubMed Central

    Yuan, Julin; Wang, Xueqin; Gu, Zhiming; Zhang, Yingying; Wang, Zaizhao

    2015-01-01

    Microcystins (MCs) are a major group of cyanotoxins with side effects in many organisms; thus, compounds in this group are recognized as potent stressors and health hazards in aquatic ecosystems. In order to assess the toxicity of MCs and detoxification mechanism of freshwater shrimp Macrobrachium nipponense, the full-length cDNAs of the glutathione S-transferase (gst) and catalase (cat) genes were isolated from the hepatopancreas. The transcription level and activity changes in the biotransformation enzyme (glutathione S-transferase (GST)) and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)) in the hepatopancreas of M. nipponense exposed to MC-LR (0.2, 1, 5, and 25 μg/L) for 12, 24, 72 and 96 h were analyzed. The results showed that the isolated full-length cDNAs of cat and gst genes from M. nipponense displayed a high similarity to other crustaceans, and their mRNAs were mainly expressed in the hepatopancreas. MC-LR caused significant increase of GST activity following 48–96 h (p < 0.05) and an increase in SOD activity especially in 24- and 48-h exposures. CAT activity was activated when exposed to MC-LR in 12-, 24- and 48-h exposures and then it was inhibited at 96-h exposure. There was no significant effect on GPx activity after the 12- and 24-h exposures, whereas it was significantly stimulated after the 72- and 96-h exposures (p < 0.05). The transcription was altered similarly to enzyme activity, but the transcriptional response was generally more immediate and had greater amplitude than enzymatic response, particularly for GST. All of the results suggested that MC-LR can induce antioxidative modulation variations in M. nipponense hepatopancreas in order to eliminate oxidative damage. PMID:26457718

  6. Changes in diarrhea, nutrients apparent digestibility, digestive enzyme activities of weaned piglets in response to chitosan-zinc chelate.

    PubMed

    Qian, Lichun; Yue, Xiaojing; Hu, Luansha; Ma, Yuanfei; Han, Xinyan

    2016-04-01

    A total of 120 weanling barrows weighing 6.11 ± 0.20 kg were randomly allotted to four treatments with three replications (i.e. pen) of ten piglets per replicate. Pigs were received corn-soybean basal diet (control) or the same basal diet supplemented with the following sources of zinc: (i) 100 mg/kg of Zn as ZnSO4 ; (ii) 100 mg/kg of Zn as chitosan-Zn chelate (CS-Zn); and (iii) 100 mg/kg of Zn as ZnSO4 mixed with chitosan (CS + ZnSO4 ). The results showed that CS-Zn could highly improve average daily gain and average daily feed intake than those of ZnSO4 or CS+ ZnSO4 (P < 0.05). The pigs fed dietary CS-Zn had lower diarrhea incidence and higher apparent digestibility of crude protein than those of the pigs fed dietary ZnSO4 (P < 0.05). The protease activities in duodenal content of the pigs receiving CS-Zn diets was higher than that of the pigs fed dietary ZnSO4 or CS + ZnSO4 (P < 0.05). The amylase activity in duodenal content of the pigs fed dietary CS-Zn was higher than that of the pigs receiving ZnSO4 diets or basal diets (P < 0.05). These results indicated that dietary CS-Zn showed different bioactivities from ZnSO4 or CS + ZnSO4 in reducing the incidence of diarrhea, improving activities of digestive enzymes and growth performance of weaned pigs. PMID:26304729

  7. Short-Term Responses of Soil Respiration and C-Cycle Enzyme Activities to Additions of Biochar and Urea in a Calcareous Soil.

    PubMed

    Song, Dali; Xi, Xiangyin; Huang, Shaomin; Liang, Guoqing; Sun, Jingwen; Zhou, Wei; Wang, Xiubin

    2016-01-01

    Biochar (BC) addition to soil is a proposed strategy to enhance soil fertility and crop productivity. However, there is limited knowledge regarding responses of soil respiration and C-cycle enzyme activities to BC and nitrogen (N) additions in a calcareous soil. A 56-day incubation experiment was conducted to investigate the combined effects of BC addition rates (0, 0.5, 1.0, 2.5 and 5.0% by mass) and urea (U) application on soil nutrients, soil respiration and C-cycle enzyme activities in a calcareous soil in the North China Plain. Our results showed soil pH values in both U-only and U plus BC treatments significantly decreased within the first 14 days and then stabilized, and CO2emission rate in all U plus BC soils decreased exponentially, while there was no significant difference in the contents of soil total organic carbon (TOC), dissolved organic carbon (DOC), total nitrogen (TN), and C/N ratio in each treatment over time. At each incubation time, soil pH, electrical conductivity (EC), TOC, TN, C/N ratio, DOC and cumulative CO2 emission significantly increased with increasing BC addition rate, while soil potential activities of the four hydrolytic enzymes increased first and then decreased with increasing BC addition rate, with the largest values in the U + 1.0%BC treatment. However, phenol oxidase activity in all U plus BC soils showed a decreasing trend with the increase of BC addition rate. Our results suggest that U plus BC application at a rate of 1% promotes increases in hydrolytic enzymes, does not highly increase C/N and C mineralization, and can improve in soil fertility. PMID:27589265

  8. Antioxidant enzyme activities of Microcystis aeruginosa in response to nonylphenols and degradation of nonylphenols by M. aeruginosa.

    PubMed

    Wang, Jingxian; Xie, Ping

    2007-10-01

    The aim of this study was to examine the effects of chemical nonylphenols (NPs) on the antioxidant system of Microcystis aeruginosa strains. The degradation and sorption of NPs by M. aeruginosa were also evaluated. High concentrations of NPs (1 and 2 mg/l) were found to cause increases in superoxidase dismutase (SOD) and glutathione-S-transferase (GST) activities and in glutathione (GSH) levels. These results suggest that toxic stress manifested by elevated SOD and GST levels and GSH contents may be responsible for the toxicity of NPs to M. aeruginosa and that the algal cells could improve their antioxidant and detoxification ability through the enhancement of enzymatic and nonenzymatic prevention substances. The observed elevations in GSH levels and GST activities were relatively higher than those in SOD activities, indicating that GSH and GST contributed more in eliminating toxic effects than SOD. Low concentrations of NPs (0.05-0.2 mg/l) enhanced cell growth and decreased GST activity in algal cells of M. aeruginosa, suggesting that NPs may have acted as a protecting factor, such as an antioxidant. The larger portion of the NPs (>60%) disappeared after 12 days of incubation, indicating the strong ability of M. aeruginosa to degrade the moderate persistent NP compounds. The sorption ratio of M. aeruginosa after a 12-day exposure to low nominal concentrations of NPs (0.02-0.5 mg/l) was relatively high (>30%). The fact that M. aeruginosa effectively resisted the toxic effects of NPs and strongly degraded these pollutants indicate that M. aeruginosa cells have a strong ability to adapt to variations in environmental conditions and that low and moderate concentrations of organic compounds may favor its survival. Further studies are needed to provide detailed information on the fate of persistent organic pollutants and the survival of algae and to determine the possible role of organic pollutants in the occurrence of water blooms in eutrophic lakes. PMID:17342429

  9. Determining Enzyme Activity by Radial Diffusion

    ERIC Educational Resources Information Center

    Davis, Bill D.

    1977-01-01

    Discusses advantages of radial diffusion assay in determining presence of enzyme and/or rough approximation of amount of enzyme activities. Procedures are included for the preparation of starch-agar plates, and the application and determination of enzyme. Techniques using plant materials (homogenates, tissues, ungerminated embryos, and seedlings)…

  10. Nutritional performance and activity of some digestive enzymes of the cotton bollworm, Helicoverpa armigera, in response to seven tested bean cultivars.

    PubMed

    Namin, Foroogh Rahimi; Naseri, Bahram; Razmjou, Jabraeil

    2014-01-01

    Nutritional performance and activity of some digestive enzymes (protease and α-amylase) of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) in response to feeding on bean (Phaseolus vulgaris L. (Fabales: Fabaceae)) cultivars (Shokufa, Akhtar, Sayyad, Naz, Pak, Daneshkadeh, and Talash) were evaluated under laboratory conditions (25 ± 1°C, 65 ± 5% RH, and a 16:8 L:D photoperiod). The highest and lowest respective values of approximate digestibility were observed when fourth, fifth, and sixth larval instar H. armigera were fed red kidney bean Akhtar and white kidney bean Daneshkadeh. The efficiency of conversion of ingested and digested food was highest when H. armigera was fed red kidney beans Akhtar and Naz and lowest when they were fed white kidney bean Pak. The highest protease activity of fifth instars was observed when they were fed red kidney bean Naz, and the highest amylase activity of fifth instars was observed when they were fed red kidney bean Sayyad. Sixth instar larvae that fed on red kidney bean Sayyad showed the highest protease activity. Larvae reared on common bean Talash and white kidney bean Pak showed the highest amylase activity. Among bean cultivars tested, red kidney bean Sayyad was the most unsuitable host for feeding H. armigera. PMID:25368049

  11. Enzyme Activity Experiments Using a Simple Spectrophotometer

    ERIC Educational Resources Information Center

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

    Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

  12. Enzyme-responsive nanoparticles for drug release and diagnostics.

    PubMed

    de la Rica, Roberto; Aili, Daniel; Stevens, Molly M

    2012-08-01

    Enzymes are key components of the bionanotechnology toolbox that possess exceptional biorecognition capabilities and outstanding catalytic properties. When combined with the unique physical properties of nanomaterials, the resulting enzyme-responsive nanoparticles can be designed to perform functions efficiently and with high specificity for the triggering stimulus. This powerful concept has been successfully applied to the fabrication of drug delivery schemes where the tissue of interest is targeted via release of cargo triggered by the biocatalytic action of an enzyme. Moreover, the chemical transformation of the carrier by the enzyme can also generate therapeutic molecules, therefore paving the way to design multimodal nanomedicines with synergistic effects. Dysregulation of enzymatic activity has been observed in a number of severe pathological conditions, and this observation is useful not only to program drug delivery in vivo but also to fabricate ultrasensitive sensors for diagnosing these diseases. In this review, several enzyme-responsive nanomaterials such as polymer-based nanoparticles, liposomes, gold nanoparticles and quantum dots are introduced, and the modulation of their physicochemical properties by enzymatic activity emphasized. When known, toxicological issues related to the utilization nanomaterials are highlighted. Key examples of enzyme-responsive nanomaterials for drug delivery and diagnostics are presented, classified by the type of effector biomolecule, including hydrolases such as proteases, lipases and glycosidases, and oxidoreductases. PMID:22266127

  13. Enzyme activities associated with oxidative stress in Metarhizium anisopliae during germination, mycelial growth, and conidiation and in response to near-UV irradiation.

    PubMed

    Miller, Charles D; Rangel, Drauzio; Braga, Gilberto U L; Flint, Stephan; Kwon, Sun-Il; Messias, Claudio L; Roberts, Donald W; Anderson, Anne J

    2004-01-01

    Metarhizium anisopliae isolates have a wide insect host range, but an impediment to their commercial use as a biocontrol agent of above-ground insects is the high susceptibility of spores to the near-UV present in solar irradiation. To understand stress responses in M. anisopliae, we initiated studies of enzymes that protect against oxidative stress in two strains selected because their spores differed in sensitivity to UV-B. Spores of the more near-UV resistant strain in M. anisopliae 324 displayed different isozyme profiles for catalase-peroxidase, glutathione reductase, and superoxide dismutase when compared with the less resistant strain 2575. A transient loss in activity of catalase-peroxidase and glutathione reductase was observed during germination of the spores, whereas the intensity of isozymes displaying superoxide dismutase did not change as the mycelium developed. Isozyme composition for catalase-peroxidases and glutathione reductase in germlings changed with growth phase. UV-B exposure from lamps reduced the activity of isozymes displaying catalase-peroxidase and glutathione reductase activities in 2575 more than in 324. The major effect of solar UV-A plus UV-B also was a reduction in catalase-peroxidases isozyme level, a finding confirmed by measurement of catalase specific activity. Impaired growth of M. anisopliae after near-UV exposure may be related to reduced abilities to handle oxidative stress. PMID:15052320

  14. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    PubMed Central

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions. PMID:8597660

  15. Antioxidant enzymes activities in obese Tunisian children

    PubMed Central

    2013-01-01

    Background The oxidant stress, expected to increase in obese adults, has an important role in the pathogenesis of many diseases. It results when free radical formation is greatly increased or protective antioxidant mechanisms are compromised. The main objective of this study is to evaluate the antioxidant response to obesity-related stress in healthy children. Methods A hundred and six healthy children (54 obese and 52 controls), aged 6–12 years old, participated in this study. The collected data included anthropometric measures, blood pressure, fasting glucose, total cholesterol, triglycerides and enzymatic antioxidants (Superoxide dismutase: SOD, Catalase: CAT and Glutathione peroxidase: GPx). Results The first step antioxidant response, estimated by the SOD activity, was significantly higher in obese children compared with normal-weight controls (p < 0.05). Mean activities of anti-radical GPx and CAT enzymes were not affected by the BMI increase. Although, total cholesterol levels were statistically higher in the obese group, there was no significant association with the SOD activity. Conclusions The obesity-related increase of the oxidant stress can be observed even in the childhood period. In addition to the complications of an increased BMI, obesity itself can be considered as an independent risk factor of free radical production resulting in an increased antioxidant response. PMID:23360568

  16. Assessment of Antioxidant Enzyme Activity and Mineral Nutrients in Response to NaCl Stress and its Amelioration Through Glutathione in Chickpea.

    PubMed

    Shankar, Vinay; Kumar, Dinesh; Agrawal, Veena

    2016-01-01

    Salinity stress has been reckoned as one of the major threat towards crop productivity as it causes significant decline in the yield. The impact of NaCl stress (0, 1, 10, 50, 100 and 200 mg L(-1)) as well as glutathione (10 mg L(-1)) either alone or in combination has been evaluated on the induction of multiple shoots, antioxidant enzymes' activity, lipid peroxidation, relative permeability, concentration of nutrients, photosynthetic pigments, protein and proline content of nodal segments of chickpea after 14 days of culture. The antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) were found to be increased under salt stress as well as glutathione-supplemented medium. A significant decrease in the concentrations of chlorophylls a, b, total chlorophyll and carotenoid was observed under salt stress. Concentrations of nitrogen, phosphorus, potassium, calcium, carbon, magnesium and sulphur showed an initial increase up to 10 mg L(-1) NaCl, but a decline was seen at higher NaCl levels. Proline content and malondialdehyde concentration were found to be increased under salt stress. Three isoforms of SOD, one of CAT and four of GPX were expressed during native polyacrylamide gel electrophoresis (PAGE) analysis. However, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the stressed nodal explants revealed the over-expression of several polypeptide bands related to NaCl stress. These findings for the first time suggest that glutathione (GSH) helps in ameliorating NaCl stress in nodal explants of chickpea by manipulating various biochemical and physiological responses of plants. PMID:26440314

  17. Characterization of Soil Samples of Enzyme Activity

    ERIC Educational Resources Information Center

    Freeland, P. W.

    1977-01-01

    Described are nine enzyme essays for distinguishing soil samples. Colorimetric methods are used to compare enzyme levels in soils from different sites. Each soil tested had its own spectrum of activity. Attention is drawn to applications of this technique in forensic science and in studies of soil fertility. (Author/AJ)

  18. Enzyme Activities in Polarized Cell Membranes

    PubMed Central

    Bass, L.; McIlroy, D. K.

    1968-01-01

    The theoretical pH dependence of enzyme activities in membranes of low dielectric constant is estimated. It is shown that in biological membranes some types of enzymes may attain a limiting pH sensitivity such that an increment of only 0.2 pH unit (sufficient to induce action potentials in squid axons) causes a relative activity change of over 25%. The transients of enzyme activity generated by membrane depolarization and by pH increments in the bathing solution are discussed in relation to the transients of nervous excitation. PMID:5641405

  19. How should enzyme activities be used in fish growth studies?

    PubMed

    Pelletier; Blier; Dutil; Guderley

    1995-01-01

    The activity of glycolytic and oxidative enzymes was monitored in the white muscle of Atlantic cod Gadus morhua experiencing different growth rates. A strong positive relationship between the activity of two glycolytic enzymes and individual growth rate was observed regardless of whether the enzyme activity was expressed as units per gram wet mass, units per gram dry mass or with respect to muscle protein and DNA content. The most sensitive response to growth rate was observed when pyruvate kinase and lactate dehydrogenase activities were expressed as units per microgram DNA, and this may be useful as an indicator of growth rate in wild fish. In contrast, no relationship between the activities of oxidative enzymes and growth rate was observed when cytochrome c oxidase and citrate synthase activities were expressed as units per gram protein. Apparently, the aerobic capacity of white muscle in cod is not specifically increased to match growth rate. PMID:9319392

  20. [Muscle enzyme activity and exercise].

    PubMed

    Gojanovic, B; Feihl, F; Gremion, G; Waeber, B

    2009-02-01

    Exercise is classically associated with muscular soreness, presenting one to two days later, delayed onset muscular soreness. Blood muscle enzymes and protein elevations are characteristic, and may cause renal failure. Creatin phosphokinase peak appears on the fourth day and depends on exercise type and individual parameters. This effect is attenuated with repeated bouts, by habituation. Metabolic complications are rare. The knowledge of this reaction, even with common exercises, allows to postpone investigations for a complex metabolic disorder, or to avoid stopping a medication for fear of a side effect, as with statins. Indeed, it is necessary to wait for seven days without any exercise before interpreting an elevated CK result. PMID:19180440

  1. Oxidative stress in deep scattering layers: Heat shock response and antioxidant enzymes activities of myctophid fishes thriving in oxygen minimum zones

    NASA Astrophysics Data System (ADS)

    Lopes, Ana Rita; Trübenbach, Katja; Teixeira, Tatiana; Lopes, Vanessa M.; Pires, Vanessa; Baptista, Miguel; Repolho, Tiago; Calado, Ricardo; Diniz, Mário; Rosa, Rui

    2013-12-01

    Diel vertical migrators, such as myctophid fishes, are known to encounter oxygen minimum zones (OMZ) during daytime in the Eastern Pacific Ocean and, therefore, have to cope with temperature and oxidative stress that arise while ascending to warmer, normoxic surface waters at night-time. The aim of this study was to investigate the antioxidant defense strategies and heat shock response (HSR) in two myctophid species, namely Triphoturus mexicanus and Benthosema panamense, at shallow and warm surface waters (21 kPa, 20-25 °C) and at hypoxic, cold (≤1 kPa, 10 °C) mesopelagic depths. More specifically, we quantified (i) heat shock protein concentrations (HSP70/HSC70) (ii) antioxidant enzyme activities [including superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST)], and (iii) lipid peroxidation [malondialdehyde (MDA) levels]. HSP70/HSC70 levels increased in both myctophid species at warmer, well-oxygenated surface waters probably to prevent cellular damage (oxidative stress) due to increased oxygen demand under elevated temperatures and reactive oxygen species (ROS) formation. On the other hand, CAT and GST activities were augmented under hypoxic conditions, probably as preparatory response to a burst of oxyradicals during the reoxygenation phase (while ascending). SOD activity decreased under hypoxia in B. panamense, but was kept unchanged in T. mexicanus. MDA levels in B. panamense did not change between the surface and deep-sea conditions, whereas T. mexicanus showed elevated MDA and HSP70/HSC70 concentrations at warmer surface waters. This indicated that T. mexicanus seems to be not so well tuned to temperature and oxidative stress associated to diel vertical migrations. The understanding of such physiological strategies that are linked to oxygen deprivation and reoxygenation phases may provide valuable information about how different species might respond to the impacts of environmental stressors (e.g. expanding mesopelagic hypoxia

  2. Modulation of innate and acquired immune responses by Escherichia coli heat-labile toxin: distinct pro- and anti-inflammatory effects of the nontoxic AB complex and the enzyme activity.

    PubMed

    Ryan, E J; McNeela, E; Pizza, M; Rappuoli, R; O'Neill, L; Mills, K H

    2000-11-15

    We have examined the roles of enzyme activity and the nontoxic AB complex of heat-labile toxin (LT) from Escherichia coli on its adjuvant and immunomodulatory properties. LTK63, an LT mutant that is completely devoid of enzyme activity, enhanced Th1 responses to coinjected Ags at low adjuvant dose. In contrast, LTR72, a partially detoxified mutant, enhanced Th2 responses and when administered intranasally to mice before infection with Bordetella pertussis suppressed Th1 responses and delayed bacterial clearance from the lungs. LTR72 or wild-type LT inhibited Ag-induced IFN-gamma production by Th1 cells, and LT enhanced IL-5 production by Th2 cells in vitro. Each of the toxins enhanced B7-1 expression on macrophages, but enhancement of B7-2 expression was dependent on enzyme activity. We also observed distinct effects of the nontoxic AB complex and enzyme activity on inflammatory cytokine production. LT and LTR72 suppressed LPS and IFN-gamma induced TNF-alpha and IL-12 production, but enhanced IL-10 secretion by macrophages in vitro and suppressed IL-12 production in vivo in a murine model of LPS-induced shock. In contrast, LTK63 augmented the production of IL-12 and TNF-alpha. Furthermore, LTK63 enhanced NF-kappaB translocation, whereas low doses of LTR72 or LT failed to activate NF-kappaB, but stimulated cAMP production. Thus, E. coli LT appears to be capable of suppressing Th1 responses and enhancing Th2 responses through the modulatory effects of enzyme activity on NF-kappaB activation and IL-12 production. In contrast, the nontoxic AB complex can stimulate acquired immune responses by activating components of the innate immune system. PMID:11067933

  3. Visualization of enzyme activities inside earthworm pores

    NASA Astrophysics Data System (ADS)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  4. Antimutagenic activity of oxidase enzymes

    SciTech Connect

    Agabeili, R.A.

    1986-11-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity.

  5. Enzyme activity in dialkyl phosphate ionic liquids.

    PubMed

    Thomas, Marie F; Li, Luen-Luen; Handley-Pendleton, Jocelyn M; van der Lelie, Daniel; Dunn, John J; Wishart, James F

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariella volvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v. PMID:22001053

  6. Enzyme activity in dialkyl phosphate ionic liquids

    SciTech Connect

    Thomas, M.F.; Dunn, J.; Li, L.-L.; Handley-Pendleton, J. M.; van der lelie, D.; Wishart, J. F.

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariellavolvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

  7. Halophilic enzyme activation induced by salts

    PubMed Central

    Ortega, Gabriel; Laín, Ana; Tadeo, Xavier; López-Méndez, Blanca; Castaño, David; Millet, Oscar

    2011-01-01

    Halophilic archea (halobacteriae) thrive in hypersaline environments, avoiding osmotic shock by increasing the ion concentration of their cytoplasm by up to 3–6 M. To remain folded and active, their constitutive proteins have evolved towards a biased amino acid composition. High salt concentration affects catalytic activity in an enzyme-dependent way and a unified molecular mechanism remains elusive. Here, we have investigated a DNA ligase from Haloferax volcanii (Hv LigN) to show that K+ triggers catalytic activity by preferentially stabilising a specific conformation in the reaction coordinate. Sodium ions, in turn, do not populate such isoform and the enzyme remains inactive in the presence of this co-solute. Our results show that the halophilic amino acid signature enhances the enzyme's thermodynamic stability, with an indirect effect on its catalytic activity. This model has been successfully applied to reengineer Hv LigN into an enzyme that is catalytically active in the presence of NaCl. PMID:22355525

  8. Activation of polyamine catabolic enzymes involved in diverse responses against epibrassinolide-induced apoptosis in LNCaP and DU145 prostate cancer cell lines.

    PubMed

    Obakan, Pinar; Arisan, Elif Damla; Calcabrini, Annarica; Agostinelli, Enzo; Bolkent, Sehnaz; Palavan-Unsal, Narçin

    2014-03-01

    Epibrassinolide (EBR) is a biologically active compound of the brassinosteroids, steroid-derived plant growth regulator family. Generally, brassinosteroids are known for their cell expansion and cell division-promoting roles. Recently, EBR was shown as a potential apoptotic inducer in various cancer cells without affecting the non-tumor cell growth. Androgen signaling controls cell proliferation through the interaction with the androgen receptor (AR) in the prostate gland. Initially, the development of prostate cancer is driven by androgens. However, in later stages, a progress to the androgen-independent stage is observed, resulting in metastatic prostate cancer. The androgen-responsive or -irresponsive cells are responsible for tumor heterogeneity, which is an obstacle to effective anti-cancer therapy. Polyamines are amine-derived organic compounds, known for their role in abnormal cell proliferation as well as during malignant transformation. Polyamine catabolism-targeting agents are being investigated against human cancers. Many chemotherapeutic agents including polyamine analogs have been demonstrated to induce polyamine catabolism that depletes polyamine levels and causes apoptosis in tumor models. In our study, we aimed to investigate the mechanism of apoptotic cell death induced by EBR, related with polyamine biosynthetic and catabolic pathways in LNCaP (AR+), DU145 (AR-) prostate cancer cell lines and PNT1a normal prostate epithelial cell line. Induction of apoptotic cell death was observed in prostate cancer cell lines after EBR treatment. In addition, EBR induced the decrease of intracellular polyamine levels, accompanied by a significant ornithine decarboxylase (ODC) down-regulation in each prostate cancer cell and also modulated ODC antizyme and antizyme inhibitor expression levels only in LNCaP cells. Catabolic enzymes SSAT and PAO expression levels were up-regulated in both cell lines; however, the specific SSAT and PAO siRNA treatments prevented the

  9. Enzyme Specific Activity in Functionalized Nanoporous Supports

    SciTech Connect

    Lei, Chenghong; Soares, Thereza A.; Shin, Yongsoon; Liu, Jun; Ackerman, Eric J.

    2008-03-26

    Enzyme specific activity can be increased or decreased to a large extent by changing protein loading density in functionalized nanoporous support, where organophosphorus hydrolase can display a constructive orientation and thus leave a completely open entrance for substrate even at higher protein loading density, but glucose oxidase can not.

  10. An NMR Study of Enzyme Activity.

    ERIC Educational Resources Information Center

    Peterman, Keith E.; And Others

    1989-01-01

    A laboratory experiment designed as a model for studying enzyme activity with a basic spectrometer is presented. Included are background information, experimental procedures, and a discussion of probable results. Stressed is the value of the use of Nuclear Magnetic Resonance in biochemistry. (CW)

  11. Enzyme-Responsive Delivery of Multiple Proteins with Spatiotemporal Control

    PubMed Central

    Zhu, Suwei; Nih, Lina; Carmichael, S. Thomas; Lu, Yunfeng; Segura, Tatiana

    2015-01-01

    The growth of tissues and organs is regulated by orchestrated signals from biomolecules such as enzymes and growth factors. The ability to deliver signal molecules in response to particular biological events (e.g., enzyme expression and activation) holds great promise towards tissue healing and regeneration. The current delivery vehicles mainly rely on hydrolysable scaffolds and thin films of protein-containing polymers, which cannot be programmed to respond to biological signals. We report herein an injectable delivery platform based on enantiomeric protein nanocapsules, which can deliver multiple proteins with spatiotemporal control in response to the tissue proteases secreted during wound healing. Exemplified by stroke and diabetic wound healing in mice, sequential delivery of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) greatly enhances tissue revascularization and vessel maturation, providing effective delivery vehicles for tissue engineering and reparative medicine. PMID:25962336

  12. Multiple plasma enzyme activities in liver disease

    PubMed Central

    Hargreaves, T.; Janota, I.; Smith, M. J. H.

    1961-01-01

    The measurement of the plasma activities of glutamic-oxaloacetic and glutamic-pyruvic transaminases, aldolase, cholinesterase, and isocitric, lactic, and phosphogluconic dehydrogenases in random samples of blood was found to be of no value in the differential diagnosis of hepatitis, obstructive jaundice, hepatic cirrhosis, and neoplastic conditions involving the liver. Serial determinations of the enzyme activities provided useful information about the course of certain hepatic disorders, particularly acute viral hepatitis. PMID:13711559

  13. Response of microbial extracellular enzyme activities and r- vs. K- selected microorganisms to elevated atmospheric CO2 depends on soil aggregate size

    NASA Astrophysics Data System (ADS)

    Dorodnikov, Maxim; Blagodatskaya, Evgenia; Blagodatskiy, Sergey; Kuzyakov, Yakov

    2014-05-01

    Increased belowground carbon (C) transfer by plant roots under elevated atmospheric CO2 and the contrasting environment in soil macro- and microaggregates could affect properties of the microbial community in the rhizosphere. We evaluated the effect of 5 years of elevated CO2 (550 ppm) on four extracellular enzymes: ß-glucosidase, chitinase, phosphatase, and sulfatase along with the contribution of fast- (r-strategists) and slow-growing microorganisms (K-strategists) in soil aggregates. We fractionated the bulk soil from the ambient and elevated CO2 treatments of FACE-Hohenheim (Stuttgart) into large macro- (>2 mm), small macro- (0.25-2.00 mm), and microaggregates (<0.25 mm) using a modified dry sieving. Microbial biomass (C-mic by SIR), the maximal specific growth rate (µ), growing microbial biomass (GMB) and lag-period (t-lag) were estimated by the kinetics of CO2 emission from bulk soil and aggregates amended with glucose and nutrients. In the bulk soil and isolated aggregates before and after activation with glucose, the actual and the potential enzyme activities were measured. Although C-org and C-mic as well as the activities of ß-glucosidase, phosphatase, and sulfatase were unaffected in bulk soil and in aggregate-size classes by elevated CO2, significant changes were observed in potential enzyme production after substrate amendment. After adding glucose, enzyme activities under elevated CO2 were 1.2-1.9-fold higher than under ambient CO2. In addition, µ values were significantly higher under elevated than ambient CO2 for bulk soil, small macroaggregates, and microaggregates. Based on changes in µ, GMB, and lag-period, we conclude that elevated atmospheric CO2 stimulated the r-selected microorganisms, especially in soil microaggregates. In contrast, significantly higher chitinase activity in bulk soil and in large macroaggregates under elevated CO2 revealed an increased contribution of fungi to turnover processes. We conclude that quantitative and

  14. Correction of Mitochondrial Enzyme Activities in the Skeletal Muscles of Old Rats in Response to Addition of Olive Oil to the Ration.

    PubMed

    Bronnikov, G E; Kulagina, T P; Aripovskii, A V; Kramarova, L I

    2015-06-01

    Activities of mitochondrial electron transport chain enzymes NADH-CoQ oxidoreductase (complex I), cytochrome C-oxidase (complex IV), and citrate synthase were measured by spectrophotometry in m. quadriceps femoris homogenate from old rats receiving olive oil with the ration. Reduced activities of complexes I and IV in old animals were restored to the level of young animals after 6-week consumption of olive oil. Activity of citrate synthase did not change with age. Positive effect of olive oil on fatty-acid composition of the muscle tissue in old animals was demonstrated. The content of summary monounsaturated fatty acids, reduced with aging, and of summary polyunsaturated ones, increasing with age, were restored in old rats to the levels virtually not differing from the levels of young animals. PMID:26087754

  15. Supplementation with sodium selenite and selenium-enriched microalgae biomass show varying effects on blood enzymes activities, antioxidant response, and accumulation in common barbel (Barbus barbus).

    PubMed

    Kouba, Antonín; Velíšek, Josef; Stará, Alžběta; Masojídek, Jiří; Kozák, Pavel

    2014-01-01

    Yearling common barbel (Barbus barbus L.) were fed four purified casein-based diets for 6 weeks in outdoor cages. Besides control diet, these were supplemented with 0.3 mg kg(-1) dw selenium (Se) from sodium selenite, or 0.3 and 1.0 mg kg(-1) from Se-enriched microalgae biomass (Chlorella), a previously untested Se source for fish. Fish mortality, growth, Se accumulation in muscle and liver, and activity of selected enzymes in blood plasma, muscle, liver, and intestine were evaluated. There was no mortality, and no differences in fish growth, among groups. Se concentrations in muscle and liver, activity of alanine aminotransferase and creatine kinase in blood plasma, glutathione reductase (GR) in muscle, and GR and catalase in muscle and liver suggested that selenium from Se-enriched Chlorella is more readily accumulated and biologically active while being less toxic than sodium selenite. PMID:24772422

  16. Supplementation with Sodium Selenite and Selenium-Enriched Microalgae Biomass Show Varying Effects on Blood Enzymes Activities, Antioxidant Response, and Accumulation in Common Barbel (Barbus barbus)

    PubMed Central

    Kouba, Antonín; Velíšek, Josef; Stará, Alžběta; Masojídek, Jiří; Kozák, Pavel

    2014-01-01

    Yearling common barbel (Barbus barbus L.) were fed four purified casein-based diets for 6 weeks in outdoor cages. Besides control diet, these were supplemented with 0.3 mg kg−1 dw selenium (Se) from sodium selenite, or 0.3 and 1.0 mg kg−1 from Se-enriched microalgae biomass (Chlorella), a previously untested Se source for fish. Fish mortality, growth, Se accumulation in muscle and liver, and activity of selected enzymes in blood plasma, muscle, liver, and intestine were evaluated. There was no mortality, and no differences in fish growth, among groups. Se concentrations in muscle and liver, activity of alanine aminotransferase and creatine kinase in blood plasma, glutathione reductase (GR) in muscle, and GR and catalase in muscle and liver suggested that selenium from Se-enriched Chlorella is more readily accumulated and biologically active while being less toxic than sodium selenite. PMID:24772422

  17. Usefulness of plasma renin activity in predicting haemodynamic and clinical responses and survival during long term converting enzyme inhibition in severe chronic heart failure. Experience in 100 consecutive patients.

    PubMed Central

    Packer, M; Medina, N; Yushak, M; Lee, W H

    1985-01-01

    The relation between plasma renin activity before treatment and the haemodynamic and clinical responses to converting enzyme inhibition was determined in 100 consecutive patients with severe chronic heart failure who were treated with captopril or enalapril. Initial doses of captopril produced significant increases in cardiac index and decreases in left ventricular filling pressure, mean arterial pressure, mean right atrial pressure, heart rate, and systemic vascular resistance that varied linearly with the pretreatment value for plasma renin activity. In contrast, there was no relation between the pretreatment activity and the magnitude of haemodynamic improvement after 1-3 months of treatment with the converting enzyme inhibitors, and, consequently, a similar proportion of patients with a high (greater than 6 ng/ml/h; greater than 4.62 mmol/l/h), intermediate (2-6 ng/ml/h; 1.54-4.62 mmol/l/h), and low (less than 2 ng/ml/h; less than 1.54 mmol/l/h) pretreatment value improved clinically during long term treatment (64%, 60%, and 64% respectively). Long term survival after one, two, and three years was similar in the three groups. Estimating the degree of activation of the renin-angiotensin system by measuring pretreatment plasma renin activity fails to predict the long term haemodynamic or clinical responses to converting enzyme inhibitors in patients with severe chronic heart failure, and thus appears to be of limited value in selecting those patients likely to benefit from treatment with these drugs. PMID:2994697

  18. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme

    PubMed Central

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

  19. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme.

    PubMed

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

  20. Activity of antioxidant enzymes in response to atmospheric pressure induced physiological stress in deep-sea hydrothermal vent mussel Bathymodiolus azoricus.

    PubMed

    Martins, Inês; Romão, Célia V; Goulart, Joana; Cerqueira, Teresa; Santos, Ricardo S; Bettencourt, Raul

    2016-03-01

    Deep sea hydrothermal Bathymodiolus azoricus mussels from Portuguese EEZ Menez Gwen hydrothermal field possess the remarkable ability to overcome decompression and survive successfully at atmospheric pressure conditions. We investigated the potential use of antioxidant defense enzymes in mussel B. azoricus as biomarkers of oxidative stress induced by long term acclimatization to atmospheric pressure conditions. Mussels collected at Menez Gwen hydrothermal field were acclimatized for two weeks in three distinct conditions suitable of promoting physiological stress, (i) in plain seawater for concomitant endosymbiont bacteria loss, (ii) in plain seawater under metal iron exposure, (iii) constant bubbling methane and pumped sulfide for endosymbiont bacteria survival. The enzymatic activities of superoxide dismutase (SOD), catalase (CAT), and iron storage proteins in addition to electrophoretic profiles were examined in vent mussel gills and digestive gland. Gills showed approximately 3 times more SOD specific activity than digestive glands. On the other hand, digestive glands showed approximately 6 times more CAT specific activity than gills. Iron storage proteins were identified in gill extracts from all experimental conditions mussels. However, in digestive gland extracts only fresh collected mussels and after 2 weeks in FeSO4 showed the presence of iron storage proteins. The differences between SOD, CAT specific activities and the presence of iron storage proteins in the examined tissues reflect dissimilar metabolic and antioxidant activities, as a result of tissue specificities and acclimatization conditions influences on the organism. PMID:26790096

  1. Biochemical identification of a neutral sphingomyelinase 1 (NSM1)-like enzyme as the major NSM activity in the DT40 B-cell line: absence of a role in the apoptotic response to endoplasmic reticulum stress.

    PubMed Central

    Fensome, Amanda C; Josephs, Michelle; Katan, Matilda; Rodrigues-Lima, Fernando

    2002-01-01

    DT40 cells have approx. 10-fold higher Mg2+-dependent neutral sphingomyelinase (NSM) activity in comparison with other B-cell lines and contain very low acidic sphingomyelinase activity. Purification of this activity from DT40 cell membranes suggested the presence of one major NSM isoform. Although complete purification of this isoform could not be achieved, partially purified fractions were examined further with regard to the known characteristics of previously partially purified NSMs and the two cloned enzymes exhibiting in vitro NSM activity (NSM1 and NSM2). For a direct comparative study, highly purified brain preparations, purified NSM1 protein and Bacillus cereus enzyme were used. Analysis of the enzymic properties of the partially purified DT40 NSM, such as cation dependence, substrate specificity, redox regulation and stimulation by phosphatidylserine, together with the localization of this enzyme to the endoplasmic reticulum (ER), suggested that this NSM from DT40 cells corresponds to NSM1. Further studies aimed to correlate presence of the high levels of this NSM1-like activity in DT40 cells with the ability of these cells to accumulate ceramide and undergo apoptosis. When DT40 cells were stimulated to apoptose by a variety of agents, including the ER stress, an increase in endogenous ceramide levels was observed. However, these responses were not enhanced compared with another B-cell line (Nalm-6), characterized by low sphingomyelinase activity. In addition, DT40 cells were not more susceptible to ceramide accumulation and apoptosis when exposed to the ER stress compared with other apoptotic agents. Inhibition of de novo synthesis of ceramide partially inhibited its accumulation, indicating that the ceramide production in DT40 cells could be complex and, under some conditions, could involve both sphingomyelin hydrolysis and ceramide synthesis. PMID:12071841

  2. Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia – Impact on enzyme activity and response to cytotoxics

    PubMed Central

    Morad, Samy A. F.; Tan, Su-Fern; Feith, David J.; Kester, Mark; Claxton, David F.; Loughran, Thomas P.; Barth, Brian M.; Fox, Todd E.; Cabot, Myles C.

    2015-01-01

    The triphenylethylene antiestrogen, tamoxifen, can be an effective inhibitor of sphingolipid metabolism. This off-target activity makes tamoxifen an interesting ancillary for boosting the apoptosis-inducing properties of ceramide, a sphingolipid with valuable tumor censoring activity. Here we show for the first time that tamoxifen and metabolite, N –desmethyltamoxifen (DMT) block ceramide glycosylation and inhibit ceramide hydrolysis (by acid ceramidase, AC) in human acute myelogenous leukemia (AML) cell lines and in AML cells derived from patients. Tamoxifen (1-10 μM) inhibition of AC in AML cells was accompanied by decreases in AC protein expression. Tamoxifen also depressed expression and activity of sphingosine kinase 1 (SphK1), the enzyme catalyzing production of mitogenic sphingosine 1-phosphate (S1-P). Results from mass spectroscopy showed that tamoxifen and DMT, i ) increased the levels of endogenous C16:0- and C24:1 ceramide molecular species, ii) nearly totally halted production of respective glucosylceramide (GC) molecular species, iii ) drastically reduced levels of sphingosine ( to 9% of control), and iv ) reduced levels of S1-P by 85%, in vincristine-resistant HL-60/VCR cells. Co-administration of tamoxifen with either N-(4-hydroxyphenyl)retinamide (4-HPR), a ceramide-generating retinoid, or a cell-deliverable form of ceramide, C6-ceramide, resulted in marked decreases in HL-60/VCR cell viability that far exceeded single agent potency. Combination treatments resulted in synergistic apoptotic cell death as gauged by increased Annexin V binding and DNA fragmentation and activation of caspase-3. These results show the versatility of adjuvant triphenylethylene with ceramide-centric therapies for magnifying therapeutic potential in AML. Such drug regimens could serve as effective strategies, even in the multidrug resistant setting. PMID:25769964

  3. Interactions of water contaminants. I. Plasma enzyme activity and response surface methodology following gavage administration of CCl4 and CHCl3 or TCE singly and in combination in the rat

    SciTech Connect

    Borzelleca, J.F.; O'Hara, T.M.; Gennings, C.; Granger, R.H.; Sheppard, M.A.; Condie, L.W. Jr. )

    1990-04-01

    The joint hepatotoxicity of CCl4 and CHCl3 or TCE in male CD rats following simultaneous oral administration has been investigated. Rats with chronic indwelling arterial cannulas were administered a single oral dose of CCl4 and CHCl3 or CCl4 and TCE in 5% Emulphor at doses of 0 to 700 mg/kg. Hepatotoxicity was evaluated by measuring the activity of AST, ALT, and SDH in plasma at 0, 3, 6, 12, 24, 36, 48, and 72 hr postgavage. Response data were analyzed for interaction using response surface methodology. CCl4 alone displayed dose-dependent toxicity. TCE demonstrated little evidence of hepatotoxicity. In combination, both CCl4/CHCl3 and CCl4/TCE displayed a synergistic (supraadditive) response for peak plasma enzyme activity.

  4. Enzyme activities by indicator of quality in organic soil

    NASA Astrophysics Data System (ADS)

    Raigon Jiménez, Mo; Fita, Ana Delores; Rodriguez Burruezo, Adrián

    2016-04-01

    The analytical determination of biochemical parameters, as soil enzyme activities and those related to the microbial biomass is growing importance by biological indicator in soil science studies. The metabolic activity in soil is responsible of important processes such as mineralization and humification of organic matter. These biological reactions will affect other key processes involved with elements like carbon, nitrogen and phosphorus , and all transformations related in soil microbial biomass. The determination of biochemical parameters is useful in studies carried out on organic soil where microbial processes that are key to their conservation can be analyzed through parameters of the metabolic activity of these soils. The main objective of this work is to apply analytical methodologies of enzyme activities in soil collections of different physicochemical characteristics. There have been selective sampling of natural soils, organic farming soils, conventional farming soils and urban soils. The soils have been properly identified conserved at 4 ° C until analysis. The enzyme activities determinations have been: catalase, urease, cellulase, dehydrogenase and alkaline phosphatase, which bring together a representative group of biological transformations that occur in the soil environment. The results indicate that for natural and agronomic soil collections, the values of the enzymatic activities are within the ranges established for forestry and agricultural soils. Organic soils are generally higher level of enzymatic, regardless activity of the enzyme involved. Soil near an urban area, levels of activities have been significantly reduced. The vegetation cover applied to organic soils, results in greater enzymatic activity. So the quality of these soils, defined as the ability to maintain their biological productivity is increased with the use of cover crops, whether or spontaneous species. The practice of cover based on legumes could be used as an ideal choice

  5. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Guoxin Lu

    2007-12-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  6. Angiotensin Converting Enzyme Activity in Alopecia Areata

    PubMed Central

    Namazi, Mohammad Reza; Handjani, Farhad; Eftekhar, Ebrahim; Kalafi, Amir

    2014-01-01

    Background. Alopecia areata (AA) is a chronic inflammatory disease of the hair follicle. The exact pathogenesis of AA remains unknown, although recent studies support a T-cell mediated autoimmune process. On the other hand, some studies have proposed that the renin-angiotensin-aldosterone system (RAAS) may play a role in autoimmunity. Therefore, we assessed serum activity of angiotensin converting enzyme (ACE), a component of this system, in AA. Methods. ACE activity was measured in the sera of 19 patients with AA and 16 healthy control subjects. In addition, the relationship between severity and duration of the disease and ACE activity was evaluated. Results. Serum ACE activity was higher in the patient group (55.81 U/L) compared to the control group (46.41 U/L), but the difference was not statistically significant (P = 0.085). Also, there was no correlation between ACE activity and severity (P = 0.13) and duration of disease (P = 0.25) in the patient group. Conclusion. The increased serum ACE activity found in this study may demonstrate local involvement of the RAAS in the pathogenesis of AA. Assessment of ACE in a study with a larger sample size as well as in tissue samples is recommended in order to further evaluate the possible role of RAAS in AA. PMID:25349723

  7. Potential enzyme activities in cryoturbated organic matter of arctic soils

    NASA Astrophysics Data System (ADS)

    Schnecker, J.; Wild, B.; Rusalimova, O.; Mikutta, R.; Guggenberger, G.; Richter, A.

    2012-12-01

    An estimated 581 Gt organic carbon is stored in arctic soils that are affected by cryoturbtion, more than in today's atmosphere (450 Gt). The high amount of organic carbon is, amongst other factors, due to topsoil organic matter (OM) that has been subducted by freeze-thaw processes. This cryoturbated OM is usually hundreds to thousands of years old, while the chemical composition remains largely unaltered. It has therefore been suggested, that the retarded decomposition rates cannot be explained by unfavourable abiotic conditions in deeper soil layers alone. Since decomposition of soil organic material is dependent on extracellular enzymes, we measured potential and actual extracellular enzyme activities in organic topsoil, mineral subsoil and cryoturbated material from three different tundra sites, in Zackenberg (Greenland) and Cherskii (North-East Siberia). In addition we analysed the microbial community structure by PLFAs. Hydrolytic enzyme activities, calculated on a per gram dry mass basis, were higher in organic topsoil horizons than in cryoturbated horizons, which in turn were higher than in mineral horizons. When calculated on per gram carbon basis, the activity of the carbon acquiring enzyme exoglucanase was not significantly different between cryoturbated and topsoil organic horizons in any of the three sites. Oxidative enzymes, i.e. phenoloxidase and peroxidase, responsible for degradation of complex organic substances, showed higher activities in topsoil organic and cryoturbated horizons than in mineral horizons, when calculated per gram dry mass. Specific activities (per g C) however were highest in mineral horizons. We also measured actual cellulase activities (by inhibiting microbial uptake of products and without substrate addition): calculated per g C, the activities were up to ten times as high in organic topsoil compared to cryoturbated and mineral horizons, the latter not being significantly different. The total amount of PLFAs, as a proxy for

  8. Enzyme Action in the Regulation of Plant Hormone Responses*

    PubMed Central

    Westfall, Corey S.; Muehler, Ashley M.; Jez, Joseph M.

    2013-01-01

    Plants synthesize a chemically diverse range of hormones that regulate growth, development, and responses to environmental stresses. The major classes of plant hormones are specialized metabolites with exquisitely tailored perception and signaling systems, but equally important are the enzymes that control the dose and exposure to the bioactive forms of these molecules. Here, we review new insights into the role of enzyme families, including the SABATH methyltransferases, the methylesterases, the GH3 acyl acid-amido synthetases, and the hormone peptidyl hydrolases, in controlling the biosynthesis and modifications of plant hormones and how these enzymes contribute to the network of chemical signals responsible for plant growth, development, and environmental adaptation. PMID:23709222

  9. Changes in enzyme activities in tissues of rats exposed to hypoxia (Short Communication)

    PubMed Central

    Cryer, Anthony; Bartley, Walter

    1973-01-01

    Rats were exposed to various degrees of hypoxia and enzyme activities in their tissues were determined. In general, oxidative metabolism was not increased in response to hypoxia, nor was anaerobic metabolism. Physiological and anatomical changes were concluded to be more important than changes in cellular enzyme activities in the overall adaptation to acute hypoxia. PMID:4357712

  10. Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity[S

    PubMed Central

    Jaishy, Bharat; Zhang, Quanjiang; Chung, Heaseung S.; Riehle, Christian; Soto, Jamie; Jenkins, Stephen; Abel, Patrick; Cowart, L. Ashley; Van Eyk, Jennifer E.; Abel, E. Dale

    2015-01-01

    Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCβII. These findings reveal a novel mechanism linking lipotoxicity with a PKCβ-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs. PMID:25529920

  11. Enzyme and root activities in surface-flow constructed wetlands.

    PubMed

    Kong, Ling; Wang, Yu-Bin; Zhao, Li-Na; Chen, Zhang-He

    2009-07-01

    Sixteen small-scale wetlands planted with four plant species were constructed for domestic wastewater purification. The objective of this study was to determine the correlations between contaminant removal and soil enzyme activity, root activity, and growth in the constructed wetlands. The results indicated that correlations between contaminant removal efficiency and enzyme activity varied depending on the contaminants. The removal efficiency of NH4+ was significantly correlated with both urease and protease activity in all wetlands, and the removal of total phosphorus and soluble reactive phosphorus was significantly correlated with phosphatase activity in most wetlands, while the removal of total nitrogen, NO3(-) , and chemical oxygen demand (COD) was significantly correlated with enzyme activity only in a few instances. Correlations between soil enzyme activity and root activity varied among species. Activities of all enzymes were significantly correlated with root activity in Vetiveria zizanioides and Phragmites australis wetlands, but not in Hymenocallis littoralis wetlands. Significant correlations between enzyme activity and root biomass and between enzyme activity and root growth were found mainly in Cyperus flabelliformis wetlands. Root activity was significantly correlated with removal efficiencies of all contaminants except NO3(-) and COD in V. zizanioides wetlands. Enzyme activities and root activity showed single-peak seasonal patterns. Activities of phosphatase, urease, and cellulase were significantly higher in the top layer of the substrate than in the deeper layers, and there were generally no significant differences between the deeper layers (deeper than 15 cm). PMID:19497608

  12. Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation.

    PubMed Central

    Aoufouchi, S; Shall, S

    1997-01-01

    Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme that is dependent on DNA breaks and nicks for its enzyme activity. These DNA nicks and breaks function as allosteric effectors of the enzyme activity. This reaction is important for efficient DNA base excision repair, although it is not a component of the elementary repair pathway itself. The physiological relevance of this reaction might be to ensure correct and efficient DNA repair. We have examined the enzyme activity of PARP in oocytes and eggs of Xenopus laevis. Although both oocytes and eggs contain approximately the same amounts of enzyme protein, there is no detectable enzyme activity in the oocytes, whereas in the eggs the enzyme is active. Enzyme activity appears during oocyte maturation, approx. 4 h after induction by progesterone. This enzyme activation coincides with the appearance of active maturation-promoting factor. Enzyme activation is accompanied by a shift in the electrophoretic mobility of the polypeptide, from an apparent molecular mass of 116 kDa to 125 kDa. Treatment with either bacterial or potato phosphatase reverses the mobility shift and abolishes enzyme activity. Incubation of maturing X. laevis eggs with radioactive inorganic phosphate and subsequent immunoprecipitation demonstrate that the PARP protein is phosphorylated in vivo. We show that maturation-promoting factor (Cyclin B/cdc2) cannot itself be responsible for the phosphorylation and activation of PARP in maturing X. laevis eggs. Together, these results demonstrate that the enzyme activity of PARP in X. laevis oocytes and eggs is regulated by post-translational, covalent phosphorylation. PMID:9230139

  13. Paradoxical control properties of enzymes within pathways: can activation cause an enzyme to have increased control?

    PubMed Central

    Kholodenko, B N; Brown, G C

    1996-01-01

    It is widely assumed that within a metabolic pathway inhibition of an enzyme causes the control exerted by that enzyme over the flux through its own reaction to increase, whereas activation causes its control to decrease. This assumption forms the basis of a number of experimental methods. For a pathway conceptually divided into two enzyme groups connected via a single metabolite we have derived a general condition under which this assumption is false, and thus the pathway shows paradoxical control behaviour, i.e. increased control with activation and decreased control with inhibition of an enzyme or group of enzymes. Paradoxical control behaviour occurs widely when enzyme activity is altered by changing Km (if an enzyme is already close to saturation by its substrate), but may also occur with changes in Vmax. when the elasticity to the linking metabolite increases with its concentration (as in some cases of sigmoidal and exponential kinetics or for reactions catalysed by isoenzymes). These findings suggest that enzymes with sigmoidal kinetics may have low control in the absence of activation, but may gain control with activation, and thus have beneficial regulatory properties. PMID:8615766

  14. The effect of dietary cricket meal (Gryllus bimaculatus) on growth performance, antioxidant enzyme activities, and haematological response of African catfish (Clarias gariepinus).

    PubMed

    Taufek, Norhidayah Mohd; Aspani, Firdaus; Muin, Hasniyati; Raji, Ameenat Abiodun; Razak, Shaharudin Abdul; Alias, Zazali

    2016-08-01

    This study was conducted to investigate the growth performance, biomarkers of oxidative stress, catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST) as well as the haematological response of African catfish after being fed with fish feed containing different levels of cricket meal. The juvenile fish were assigned to three different treatments with isonitrogenous (35 %) and isoenergetic (19 kJ g(-1)) diets containing 100 % cricket meal (100 % CM), 75 % cricket meal (75 % CM), and 100 % fishmeal (100 % FM) as control groups for 7 weeks. The results indicated that a diet containing 100 % CM and 75 % CM improved growth performance in terms of body weight gain and specific growth rate, when compared to 100 % FM. The feed conversion ratio (FCR) and protein efficiency ratio (PER) did not differ significantly between all diets, but reduced FCR and increased PER were observed with a higher inclusion of cricket meal. A haematological examination of fish demonstrated no significant difference of red blood cells in all diets and white blood cells showed a significantly higher value in fishmeal-fed fish. On the other hand, haemoglobin and haematocrit significantly increased with increasing amounts of cricket meal in the diet. Antioxidant activity of CAT was higher in the 100 % CM group compared to fish fed other diets, whereas GST and SOD showed increasing trends with a higher incorporation of cricket, although insignificant differences were observed between all diets. These results suggest that cricket meal could be an alternative to fishmeal as a protein source in the African catfish diet. PMID:26886132

  15. Spatial distribution of enzyme activities in the rhizosphere

    NASA Astrophysics Data System (ADS)

    Razavi, Bahar S.; Zarebanadkouki, Mohsen; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    The rhizosphere, the tiny zone of soil surrounding roots, certainly represents one of the most dynamic habitat and interfaces on Earth. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. That is why there is an urgent need in spatially explicit methods for the determination of the rhizosphere extension and enzyme distribution. Recently, zymography as a new technique based on diffusion of enzymes through the 1 mm gel plate for analysis has been introduced (Spohn & Kuzyakov, 2013). We developed the zymography technique to visualize the enzyme activities with a higher spatial resolution. For the first time, we aimed at quantitative imaging of enzyme activities as a function of distance from the root tip and the root surface in the soil. We visualized the two dimensional distribution of the activity of three enzymes: β-glucosidase, phosphatase and leucine amino peptidase in the rhizosphere of maize using fluorogenically labelled substrates. Spatial-resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography visualized heterogeneity of enzyme activities along the roots. The activity of all enzymes was the highest at the apical parts of individual roots. Across the roots, the enzyme activities were higher at immediate vicinity of the roots (1.5 mm) and gradually decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify spatial distribution of enzyme activities in the rhizosphere hotspots. References Spohn, M., Kuzyakov, Y., 2013. Phosphorus mineralization can be driven by microbial need for carbon. Soil Biology & Biochemistry 61: 69-75

  16. Self-Assembly of Amyloid Fibrils That Display Active Enzymes

    PubMed Central

    Zhou, Xiao-Ming; Entwistle, Aiman; Zhang, Hong; Jackson, Antony P; Mason, Thomas O; Shimanovich, Ulyana; Knowles, Tuomas P J; Smith, Andrew T; Sawyer, Elizabeth B; Perrett, Sarah

    2014-01-01

    Enzyme immobilization is an important strategy to enhance the stability and recoverability of enzymes and to facilitate the separation of enzymes from reaction products. However, enzyme purification followed by separate chemical steps to allow immobilization on a solid support reduces the efficiency and yield of the active enzyme. Here we describe polypeptide constructs that self-assemble spontaneously into nanofibrils with fused active enzyme subunits displayed on the amyloid fibril surface. We measured the steady-state kinetic parameters for the appended enzymes in situ within fibrils and compare these with the identical protein constructs in solution. Finally, we demonstrated that the fibrils can be recycled and reused in functional assays both in conventional batch processes and in a continuous-flow microreactor. PMID:25937845

  17. Intracellular localization of mevalonate-activating enzymes in plant cells

    PubMed Central

    Rogers, L. J.; Shah, S. P. J.; Goodwin, T. W.

    1966-01-01

    Mevalonate-activating enzymes are shown to be present in the chloroplasts of French-bean leaves. The chloroplast membrane is impermeable to mevalonic acid. Mevalonate-activating enzymes also appear to be found outside the chloroplast. These results support the view that terpenoid biosynthesis in the plant cell is controlled by a combination of enzyme segregation and specific membrane permeability. ImagesFig. 1.Fig. 2. PMID:5947149

  18. Plasma lysosomal enzyme activity in acute myocardial infarction.

    PubMed

    Welman, E; Selwyn, A P; Peters, T J; Colbeck, J F; Fox, K M

    1978-02-01

    N-acetyl-beta-glucosaminidase (EC 3.2.1.30, recommended name beta-N-Acetylglucosaminidase) was found to be a constituent of human cardiac lysosomes. beta-glucuronidase was also found in this tissue, while lysozyme, an enzyme present in leucocyte lysosomes, was not detectable in the heart. The activities of both N-acetyl-beta-glucosaminidase and beta-glucuronidase were elevated in plasma during the first 24 h after the onset of chest pain in patients with acute myocardial infarction and the peak levels of N-acetyl-beta-glucosaminidase correlated well with those of creatine kinase. N-acetyl-beta-glucosaminidase showed a further rise in plasma activity which gave a peak at 72 h after the onset of chest pain and this was accompanied by a rise in lysozyme activity. It is suggested that lysosome disruption caused by myocardial cell necrosis was responsible for the initial rise in plasma lysosomal enzyme activity and that the subsequent inflammatory reaction gave rise to the second peak. PMID:647716

  19. Enzyme family-specific and activity-based screening of chemical libraries using enzyme microarrays.

    PubMed

    Funeriu, Daniel P; Eppinger, Jörg; Denizot, Lucile; Miyake, Masato; Miyake, Jun

    2005-05-01

    The potential of protein microarrays in high-throughput screening (HTS) still remains largely unfulfilled, essentially because of the difficulty of extracting meaningful, quantitative data from such experiments. In the particular case of enzyme microarrays, low-molecular-weight fluorescent affinity labels (FALs) can function as ideally suited activity probes of the microarrayed enzymes. FALs form covalent bonds with enzymes in an activity-dependent manner and therefore can be used to characterize enzyme activity at each enzyme's address, as predetermined by the microarraying process. Relying on this principle, we introduce herein thematic enzyme microarrays (TEMA). In a kinetic setup we used TEMAs to determine the full set of kinetic constants and the reaction mechanism between the microarrayed enzymes (the theme of the microarray) and a family-wide FAL. Based on this kinetic understanding, in an HTS setup we established the practical and theoretical methodology for quantitative, multiplexed determination of the inhibition profile of compounds from a chemical library against each microarrayed enzyme. Finally, in a validation setup, K(i)(app) values and inhibitor profiles were confirmed and refined. PMID:15821728

  20. A Simple and Accurate Method for Measuring Enzyme Activity.

    ERIC Educational Resources Information Center

    Yip, Din-Yan

    1997-01-01

    Presents methods commonly used for investigating enzyme activity using catalase and presents a new method for measuring catalase activity that is more reliable and accurate. Provides results that are readily reproduced and quantified. Can also be used for investigations of enzyme properties such as the effects of temperature, pH, inhibitors,…

  1. Activity of cholinesterases, pyruvate kinase and adenosine deaminase in rats experimentally infected by Fasciola hepatica: Influences of these enzymes on inflammatory response and pathological findings.

    PubMed

    Baldissera, Matheus D; Bottari, Nathieli B; Mendes, Ricardo E; Schwertz, Claiton I; Lucca, Neuber J; Dalenogare, Diessica; Bochi, Guilherme V; Moresco, Rafael N; Morsch, Vera M; Schetinger, Maria R C; Rech, Virginia C; Jaques, Jeandre A; Da Silva, Aleksandro S

    2015-11-01

    The aim of this study was to investigate acetylcholinesterase (AChE) in total blood and liver tissue; butyrylcholinesterase (BChE) in serum and liver tissue; adenosine deaminase (ADA) in serum and liver tissue; and pyruvate kinase (PK) in liver tissue of rats experimentally infected by Fasciola hepatica. Animals were divided into two groups with 12 animals each, as follows: group A (uninfected) and group B (infected). Samples were collected at 20 (A1 and B1;n=6 each) and 150 (A2 and B2; n=6 each) days post-infection (PI). Infected animals showed an increase in AChE activity in whole blood and a decrease in AChE activity in liver homogenates (P<0.05) at 20 and 150 days PI. BChE and PK activities were decreased (P<0.05) in serum and liver homogenates of infected animals at 150 days PI. ADA activity was decreased in serum at 20 and 150 days PI, while in liver homogenates it was only decreased at 150 days PI (P<0.05). Aspartate aminotransferase and alanine aminotransferase activities in serum were increased (P<0.05), while concentrations of total protein and albumin were decreased (P<0.05) when compared to control. The histological analysis revealed fibrous perihepatitis and necrosis. Therefore, we conclude that the liver fluke is associated with cholinergic and purinergic dysfunctions, which in turn may influence the pathogenesis of the disease. PMID:26452485

  2. Enzyme-responsive protein/polysaccharide supramolecular nanoparticles.

    PubMed

    Hou, Xiao-Fang; Chen, Yong; Liu, Yu

    2015-03-28

    Biocompatible and enzyme-responsive supramolecular assemblies have attracted more and more interest in biomaterial fields, and find many feasible applications especially in the controlled drug release at specific sites where the target enzyme is located. In this work, novel supramolecular nanoparticles were successfully constructed from two biocompatible materials, i.e. a cyclic polysaccharide named sulfato-β-cyclodextrin (SCD) and a protein named protamine, through non-covalent association, and fully characterized by means of atomic force microscopy (AFM) and high-resolution transmission electron microscopy (TEM). Significantly, the disassembly of the resulting nanoparticles can respond especially to trypsin over other enzymes. Owing to their trypsin-triggered disassembly behaviors, these nanoparticles can efficiently release the encapsulated model substrate in a controlled manner. That is, the model substrate can be encapsulated inside the nanoparticles with a high stability and released when treated with trypsin. PMID:25679755

  3. Oltipraz-induced phase 2 enzyme response conserved in cells lacking mitochondrial DNA.

    PubMed

    Chua, Yee Liu; Zhang, Dawei; Boelsterli, Urs; Moore, Philip K; Whiteman, Matthew; Armstrong, Jeffrey S

    2005-11-11

    Oltipraz, a member of a class of 1,2-dithiolethiones, is a potent phase 2 enzyme inducing agent used as a cancer chemopreventive. In this study, we investigated regulation of the phase 2 enzyme response and protection against endogenous oxidative stress in lymphoblastic leukemic parental CEM cells and cells lacking mitochondrial DNA (mtDNA) (rho0) by oltipraz. Glutathione (GSH) levels (total and mitochondrial) and glutathione S-transferase (GST) activity were significantly increased after pretreatment with oltipraz in both parental (rho+) and rho0 cells, and both cell lines were resistant to mitochondrial oxidation, loss of mitochondrial membrane potential, and cell death in response to the GSH depleting agent diethylmaleate. These results show that the phase 2 enzyme response, by enhancing GSH-dependent systems involved in xenobiotic metabolism, blocks endogenous oxidative stress and cell death, and that this response is intact in cells lacking mtDNA. PMID:16188238

  4. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  5. Enzyme Activity Profiles during Fruit Development in Tomato Cultivars and Solanum pennellii1[W][OA

    PubMed Central

    Steinhauser, Marie-Caroline; Steinhauser, Dirk; Koehl, Karin; Carrari, Fernando; Gibon, Yves; Fernie, Alisdair R.; Stitt, Mark

    2010-01-01

    Enzymes interact to generate metabolic networks. The activities of more than 22 enzymes from central metabolism were profiled during the development of fruit of the modern tomato cultivar Solanum lycopersicum ‘M82’ and its wild relative Solanum pennellii (LA0716). In S. pennellii, the mature fruit remains green and contains lower sugar and higher organic acid levels. These genotypes are the parents of a widely used near introgression line population. Enzymes were also profiled in a second cultivar, S. lycopersicum ‘Moneymaker’, for which data sets for the developmental changes of metabolites and transcripts are available. Whereas most enzyme activities declined during fruit development in the modern S. lycopersicum cultivars, they remained high or even increased in S. pennellii, especially enzymes required for organic acid synthesis. The enzyme profiles were sufficiently characteristic to allow stages of development and cultivars and the wild species to be distinguished by principal component analysis and clustering. Many enzymes showed coordinated changes during fruit development of a given genotype. Comparison of the correlation matrices revealed a large overlap between the two modern cultivars and considerable overlap with S. pennellii, indicating that despite the very different development responses, some basic modules are retained. Comparison of enzyme activity, metabolite profiles, and transcript profiles in S. lycopersicum ‘Moneymaker’ revealed remarkably little connectivity between the developmental changes of transcripts and enzymes and even less between enzymes and metabolites. We discuss the concept that the metabolite profile is an emergent property that is generated by complex network interactions. PMID:20335402

  6. Long-term soil microbial community and enzyme activity responses to an integrated cropping-livestock system in a semi-arid region

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study is part of a larger long-term project to develop and evaluate integrated crop and livestock systems in order to reduce dependence on underground water sources by optimizing cotton (Gossypium hirsutum) production in the Texas High Plains of U.S. Microbial communities and activities were e...

  7. Enzyme activities along a latitudinal transect in Western Siberia

    NASA Astrophysics Data System (ADS)

    Schnecker, Jörg; Wild, Birgit; Eloy Alves, Ricardo J.; Gentsch, Norman; Gittel, Antje; Knoltsch, Anna; Lashchinskiy, Nikolay; Mikutta, Robert; Takriti, Mounir; Richter, Andreas

    2014-05-01

    Decomposition of soil organic matter (SOM) and thus carbon and nutrient cycling in soils is mediated by the activity of extracellular enzymes. The specific activities of these enzymes and their ratios to each other represent the link between the composition of soil organic matter and the nutrient demand of the microbial community. Depending on the difference between microbial nutrient demand and substrate availability, extracellular enzymes can enhance or slow down different nutrient cycles in the soil. We investigated activities of six extracellular enzymes (cellobiohydrolase, leucine-amino-peptidase, N-acetylglucosaminidase, chitotriosidase, phosphatase and phenoloxidase) in the topsoil organic horizon, topsoil mineral horizon and subsoil horizon in seven ecosystems along a 1,500 km-long North-South transect in Western Siberia. The transect included sites in the southern tundra, northern taiga, middle taiga, southern taiga, forest-steppe (in forested patches as well as in adjacent meadows) and Steppe. We found that enzyme patterns varied stronger with soil depth than between ecosystems. Differences between horizons were mainly based on the increasing ratio of oxidative enzymes to hydrolytic enzymes. Differences between sites were more pronounced in topsoil than in subsoil mineral horizons, but did not reflect the north-south transect and the related gradients in temperature and precipitation. The observed differences between sites in topsoil horizons might therefore result from differences in vegetation rather than climatic factors. The decreasing variability in the enzyme pattern with depth might also indicate that the composition of soil organic matter becomes more similar with soil depth, most likely by an increasing proportion of microbial remains compared to plant derived constituents of SOM. This also indicates, that SOM becomes less divers the more it is processed by soil microorganisms. Our findings highlight the importance of soil depth on enzyme

  8. Activation and stabilization of enzymes in ionic liquids.

    PubMed

    Moniruzzaman, Muhammad; Kamiya, Noriho; Goto, Masahiro

    2010-06-28

    As environmentally benign "green" solvents, room temperature ionic liquids (ILs) have been used as solvents or (co)solvents in biocatalytic reactions and processes for a decade. The technological utility of enzymes can be enhanced greatly by their use in ionic liquids (ILs) rather than in conventional organic solvents or in their natural aqueous reaction media. In fact, the combination of green properties and unique tailor-made physicochemical properties make ILs excellent non-aqueous solvents for enzymatic catalysis with numerous advantages over other solvents, including high conversion rates, high selectivity, better enzyme stability, as well as better recoverability and recyclability. However, in many cases, particularly in hydrophilic ILs, enzymes show relative instability and/or lower activity compared with conventional solvents. To improve the enzyme activity as well as stability in ILs, various attempts have been made by modifying the form of the enzymes. Examples are enzyme immobilization onto support materials via adsorption or multipoint attachment, lyophilization in the presence of stabilizing agents, chemical modification with stabilizing agents, formation of cross-linked enzyme aggregates, pretreatment with polar organic solvents or enzymes combined with suitable surfactants to form microemulsions. The use of these enzyme preparations in ILs can dramatically increase the solvent tolerance, enhance activity as well as stability, and improve enantioselectivity. This perspective highlights a number of pronounced strategies being used successfully for activation and stabilization of enzymes in non-aqueous ILs media. This review is not intended to be comprehensive, but rather to present a general overview of the potential approaches to activate enzymes for diverse enzymatic processes and biotransformations in ILs. PMID:20445940

  9. Changes in the spectrum and rates of extracellular enzyme activities in seawater following aggregate formation

    NASA Astrophysics Data System (ADS)

    Ziervogel, K.; Steen, A. D.; Arnosti, C.

    2010-03-01

    Marine snow aggregates are heavily colonized by heterotrophic microorganisms that express high levels of hydrolytic activities, making aggregates hotspots for carbon remineralization in the ocean. To assess how aggregate formation influences the ability of seawater microbial communities to access organic carbon, we compared hydrolysis rates of six polysaccharides in coastal seawater after aggregates had been formed (via incubation on a roller table) with hydrolysis rates in seawater from the same site that had not incubated on a roller table (referred to as whole seawater). Hydrolysis rates in the aggregates themselves were up to three orders of magnitude higher on a volume basis than in whole seawater. The enhancement of enzyme activity in aggregates relative to whole seawater differed by substrate, suggesting that the enhancement was under cellular control, rather than due to factors such as lysis or grazing. A comparison of hydrolysis rates in whole seawater with those in aggregate-free seawater, i.e. the fraction of water from the roller bottles that did not contain aggregates, demonstrated a nuanced microbial response to aggregate formation. Activities of laminarinase and xylanase enzymes in aggregate-free seawater were higher than in whole seawater, while activities of chondroitin, fucoidan, and arabinogalactan hydrolyzing enzymes were lower than in whole seawater. These data suggest that aggregate formation enhanced production of laminarinase and xylanase enzymes, and the enhancement also affected the surrounding seawater. Decreased activities of chondroitin, fucoidan, and arabinoglactan-hydrolyzing enzymes in aggregate-free seawaters relative to whole seawater are likely due to shifts in enzyme production by the aggregate-associated community, coupled with the effects of enzyme degradation. Enhanced activities of laminarin- and xylan-hydrolyzing enzymes in aggregate-free seawater were due at least in part to cell-free enzymes. Measurements of enzyme

  10. TREATABILITY STUDY BULLETIN: ENZYME-ACTIVATED CELLULOSE TECHNOLOGY - THORNECO, INC

    EPA Science Inventory

    The Enzyme-Activated Cellulose Technology developed by Thorneco, Inc. uses cellulose placed into one or more cylindrical towers to remove metals and organic compounds from an aqueous solution. The cellulose is coated with a proprietary enzyme. Operating parameters that can affe...

  11. Photoreactivating enzyme activity in the rat tapeworm, Hymenolepis diminuta

    SciTech Connect

    Woodhead, A.D.; Achey, P.M.

    1981-01-01

    There has been considerable speculation about the occurrence of photoreactivating enzyme in different organisms and about its biologic purpose. We have developed a simple, sensitive assay for estimating pyrimidine dimers in DNA which is useful in making a rapid survey for the presence of the enzyme. Using this method, we have found photoreactivating enzyme activity in the tissues of the rat tapeworm, Hymenolepis diminuta. This parasite spends the majority of its life span in the bodies of its definitive or intermediate hosts, but a period is spent externally. We suggest that photoreactivating enzyme may be important in perserving the integrity of embryonic DNA during this free-living stage.

  12. Photoreactivating enzyme activity in the rat tapeworm, Hymenolepis diminuta

    SciTech Connect

    Woodhead, A.D.; Achey, P.M.

    1981-06-01

    There has been considerable speculation about the occurrence of photoreactivating enzyme in different organisms and about its biological purpose. We have developed a simple, sensitive assay for estimating pyrimidine dimers in DNA which is useful in making a rapid survey for the presence of the enzyme. Using this method, we have found photoreactivating enzyme activity in the tissues of the rat tapeworm Hymenolepis diminuta. This parasite spends the majority of its life span in the bodies of its definitive or intermediate hosts, but a period is spent externally. We suggest that photoreactivating enzyme may be important in preserving the integrity of embryonic DNA during this free-living stage.

  13. Activation of immobilized enzymes by acoustic wave resonance oscillation.

    PubMed

    Nishiyama, Hiroshi; Watanabe, Tomoya; Inoue, Yasunobu

    2014-12-01

    Acoustic wave resonance oscillation has been used successfully in the development of methods to activate immobilized enzyme catalysts. In this study, resonance oscillation effects were demonstrated for enzyme reactions on galactose oxidase (GAD), D-amino acid oxidase (DAAO), and L-amino acid oxidase (LAAO), all of which were immobilized covalently on a ferroelectric lead zirconate titanate (PZT) device that could generate thickness-extensional resonance oscillations (TERO) of acoustic waves. For galactose oxidation on immobilized GAD in a microreactor, TERO generation immediately increased enzyme activity 2- to 3-fold. Eliminating TERO caused a slight decrease in the activity, with ∼90% of the enhanced activity retained while the reaction proceeded. Contact of the enhanced enzyme with a galactose-free solution caused almost complete reversion of the activity to the original low level before TERO generation, indicating that, not only TERO-induced GAD activation, but also preservation of the increased activity, required a galactose substrate. Similar activity changes with TERO were observed for enzyme reactions on DAAO and LAAO. Kinetic analysis demonstrated that TERO helped strengthen the interactions of the immobilized enzyme with the reactant substrate and promoted formation of an activation complex. PMID:25442945

  14. Analysis of five rice 4-coumarate:coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice

    SciTech Connect

    Sun, Haiyan; Li, Ying; Feng, Shengqiu; Zou, Weihua; Guo, Kai; Fan, Chunfen; Si, Shengli; and others

    2013-01-18

    Highlights: ► 4CLs play important roles in both lignin and flavonoids biosynthesis. ► PA and FA are the two main substrates of 4CL (Os4CL1/3/4/5) for lignin biosynthesis. ► Os4CL2 is suggested for flavonoid formation in defense against UV radiation. -- Abstract: 4-Coumarate:coenzyme A ligase (4CL) catalyzes the conversion of hydroxycinnamates into corresponding CoA esters for biosynthesis of flavonoids and lignin. In this study, five members of the 4CL gene family from rice were cloned and analyzed. Recombinant 4CL data revealed that 4-coumaric acid and ferulic acid were the two main substrates of 4CL (Os4CL1/3/4/5) for monolignol biosynthesis in rice. Os4CL2 was specifically expressed in the anther and was strongly activated by UV irradiation, suggesting its potential involvement in flavonoid formation. Moreover, bioinformatics analysis showed that the existence of valine residue at the substrate-binding pocket may mainly affect rice 4CL activities toward sinapic acid.

  15. Effects of ursodeoxycholic acid on serum liver enzymes and bile acid metabolism in chronic active hepatitis: a dose-response study.

    PubMed

    Crosignani, A; Battezzati, P M; Setchell, K D; Camisasca, M; Bertolini, E; Roda, A; Zuin, M; Podda, M

    1991-02-01

    The effect of ursodeoxycholic acid administration on liver function tests and on bile acid metabolism was investigated in 18 patients with chronic active hepatitis. Three different doses of ursodeoxycholic acid--250 mg, 500 mg and 750 mg--were administered daily to each patient for consecutive 2-mo periods. The order of doses was randomly assigned according to a replicated Latin-square design. A significant decrease in serum transaminases and gamma-glutamyl transpeptidase occurred with the lowest dose of ursodeoxycholic acid, which corresponded to 4 mg/kg body wt/day, and no further significant decrease with the higher doses was seen. Biliary bile acid composition was determined by high-performance liquid chromatography and gas chromatography-mass spectrometry. At entry the relative proportions of major bile acids were similar to those observed in normal individuals. During treatment the mean percentage of ursodeoxycholic acid in bile (22% with the 250 mg dose, 32% with the 500 mg dose and 34% with the 750 mg dose) was lower than values previously reported for patients with gallstones and normal liver function. The major bile acids were cholic, chenodeoxycholic and deoxycholic acids. A number of unusual bile acids were identified by gas chromatography-mass spectrometry, but these accounted for only 3% to 5% of the total and did not change during ursodeoxycholic acid therapy. No correlation between the improvement in liver function tests and the percentage of ursodeoxycholic acid in bile existed. These data suggest that even a slight enrichment of bile with ursodeoxycholic acid, as is attained with 250 mg/day, is effective in improving biochemical markers of liver function in patients with chronic active hepatitis. PMID:1671665

  16. Hawthorn (Crataegus oxyacantha L.) bark extract regulates antioxidant response element (ARE)-mediated enzyme expression via Nrf2 pathway activation in normal hepatocyte cell line.

    PubMed

    Krajka-Kuźniak, Violetta; Paluszczak, Jarosław; Oszmiański, Jan; Baer-Dubowska, Wanda

    2014-04-01

    Hawthorn (Crataegus oxyacantha L.), a plant used in traditional medicine, is a rich source of procyanidins which have been reported to exhibit antioxidant and anti-carcinogenic activity. In this study, we assessed the effect of hawthorn bark extract (HBE) on Nrf2 pathway activation in THLE-2 and HepG2 cells. Treatment with 1.1 µg/mL, 5.5 µg/mL and 11 µg/mL of HBE resulted in the translocation of Nrf2 from the cytosol to the nucleus in both cell lines; however, the accumulation of phosphorylated Nrf2 was observed only in THLE-2. Accordingly, treatment of cells with HBE was associated with an increase in the mRNA and protein level of such Nrf2-dependent genes as glutathione S-transferases (GSTA, GSTP, GSTM, GSTT), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) (0.2-1.1-fold change, p < 0.05), however, only in normal THLE-2 hepatocytes. The induction of NQO1 correlated with an increased level of p53 (0.21-0.42-fold change, p < 0.05). These effects may be related to induction of phosphorylation of upstream ERK and JNK kinases. Collectively, the results suggest that the Nrf2/ARE pathway may play an important role in the regulation of procyanidin-mediated antioxidant/detoxifying effects in hepatocytes, and this may explain the hepatoprotective and chemopreventive properties of these phytochemicals. PMID:23843400

  17. Lipid metabolizing enzyme activities modulated by phospholipid substrate lateral distribution.

    PubMed

    Salinas, Dino G; Reyes, Juan G; De la Fuente, Milton

    2011-09-01

    Biological membranes contain many domains enriched in phospholipid lipids and there is not yet clear explanation about how these domains can control the activity of phospholipid metabolizing enzymes. Here we used the surface dilution kinetic theory to derive general equations describing how complex substrate distributions affect the activity of enzymes following either the phospholipid binding kinetic model (which assumes that the enzyme molecules directly bind the phospholipid substrate molecules), or the surface-binding kinetic model (which assumes that the enzyme molecules bind to the membrane before binding the phospholipid substrate). Our results strongly suggest that, if the enzyme follows the phospholipid binding kinetic model, any substrate redistribution would increase the enzyme activity over than observed for a homogeneous distribution of substrate. Besides, enzymes following the surface-binding model would be independent of the substrate distribution. Given that the distribution of substrate in a population of micelles (each of them a lipid domain) should follow a Poisson law, we demonstrate that the general equations give an excellent fit to experimental data of lipases acting on micelles, providing reasonable values for kinetic parameters--without invoking special effects such as cooperative phenomena. Our theory will allow a better understanding of the cellular-metabolism control in membranes, as well as a more simple analysis of the mechanisms of membrane acting enzymes. PMID:21108012

  18. Function and biotechnology of extremophilic enzymes in low water activity

    PubMed Central

    2012-01-01

    Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology. PMID:22480329

  19. Sustained gastrointestinal activity of dendronized polymer-enzyme conjugates

    NASA Astrophysics Data System (ADS)

    Fuhrmann, Gregor; Grotzky, Andrea; Lukić, Ružica; Matoori, Simon; Luciani, Paola; Yu, Hao; Zhang, Baozhong; Walde, Peter; Schlüter, A. Dieter; Gauthier, Marc A.; Leroux, Jean-Christophe

    2013-07-01

    Methods to stabilize and retain enzyme activity in the gastrointestinal tract are investigated rarely because of the difficulty of protecting proteins from an environment that has evolved to promote their digestion. Preventing the degradation of enzymes under these conditions, however, is critical for the development of new protein-based oral therapies. Here we show that covalent conjugation to polymers can stabilize orally administered therapeutic enzymes at different locations in the gastrointestinal tract. Architecturally and functionally diverse polymers are used to protect enzymes sterically from inactivation and to promote interactions with mucin on the stomach wall. Using this approach the in vivo activity of enzymes can be sustained for several hours in the stomach and/or in the small intestine. These findings provide new insight and a firm basis for the development of new therapeutic and imaging strategies based on orally administered proteins using a simple and accessible technology.

  20. Using shotgun sequence data to find active restriction enzyme genes.

    PubMed

    Zheng, Yu; Posfai, Janos; Morgan, Richard D; Vincze, Tamas; Roberts, Richard J

    2009-01-01

    Whole genome shotgun sequence analysis has become the standard method for beginning to determine a genome sequence. The preparation of the shotgun sequence clones is, in fact, a biological experiment. It determines which segments of the genome can be cloned into Escherichia coli and which cannot. By analyzing the complete set of sequences from such an experiment, it is possible to identify genes lethal to E. coli. Among this set are genes encoding restriction enzymes which, when active in E. coli, lead to cell death by cleaving the E. coli genome at the restriction enzyme recognition sites. By analyzing shotgun sequence data sets we show that this is a reliable method to detect active restriction enzyme genes in newly sequenced genomes, thereby facilitating functional annotation. Active restriction enzyme genes have been identified, and their activity demonstrated biochemically, in the sequenced genomes of Methanocaldococcus jannaschii, Bacillus cereus ATCC 10987 and Methylococcus capsulatus. PMID:18988632

  1. Silk microgels formed by proteolytic enzyme activity.

    PubMed

    Samal, Sangram K; Dash, Mamoni; Chiellini, Federica; Kaplan, David L; Chiellini, Emo

    2013-09-01

    The proteolytic enzyme α-chymotrypsin selectively cleaves the amorphous regions of silk fibroin protein (SFP) and allows the crystalline regions to self-assemble into silk microgels (SMGs) at physiological temperature. These microgels consist of lamellar crystals in the micrometer scale, in contrast to the nanometer-scaled crystals in native silkworm fibers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zeta potential results demonstrated that α-chymotrypsin utilized only the non-amorphous domains or segments of the heavy chain of SFP to form negatively charged SMGs. The SMGs were characterized in terms of size, charge, structure, morphology, crystallinity, swelling kinetics, water content and thermal properties. The results suggest that the present technique of preparing SMGs by α-chymotrypsin is simple and efficient, and that the prepared SMGs have useful features for studies related to biomaterial and pharmaceutical needs. This process is also an easy way to obtain the amorphous peptide chains for further study. PMID:23756227

  2. Changes in the spectrum and rates of extracellular enzyme activities in seawater following aggregate formation

    NASA Astrophysics Data System (ADS)

    Ziervogel, K.; Steen, A. D.; Arnosti, C.

    2009-12-01

    Marine snow aggregates are heavily colonized by heterotrophic microorganisms that express high levels of hydrolytic activities, making aggregates hotspots for carbon remineralization in the ocean. To assess how aggregate formation influences the ability of seawater microbial communities to access organic carbon, we compared hydrolysis rates of six polysaccharides in coastal seawater after aggregates had been formed (via incubation on a roller table) with hydrolysis rates in seawater from the same site that had not incubated on a roller table (referred to as whole seawater). Hydrolysis rates in the aggregates themselves were up to three orders of magnitude higher on a volume basis than in whole seawater. The enhancement of enzyme activity in aggregates relative to whole seawater differed by substrate, suggesting that the enhancement was under cellular control, rather than due to factors such as lysis or grazing. A comparison of hydrolysis rates in whole seawater with those in aggregate-free seawater, i.e. the fraction of water from the roller bottles that did not contain aggregates, demonstrated a nuanced microbial response to aggregate formation. Activities of laminarinase and xylanase enzymes in aggregate-free seawater were higher than in whole seawater, while activities of chondroitin, fucoidan, and arabinogalactan hydrolyzing enzymes were lower than in whole seawater. These data suggest that aggregate formation enhanced production of laminarinase and xylanase enzymes, and the enhancement also affected the surrounding seawater. Decreased activities of chondroitin, fucoidan, and arabinoglactan-hydrolyzing enzymes in aggregate-free seawater relative to whole seawater are likely due to shifts in enzyme production by the aggregate-associated community, coupled with the effects of enzyme degradation. Enhanced activities of laminarin- and xylan-hydrolyzing enzymes in aggregate-free seawater were due at least in part to cell-free enzymes. Measurements of enzyme lifetime

  3. Synergetic Effects of Nanoporous Support and Urea on Enzyme Activity

    SciTech Connect

    Lei, Chenghong; Shin, Yongsoon; Liu, Jun; Ackerman, Eric J.

    2007-02-01

    Here we report that synergetic effects of functionalized nanoporous support and urea on enzyme activity enhancement. Even in 8.0 M urea, the specific activity of GI entrapped in FMS was still higher than the highest specific activity of GI free in solution, indicating the strong tolerance of GI in FMS to the high concentration of urea.

  4. Supramolecular sensing: Enzyme activity with a twist

    NASA Astrophysics Data System (ADS)

    Amabilino, David B.

    2015-04-01

    A supramolecular polymer comprising stacked artificial chromophores to which zinc(II) complexes are appended is able to respond to enzymatic hydrolysis in aqueous solution. The assembly of molecules can twist reversibly and quickly in response to changes in the type of adenosine phosphate present.

  5. Photoregulation of Biological Activity by Photochromic Reagents, IV. A Model for Diurnal Variation of Enzymic Activity*

    PubMed Central

    Bieth, Joseph; Wassermann, Norbert; Vratsanos, Spyros M.; Erlanger, Bernard F.

    1970-01-01

    Levels of acetylcholinesterase activity can be made to vary in response to the presence or absence of sunlight in a system that can be considered as a model for photoperiodic processes found in nature. The enzyme is rendered photosensitive by the presence of a photochromic inhibitor, N-p-phenylazophenylcarbamyl choline, which changes from a trans to a cis isomer under the influence of the light of the sun and reverts back to the trans isomer in the dark. The two isomers differ in their ability acetylcholinesterase, thus rendering the enzyme system responsive to sunlight. The relationship of this system to photoresponsive processes in nature is discussed, and a possible role in photoregulation is suggested for naturally occurring carotenoids. PMID:5269248

  6. Inhibition of existing denitrification enzyme activity by chloramphenicol

    USGS Publications Warehouse

    Brooks, M.H.; Smith, R.L.; Macalady, D.L.

    1992-01-01

    Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (>50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log- phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.

  7. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  8. Activity of selected hydrolytic enzymes in Allium sativum L. anthers.

    PubMed

    Winiarczyk, Krystyna; Gębura, Joanna

    2016-05-01

    The aim of the study was to determine enzymatic activity in sterile Allium sativum anthers in the final stages of male gametophyte development (the stages of tetrads and free microspores). The analysed enzymes were shown to occur in the form of numerous isoforms. In the tetrad stage, esterase activity was predominant, which was manifested by the greater number of isoforms of the enzyme. In turn, in the microspore stage, higher numbers of isoforms of acid phosphatases and proteases were detected. The development of sterile pollen grains in garlic is associated with a high level of protease and acid phosphatase activity and lower level of esterase activities in the anther locule. Probably this is the first description of the enzymes activity (ACPH, EST, PRO) in the consecutives stages of cell wall formation which is considered to be one of the causes of male sterility in flowering plant. PMID:26901781

  9. Interfacial activation-based molecular bioimprinting of lipolytic enzymes.

    PubMed Central

    Mingarro, I; Abad, C; Braco, L

    1995-01-01

    Interfacial activation-based molecular (bio)-imprinting (IAMI) has been developed to rationally improve the performance of lipolytic enzymes in nonaqueous environments. The strategy combinedly exploits (i) the known dramatic enhancement of the protein conformational rigidity in a water-restricted milieu and (ii) the reported conformational changes associated with the activation of these enzymes at lipid-water interfaces, which basically involves an increased substrate accessibility to the active site and/or an induction of a more competent catalytic machinery. Six model enzymes have been assayed in several model reactions in nonaqueous media. The results, rationalized in light of the present biochemical and structural knowledge, show that the IAMI approach represents a straightforward, versatile method to generate manageable, activated (kinetically trapped) forms of lipolytic enzymes, providing under optimal conditions nonaqueous rate enhancements of up to two orders of magnitude. It is also shown that imprintability of lipolytic enzymes depends not only on the nature of the enzyme but also on the "quality" of the interface used as the template. PMID:7724558

  10. Salt stress induced lipid accumulation in heterotrophic culture cells of Chlorella protothecoides: Mechanisms based on the multi-level analysis of oxidative response, key enzyme activity and biochemical alteration.

    PubMed

    Wang, Tao; Ge, Haiyan; Liu, Tingting; Tian, Xiwei; Wang, Zejian; Guo, Meijin; Chu, Ju; Zhuang, Yingping

    2016-06-20

    Salt stress as an effective stress factor that could improve the lipid content and lipid yield of glucose in the heterotrophic culture cells of Chlorella protothecoides was demonstrated in this study. The highest lipid content of 41.2% and lipid yield of 185.8mg/g were obtained when C. protothecoides was stressed under 30g/L NaCl condition at its late logarithmic growth phase. Moreover, the effects of salt and osmotic stress on lipid accumulation were comparatively analyzed, and it was found that the effects of NaCl and KCl stress had no significant differences at the same osmolarity level of 1150mOsm/kg with lipid contents of 41.7 and 40.8% as well as lipid yields of 192.9 and 186.8mg/g, respectively, whereas these results were obviously higher than those obtained under the iso-osmotic glycerol and sorbitol stresses. Furthermore, basing on the multi-level analysis of oxidative response, key enzyme activity and biochemical alteration, the superior performance of salt stress driving lipid over-synthesis was probably ascribed to the more ROS production as a result of additional ion effect besides the osmotic effect, subsequently mediating the alteration from carbohydrate storage to lipid accumulation in signal transduction process of C. protothecoides. PMID:27085889

  11. Screening for enzyme activity in turbid suspensions with scattered light.

    PubMed

    Huber, Robert; Wulfhorst, Helene; Maksym, Lukas; Stehr, Regina; Pöhnlein, Martin; Jäger, Gernot; Spiess, Antje C; Büchs, Jochen

    2011-01-01

    New screening techniques for improved enzyme variants in turbid media are urgently required in many industries such as the detergent and food industry. Here, a new method is presented to measure enzyme activity in different types of substrate suspensions. This method allows a semiquantitative determination of protease activity using native protein substrates. Unlike conventional techniques for measurement of enzyme activity, the BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of samples in microtiter plates. The BioLector technique is hereby used to monitor the hydrolysis of an insoluble protein substrate by measuring the decrease of scattered light. The kinetic parameters for the enzyme reaction (V(max,app) and K(m,app)) are determined from the scattered light curves. Moreover, the influence of pH on the protease activity is investigated. The optimal pH value for protease activity was determined to be between pH 8 to 11 and the activities of five subtilisin serine proteases with variations in the amino acid sequence were compared. The presented method enables proteases from genetically modified strains to be easily characterized and compared. Moreover, this method can be applied to other enzyme systems that catalyze various reactions such as cellulose decomposition. PMID:21302369

  12. Enzyme-polymer composites with high biocatalytic activity and stability

    SciTech Connect

    Kim, Jungbae; Kosto, Timothy J.; Manimala, Joseph C.; Nauman, E B.; Dordick, Jonathan S.

    2004-08-22

    We have applied vacuum-spraying and electrospinning to incorporate an enzyme into a polymer matrix, creating a novel and highly active biocatalytic composite. As a unique technical approach, enzymes were co-dissolved in toluene with polymers, and the solvent was then rapidly removed by injecting the mixture into a vacuum chamber or by electrospinning. Subsequent crosslinking of the enzyme with glutaraldehyde resulted in stable entrapped enzyme within the polymeric matrices. For example, an amorphous composite of alpha-chymotrypsin and polyethylene showed no significant loss of enzymatic activity in aqueous buffer for one month. Nanofibers of alpha-chymotrypsin and polystyrene also showed no decrease in activity for more than two weeks. The normalized activity of amorphous composite in organic solvents was 3-13 times higher than that of native alpha-chymotrypsin. The activity of nanofibers was 5-7 times higher than that of amorphous composite in aqueous buffer solution. The composites of alpha-chymotrypsin and polymers demonstrate the feasibility of obtaining a wide variety of active and stable biocatalytic materials with many combinations of enzymes and polymers.

  13. Enzyme-enabled responsive surfaces for anti-contamination materials.

    PubMed

    Wu, Songtao; Buthe, Andreas; Jia, Hongfei; Zhang, Minjuan; Ishii, Masahiko; Wang, Ping

    2013-06-01

    Many real-life stains have origins from biological matters including proteins, lipids, and carbohydrates that act as gluing agents binding along with other particulates or microbes to exposed surfaces of automobiles, furniture, and fabrics. Mimicking naturally occurring self-defensive processes, we demonstrate in this work that a solid surface carrying partially exposed enzyme granules protected the surface in situ from contamination by biological stains and fingerprints. Attributed to the activities of enzymes which can be made compatible with a wide range of materials, such anti-contamination and self-cleaning functionalities are highly selective and efficient toward sticky chemicals. This observation promises a new mechanism in developing smart materials with desired anti-microbial, self-reporting, self-cleaning, or self-healing functions. PMID:23335427

  14. Chimeric enzymes with improved cellulase activities

    SciTech Connect

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  15. Improving Activity of Salt-Lyophilized Enzymes in Organic Media

    NASA Astrophysics Data System (ADS)

    Borole, Abhijeet P.; Davison, Brian H.

    Lyophilization with salts has been identified as an important method of activating enzymes in organic media. Using salt-activated enzymes to transform molecules tethered to solid surfaces in organic phase requires solubilization of enzymes in the solvents. Methods of improving performance of salt-lyophilized enzymes, further, via chemical modification, and use of surfactants and surfactants to create fine emulsions prior to lyophilization are investigated. The reaction system used is transesterification of N-acetyl phenylalanine ethyl ester with methanol or propanol. Initial rate of formation of amino acid esters by subtilisin Carlsberg (SC) was studied and found to increase two to sevenfold by either chemical modification or addition of surfactants in certain solvents, relative to the salt (only)-lyophilized enzyme. The method to prepare highly dispersed enzymes in a salt-surfactant milieu also improved activity by two to threefold. To test the effect of chemical modification on derivatization of drug molecules, acylation of bergenin was investigated using chemically modified SC.

  16. Distribution and activity of hydrogenase enzymes in subsurface sediments

    NASA Astrophysics Data System (ADS)

    Adhikari, R.; Nickel, J.; Glombitza, C.; Spivack, A. J.; D'Hondt, S. L.; Kallmeyer, J.

    2013-12-01

    Metabolically active microbial communities are present in a wide range of subsurface environments. Techniques like enumeration of microbial cells, activity measurements with radiotracer assays and the analysis of porewater constituents are currently being used to explore the subsurface biosphere, alongside with molecular biological analyses. However, many of these techniques reach their detection limits due to low microbial activity and abundance. Direct measurements of microbial turnover not just face issues of insufficient sensitivity, they only provide information about a single specific process rather than an overall microbial activity. Since hydrogenase enzymes are intracellular and ubiquitous in subsurface microbial communities, the enzyme activity represents a measure of total activity of the entire microbial community. A hydrogenase activity assay could quantify total metabolic activity without having to identify specific processes. This would be a major advantage in subsurface biosphere studies, where several metabolic processes can occur simultaneously. We quantified hydrogenase enzyme activity and distribution in sediment samples from different aquatic subsurface environments (Lake Van, Barents Sea, Equatorial Pacific and Gulf of Mexico) using a tritium-based assay. We found enzyme activity at all sites and depths. Volumetric hydrogenase activity did not show much variability between sites and sampling depths, whereas cell-specific activity ranged from 10-5 to 1 nmol H2 cell-1 d-1. Activity was lowest in sediment layers where nitrate was detected. Higher activity was associated with samples in which sulfate was the predominant electron acceptor. We found highest activity in samples from environments with >10 ppm methane in the pore water. The results show that cell-specific hydrogenase enzyme activity increases with decreasing energy yield of the electron acceptor used. It is not possible to convert volumetric or cell-specific hydrogenase activity into a

  17. Patterns of functional enzyme activity in fungus farming ambrosia beetles

    PubMed Central

    2012-01-01

    Introduction In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose

  18. Hepatic biotransformation and antioxidant enzyme activities in Mediterranean fish from different habitat depths.

    PubMed

    Ribalta, C; Sanchez-Hernandez, J C; Sole, M

    2015-11-01

    Marine fish are threatened by anthropogenic chemical discharges. However, knowledge on adverse effects on deep-sea fish or their detoxification capabilities is limited. Herein, we compared the basal activities of selected hepatic detoxification enzymes in several species (Solea solea, Dicentrarchus labrax, Trachyrhynchus scabrus, Mora moro, Cataetix laticeps and Alepocehalus rostratus) collected from the coast, middle and lower slopes of the Blanes Canyon region (Catalan continental margin, NW Mediterranean Sea). The xenobiotic-detoxifying enzymes analysed were the phase-I carboxylesterases (CbEs), and the phase-II conjugation activities uridine diphosphate glucuronyltransferase (UDPGT) and glutathione S-transferase (GST). Moreover, some antioxidant enzyme activities, i.e., catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR), were also included in this comparative study. Because CbE activity is represented by multiple isoforms, the substrates α-naphthyl acetate (αNA) and ρ-nitrophenyl acetate (ρNPA) were used in the enzyme assays, and in vitro inhibition kinetics with dichlorvos were performed to compare interspecific CbE sensitivity. Activity of xenobiotic detoxification enzymes varied among the species, following a trend with habitat depth and body size. Thus, UDPGT and some antioxidant enzyme activities decreased in fish inhabiting lower slopes of deep-sea, whereas UDPGT and αNA-CbE activities were negatively related to fish size. A trend between CbE activities and the IC50 values for dichlorvos suggested S. solea and M. moro as potentially more sensitive to anticholinesterasic pesticides, and T. scabrus as the most resistant one. A principal component analysis considering all enzyme activities clearly identified the species but this grouping was not related to habitat depth or phylogeny. Although these results can be taken as baseline levels of the main xenobiotic detoxification enzymes in Mediterranean fish, further research is

  19. Chemoproteomic profiling of host and pathogen enzymes active in cholera

    PubMed Central

    Hatzios, Stavroula K.; Hubbard, Troy; Sasabe, Jumpei; Munera, Diana; Clark, Lars; Bachovchin, Daniel A.; Qadri, Firdausi; Ryan, Edward T.; Davis, Brigid M.; Weerapana, Eranthie; Waldor, Matthew K.

    2016-01-01

    Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human cholera stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, while genetic disruption of all four proteases increased the abundance and binding of an intestinal lectin—intelectin—to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialogue in an animal model of infection. PMID:26900865

  20. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    SciTech Connect

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.L.; Bolon, Daniel N.A.; Schiffer, Celia A.

    2012-09-11

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

  1. A DNA enzyme with N-glycosylase activity

    NASA Technical Reports Server (NTRS)

    Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F.

    2000-01-01

    In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

  2. Activation Energy of Extracellular Enzymes in Soils from Different Biomes

    PubMed Central

    Steinweg, J. Megan; Jagadamma, Sindhu; Frerichs, Joshua; Mayes, Melanie A.

    2013-01-01

    Enzyme dynamics are being incorporated into soil carbon cycling models and accurate representation of enzyme kinetics is an important step in predicting belowground nutrient dynamics. A scarce number of studies have measured activation energy (Ea) in soils and fewer studies have measured Ea in arctic and tropical soils, or in subsurface soils. We determined the Ea for four typical lignocellulose degrading enzymes in the A and B horizons of seven soils covering six different soil orders. We also elucidated which soil properties predicted any measurable differences in Ea. β-glucosidase, cellobiohydrolase, phenol oxidase and peroxidase activities were measured at five temperatures, 4, 21, 30, 40, and 60°C. Ea was calculated using the Arrhenius equation. β-glucosidase and cellobiohydrolase Ea values for both A and B horizons in this study were similar to previously reported values, however we could not make a direct comparison for B horizon soils because of the lack of data. There was no consistent relationship between hydrolase enzyme Ea and the environmental variables we measured. Phenol oxidase was the only enzyme that had a consistent positive relationship between Ea and pH in both horizons. The Ea in the arctic and subarctic zones for peroxidase was lower than the hydrolases and phenol oxidase values, indicating peroxidase may be a rate limited enzyme in environments under warming conditions. By including these six soil types we have increased the number of soil oxidative enzyme Ea values reported in the literature by 50%. This study is a step towards better quantifying enzyme kinetics in different climate zones. PMID:23536898

  3. Characterization of amylolytic enzyme activities associated with the hyperthermophilic archaebacterium Pyrococcus furiosus

    SciTech Connect

    Brown, S.H.; Costantino, H.R.; Kelly, R.M. Univ. of Maryland, Baltimore )

    1990-07-01

    The hyperthermophilic archaebacterium Pyrococcus furiosus produces several amylolytic enzymes in response to the presence of complex carbohydrates in the growth medium. These enzyme activities, {alpha}-glucosidase, pullulanase, and {alpha}-amylase, were detected in both cell extracts and culture supernatants. All activities were characterized by temperature optima of at least 100{degree}C as well as a high degree of thermostability. The existence of this collection of activities in P. furiosus suggests that polysaccharide availability in its growth environment is a significant aspect of the niche from which it was isolated.

  4. Modulating enzyme activity using ionic liquids or surfactants.

    PubMed

    Goldfeder, Mor; Fishman, Ayelet

    2014-01-01

    One of the important strategies for modulating enzyme activity is the use of additives to affect their microenvironment and subsequently make them suitable for use in different industrial processes. Ionic liquids (ILs) have been investigated extensively in recent years as such additives. They are a class of solvents with peculiar properties and a "green" reputation in comparison to classical organic solvents. ILs as co-solvents in aqueous systems have an effect on substrate solubility, enzyme structure and on enzyme-water interactions. These effects can lead to higher reaction yields, improved selectivity, and changes in substrate specificity, and thus there is great potential for IL incorporation in biocatalysis. The use of surfactants, which are usually denaturating agents, as additives in enzymatic reactions is less reviewed in recent years. However, interesting modulations in enzyme activity in their presence have been reported. In the case of surfactants there is a more pronounced effect on the enzyme structure, as can be observed in a number of crystal structures obtained in their presence. For each additive and enzymatic process, a specific optimization process is needed and there is no one-fits-all solution. Combining ILs and surfactants in either mixed micelles or water-in-IL microemulsions for use in enzymatic reaction systems is a promising direction which may further expand the range of enzyme applications in industrial processes. While many reviews exist on the use of ILs in biocatalysis, the present review centers on systems in which ILs or surfactants were able to modulate and improve the natural activity of enzymes in aqueous systems. PMID:24281758

  5. Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS-917

    PubMed Central

    Kubota, Kazuishi; Inaba, Shin-ichi; Nakano, Rika; Watanabe, Mihoko; Sakurai, Hidetaka; Fukushima, Yumiko; Ichikawa, Kimihisa; Takahashi, Tohru; Izumi, Takashi; Shinagawa, Akira

    2015-01-01

    CS-917 (MB06322) is a selective small compound inhibitor of fructose 1,6-bisphosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS-917 is a bisamidate prodrug and activation of CS-917 requires a two-step enzyme catalyzed reaction. The first-step enzyme, esterase, catalyzes the conversion of CS-917 into the intermediate form (R-134450) and the second-step enzyme, phosphoramidase, catalyzes the conversion of R-134450 into the active form (R-125338). In this study, we biochemically purified the CS-917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS-917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R-134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid-like 3A (SMPADL3A), as an R-134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs. PMID:26171222

  6. A 19F NMR Study of Enzyme Activity

    NASA Astrophysics Data System (ADS)

    Peterman, Keith E.; Lentz, Kevin; Duncan, Jeffery

    1998-10-01

    This basic enzyme activity laboratory experiment demonstrates how 19F NMR can be used in biochemical studies and presents the advantages of 19F NMR over 1H NMR for studies of this nature. N-Trifluoroacetylglycine was selected as a commercially available model fluorine-tagged substrate that readily undergoes acylase I-catalyzed hydrolysis to produce trifluoroacetic acid and glycine. Progress of the reaction was monitored by following conversion of the trifluoroacetyl moiety peak of N-trifluoroacetylglycine to trifluoroacetic acid. The extent of hydrolysis was determined by comparing integrated ratios of the two 19F NMR peaks. A plot of percent hydrolysis versus enzyme concentration was used to calculate unit activity of the enzyme. This is a viable laboratory experiment for junior/senior-level courses in instrumental analytical chemistry, biochemistry, molecular biology, or spectroscopy.

  7. Interdomain communications in bifunctional enzymes: how are different activities coordinated?

    PubMed

    Nagradova, Natalya

    2003-08-01

    Although bifunctional enzymes containing two different active centers located within separate domains are quite common in living systems, the significance of this bifunctionality is not always clear, and the molecular mechanisms of site-site interactions in such complex systems have come under the scrutiny of science only in recent years. This review summarizes recent data on the mechanisms of communication between active centers in bifunctional enzymes. Three types of enzymes are considered: (1) those catalyzing consecutive reactions of a metabolic pathway and exhibiting substrate channeling (glutamate synthase and imidazole glycerol phosphate synthase), (2) those catalyzing consecutive reactions without substrate channeling (lysine-ketoglutarate reductase/saccharopine dehydrogenase), and (3) those catalyzing opposed reactions (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase). The functional role of interdomain communications is briefly discussed. PMID:14609201

  8. Stimulus-responsive Controlled Release System by Covalent Immobilization of an Enzyme into Mesoporous Silica Nanoparticles

    PubMed Central

    Méndez, Jessica; Monteagudo, Alina; Griebenow, Kai

    2012-01-01

    Mesoporous silica nanoparticles (MSN) have emerged as an attractive class of drug delivery carriers for therapeutic agents. Herein, we explored the covalent immobilization of proteins into MSN to generate a stimulus-responsive controlled release system. First, MSN were functionalized with thiol groups using (mercaptopropyl)-trimethoxysilane (MPTMS). Functionalization was verified by X-ray photoelectron spectroscopy (XP), Fourier-transform infrared (FTIR) spectroscopy, and dynamic light scattering. The model enzyme carbonic anhydrase (CA) was coupled to sulfosuccinimidyl 6-[3'(2-pyridyldithio)-propionamido]hexanoate (Sulfo-LC-SPDP) at a low ratio of 1:1 to prevent enzyme inactivation and subsequently covalently immobilized into MSN via thiol-disulfide interchange. The enzyme could be released from MSN with 10 mM glutathione which represents intra-cellular redox conditions while it remained bound to the MSN at extra-cellular redox conditions represented by 1 μM glutathione. The activity of the released enzyme was >80% demonstrating that the enzyme was still largely functional and active after immobilization and release. Human cervical cancer (HeLa) cells were incubated with the MSN-CA bioconjugates at various concentrations for 24 h and the data show good biocompatibility. In summary, we demonstrate the potential of MSN as potential drug delivery systems for proteins. PMID:22375899

  9. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

    PubMed

    Harlan, Fiona Karen; Lusk, Jason Scott; Mohr, Breanna Michelle; Guzikowski, Anthony Peter; Batchelor, Robert Hardy; Jiang, Ying; Naleway, John Joseph

    2016-01-01

    Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and

  10. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities

    PubMed Central

    Harlan, Fiona Karen; Lusk, Jason Scott; Mohr, Breanna Michelle; Guzikowski, Anthony Peter; Batchelor, Robert Hardy; Jiang, Ying

    2016-01-01

    Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson’s Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and

  11. A study on the flexibility of enzyme active sites

    PubMed Central

    2011-01-01

    Background A common assumption about enzyme active sites is that their structures are highly conserved to specifically distinguish between closely similar compounds. However, with the discovery of distinct enzymes with similar reaction chemistries, more and more studies discussing the structural flexibility of the active site have been conducted. Results Most of the existing works on the flexibility of active sites focuses on a set of pre-selected active sites that were already known to be flexible. This study, on the other hand, proposes an analysis framework composed of a new data collecting strategy, a local structure alignment tool and several physicochemical measures derived from the alignments. The method proposed to identify flexible active sites is highly automated and robust so that more extensive studies will be feasible in the future. The experimental results show the proposed method is (a) consistent with previous works based on manually identified flexible active sites and (b) capable of identifying potentially new flexible active sites. Conclusions This proposed analysis framework and the former analyses on flexibility have their own advantages and disadvantage, depending on the cause of the flexibility. In this regard, this study proposes an alternative that complements previous studies and helps to construct a more comprehensive view of the flexibility of enzyme active sites. PMID:21342563

  12. Angiotensin-Converting Enzyme Inhibitors and Active Tuberculosis

    PubMed Central

    Wu, Jiunn-Yih; Lee, Meng-Tse Gabriel; Lee, Si-Huei; Lee, Shih-Hao; Tsai, Yi-Wen; Hsu, Shou-Chien; Chang, Shy-Shin; Lee, Chien-Chang

    2016-01-01

    Abstract Numerous epidemiological data suggest that the use of angiotensin-converting enzyme inhibitors (ACEis) can improve the clinical outcomes of pneumonia. Tuberculosis (TB) is an airborne bacteria like pneumonia, and we aimed to find out whether the use of ACEis can decrease the risk of active TB. We conducted a nested case–control analysis by using a 1 million longitudinally followed cohort, from Taiwan national health insurance research database. The rate ratios (RRs) for TB were estimated by conditional logistic regression, and adjusted using a TB-specific disease risk score (DRS) with 71 TB-related covariates. From January, 1997 to December, 2011, a total of 75,536 users of ACEis, and 7720 cases of new active TB were identified. Current use (DRS adjusted RR, 0.87 [95% CI, 0.78–0.97]), but not recent and past use of ACEis, was associated with a decrease in risk of active TB. Interestingly, it was found that chronic use (>90 days) of ACEis was associated with a further decrease in the risk of TB (aRR, 0.74, [95% CI, 0.66–0.83]). There was also a duration response effect, correlating decrease in TB risk with longer duration of ACEis use. The decrease in TB risk was also consistent across all patient subgroups (age, sex, heart failure, cerebrovascular diseases, myocardial infraction, renal diseases, and diabetes) and patients receiving other cardiovascular medicine. In this large population-based study, we found that subjects with recent and chronic use of ACEis were associated with decrease in TB risk. PMID:27175655

  13. Odorant Metabolism Catalyzed by Olfactory Mucosal Enzymes Influences Peripheral Olfactory Responses in Rats

    PubMed Central

    Thiebaud, Nicolas; Veloso Da Silva, Stéphanie; Jakob, Ingrid; Sicard, Gilles; Chevalier, Joëlle; Ménétrier, Franck; Berdeaux, Olivier; Artur, Yves; Heydel, Jean-Marie; Le Bon, Anne-Marie

    2013-01-01

    A large set of xenobiotic-metabolizing enzymes (XMEs), such as the cytochrome P450 monooxygenases (CYPs), esterases and transferases, are highly expressed in mammalian olfactory mucosa (OM). These enzymes are known to catalyze the biotransformation of exogenous compounds to facilitate elimination. However, the functions of these enzymes in the olfactory epithelium are not clearly understood. In addition to protecting against inhaled toxic compounds, these enzymes could also metabolize odorant molecules, and thus modify their stimulating properties or inactivate them. In the present study, we investigated the in vitro biotransformation of odorant molecules in the rat OM and assessed the impact of this metabolism on peripheral olfactory responses. Rat OM was found to efficiently metabolize quinoline, coumarin and isoamyl acetate. Quinoline and coumarin are metabolized by CYPs whereas isoamyl acetate is hydrolyzed by carboxylesterases. Electro-olfactogram (EOG) recordings revealed that the hydroxylated metabolites derived from these odorants elicited lower olfactory response amplitudes than the parent molecules. We also observed that glucurono-conjugated derivatives induced no olfactory signal. Furthermore, we demonstrated that the local application of a CYP inhibitor on rat olfactory epithelium increased EOG responses elicited by quinoline and coumarin. Similarly, the application of a carboxylesterase inhibitor increased the EOG response elicited by isoamyl acetate. This increase in EOG amplitude provoked by XME inhibitors is likely due to enhanced olfactory sensory neuron activation in response to odorant accumulation. Taken together, these findings strongly suggest that biotransformation of odorant molecules by enzymes localized to the olfactory mucosa may change the odorant’s stimulating properties and may facilitate the clearance of odorants to avoid receptor saturation. PMID:23555703

  14. MICROBIAL COMMUNITY STRUCTURE AND ENZYME ACTIVITIES IN SEMIARID AGRICULTURAL SOILS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of management on the microbial community structure and enzyme activities of three semiarid soils from Southern High Plains of Texas were investigated. The soils (sandy clay loam, fine sandy loam and loam) were under continuous cotton (Gossypium hirsutum L.) or in cotton -peanut (Arachis h...

  15. Carbohydrate active enzymes revealed in Coptotermes formosanus transcriptome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A normalized cDNA library of Coptotermes formosanus was constructed using mixed RNA isolated from workers, soldiers, nymphs and alates of both sexes. Sequencing of this library generated 131,637 EST and 25,939 unigenes were assembled. Carbohydrate active enzymes (CAZymes) revealed in this library we...

  16. MICROBIAL SUCCESSION AND INTESTINAL ENZYME ACTIVITIES IN THE DEVELOPING RAT

    EPA Science Inventory

    The succession of gastrointestinal flora in the developing rat was studied, concomitant with studies of intestinal enzyme activity. Aerobes and anaerobes were identified as members of 4 major bacterial groups, i.e., Lactobacilli spp., Gram positive enterococci, Gram negative rods...

  17. Variation in Soil Enzyme Activities in a Temperate Agroforestry Watershed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Integration of agroforestry and grass buffers into row crop watersheds improves overall environmental quality, including soil quality. The objective of this study was to examine management and landscape effects on soil carbon, soil nitrogen, microbial diversity, enzyme activity, and DNA concentrati...

  18. Chemoprotective activity of boldine: modulation of drug-metabolizing enzymes.

    PubMed

    Kubínová, R; Machala, M; Minksová, K; Neca, J; Suchý, V

    2001-03-01

    Possible chemoprotective effects of the naturally occurring alkaloid boldine, a major alkaloid of boldo (Peumus boldus Mol.) leaves and bark, including in vitro modulations of drug-metabolizing enzymes in mouse hepatoma Hepa-1 cell line and mouse hepatic microsomes, were investigated. Boldine manifested inhibition activity on hepatic microsomal CYP1A-dependent 7-ethoxyresorufin O-deethylase and CYP3A-dependent testosterone 6 beta-hydroxylase activities and stimulated glutathione S-transferase activity in Hepa-1 cells. In addition to the known antioxidant activity, boldine could decrease the metabolic activation of other xenobiotics including chemical mutagens. PMID:11265593

  19. Tandem antioxidant enzymes confer synergistic protective responses in experimental filariasis.

    PubMed

    Prince, P R; Madhumathi, J; Anugraha, G; Jeyaprita, P J; Reddy, M V R; Kaliraj, P

    2014-12-01

    Helminth parasites use antioxidant defence strategies for survival during oxidative stress due to free radicals in the host. Accordingly, tissue-dwelling filarial parasites counteract host responses by releasing a number of antioxidants. Targeting these redox regulation proteins together, would facilitate effective parasite clearance. Here, we report the combined effect of protective immune responses trigged by recombinant Wuchereria bancrofti thioredoxin (WbTRX) and thioredoxin peroxidase (WbTPX) in an experimental filarial model. The expression of WbTRX and WbTPX in different stages of the parasite and their cross-reactivity were analysed by enzyme-linked immunosorbent assay (ELISA). The immunogenicity of recombinant proteins and their protective efficacy were studied in animal models when immunized in single or cocktail mode. The antigens showed cross-reactive epitopes and induced high humoral and cellular immune responses in mice. Further, parasite challenge against Brugia malayi L3 larvae in Mastomys coucha conferred significant protection of 57% and 62% against WbTRX and WbTPX respectively. The efficacy of L3 clearance was significantly higher (71%) (P <  0.001) when the antigens were immunized together, showing a synergistic effect in multiple-mode vaccination. Hence, the study suggests WbTRX and WbTPX to be attractive vaccine candidates when immunized together and provides a tandem block for parasite elimination in the control of lymphatic filariasis. PMID:23676147

  20. Optimization of enzyme assisted extraction of Fructus Mori polysaccharides and its activities on antioxidant and alcohol dehydrogenase.

    PubMed

    Deng, Qingfang; Zhou, Xin; Chen, Huaguo

    2014-10-13

    In the present study, enzyme assisted extraction of Fructus Mori polysaccharides (FMPS) from F. mori using four kinds of enzymes and three compound enzymes were examined. Research found that glucose oxidase offered a better performance in enhancement of the extraction yields of FMPS, antioxidant and activate alcohol dehydrogenase activities. The glucose oxidase assisted extraction process was further optimized by using response surface method (RSM) to obtain maximum yield of crude FMPS. The results showed that optimized extraction conditions were ratio of enzyme amount 0.40%, enzyme treated time 38 min, treated temperature 58 °C and liquid-solid radio 11.0. Under these conditions, the mean experimental value of extraction yield (16.16 ± 0.14%) corresponded well with the predicted values and increased 160% than none enzyme treated ones. Pharmacological verification tests showed that F. mori crude polysaccharides had good antioxidant and activate alcohol dehydrogenase activities in vitro. PMID:25037415

  1. A Metal-Based Inhibitor of NEDD8-Activating Enzyme

    PubMed Central

    Chan, Daniel Shiu-Hin; Leung, Chung-Hang; Wang, Hui-Min; Ma, Dik-Lung

    2012-01-01

    A cyclometallated rhodium(III) complex [Rh(ppy)2(dppz)]+ (1) (where ppy = 2-phenylpyridine and dppz = dipyrido[3,2-a:2′,3′-c]phenazine dipyridophenazine) has been prepared and identified as an inhibitor of NEDD8-activating enzyme (NAE). The complex inhibited NAE activity in cell-free and cell-based assays, and suppressed the CRL-regulated substrate degradation and NF-κB activation in human cancer cells with potency comparable to known NAE inhibitor MLN4924. Molecular modeling analysis suggested that the overall binding mode of 1 within the binding pocket of the APPBP1/UBA3 heterodimer resembled that for MLN4924. Complex 1 is the first metal complex reported to suppress the NEDDylation pathway via inhibition of the NEDD8-activating enzyme. PMID:23185368

  2. Production of Cell Wall Hydrolyzing Enzymes by Barley Aleurone Layers in Response to Gibberellic Acid 1

    PubMed Central

    Taiz, Lincoln; Honigman, William A.

    1976-01-01

    The cell walls of barley (Hordeum vulgare var. Himalaya) aleurone layers undergo extensive degradation during the tissue's response to gibberellic acid. Previous work had shown that these cell walls consist almost entirely of arabinoxylan. In this study we show that gibberellic acid stimulates endo-β-1,4-xylanase activity in isolated aleurone layers. In addition, gibberellic acid enhances the activity of two glycosidases: β-xylopyranosidase and α-arabinofuranosidase. No gibberellic acid-stimulated cellulase activity was detected. Germination studies showed a similar pattern of enzyme development in intact seeds. Images PMID:16659683

  3. Influence of Molting and Starvation on Digestive Enzyme Activities and Energy Storage in Gammarus fossarum

    PubMed Central

    Charron, Laetitia; Geffard, Olivier; Chaumot, Arnaud; Coulaud, Romain; Jaffal, Ali; Gaillet, Véronique; Dedourge-Geffard, Odile; Geffard, Alain

    2014-01-01

    Among the many biological responses studied in ecotoxicology, energy-based biomarkers such as digestive enzyme activities and energy reserves appear to be useful predictive tools for detecting physiological disturbances in organisms. However, the use of these biological responses as biomarkers could be limited by the effects of confounding factors (biotic and abiotic) and physiological processes, such as the reproductive cycle. Thus, the optimal use of these biomarkers will be facilitated by understanding the effects of these factors on the energy metabolism of the sentinel species being studied. We considered abiotic factors (temperature and conductivity) in a previous study, whereas the present study investigated the effects of gender, the female reproductive stage, and food availability on the digestive enzyme activities and energy storage of Gammarus fossarum. The results indicated that, during the female reproductive cycle, the activities of digestive enzymes (amylase, cellulase, and trypsin) decreased significantly, whereas the levels of reserves (proteins, lipids, and sugar) increased until the last premolt stage. Restricted food diets only led to decreased amylase activities in both sexes. Food starvation also induced a decrease in the energy outcomes in females, whereas there were no effects in males. In general, the biochemical (digestive enzyme activities) and physiological (energy reserves) responses were more stable in males than in females. These results support the use of males fed ad libitum to limit the effects of confounding factors when using these energy biomarkers in Gammarus fossarum during biomonitoring programs. PMID:24788197

  4. Heterogeneity of hydrolytic enzyme activities under drought: imaging and quantitative analysis

    NASA Astrophysics Data System (ADS)

    Sanaullah, Muhammad; Razavi, Bahar S.; Kuzyakov, Yakov

    2015-04-01

    The zymography-based "snap-shoot" of enzyme activities in the rhizosphere is challenging to detect the in situ microbial response to global climate change. We developed in situ soil zymography and used it for identification and localization of hotspots of β-glucosidase activity in the rhizosphere of maize under drought stress (30% of field capacity). The zymographic signals were especially high at root tips and were much stronger for activity of β-glucosidase under drought as compared with optimal moisture (70% of field capacity). This distribution of enzyme activity was confirmed by fluorogenically labelled substrates applied directly to the root exudates. The activity of β-glucosidase in root exudates (produced by root and microorganism associated on the root surface), sampled within 1 hour after zymography was significantly higher by drought stressed plants as compared with optimal moisture. In contrast, the β-glucosidase activity in destructively sampled rhizosphere soil was lower under drought stress compared with optimal moisture. Furthermore, drought stress did not affected β-glucosidase activity in bulk soil, away from rhizosphere. Consequently, we conclude that higher release of mucilage by roots und drought stimulated β-glucosidase activity in the rhizosphere. Thus, the zymography revealed plant-mediated mechanisms accelerating β-glucosidase activity under drought at the root-soil interface. So, coupling of zymography and enzyme assays in the rhizosphere and non-rhizosphere soil enables precise mapping the changes in two-dimensional distribution of enzyme activities due to climate change within dynamic soil interfaces.

  5. Host suitability and diet mixing influence activities of detoxification enzymes in adult Japanese beetles.

    PubMed

    Adesanya, Adekunle; Liu, Nannan; Held, David W

    2016-05-01

    Induction of cytochrome P450, glutathione S transferase (GST), and carboxylesterase (CoE) activity was measured in guts of the scarab Popillia japonica Newman, after consumption of single or mixed plant diets of previously ranked preferred (rose, Virginia creeper, crape myrtle and sassafras) or non-preferred hosts (boxelder, riverbirch and red oak). The goal of this study was to quantify activities of P450, GST and CoE enzymes in the midgut of adult P. japonica using multiple substrates in response to host plant suitability (preferred host vs non-preferred hosts), and single and mixed diets. Non-preferred hosts were only sparingly fed upon, and as a group induced higher activities of P450, GST and CoE than did preferred hosts. However, enzyme activities for some individual plant species were similar across categories of host suitability. Similarly, beetles tended to have greater enzyme activities after feeding on a mixture of plants compared to a single plant type, but mixing per se does not seem as important as the species represented in the mix. Induction of detoxification enzymes on non-preferred hosts, or when switching between hosts, may explain, in part, the perceived feeding preferences of this polyphagous insect. The potential consequences of induced enzyme activities on the ecology of adult Japanese beetles are discussed. PMID:26964493

  6. Micropollutant degradation via extracted native enzymes from activated sludge.

    PubMed

    Krah, Daniel; Ghattas, Ann-Kathrin; Wick, Arne; Bröder, Kathrin; Ternes, Thomas A

    2016-05-15

    A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the

  7. [Activity of hydrogen sulfide production enzymes in kidneys of rats].

    PubMed

    Mel'nyk, A V; Pentiuk, O O

    2009-01-01

    An experimental research of activity and kinetic descriptions of enzymes participating in formation of hydrogen sulfide in the kidney of rats has been carried out. It was established that cystein, homocystein and thiosulphate are the basic substrates for hydrogen sulfide synthesis. The higest activity for hydrogen sulfide production belongs to thiosulfate-dithiolsulfurtransferase and cysteine aminotransferase, less activity is characteristic of cystathionine beta-synthase and cystathio-nine gamma-lyase. The highest affinity to substrate is registered for thiosulfate-dithiolsulfurtransferase and cystathionine gamma-lyase. It is discovered that the substrate inhibition is typical of all hydrogen sulfide formation enzymes, although this characteristic is the most expressed thiosulfat-dithiolsulfurtransferase. PMID:20387629

  8. A biologically active surface enzyme assembly that attenuates thrombus formation

    PubMed Central

    Qu, Zheng; Muthukrishnan, Sharmila; Urlam, Murali K.; Haller, Carolyn A.; Jordan, Sumanas W.; Kumar, Vivek A.; Marzec, Ulla M.; Elkasabi, Yaseen; Lahann, Joerg; Hanson, Stephen R.

    2013-01-01

    Activation of hemostatic pathways by blood-contacting materials remains a major hurdle in the development of clinically durable artificial organs and implantable devices. We postulate that surface-induced thrombosis may be attenuated by the reconstitution onto blood contacting surfaces of bioactive enzymes that regulate the production of thrombin, a central mediator of both clotting and platelet activation cascades. Thrombomodulin (TM), a transmembrane protein expressed by endothelial cells, is an established negative regulator of thrombin generation in the circulatory system. Traditional techniques to covalently immobilize enzymes on solid supports may modify residues contained within or near the catalytic site, thus reducing the bioactivity of surface enzyme assemblies. In this report, we present a molecular engineering and bioorthogonal chemistry approach to site-specifically immobilize a biologically active recombinant human TM fragment onto the luminal surface of small diameter prosthetic vascular grafts. Bioactivity and biostability of TM modified grafts is confirmed in vitro and the capacity of modified grafts to reduce platelet activation is demonstrated using a non-human primate model. These studies indicate that molecularly engineered interfaces that display TM actively limit surface-induced thrombus formation. PMID:23532366

  9. Microbial Community Structure and Enzyme Activities in Semiarid Agricultural Soils

    NASA Astrophysics Data System (ADS)

    Acosta-Martinez, V. A.; Zobeck, T. M.; Gill, T. E.; Kennedy, A. C.

    2002-12-01

    The effect of agricultural management practices on the microbial community structure and enzyme activities of semiarid soils of different textures in the Southern High Plains of Texas were investigated. The soils (sandy clay loam, fine sandy loam and loam) were under continuous cotton (Gossypium hirsutum L.) or in rotations with peanut (Arachis hypogaea L.), sorghum (Sorghum bicolor L.) or wheat (Triticum aestivum L.), and had different water management (irrigated or dryland) and tillage (conservation or conventional). Microbial community structure was investigated using fatty acid methyl ester (FAME) analysis by gas chromatography and enzyme activities, involved in C, N, P and S cycling of soils, were measured (mg product released per kg soil per h). The activities of b-glucosidase, b-glucosaminidase, alkaline phosphatase, and arylsulfatase were significantly (P<0.05) increased in soils under cotton rotated with sorghum or wheat, and due to conservation tillage in comparison to continuous cotton under conventional tillage. Principal component analysis showed FAME profiles of these soils separated distinctly along PC1 (20 %) and PC2 (13 %) due to their differences in soil texture and management. No significant differences were detected in FAME profiles due to management practices for the same soils in this sampling period. Enzyme activities provide early indications of the benefits in microbial populations and activities and soil organic matter under crop rotations and conservation tillage in comparison to the typical practices in semiarid regions of continuous cotton and conventional tillage.

  10. Stoichiometry of soil enzyme activity at global scale.

    PubMed

    Sinsabaugh, Robert L; Lauber, Christian L; Weintraub, Michael N; Ahmed, Bony; Allison, Steven D; Crenshaw, Chelsea; Contosta, Alexandra R; Cusack, Daniela; Frey, Serita; Gallo, Marcy E; Gartner, Tracy B; Hobbie, Sarah E; Holland, Keri; Keeler, Bonnie L; Powers, Jennifer S; Stursova, Martina; Takacs-Vesbach, Cristina; Waldrop, Mark P; Wallenstein, Matthew D; Zak, Donald R; Zeglin, Lydia H

    2008-11-01

    Extracellular enzymes are the proximate agents of organic matter decomposition and measures of these activities can be used as indicators of microbial nutrient demand. We conducted a global-scale meta-analysis of the seven-most widely measured soil enzyme activities, using data from 40 ecosystems. The activities of beta-1,4-glucosidase, cellobiohydrolase, beta-1,4-N-acetylglucosaminidase and phosphatase g(-1) soil increased with organic matter concentration; leucine aminopeptidase, phenol oxidase and peroxidase activities showed no relationship. All activities were significantly related to soil pH. Specific activities, i.e. activity g(-1) soil organic matter, also varied in relation to soil pH for all enzymes. Relationships with mean annual temperature (MAT) and precipitation (MAP) were generally weak. For hydrolases, ratios of specific C, N and P acquisition activities converged on 1 : 1 : 1 but across ecosystems, the ratio of C : P acquisition was inversely related to MAP and MAT while the ratio of C : N acquisition increased with MAP. Oxidative activities were more variable than hydrolytic activities and increased with soil pH. Our analyses indicate that the enzymatic potential for hydrolyzing the labile components of soil organic matter is tied to substrate availability, soil pH and the stoichiometry of microbial nutrient demand. The enzymatic potential for oxidizing the recalcitrant fractions of soil organic material, which is a proximate control on soil organic matter accumulation, is most strongly related to soil pH. These trends provide insight into the biogeochemical processes that create global patterns in ecological stoichiometry and organic matter storage. PMID:18823393

  11. Activities of N-mineralization enzymes associated with soil aggregates in three different tillage systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil enzymes released by microorganisms play a significant role in N mineralization process that determines N availability for plant growth. Soil aggregates of different sizes provide diverse microhabitats for microorganisms and therefore influence soil enzyme activities. We hypothesize that enzyme ...

  12. The cystathionine-β-synthase domains on the guanosine 5''-monophosphate reductase and inosine 5'-monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels.

    PubMed

    Smith, Sabrina; Boitz, Jan; Chidambaram, Ehzilan Subramanian; Chatterjee, Abhishek; Ait-Tihyaty, Maria; Ullman, Buddy; Jardim, Armando

    2016-06-01

    The Leishmania guanosine 5'-monophosphate reductase (GMPR) and inosine 5'-monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine-β-synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in Escherichia coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH-dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the Km values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP Km value 10-fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of the IMPDH and GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools. PMID:26853689

  13. Altered Erythrocyte Glycolytic Enzyme Activities in Type-II Diabetes.

    PubMed

    Mali, Aniket V; Bhise, Sunita S; Hegde, Mahabaleshwar V; Katyare, Surendra S

    2016-07-01

    The activity of enzymes of glycolysis has been studied in erythrocytes from type-II diabetic patients in comparison with control. RBC lysate was the source of enzymes. In the diabetics the hexokinase (HK) activity increased 50 % while activities of phosphoglucoisomerase (PGI), phosphofructokinase (PFK) and aldolase (ALD) decreased by 37, 75 and 64 % respectively but were still several folds higher than that of HK. Hence, it is possible that in the diabetic erythrocytes the process of glycolysis could proceed in an unimpaired or in fact may be augmented due to increased levels of G6P. The lactate dehydrogenase (LDH) activity was comparatively high in both the groups; the diabetic group showed 85 % increase. In control group the HK, PFK and ALD activities showed strong positive correlation with blood sugar level while PGI activity did not show any correlation. In the diabetic group only PFK activity showed positive correlation. The LDH activity only in the control group showed positive correlation with marginal increase with increasing concentrations of glucose. PMID:27382204

  14. The role of conserved Cys residues in Brassica rapa auxin amidohydrolase: Cys139 is crucial for the enzyme activity and Cys320 regulates enzyme stability.

    PubMed

    Smolko, Ana; Šupljika, Filip; Martinčić, Jelena; Jajčanin-Jozić, Nina; Grabar-Branilović, Marina; Tomić, Sanja; Ludwig-Müller, Jutta; Piantanida, Ivo; Salopek-Sondi, Branka

    2016-04-01

    Brassica rapa auxin amidohydrolase (BrILL2) participates in the homeostasis of the plant hormones auxins by hydrolyzing the amino acid conjugates of auxins, thereby releasing the free active form of hormones. Herein, the potential role of the two conserved Cys residues of BrILL2 (at sequence positions 139 and 320) has been investigated by using interdisciplinary approaches and methods of molecular biology, biochemistry, biophysics and molecular modelling. The obtained results show that both Cys residues participate in the regulation of enzyme activity. Cys320 located in the satellite domain of the enzyme is mainly responsible for protein stability and regulation of enzyme activity through polymer formation, as has been revealed by enzyme kinetics and differential scanning calorimetry analysis of the BrILL2 wild type and mutants C320S and C139S. Cys139 positioned in the active site of the catalytic domain is involved in the coordination of one Mn(2+) ion of the bimetal center and is crucial for the enzymatic activity. Although the point mutation Cys139 to Ser causes the loss of enzyme activity, it does not affect the metal binding to the BrILL2 enzyme, as has been shown by isothermal titration calorimetry, circular dichroism spectropolarimetry and differential scanning calorimetry data. MD simulations (200 ns) revealed a different active site architecture of the BrILL2C139S mutant in comparison to the wild type enzyme. Additional possible reasons for the inactivity of the BrILL2C139S mutant have been discussed based on MD simulations and MM-PBSA free energy calculations of BrILL2 enzyme complexes (wt and C139S mutant) with IPA-Ala as a substrate. PMID:26959939

  15. Analysis of enzyme activity regulation by non-denaturing electrophoresis and application of this regulation for enzyme reactor production.

    PubMed

    Shimazaki, Youji; Miki, Shizuka

    2013-10-01

    Non-denaturing electrophoresis can be used to screen enzymes that self-regulate their activities by using a combination of enzymes and their inhibitors. Furthermore, this technique can be applied to develop enzyme reactors that self-regulate their activities. After separation of proteins from mouse liver cytosol by non-denaturing isoelectric focusing, lactate dehydrogense (LDH) and esterase activities were qualitatively and quantitatively examined using a combination of two-dimensional electrophoresis (2-DE) and non-denaturing stacking gel electrophoresis. Activities of mouse liver-derived LDH and carboxylesterase were reversibly inhibited by oxamate and 6,9-diamino-2-ethoxyacridine (acrinol), respectively, in the stacking gels and recovered when the enzymes migrated towards the separation gels. After separation and immobilization of the enzymes, their activities were inhibited by inhibitors and recovered after inhibitor removal. These results indicate that non-denaturing electrophoresis can be applied to select enzymes that self-regulate their activities and subsequently aid in the development of enzyme reactors that can control the enzyme activities. PMID:22803677

  16. A DNA enzyme with Mg(2+)-Dependent RNA Phosphoesterase Activity

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approx. 10(exp -4)/ min in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 C. The Mg(2+) - dependent reaction is more difficult, with an uncatalyzed rate of approx. 10(exp -7)/ min under comparable conditions. Mg(2+) - dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+) - dependent reaction. Results: We generated a population of greater than 10(exp 13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg(2+), Mn(2+), Zn(2+) or Pb(2+). Examination of individual clones from the Mg(2+) lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction.This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01/ min. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. Conclusions: We have generated a Mg(2+) - dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approx. 10(exp 5) - fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

  17. Effects of Recurring Droughts on Extracellular Enzyme Activity in Mountain Grassland

    NASA Astrophysics Data System (ADS)

    Fuchslueger, L.; Bahn, M.; Kienzl, S.; Hofhansl, F.; Schnecker, J.; Richter, A.

    2015-12-01

    Water availability is a key factor for biogeochemical processes and determines microbial activity and functioning, and thereby organic matter decomposition in soils by affecting the osmotic potential, soil pore connectivity, substrate diffusion and nutrient availability. Low water availability during drought periods therefore directly affects microbial activity. Recurring drought periods likely induce shifts in microbial structure that might be reflected in altered responses of microbial turnover of organic matter by extracellular enzymes. To study this we measured a set of potential extracellular enzyme activity rates (cellobiohydrolase CBH; leucine-amino-peptidase LAP; phosphatase PHOS; phenoloxidase POX), in grassland soils that were exposed to extreme experimental droughts during the growing seasons of up to five subsequent years. During the first drought period after eight weeks of rain exclusion all measured potential enzyme activities were significantly decreased. In parallel, soil extractable organic carbon and nitrogen concentrations increased and microbial community structure, determined by phospholipid fatty acid analysis, changed. In soils that were exposed to two and three drought periods only PHOS decreased. After four years of drought again CBH, PHOS and POX decreased, while LAP was unaffected; after five years of drought PHOS and POX decreased and CBH and LAP remained stable. Thus, our results suggest that recurring extreme drought events can cause different responses of extracellular enzyme activities and that the responses change over time. We will discuss whether and to what degree these changes were related to shifts in microbial community composition. However, independent of whether a solitary or a recurrent drought was imposed, in cases when enzyme activity rates were altered during drought, they quickly recovered after rewetting. Overall, our data suggest that microbial functioning in mountain grassland is sensitive to drought, but highly

  18. Effects of ionizing radiation on the enzyme activities and ultrastructural changes of poultry

    NASA Astrophysics Data System (ADS)

    Hwang, H.-I.; Hau, L.-B.

    1995-02-01

    Enzyme-catalyzed changes are generally recognized as one of the major reasons for fresh meat deterioration after irradiation. In this study, the effects of ionizing radiation and storage on the enzyme activities of poultry as well as the ultrastructural change of muscle were evaluated. When chicken breasts were irradiated at 4°C and -20°C, both Ca 2+-dependent protease and cathepsin D showed some degree of resistance to irradiation. The activities of those two enzymes decreased with the increase of irradiation doses. During storage, Ca 2+-dependent proteases showed a marked decrease in activity. On the other hand, the cathepsin D activity was not significantly changed at either 4°C or -20°C after 20 days. Transmission electron microscope examination showed no structural changes of the myofibrils with a radiation dose of up to 10 kGy at either 4°C or -20°C. Freezing protected the irradiated chicken breasts from autolytic enzymes damage during storage. In contrast, considerable sarcomere degradation occurred in Z-line for irradiated samples when stored at 4°C for 20 days. The action of the proteolytic enzymes may have been responsible for the sarcomere degradation in irradiated chicken breasts.

  19. Age composition and antioxidant enzyme activities in blood of Black Sea teleosts.

    PubMed

    Rudneva, Irina I; Skuratovskaya, Ekaterina N; Kuzminova, Natalya S; Kovyrshina, Tatyana B

    2010-03-01

    Age composition and age-related trends of antioxidant enzyme activities superoxide dismutase (SOD), catalase (CAT), peroxidase (PER), glutathione reductase (GR) and glutathione-S-transferase (GST) in the blood of seven Black Sea teleosts (Carangidae, Centracanthidae, Gadidae, Mullidae, Gobiidae and Scorpaenidae) collected in marine coastal area of Sevastopol (Ukraine) were studied. In the catches the animals of 1-2 years of age dominated while in the Scorpaena porcus population the number of relatively elder individuals belonging to classes of 3-4 years was the highest. The trends of antioxidant enzyme activities in blood were not uniform. Three types of age-dependent responses were indicated in fish blood: 1. enzymatic activity did not change with age; 2. enzymatic activity decreased with age and 3. enzyme activity increased with age or varied unclearly. The interspecies differences of age-related enzymatic activities associated with the specificity of fish biology and ecology were indicated. Despite no clear evidence of age-related differences between fish species belonging to different ecological groups both benthic forms exhibited similar age-dependent trends of SOD and PER. The correlations between blood antioxidant enzyme activities in fish belonging to suprabenthic and benthic/pelagic groups demonstrated the intermediate values as compared to the benthic and pelagic forms. The results suggest the importance of age trends for biomarkers in fish monitoring studies. PMID:19897051

  20. 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

  1. Chemoproteomic profiling of host and pathogen enzymes active in cholera.

    PubMed

    Hatzios, Stavroula K; Abel, Sören; Martell, Julianne; Hubbard, Troy; Sasabe, Jumpei; Munera, Diana; Clark, Lars; Bachovchin, Daniel A; Qadri, Firdausi; Ryan, Edward T; Davis, Brigid M; Weerapana, Eranthie; Waldor, Matthew K

    2016-04-01

    Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human choleric stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, and genetic disruption of all four proteases increased the abundance of intelectin, an intestinal lectin, and its binding to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting that it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialog in an animal model of infection. PMID:26900865

  2. Exploring the sheep rumen microbiome for carbohydrate-active enzymes.

    PubMed

    Lopes, Lucas Dantas; de Souza Lima, André Oliveira; Taketani, Rodrigo Gouvêa; Darias, Phillip; da Silva, Lília Raquel Fé; Romagnoli, Emiliana Manesco; Louvandini, Helder; Abdalla, Adibe Luiz; Mendes, Rodrigo

    2015-07-01

    The rumen is a complex ecosystem enriched for microorganisms able to degrade biomass during the animal's digestion process. The recovery of new enzymes from naturally evolved biomass-degrading microbial communities is a promising strategy to overcome the inefficient enzymatic plant destruction in industrial production of biofuels. In this context, this study aimed to describe the bacterial composition and functions in the sheep rumen microbiome, focusing on carbohydrate-active enzymes (CAE). Here, we used phylogenetic profiling analysis (inventory of 16S rRNA genes) combined with metagenomics to access the rumen microbiome of four sheep and explore its potential to identify fibrolytic enzymes. The bacterial community was dominated by Bacteroidetes and Firmicutes, followed by Proteobacteria. As observed for other ruminants, Prevotella was the dominant genus in the microbiome, comprising more than 30 % of the total bacterial community. Multivariate analysis of the phylogenetic profiling data and chemical parameters showed a positive correlation between the abundance of Prevotellaceae (Bacteroidetes phylum) and organic matter degradability. A negative correlation was observed between Succinivibrionaceae (Proteobacteria phylum) and methane production. An average of 2 % of the shotgun metagenomic reads was assigned to putative CAE when considering nine protein databases. In addition, assembled contigs allowed recognition of 67 putative partial CAE (NCBI-Refseq) representing 12 glycosyl hydrolase families (Pfam database). Overall, we identified a total of 28 lignocellulases, 22 amylases and 9 other putative CAE, showing the sheep rumen microbiome as a promising source of new fibrolytic enzymes. PMID:25900454

  3. In vivo enzyme activity in inborn errors of metabolism

    SciTech Connect

    Thompson, G.N.; Walter, J.H.; Leonard, J.V.; Halliday, D. )

    1990-08-01

    Low-dose continuous infusions of (2H5)phenylalanine, (1-13C)propionate, and (1-13C)leucine were used to quantitate phenylalanine hydroxylation in phenylketonuria (PKU, four subjects), propionate oxidation in methylmalonic acidaemia (MMA, four subjects), and propionic acidaemia (PA, four subjects) and leucine oxidation in maple syrup urine disease (MSUD, four subjects). In vivo enzyme activity in PKU, MMA, and PA subjects was similar to or in excess of that in adult controls (range of phenylalanine hydroxylation in PKU, 3.7 to 6.5 mumol/kg/h, control 3.2 to 7.9, n = 7; propionate oxidation in MMA, 15.2 to 64.8 mumol/kg/h, and in PA, 11.1 to 36.0, control 5.1 to 19.0, n = 5). By contrast, in vivo leucine oxidation was undetectable in three of the four MSUD subjects (less than 0.5 mumol/kg/h) and negligible in the remaining subject (2 mumol/kg/h, control 10.4 to 15.7, n = 6). These results suggest that significant substrate removal can be achieved in some inborn metabolic errors either through stimulation of residual enzyme activity in defective enzyme systems or by activation of alternate metabolic pathways. Both possibilities almost certainly depend on gross elevation of substrate concentrations. By contrast, only minimal in vivo oxidation of leucine appears possible in MSUD.

  4. Evaluation of pancreatin stability through enzyme activity determination.

    PubMed

    Terra, Gleysson De Paula; Vinícius De Farias, Marcus; Trevisan, Marcello Garcia; Garcia, Jerusa Simone

    2016-09-01

    Pancreatin is a biotechnological product containing an enzyme complex, obtained from porcine pancreas, that is employed in treating pancreatic diseases. Experiments regarding the stability of the pharmaceutical formulation containing pancreatin were performed using standard binary mixtures with 6 excipients in a 1:1 ratio (m/m) and a commercial formulation. To accomplish these goals, samples were stored for 1, 3 and 6 months at 40 ± 1 °C and 75 ± 5 % relative humidity (RH) and 40 ± 1 °C and 0 % RH. Stress testing was also performed. All samples were analyzed to evaluate the α-amylase, lipase and protease activities through UV/Vis spectrophotometry. The results revealed that the excipient proprieties and the storage conditions affected enzyme stability. Humidity was a strong influencing factor in the reduction of α-amylase and protease activities. Stress testing indicated that pH 9.0 and UV light did not induce substantial alterations in enzyme activity. PMID:27383890

  5. Digestive enzyme activities in larvae of sharpsnout seabream (Diplodus puntazzo).

    PubMed

    Suzer, Cüneyt; Aktülün, Sevim; Coban, Deniz; Okan Kamaci, H; Saka, Sahin; Firat, Kürşat; Alpbaz, Atilla

    2007-10-01

    The ontogenesis and specific activities of pancreatic and intestinal enzymes were investigated in sharpsnout sea bream, Diplodus puntazzo, during larval development until the end of weaning on day 50. The green-water technique was carried out for larval rearing in triplicate. Trypsin was first detected as early as hatching and sharply increased related to age and exogenous feeding until day 25, but a sharp decrease was observed towards the end of the experiment. Amylase was determined 2 days after hatching (DAH) and sharply increased to 10 DAH. Afterwards, slight decreases were found between 10 and 20 DAH and then slow alterations were continued until end of the experiment. Lipase was measured for the first time on day 4, and then slight increase was found to 25 DAH. After this date, slow variations were maintained until end of the experiment. Pepsin was firstly assayed 32 DAH related with stomach formation and sharply increased to 40 DAH. Then it was fluctuated until end of the experiment. Enzymes of brush border membranes, alkaline phosphatase and aminopeptidase N, showed similar pattern on specific activities during the first 10 days. Thereafter, while specific activity of alkaline phosphatase slightly decreased to 15 DAH and fluctuated until 20 DAH, aminopeptidase N activity slowly declined to 20 DAH. Afterwards, activity of alkaline phosphatase and aminopeptidase N were sharply increased to 30 DAH, showing maturation of the intestinal digestive process and also these activities continued to slight increase until end of the experiment. The specific activity of cytosolic peptidase, leucine-alanine peptidase sharply increased to on day 8, then suddenly declined to 12 DAH and further decreased until 20 DAH. After this date, in contrast to enzymes of brush border membranes, it sharply decreased to 25 DAH and continued to gradually decline until the end of the experiment. These converse expressions were indicative of a maturation of enterocytes and the transition to

  6. Molecular Imprint of Enzyme Active Site by Camel Nanobodies

    PubMed Central

    Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

    2012-01-01

    Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach. PMID:22374998

  7. Effect of cold adaptation on activities of relevant enzymes and antioxidant system in rats

    PubMed Central

    Xing, Ji-Qing; Zhou, Yang; Chen, Jian-Feng; Li, Shang-Bin; Fang, Wei; Yang, Jun

    2014-01-01

    Exercise in cold environments can cause significant metabolic regulation and antioxidant behavior. For discussing enzymatic responses towards cold adaptation, we investigated enzyme activities of adenylate cyclase (AC) and phosphodiesterase (PDE) in liver, skeletal muscle, and brown adipose tissue (BAT), as well as Na+·K+ ATPase and Na+/K+ ratio in blood. Malondialdehyde (MDA) and superoxide dismutase (SOD) activity in blood were also studied to address the effect of cold adaptation on oxidative damage and antioxidant system. Experimental results indicated that enzyme activities in liver, skeletal muscle and BAT maintained relatively constant for the control group. For the cold adaptation group, enzyme activities in liver and skeletal muscle were in high levels at the beginning, and then gradually decreased to similar values with the control group. However, enzyme activities in BAT performed an increasing trend and significantly higher than the control at the end. In addition, decreased oxidative damage and activated antioxidant system was observed along with the cold adaptation process. PMID:25550936

  8. Substrate-competitive activity-based profiling of ester prodrug activating enzymes

    PubMed Central

    Xu, Hao; Majmudar, Jaimeen D.; Davda, Dahvid; Ghanakota, Phani; Kim, Ki H.; Carlson, Heather A.; Showalter, Hollis D.; Martin, Brent R.; Amidon, Gordon L.

    2015-01-01

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating pre-clinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a 4-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse, but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design and preclinical

  9. Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes.

    PubMed

    Kriechbaumer, Verena; Seo, Hyesu; Park, Woong June; Hawes, Chris

    2015-09-01

    Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized. PMID:26139824

  10. Extracellular enzyme activities and nutrient availability during artificial groundwater recharge.

    PubMed

    Kolehmainen, Reija E; Korpela, Jaana P; Münster, Uwe; Puhakka, Jaakko A; Tuovinen, Olli H

    2009-02-01

    Natural organic matter (NOM) removal is the main objective of artificial groundwater recharge (AGR) for drinking water production and biodegradation plays a substantial role in this process. This study focused on the biodegradation of NOM and nutrient availability for microorganisms in AGR by the determination of extracellular enzyme activities (EEAs) and nutrient concentrations along a flow path in an AGR aquifer (Tuusula Water Works, Finland). Natural groundwater in the same area but outside the influence of recharge was used as a reference. Determination of the specific alpha-d-glucosidase (alpha-Glu), beta-d-glucosidase (beta-Glu), phosphomonoesterase (PME), leucine aminopeptidase (LAP) and acetate esterase (AEST) activities by fluorogenic model substrates revealed major increases in the enzymatic hydrolysis rates in the aquifer within a 10m distance from the basin. The changes in the EEAs along the flow path occurred simultaneously with decreases in nutrient concentrations. The results support the assumption that the synthesis of extracellular enzymes in aquatic environments is up and down regulated by nutrient availability. The EEAs in the basin sediment and pore water samples (down to 10cm) were in the same order of magnitude as in the basin water, suggesting similar nutritional conditions. Phosphorus was likely to be the limiting nutrient at this particular AGR site. Furthermore, the extracellular enzymes functioned in a synergistic and cooperative way. PMID:19028394

  11. Aptamer and PNIPAAm co-conjugated nanoparticles regulate activity of enzyme with different temperature.

    PubMed

    Yu, Jiemiao; Yang, Liangrong; Liang, Xiangfeng; Dong, Tingting; Qu, Hongnan; Rong, Meng; Liu, Huizhou

    2016-10-01

    In this paper, we described a temperature responsive nano-system that can regulate activity of enzyme with different temperature. Temperature responsive polymer poly(N-isopropylacrylamide) (PNIPAAm), with low critical solution temperature of 32°C, was synthesized with thiol modification. PNIPAAm and thrombin aptamer were co-functionalized on the surface of gold nanoparticles for effective regulation of thrombin activity with different temperature. On the one hand, we studied the thermal responsive properties of this inhibitor via UV-visible spectroscopy. On the other hand, we investigated the regulation of thrombin activity by this platform with different temperature. The PNIPAAm chains could extend and shrink with different temperature, which suggested that PNIPAAm on the surface of gold nanoparticles could regulate interaction between thrombin and aptamer according to temperature changing. At 25°C, PNIPAAm was hydrophilic extended state, which blocked the interaction between thrombin and aptamer on the surface of gold nanoparticles, therefore thrombin activity had no change. On the contrary, at 37°C, PNIPAAm transformed from hydrophilic extended state to hydrophobic shrank state, allowing the aptamer to capture thrombin, inhibiting the activity of thrombin. More interestingly, this regulation was reverse to normal condition, where 37°C was always the optimum reaction temperature for most of human enzymes. This system we prepared was opposite, which was capable of inhibiting the thrombin activity at 37°C. Furthermore, this was the first report of regulation of thrombin activity using this temperature responsive platform. PMID:27474278

  12. Portable Enzyme-Paper Biosensors Based on Redox-Active CeO2 Nanoparticles.

    PubMed

    Karimi, A; Othman, A; Andreescu, S

    2016-01-01

    Portable, nanoparticle (NP)-enhanced enzyme sensors have emerged as powerful devices for qualitative and quantitative analysis of a variety of analytes for biomedicine, environmental applications, and pharmaceutical fields. This chapter describes a method for the fabrication of a portable, paper-based, inexpensive, robust enzyme biosensor for the detection of substrates of oxidase enzymes. The method utilizes redox-active NPs of cerium oxide (CeO2) as a sensing platform which produces color in response to H2O2 generated by the action of oxidase enzymes on their corresponding substrates. This avoids the use of peroxidases which are routinely used in conjunction with glucose oxidase. The CeO2 particles serve dual roles, as high surface area supports to anchor high loadings of the enzyme as well as a color generation reagent, and the particles are recycled multiple times for the reuse of the biosensor. These sensors are small, light, disposable, inexpensive, and they can be mass produced by standard, low-cost printing methods. All reagents needed for the analysis are embedded within the paper matrix, and sensors stored over extended periods of time without performance loss. This novel sensor is a general platform for the in-field detection of analytes that are substrates for oxidase enzymes in clinical, food, and environmental samples. PMID:27112400

  13. Prolidase Enzyme Activity in Conjunctiva and Pterygium Tissues

    PubMed Central

    Yıldırım, Yıldıray; Kaya, Abdullah; Kar, Taner; Muftuoglu, Tuba; Ayata, Ali

    2015-01-01

    Background The aim of this study was to determine prolidase activity in conjunctival tissue and its relationship with pterygium. Material/Methods Prolidase activity was measured in 23 pterygium and 25 healthy conjunctival tissues and the 2 groups were compared. Results Prolidase enzyme activity could not be measured in either the healthy conjunctival or in pterygium tissues. The mean serum prolidase levels of the control and pterygium groups were 967.46±353.64 and 858.29±301.83, respectively. Statistically, there was no significant difference between the groups with regard to serum prolidase levels (p>0.05). Conclusions In conclusion, absence of prolidase activity in pterygium tissue indicates that there is no collagen turnover in this tissue. We may explain this finding with the elastin-rich structure of the conjunctiva. PMID:26509313

  14. A Computational Methodology to Screen Activities of Enzyme Variants

    PubMed Central

    Hediger, Martin R.; De Vico, Luca; Svendsen, Allan; Besenmatter, Werner; Jensen, Jan H.

    2012-01-01

    We present a fast computational method to efficiently screen enzyme activity. In the presented method, the effect of mutations on the barrier height of an enzyme-catalysed reaction can be computed within 24 hours on roughly 10 processors. The methodology is based on the PM6 and MOZYME methods as implemented in MOPAC2009, and is tested on the first step of the amide hydrolysis reaction catalyzed by the Candida Antarctica lipase B (CalB) enzyme. The barrier heights are estimated using adiabatic mapping and shown to give barrier heights to within 3 kcal/mol of B3LYP/6-31G(d)//RHF/3-21G results for a small model system. Relatively strict convergence criteria (0.5 kcal/(molÅ)), long NDDO cutoff distances within the MOZYME method (15 Å) and single point evaluations using conventional PM6 are needed for reliable results. The generation of mutant structures and subsequent setup of the semiempirical calculations are automated so that the effect on barrier heights can be estimated for hundreds of mutants in a matter of weeks using high performance computing. PMID:23284627

  15. Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles.

    PubMed

    Khodashenas, Bahareh; Ghorbani, Hamid Reza

    2016-06-01

    Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO(3)) concentration and enzyme amount on nanoparticles synthesis were studied. Finally, the produced nanoparticles were studied using ultraviolet -visible (UV-Vis) spectrophotometer, dynamic light scattering technique, and transmission electron microscopy. UV-Visible spectrophotometric study showed the characteristic peak for Ag NPs at wavelength 405-420 nm for 1 mM metal precursor solution (AgNO(3)) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO(3) with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO(3) concentration and the size of produced Ag NPs. PMID:27256897

  16. Tissue enzyme activities in the loggerhead sea turtle (Caretta caretta).

    PubMed

    Anderson, Eric T; Socha, Victoria L; Gardner, Jennifer; Byrd, Lynne; Manire, Charles A

    2013-03-01

    The loggerhead sea turtle, Caretta caretta, one of the seven species of threatened or endangered sea turtles worldwide, is one of the most commonly encountered marine turtles off the eastern coast of the United States and Gulf of Mexico. Although biochemical reference ranges have been evaluated for several species of sea turtles, tissue specificity of the commonly used plasma enzymes is lacking. This study evaluated the tissue specificity of eight enzymes, including amylase, lipase, creatine kinase (CK), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), in 30 tissues from five stranded loggerhead sea turtles with no evidence of infectious disease. Amylase and lipase showed the greatest tissue specificity, with activity found only in pancreatic samples. Creatine kinase had high levels present in skeletal and cardiac muscle, and moderate levels in central nervous system and gastrointestinal samples. Gamma-glutamyl transferase was found in kidney samples, but only in very low levels. Creatine kinase, ALP, AST, and LDH were found in all tissues evaluated and ALT was found in most, indicating low tissue specificity for these enzymes in the loggerhead. PMID:23505704

  17. The carbohydrate-active enzymes database (CAZy) in 2013.

    PubMed

    Lombard, Vincent; Golaconda Ramulu, Hemalatha; Drula, Elodie; Coutinho, Pedro M; Henrissat, Bernard

    2014-01-01

    The Carbohydrate-Active Enzymes database (CAZy; http://www.cazy.org) provides online and continuously updated access to a sequence-based family classification linking the sequence to the specificity and 3D structure of the enzymes that assemble, modify and breakdown oligo- and polysaccharides. Functional and 3D structural information is added and curated on a regular basis based on the available literature. In addition to the use of the database by enzymologists seeking curated information on CAZymes, the dissemination of a stable nomenclature for these enzymes is probably a major contribution of CAZy. The past few years have seen the expansion of the CAZy classification scheme to new families, the development of subfamilies in several families and the power of CAZy for the analysis of genomes and metagenomes. This article outlines the changes that have occurred in CAZy during the past 5 years and presents our novel effort to display the resolution and the carbohydrate ligands in crystallographic complexes of CAZymes. PMID:24270786

  18. Characterization of cytidylyltransferase enzyme activity through high performance liquid chromatography.

    PubMed

    Brault, James P; Friesen, Jon A

    2016-10-01

    The cytidylyltransferases are a family of enzymes that utilize cytidine 5'-triphosphate (CTP) to synthesize molecules that are typically precursors to membrane phospholipids. The most extensively studied cytidylyltransferase is CTP:phosphocholine cytidylyltransferase (CCT), which catalyzes conversion of phosphocholine and CTP to cytidine diphosphocholine (CDP-choline), a step critical for synthesis of the membrane phospholipid phosphatidylcholine (PC). The current method used to determine catalytic activity of CCT measures production of radiolabeled CDP-choline from (14)C-labeled phosphocholine. The goal of this research was to develop a CCT enzyme assay that employed separation of non-radioactive CDP-choline from CTP. A C18 reverse phase column with a mobile phase of 0.1 M ammonium bicarbonate (98%) and acetonitrile (2%) (pH 7.4) resulted in separation of solutions of the substrate CTP from the product CDP-choline. A previously characterized truncated version of rat CCTα (denoted CCTα236) was used to test the HPLC enzyme assay by measuring CDP-choline product formation. The Vmax for CCTα236 was 3850 nmol/min/mg and K0.5 values for CTP and phosphocholine were 4.07 mM and 2.49 mM, respectively. The HPLC method was applied to glycerol 3-phosphate cytidylyltransferase (GCT) and CTP:2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), members of the cytidylyltransferase family that produce CDP-glycerol and CDP-methylerythritol, respectively. PMID:27443959

  19. Responses to converting-enzyme inhibition and hemorrhage in newborn lambs and adult sheep

    SciTech Connect

    Rose, J.C.; Block, S.M.; Flowe, K.; Morris, M.; South, S.; Sundberg, D.K.; Zimmerman, C.

    1987-02-01

    The authors compared the cardiovascular and hormonal responses to angiotensin converting enzyme inhibition and hemorrhage of 20% of blood volume in chronically instrumented unanesthetized newborn lambs and adult sheep. Administration of the nonsulfhydryl-containing converting-enzyme inhibitor enalapril reduced mean arterial pressure in the newborn but not in the adult animals. Blood pressure fell in both age groups after hemorrhage, and the hemorrhage-induced fall in blood pressure, integrated over the period of hypovolemia, was more pronounced when converting-enzyme inhibition was present in the lambs. This was not observed in the adults. Cardiac output fell following hemorrhage in both age groups, and the fall was greater when enalapril was present in the lambs, but this was not the case in the adults. Hemorrhage increased plasma renin activity in both groups, and enalapril augmented this increase. Plasma concentrations of vasopressin, measured by radioimmunoassay, and catecholamines measured by radio enzymatic assay, increased following hemorrhage within and between groups. Taken together these data suggest that the renin-angiotensin systems plays a more important role in the maintenance of cardiovascular homeostasis in newborn lambs than it does in adult sheep, and catecholamine and vasopressin responses to volume loss can occur in the presence of blockade of the renin-angiotensin system.

  20. Activity of enzyme immobilized on silanized Co-Cr-Mo.

    PubMed

    Puleo, D A

    1995-08-01

    The surface of an orthopedic biomaterial was modified by the covalent immobilization of biomolecules. Derivatization of Co-Cr-Mo samples with organic and aqueous solutions of gamma-aminopropyltriethoxysilane (APS) resulted in a concentration-dependent number of reactive NH2 groups on the surface available for coupling to protein. The enzyme trypsin was used as a model biomolecule to investigate the effect of immobilization on proteolytic activity. Trypsin was coupled to the silanized samples by formation of Schiff's base linkages via glutaraldehyde. The nature of the interaction between trypsin and biomaterial was then probed by treatment with concentrated guanidine hydrochloride (GuHCl) and urea. Residual activity (following treatment with chaotropic agents) of trypsin immobilized on silanized Co-Cr-Mo was dependent both on the nature of the silane solution and on the type of chaotropic agent. Organic silanization with APS required a minimum density of approximately 49 NH2 per nm2 of nominal surface area (> 0.021 M APS) for residual activity of immobilized trypsin. For aqueous silanization, approximately 5.4 NH2/nm2 (0.51 M APS) resulted in maximal residual trypsin activity. Treatment with GuHCl removed more trypsin activity from Co-Cr-Mo samples silanized with organic solutions of APS than did treatment with urea. On the contrary, with aqueous silanization the samples possessed greater residual activity following treatment with GuHCl than following urea. Compared to simple adsorption with protein onto Co-Cr-Mo, both methods of silanization with APS resulted in superior residual immobilized enzyme activity. PMID:7593038

  1. Global Profiling of Carbohydrate Active Enzymes in Human Gut Microbiome

    PubMed Central

    Mande, Sharmila S.

    2015-01-01

    Motivation Carbohydrate Active enzyme (CAZyme) families, encoded by human gut microflora, play a crucial role in breakdown of complex dietary carbohydrates into components that can be absorbed by our intestinal epithelium. Since nutritional wellbeing of an individual is dependent on the nutrient harvesting capability of the gut microbiome, it is important to understand how CAZyme repertoire in the gut is influenced by factors like age, geography and food habits. Results This study reports a comprehensive in-silico analysis of CAZyme profiles in the gut microbiomes of 448 individuals belonging to different geographies, using similarity searches of the corresponding gut metagenomic contigs against the carbohydrate active enzymes database. The study identifies a core group of 89 CAZyme families that are present across 85% of the gut microbiomes. The study detects several geography/age-specific trends in gut CAZyme repertoires of the individuals. Notably, a group of CAZymes having a positive correlation with BMI has been identified. Further this group of BMI-associated CAZymes is observed to be specifically abundant in the Firmicutes phyla. One of the major findings from this study is identification of three distinct groups of individuals, referred to as 'CAZotypes', having similar CAZyme profiles. Distinct taxonomic drivers for these CAZotypes as well as the probable dietary basis for such trends have also been elucidated. The results of this study provide a global view of CAZyme profiles across individuals of various geographies and age-groups. These results re-iterate the need of a more precise understanding of the role of carbohydrate active enzymes in human nutrition. PMID:26544883

  2. Phosphofructokinase from Fasciola hepatica: activation by phosphorylation and other regulatory properties distinct from the mammalian enzyme.

    PubMed

    Kamemoto, E S; Iltzsch, M H; Lan, L; Mansour, T E

    1987-10-01

    Phosphofructokinase from the liver fluke, Fasciola hepatica, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase isolated from this organism. Phosphorylated fluke phosphofructokinase had a sevenfold lower apparent Km for its substrate, Fru-6-P, and an eightfold higher 0.5 Vopt for ATP, the enzyme's primary inhibitor, than native phosphofructokinase. Activation of fluke phosphofructokinase following phorphorylation by a mammalian protein kinase catalytic subunit was previously reported (E. S. Kamemoto and T. E. Mansour (1986) J. Biol. Chem. 261, 4346-4351). The catalytic subunit of protein kinase isolated from the liver fluke phosphorylated sites on fluke phosphofructokinase similar to those phosphorylated by the mammalian enzyme. Maximal phosphate incorporation was 0.3 mol P/mol of protomer. The native enzyme was found to contain 1.3 mol P/mol of protomer. In contrast to fluke phosphofructokinase, activity of the mammalian heart enzyme was slightly decreased following phosphorylation. The dependence of allosteric interaction on an acidic pH observed with the mammalian phosphofructokinase was not observed with the fluke enzyme. Unlike mammalian phosphofructokinase, allosteric kinetics of the fluke enzyme was observed at alkaline pH (8.0). Fluke phosphofructokinase was found to be relatively insensitive to inhibition by citrate, a known potent inhibitor of the mammalian enzyme. Fru-2,6-P2, a potent modifier of phosphofructokinase from a variety of sources, was found to activate both native and phosphorylated fluke phosphofructokinase. The most potent activators of fluke phosphofructokinase were found to be Fru-2,6-P2, AMP, and phosphorylation. The endogenous level of Fru-2,6-P2 in the flukes was determined to be 29 +/- 1.3 nmol/g wet wt, a level that may well modulate enzyme activity. Fru-6-P,2-kinase, the enzyme responsible for synthesis of Fru-2,6-P2, was found to be present in the flukes. Our results suggest physiological roles for

  3. Optimizing the salt-induced activation of enzymes in organic solvents: effects of lyophilization time and water content.

    PubMed

    Ru, M T; Dordick, J S; Reimer, J A; Clark, D S

    1999-04-20

    The addition of simple inorganic salts to aqueous enzyme solutions prior to lyophilization results in a dramatic activation of the dried powder in organic media relative to enzyme with no added salt. Activation of both the serine protease subtilisin Carlsberg and lipase from Mucor javanicus resulting from lyophilization in the presence of KCl was highly sensitive to the lyophilization time and water content of the sample. Specifically, for a preparation containing 98% (w/w) KCl, 1% (w/w) phosphate buffer, and 1% (w/w) enzyme, varying the lyophilization time showed a direct correlation between water content and activity up to an optimum, beyond which the activity decreased with increasing lyophilization time. The catalytic efficiency in hexane varied as much as 13-fold for subtilisin Carlsberg and 11-fold for lipase depending on the lyophilization time. This dependence was apparently a consequence of including the salt, as a similar result was not observed for the enzyme freeze-dried without KCl. In the case of subtilisin Carlsberg, the salt-induced optimum value of kcat/Km for transesterification in hexane was over 20,000-fold higher than that for salt-free enzyme, a substantial improvement over the previously reported enhancement of 3750-fold (Khmelnitsky, 1994). As was found previously for pure enzyme, the salt-activated enzyme exhibited greatest activity when lyophilized from a solution of pH equal to the pH for optimal activity in water. The active-site content of the lyophilized enzyme samples also depended upon lyophilization time and inclusion of salt, with opposite trends in this dependence observed for the solvents hexane and tetrahydrofuran. Finally, substrate selectivity experiments suggested that mechanism(s) other than selective partitioning of substrate into the enzyme-salt matrix are responsible for salt-induced activation of enzymes in organic solvents. PMID:10099600

  4. Optimizing the salt-induced activation of enzymes in organic solvents: Effects of lyophilization time and water content

    SciTech Connect

    Ru, M.T.; Reimer, J.A.; Clark, D.S.; Dordick, J.S.

    1999-04-20

    The addition of simple inorganic salts to aqueous enzyme solutions prior to lyophilization results in a dramatic activation of the dried powder in organic media relative to enzyme with no added salt. Activation of both the serine protease subtilisin Carlsberg and lipase from Mucor javanicus resulting from lyophilization in the presence of KCl was highly sensitive to the lyophilization time and water content of the sample. Specifically, for a preparation containing 98% (w/w) KCl, 1% (w/w) phosphate buffer, and 1% (w/w) enzyme, varying the lyophilization time showed a direct correlation between water content and activity up to an optimum, beyond which the activity decreased with increasing lyophilization time. The catalytic efficiency in hexane varied as much as 13-fold for subtilisin Carlsberg and 11-fold for lipase depending on the lyophilization time. This dependence was apparently a consequence of including the salt, as a similar result was not observed for the enzyme freeze-dried without KCl. In the case of subtilisin Carlsberg, the salt-induced optimum value of k{sub cat}/K{sub m} for transesterification in hexane was over 20,000-fold higher than that for salt-free enzyme, a substantial improvement over the previously reported enhancement of 3750-fold. As was found previously for pure enzyme, the salt-activated enzyme exhibited greatest activity when lyophilized from a solution of pH equal to the pH for optimal activity in water. The active-site content of the lyophilized enzyme samples also depended upon lyophilization time and inclusion of salt, with opposite trends in this dependence observed for the solvents hexane and tetrahydrofuran. Finally, substrate selectivity experiments suggested that mechanism(s) other than selective partitioning of substrate into the enzyme-salt matrix are responsible for salt-induced activation of enzymes in organic solvents.

  5. Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities

    PubMed Central

    Hartley, Meredith D.; Schneggenburger, Philipp E.; Imperiali, Barbara

    2013-01-01

    Membrane-bound polyprenol-dependent pathways are important for the assembly of essential glycoconjugates in all domains of life. However, despite their prevalence, the functional significance of the extended linear polyprenyl groups in the interactions of the glycan substrates, the biosynthetic enzymes that act upon them, and the membrane bilayer in which they are embedded remains a mystery. These interactions are investigated simultaneously and uniquely through application of the nanodisc membrane technology. The Campylobacter jejuni N-linked glycosylation pathway has been chosen as a model pathway in which all of the enzymes and substrates are biochemically accessible. We present the functional reconstitution of two enzymes responsible for the early membrane-committed steps in glycan assembly. Protein stoichiometry analysis, fluorescence-based approaches, and biochemical activity assays are used to demonstrate the colocalization of the two enzymes in nanodiscs. Isotopic labeling of the substrates reveals that undecaprenyl-phosphate is coincorporated into discs with the two enzymes, and furthermore, that both enzymes are functionally reconstituted and can sequentially convert the coembedded undecaprenyl-phosphate into undecaprenyl-diphosphate-linked disaccharide. These studies provide a proof-of-concept demonstrating that the nanodisc model membrane system represents a promising experimental platform for analyzing the multifaceted interactions among the enzymes involved in polyprenol-dependent glycan assembly pathways, the membrane-associated substrates, and the lipid bilayer. The stage is now set for exploration of the roles of the conserved polyprenols in promoting protein–protein interactions among pathway enzymes and processing of substrates through sequential steps in membrane-associated glycan assembly. PMID:24302767

  6. Nitric Oxide Measurement from Purified Enzymes and Estimation of Scavenging Activity by Gas Phase Chemiluminescence Method.

    PubMed

    Kumari, Aprajita; Gupta, Alok Kumar; Mishra, Sonal; Wany, Aakanksha; Gupta, Kapuganti Jagadis

    2016-01-01

    In plants, nitrate reductase (NR) is a key enzyme that produces nitric oxide (NO) using nitrite as a substrate. Lower plants such as algae are shown to have nitric oxide synthase enzyme and higher plants contain NOS activity but enzyme responsible for NO production in higher plants is subjected to debate. In plant nitric oxide research, it is very important to measure NO very precisely in order to determine its functional role. A significant amount of NO is being scavenged by various cell components. The net NO production depends in production minus scavenging. Here, we describe methods to measure NO from purified NR and inducible nitric oxide synthase from mouse (iNOS), we also describe a method of measure NO scavenging by tobacco cell suspensions and mitochondria from roots. PMID:27094408

  7. Soil enzyme activities as affected by anthropogenic alterations: intensive agricultural practices and organic pollution.

    PubMed

    Gianfreda, Liliana; Antonietta Rao, Maria; Piotrowska, Anna; Palumbo, Giuseppe; Colombo, Claudio

    2005-04-01

    The activity of a range of enzymes related to the cycling of the main biologically important nutrients C, N, P and S was investigated in cultivated and non-cultivated soils from various parts of Europe. Two agricultural sites from North Italy under continuous corn (Zea mays L.) with and without organic fertilization were compared. Two other agricultural sites from South Italy under hazel (Corylus avellana L.) never flooded or repeatedly flooded over by uncontrolled urban and industrial wastes were investigated. The non-cultivated soils were from Middle and South Europe with different pollution history such as no-pollution and pollution with organic contaminants, which is phenanthrene and other polycyclic aromatic hydrocarbons (PAHs). Agricultural soils showed significant differences in some of physical-chemical properties (i.e. organic C, total and labile phosphate contents, available Ca and Mg) between the two sites studied. Enzyme activities of hazel sites periodically flooded by wastes were mainly higher than in the hazel sites never flooded. Sites under many years of continuous corn showed dehydrogenase, invertase, arylsulphatase and beta-glucosidase activities generally lower than the soils under hazel either flooded or not by wastes. As compared to agricultural soils, non-cultivated soils heavily or moderately polluted by organic contaminants displayed much lower values or complete absence of enzymatic activities. Dissimilar, contradictory correlations between soil enzyme activities and the majority of soil properties were observed separately in the two groups of soils. When the whole set of enzyme activities and soil properties were considered, all significant correlations found separately for the groups of soils were lost. The overall results seem to confirm that no direct cause-effect relationships can be derived between the changes of a soil in response to a given factor and both the variations of the activity and the behaviour of the enzymes in soil

  8. Fluorescent profiling of modular biosynthetic enzymes by complementary metabolic and activity based probes.

    PubMed

    Meier, Jordan L; Mercer, Andrew C; Burkart, Michael D

    2008-04-23

    The study of the enzymes responsible for natural product biosynthesis has proven a valuable source of new enzymatic activities and been applied to a number of biotechnology applications. Protein profiling could prove highly complementary to genetics based approaches by allowing us to understand the activity, transcriptional control, and post-translational modification of these enzymes in their native and dynamic proteomic environments. Here we present a method for the fluorescent profiling of PKS, NRPS, and FAS multidomain modular synthases in their whole proteomes using complementary metabolic and activity based probes. After first examining the reactivity of these activity based probes with a variety of purified recombinant PKS, NRPS, and FAS enzymes in vitro, we apply this duel labeling strategy to the analysis of modular synthases in a human breast cancer cell line and two strains of the natural product producer Bacillus subtilis. Collectively, these studies demonstrate that complementary protein profiling approaches can prove highly useful in the identification and assignment of inhibitor specificity and domain structure of these modular biosynthetic enzymes. PMID:18376827

  9. Glutathione and GSH-dependent enzymes in bronchoalveolar lavage fluid cells in response to ozone

    SciTech Connect

    Boehme, D.S.; Hotchkiss, J.A.; Henderson, R.F. )

    1992-02-01

    The purpose of this study was to determine if in vivo ozone exposure results in elevations in the levels of glutathione and glutathione-dependent enzymes in cells derived from bronchoalveolar lavage fluid (BALF). Our hypothesis was that, as part of a defense mechanism against oxygen toxicity, such cells would have increased levels of glutathione (GSH) in response to an oxidant stress. Female F344/N rats were exposed to 0.8 ppm ozone, 6 hr/day, for 1, 3, or 7 days, after which cells were collected by lung lavage. The GSH and GSH-peroxidase activity per milligram of protein in the cellular fraction, both necessary for reducing cellular peroxides, were elevated after 3 days of ozone exposure. After 7 days of exposure, cellular GSH had returned to control values, but the activity of glutathione reductase, the enzyme that reduces oxidized glutathione to GSH, was increased. Extracellular GSH concentration and glutathione reductase activity in BALF were also increased after 7 days of exposure. The total glutathione equivalents (GSH and GSSG, both cellular and extracellular) in BALF increased throughout the 7-day exposure, with GSH increasing first in the cells, and then in the extracellular fluid. This study demonstrated that the glutathione anti-oxidant system of BALF cells is stimulated by exposure to ozone. This response may serve to protect cells from the toxic effects of oxidant stress.

  10. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, S. C.; Brown, C. S. (Principal Investigator)

    2000-01-01

    Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.

  11. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, Eric E.; Roessler, Paul G.

    1999-01-01

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

  12. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, E.E.; Roessler, P.G.

    1999-07-27

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

  13. Glucocerebrosidase enzyme activity in GBA mutation Parkinson's disease.

    PubMed

    Ortega, Roberto A; Torres, Paola A; Swan, Matthew; Nichols, William; Boschung, Sarah; Raymond, Deborah; Barrett, Matthew J; Johannes, Brooke A; Severt, Lawrence; Shanker, Vicki; Hunt, Ann L; Bressman, Susan; Pastores, Gregory M; Saunders-Pullman, Rachel

    2016-06-01

    Mutations in the glucocerebrosidase (GBA1) gene, the most common genetic contributor to Parkinson's disease (PD), are associated with an increased risk of PD in heterozygous and homozygous carriers. While glucocerebrosidase enzyme (GCase) activity is consistently low in Gaucher disease, there is a range of leukocyte GCase activity in healthy heterozygous GBA1 mutation carriers. To determine whether GCase activity may be a marker for PD with heterozygous GBA1 mutations (GBA1 mutation PD, GBA PD), GBA PD patients (n=15) were compared to PD patients without heterozygous GBA1 mutations (idiopathic PD; n=8), heterozygous GBA1 carriers without PD (asymptomatic carriers; n=4), and biallelic mutation carriers with PD (Gaucher disease with PD, GD1 PD; n=3) in a pilot study. GCase activity (nmol/mg protein/hour) in GD1 PD (median [interquartile range]; minimum-maximum: 6.4 [5.7]; 5.3-11) was lower than that of GBA PD (16.0 [7.0]; 11-40) (p=0.01), while GCase activity in GBA PD was lower than idiopathic PD (28.5 [15.0]; 16-56) (p=0.01) and asymptomatic carriers (25.5 [2.5]; 23-27) (p=0.04). Therefore, GCase activity appears to be a possible marker of heterozygous GBA1 mutation PD, and larger studies are warranted. Prospective studies are also necessary to determine whether lower GCase activity precedes development of PD. PMID:26857292

  14. Carbon-Degrading Enzyme Activities Stimulated by Increased Nutrient Availability in Arctic Tundra Soils

    PubMed Central

    Koyama, Akihiro; Wallenstein, Matthew D.; Simpson, Rodney T.; Moore, John C.

    2013-01-01

    Climate-induced warming of the Arctic tundra is expected to increase nutrient availability to soil microbes, which in turn may accelerate soil organic matter (SOM) decomposition. We increased nutrient availability via fertilization to investigate the microbial response via soil enzyme activities. Specifically, we measured potential activities of seven enzymes at four temperatures in three soil profiles (organic, organic/mineral interface, and mineral) from untreated native soils and from soils which had been fertilized with nitrogen (N) and phosphorus (P) since 1989 (23 years) and 2006 (six years). Fertilized plots within the 1989 site received annual additions of 10 g N⋅m-2⋅year-1 and 5 g P⋅m-2⋅year-1. Within the 2006 site, two fertilizer regimes were established – one in which plots received 5 g N⋅m-2⋅year-1 and 2.5 g P⋅m-2⋅year-1 and one in which plots received 10 g N⋅m-2⋅year-1 and 5 g P⋅m-2⋅year-1. The fertilization treatments increased activities of enzymes hydrolyzing carbon (C)-rich compounds but decreased phosphatase activities, especially in the organic soils. Activities of two enzymes that degrade N-rich compounds were not affected by the fertilization treatments. The fertilization treatments increased ratios of enzyme activities degrading C-rich compounds to those for N-rich compounds or phosphate, which could lead to changes in SOM chemistry over the long term and to losses of soil C. Accelerated SOM decomposition caused by increased nutrient availability could significantly offset predicted increased C fixation via stimulated net primary productivity in Arctic tundra ecosystems. PMID:24204773

  15. Carbon-degrading enzyme activities stimulated by increased nutrient availability in Arctic tundra soils.

    PubMed

    Koyama, Akihiro; Wallenstein, Matthew D; Simpson, Rodney T; Moore, John C

    2013-01-01

    Climate-induced warming of the Arctic tundra is expected to increase nutrient availability to soil microbes, which in turn may accelerate soil organic matter (SOM) decomposition. We increased nutrient availability via fertilization to investigate the microbial response via soil enzyme activities. Specifically, we measured potential activities of seven enzymes at four temperatures in three soil profiles (organic, organic/mineral interface, and mineral) from untreated native soils and from soils which had been fertilized with nitrogen (N) and phosphorus (P) since 1989 (23 years) and 2006 (six years). Fertilized plots within the 1989 site received annual additions of 10 g N · m(-2) · year(-1) and 5 g P · m(-2) · year(-1). Within the 2006 site, two fertilizer regimes were established--one in which plots received 5 g N · m(-2) · year(-1) and 2.5 g P · m(-2) · year(-1) and one in which plots received 10 g N · m(-2) · year(-1) and 5 g P · m(-2) · year(-1). The fertilization treatments increased activities of enzymes hydrolyzing carbon (C)-rich compounds but decreased phosphatase activities, especially in the organic soils. Activities of two enzymes that degrade N-rich compounds were not affected by the fertilization treatments. The fertilization treatments increased ratios of enzyme activities degrading C-rich compounds to those for N-rich compounds or phosphate, which could lead to changes in SOM chemistry over the long term and to losses of soil C. Accelerated SOM decomposition caused by increased nutrient availability could significantly offset predicted increased C fixation via stimulated net primary productivity in Arctic tundra ecosystems. PMID:24204773

  16. Engineering Enzymes in Energy Crops: Conditionally Activated Enzymes Expressed in Cellulosic Energy Crops

    SciTech Connect

    2010-01-15

    Broad Funding Opportunity Announcement Project: Enzymes are required to break plant biomass down into the fermentable sugars that are used to create biofuel. Currently, costly enzymes must be added to the biofuel production process. Engineering crops to already contain these enzymes will reduce costs and produce biomass that is more easily digested. In fact, enzyme costs alone account for $0.50-$0.75/gallon of the cost of a biomass-derived biofuel like ethanol. Agrivida is genetically engineering plants to contain high concentrations of enzymes that break down cell walls. These enzymes can be “switched on” after harvest so they won’t damage the plant while it’s growing.

  17. Real-Time Drug Release Analysis of Enzyme and pH Responsive Polysaccharide Nanovesicles.

    PubMed

    Pramod, Poothayil Subash; Deshpande, Nilesh Umakant; Jayakannan, Manickam

    2015-08-20

    The accurate estimation of drug release kinetics of polymeric vehicles is an indispensable prerequisite for the developments of successful drug carriers for cancer therapy. The present investigation reports the development of time-resolved fluorescence spectroscopic approach for the real-time release kinetics of fluorophore loaded polysaccharide vesicles that are potential vectors in cancer treatment. The polysaccharide vesicles were custom designed with appropriate enzyme and pH responsiveness and loaded with water-soluble biocompatible fluorophore Rhodamine B (Rh-B). The semipermeable membrane dialysis method along with steady state absorbance spectroscopic technique was found to be inaccurate for the estimation of drug release. Time correlated single photon counting (TCSPC) technique was found to exhibit significant difference in excited state decay profiles and fluorescent lifetime of Rh-B in the free and polymer bound states. This enabled the establishment of real-time drug release protocols by TCSPC method for polysaccharide vesicles that are responsible to pH and enzyme with respect to intracellular compartments. Real-time analysis predicted the release kinetics 20-25% higher accuracy when compared to the dialysis method under in vitro conditions. Moreover, the ability of enzyme to cleave the polysaccharide vesicles was further validated by docking studies. The positioning of the molecules in active site of enzyme and the binding energy data were generated using AUTODOCK program to study the rupture of polysaccharide vesicles. This new TCSPC technique could be very useful for studying the drug release pattern of synthetic polymer vesicles loaded with Rh-B fluorophore. PMID:26237375

  18. Stable Colloidal Drug Aggregates Catch and Release Active Enzymes.

    PubMed

    McLaughlin, Christopher K; Duan, Da; Ganesh, Ahil N; Torosyan, Hayarpi; Shoichet, Brian K; Shoichet, Molly S

    2016-04-15

    Small molecule aggregates are considered nuisance compounds in drug discovery, but their unusual properties as colloids could be exploited to form stable vehicles to preserve protein activity. We investigated the coaggregation of seven molecules chosen because they had been previously intensely studied as colloidal aggregators, coformulating them with bis-azo dyes. The coformulation reduced colloid sizes to <100 nm and improved uniformity of the particle size distribution. The new colloid formulations are more stable than previous aggregator particles. Specifically, coaggregation of Congo Red with sorafenib, tetraiodophenolphthalein (TIPT), or vemurafenib produced particles that are stable in solutions of high ionic strength and high protein concentrations. Like traditional, single compound colloidal aggregates, the stabilized colloids adsorbed and inhibited enzymes like β-lactamase, malate dehydrogenase, and trypsin. Unlike traditional aggregates, the coformulated colloid-protein particles could be centrifuged and resuspended multiple times, and from resuspended particles, active trypsin could be released up to 72 h after adsorption. Unexpectedly, the stable colloidal formulations can sequester, stabilize, and isolate enzymes by spin-down, resuspension, and release. PMID:26741163

  19. Activity of extracellular enzymes on the marine beach differing in the level of antropopressure.

    PubMed

    Perliński, P; Mudryk, Z J

    2016-03-01

    The level of activity of extracellular enzymes was determined on two transects characterised by different anthropic pressure on a sandy beach in Ustka, the southern coast of the Baltic Sea. Generally, the level of activity of the studied enzymes was higher on the transect characterised by high anthropic pressure. The ranking order of the mean enzyme activity rates in the sand was as follows: lipase > phosphatase > aminopeptidase > β-glucosidase > α-glucosidase > chitinase. Each enzyme had its characteristic horizontal profile of activity. The levels of activity of the studied enzymes were slightly higher in the surface than subsurface sand layer. Extracellular enzymatic activities were strongly influenced by the season. PMID:26911592

  20. Evolution of an Antibiotic Resistance Enzyme Constrained by Stability and Activity Trade-offs

    SciTech Connect

    Wang, Xiaojun; Minasov, George; Shoichet, Brian K.

    2010-03-08

    Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the {beta}-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182 {yields} Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity.

  1. Effect of sulfite intake on intestinal enzyme activity in rats.

    PubMed

    Rodriguez Vieytes, M; Martinez-Sapiña, J; Taboada Montero, C; Lamas Aneiros, M

    1994-01-01

    Sulfites are usually added to food, beverages and pharmaceuticals as preservative antioxidants, bleaching agents, and dough conditioning agents. Ingestion of foods containing sulfites can cause abdominal pain, diarrhoea, seizures and death. Sulfite can react with cellular components and can cause toxicity. Changes in mucosal disaccharidases and phosphatase alkaline after sodium metabisulfite administration were investigated in the small intestine of rats. Female Wistar rats were given a diet supplemented with 0.25 or 2.5% sodium metabisulfite for 5 weeks. Sucrase, maltase, lactase and alkaline phosphatase were assayed in intestinal homogenates and in brush border membrane fractions. The intake of only 2.5% sulfite induced an increase in the specific activities of sucrase, maltase, and alkaline phosphatase compared to control levels (P < 0.05). Lactase levels were affected in a variable manner. The origin of such altered enzyme activities is still unknown. PMID:7958644

  2. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes.

    PubMed

    Chu, Wen-Ting; Wang, Jin

    2016-01-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the "hot-spot" within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design. PMID:27298067

  3. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    PubMed Central

    Chu, Wen-Ting; Wang, Jin

    2016-01-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design. PMID:27298067

  4. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    NASA Astrophysics Data System (ADS)

    Chu, Wen-Ting; Wang, Jin

    2016-06-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

  5. Cleome rutidosperma and Euphorbia thymifolia Suppress Inflammatory Response via Upregulation of Phase II Enzymes and Modulation of NF-κB and JNK Activation in LPS-Stimulated BV2 Microglia.

    PubMed

    Ding, Hsiou-Yu; Wu, Pei-Shan; Wu, Ming-Jiuan

    2016-01-01

    Cleome rutidosperma DC. and Euphorbia thymifolia L. are herbal medicines used in traditional Indian and Chinese medicine to treat various illnesses. Reports document that they have antioxidant and anti-inflammatory activities; nonetheless, the molecular mechanisms involved in their anti-inflammatory actions have not yet been elucidated. The anti-neuroinflammatory activities and underlying mechanisms of ethanol extracts of Cleome rutidosperma (CR) and Euphorbia thymifolia (ET) were studied using lipopolysaccharide (LPS)-stimulated microglial cell line BV2. The morphology changes and production of pro-inflammatory mediators were assayed. Gene expression of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-1β, and CC chemokine ligand (CCL)-2, as well as phase II enzymes such as heme oxygenase (HO)-1, the modifier subunit of glutamate cysteine ligase (GCLM) and NAD(P)H quinone dehydrogenase 1 (NQO1), were further investigated using reverse transcription quantitative-PCR (RT-Q-PCR) and Western blotting. The effects of CR and ET on mitogen activated protein kinases (MAPKs) and nuclear factor (NF)-κB signaling pathways were examined using Western blotting and specific inhibitors. CR and ET suppressed BV2 activation, down-regulated iNOS and COX-2 expression and inhibited nitric oxide (NO) overproduction without affecting cell viability. They reduced LPS-mediated tumor necrosis factor (TNF) and IL-6 production, attenuated IL-1β and CCL2 expression, but upregulated HO-1, GCLM and NQO1 expression. They also inhibited p65 NF-κB phosphorylation and modulated Jun-N terminal kinase (JNK) activation in BV2 cells. SP600125, the JNK inhibitor, significantly augmented the anti-IL-6 activity of ET. NF-κB inhibitor, Bay 11-7082, enhanced the anti-IL-6 effects of both CR and ET. Znpp, a competitive inhibitor of HO-1, attenuated the anti-NO effects of CR and ET. Our results show that CR and ET exhibit anti

  6. UDP-glucuronosyltransferase 1a enzymes are present and active in the mouse blastocyst.

    PubMed

    Collier, Abby C; Yamauchi, Yasuhiro; Sato, Brittany L M; Rougée, Luc R A; Ward, Monika A

    2014-11-01

    The UDP-glucuronosyltransferase (UGT) enzymes are critical for regulating nutrients, hormones, and endobiotics, as well as for detoxifying xenobiotics. Human and murine fetuses are known to express glucuronidation enzymes, but there are currently no data prior to implantation. Here we addressed this gap in knowledge and tested whether Ugt enzymes are already present in preimplantation-stage embryos. Blastocysts were obtained after in vitro fertilization with gametes from B6D2F1 hybrid mice and from embryo culture. Protein expression and localization were determined using pan-specific UGT1A and UGT2B, as well as anti-human isoform-specific antibodies. Immunofluorescence analysis showed that blastocysts expressed Ugt1a globally, in the cytoplasm and nuclei of all of the cells. Western blots demonstrated the presence of Ugt1a6 but not Ugt1a1, Ugt1a3, Ugt1a4, or Ugt1a9. The Ugt2b proteins were not detected by either assay. The level of Ugt activity in murine blastocysts was comparable with that of the adult human liver (per milligram of protein), but the activity of β-glucuronidase, an Ugt-partnering enzyme responsible for substrate regeneration, was lower. Altogether, these data confirm that Ugt1a proteins are present and active in preimplantation murine embryos and point to a potential role for these proteins in implantation and early embryonic and fetal development. PMID:25200869

  7. UDP-Glucuronosyltransferase 1a Enzymes Are Present and Active in the Mouse Blastocyst

    PubMed Central

    Yamauchi, Yasuhiro; Sato, Brittany L.M.; Rougée, Luc R.A.; Ward, Monika A.

    2014-01-01

    The UDP-glucuronosyltransferase (UGT) enzymes are critical for regulating nutrients, hormones, and endobiotics, as well as for detoxifying xenobiotics. Human and murine fetuses are known to express glucuronidation enzymes, but there are currently no data prior to implantation. Here we addressed this gap in knowledge and tested whether Ugt enzymes are already present in preimplantation-stage embryos. Blastocysts were obtained after in vitro fertilization with gametes from B6D2F1 hybrid mice and from embryo culture. Protein expression and localization were determined using pan-specific UGT1A and UGT2B, as well as anti-human isoform-specific antibodies. Immunofluorescence analysis showed that blastocysts expressed Ugt1a globally, in the cytoplasm and nuclei of all of the cells. Western blots demonstrated the presence of Ugt1a6 but not Ugt1a1, Ugt1a3, Ugt1a4, or Ugt1a9. The Ugt2b proteins were not detected by either assay. The level of Ugt activity in murine blastocysts was comparable with that of the adult human liver (per milligram of protein), but the activity of β-glucuronidase, an Ugt-partnering enzyme responsible for substrate regeneration, was lower. Altogether, these data confirm that Ugt1a proteins are present and active in preimplantation murine embryos and point to a potential role for these proteins in implantation and early embryonic and fetal development. PMID:25200869

  8. Solar simulated irradiation modulates gene expression and activity of antioxidant enzymes in cultured human dermal fibroblasts.

    PubMed

    Leccia, M T; Yaar, M; Allen, N; Gleason, M; Gilchrest, B A

    2001-08-01

    Exposure of skin to solar irradiation generates reactive oxygen species that damage DNA, membranes, mitochondria and proteins. To protect against such damage, skin cells have evolved antioxidant enzymes including glutathione peroxidase (GSH-Px), copper and zinc-dependent superoxide dismutase (SOD1), the mitochondrial manganese-dependent superoxide dismutase (SOD2), and catalase. This report examines the effect of a single low or moderate dose exposure to solar-simulating combined UVB and UVA irradiation on the gene expression and activities of these antioxidant enzymes in cultured normal human fibroblasts. We find that both doses initially decrease GSH-Px, SOD2 and catalase activities, but within 5 days after irradiation the activities of the enzymes return to pre-irradiation level (catalase) or are induced slightly (SOD1, GSH-Px) or substantially (SOD2) above the basal level. For SOD1, SOD2 and catalase, the higher dose also detectably modulates the mRNA level of these enzymes. Our results indicate that the effects of a single physiologic solar simulated irradiation dose persist for at least several days and suggest that skin cells prepare for subsequent exposure to damaging irradiation by upregulating this antioxidant defense system, in particular the mitochondrial SOD2. Our findings are consistent with the existence of a broad-based SOS-like response in irradiated human skin. PMID:11493316

  9. Systematic genetic and genomic analysis of cytochrome P450 enzyme activities in human liver

    PubMed Central

    Yang, Xia; Zhang, Bin; Molony, Cliona; Chudin, Eugene; Hao, Ke; Zhu, Jun; Gaedigk, Andrea; Suver, Christine; Zhong, Hua; Leeder, J. Steven; Guengerich, F. Peter; Strom, Stephen C.; Schuetz, Erin; Rushmore, Thomas H.; Ulrich, Roger G.; Slatter, J. Greg; Schadt, Eric E.; Kasarskis, Andrew; Lum, Pek Yee

    2010-01-01

    Liver cytochrome P450s (P450s) play critical roles in drug metabolism, toxicology, and metabolic processes. Despite rapid progress in the understanding of these enzymes, a systematic investigation of the full spectrum of functionality of individual P450s, the interrelationship or networks connecting them, and the genetic control of each gene/enzyme is lacking. To this end, we genotyped, expression-profiled, and measured P450 activities of 466 human liver samples and applied a systems biology approach via the integration of genetics, gene expression, and enzyme activity measurements. We found that most P450s were positively correlated among themselves and were highly correlated with known regulators as well as thousands of other genes enriched for pathways relevant to the metabolism of drugs, fatty acids, amino acids, and steroids. Genome-wide association analyses between genetic polymorphisms and P450 expression or enzyme activities revealed sets of SNPs associated with P450 traits, and suggested the existence of both cis-regulation of P450 expression (especially for CYP2D6) and more complex trans-regulation of P450 activity. Several novel SNPs associated with CYP2D6 expression and enzyme activity were validated in an independent human cohort. By constructing a weighted coexpression network and a Bayesian regulatory network, we defined the human liver transcriptional network structure, uncovered subnetworks representative of the P450 regulatory system, and identified novel candidate regulatory genes, namely, EHHADH, SLC10A1, and AKR1D1. The P450 subnetworks were then validated using gene signatures responsive to ligands of known P450 regulators in mouse and rat. This systematic survey provides a comprehensive view of the functionality, genetic control, and interactions of P450s. PMID:20538623

  10. Microbial responses to multi-factor climate change: effects on soil enzymes

    PubMed Central

    Steinweg, J. Megan; Dukes, Jeffrey S.; Paul, Eldor A.; Wallenstein, Matthew D.

    2013-01-01

    The activities of extracellular enzymes, the proximate agents of decomposition in soils, are known to depend strongly on temperature, but less is known about how they respond to changes in precipitation patterns, and the interaction of these two components of climate change. Both enzyme production and turnover can be affected by changes in temperature and soil moisture, thus it is difficult to predict how enzyme pool size may respond to altered climate. Soils from the Boston-Area Climate Experiment (BACE), which is located in an old field (on abandoned farmland), were used to examine how climate variables affect enzyme activities and microbial biomass carbon (MBC) in different seasons and in soils exposed to a combination of three levels of precipitation treatments (ambient, 150% of ambient during growing season, and 50% of ambient year-round) and four levels of warming treatments (unwarmed to ~4°C above ambient) over the course of a year. Warming, precipitation and season had very little effect on potential enzyme activity. Most models assume that enzyme dynamics follow microbial biomass, because enzyme production should be directly controlled by the size and activity of microbial biomass. We observed differences among seasons and treatments in mass-specific potential enzyme activity, suggesting that this assumption is invalid. In June 2009, mass-specific potential enzyme activity, using chloroform fumigation-extraction MBC, increased with temperature, peaking under medium warming and then declining under the highest warming. This finding suggests that either enzyme production increased with temperature or turnover rates decreased. Increased maintenance costs associated with warming may have resulted in increased mass-specific enzyme activities due to increased nutrient demand. Our research suggests that allocation of resources to enzyme production could be affected by climate-induced changes in microbial efficiency and maintenance costs. PMID:23781218

  11. County-Scale Spatial Distribution of Soil Enzyme Activities and Enzyme Activity Indices in Agricultural Land: Implications for Soil Quality Assessment

    PubMed Central

    Xie, Baoni; Wang, Junxing; He, Wenxiang; Wang, Xudong; Wei, Gehong

    2014-01-01

    Here the spatial distribution of soil enzymatic properties in agricultural land was evaluated on a county-wide (567 km2) scale in Changwu, Shaanxi Province, China. The spatial variations in activities of five hydrolytic enzymes were examined using geostatistical methods. The relationships between soil enzyme activities and other soil properties were evaluated using both an integrated total enzyme activity index (TEI) and the geometric mean of enzyme activities (GME). At the county scale, soil invertase, phosphatase, and catalase activities were moderately spatially correlated, whereas urease and dehydrogenase activities were weakly spatially correlated. Correlation analysis showed that both TEI and GME were better correlated with selected soil physicochemical properties than single enzyme activities. Multivariate regression analysis showed that soil OM content had the strongest positive effect while soil pH had a negative effect on the two enzyme activity indices. In addition, total phosphorous content had a positive effect on TEI and GME in orchard soils, whereas alkali-hydrolyzable nitrogen and available potassium contents, respectively, had negative and positive effects on these two enzyme indices in cropland soils. The results indicate that land use changes strongly affect soil enzyme activities in agricultural land, where TEI provides a sensitive biological indicator for soil quality. PMID:25610908

  12. SALT SENSITIVITY IN RESPONSE TO RENAL INJURY REQUIRES RENAL ANGIOTENSIN-CONVERTING ENZYME

    PubMed Central

    Giani, Jorge F.; Bernstein, Kenneth E.; Janjulia, Tea; Han, Jiyang; Toblli, Jorge E.; Shen, Xiao Z.; Rodriguez-Iturbe, Bernardo; McDonough, Alicia A.; Gonzalez-Villalobos, Romer A.

    2015-01-01

    Recent evidence indicates that salt-sensitive hypertension can result from a subclinical injury that impairs the kidneys’ capacity to properly respond to a high salt diet. However, how this occurs is not well understood. Here, we showed that while previously salt resistant wild-type mice became salt-sensitive after the induction of renal injury with the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME); mice lacking renal angiotensin-converting enzyme, exposed to the same insult, did not become hypertensive when faced with a sodium load. This is because the activity of renal angiotensin-converting enzyme plays a critical role in: 1) augmenting the local pool of angiotensin II and, 2) the establishment of the anti-natriuretic state via modulation of glomerular filtration rate and sodium tubular transport. Thus, this study demonstrates that the presence of renal angiotensin-converting enzyme plays a pivotal role in the development of salt sensitivity in response to renal injury. PMID:26150439

  13. Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production.

    PubMed

    Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

    2015-12-01

    Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireA-repressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes. PMID:26496881

  14. Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy.

    PubMed

    Guo, Qing; He, Yufan; Lu, H Peter

    2015-11-10

    Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure-function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2-amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme-substrate interactions, enzyme-substrate active complex formation, and protein folding-binding interactions. PMID:26512103

  15. Phlorotannins from Alaskan Seaweed Inhibit Carbolytic Enzyme Activity

    PubMed Central

    Kellogg, Joshua; Grace, Mary H.; Lila, Mary Ann

    2014-01-01

    Global incidence of type 2 diabetes has escalated over the past few decades, necessitating a continued search for natural sources of enzyme inhibitors to offset postprandial hyperglycemia. The objective of this study was to evaluate coastal Alaskan seaweed inhibition of α-glucosidase and α-amylase, two carbolytic enzymes involved in serum glucose regulation. Of the six species initially screened, the brown seaweeds Fucus distichus and Alaria marginata possessed the strongest inhibitory effects. F. distichus fractions were potent mixed-mode inhibitors of α-glucosidase and α-amylase, with IC50 values of 0.89 and 13.9 μg/mL, respectively; significantly more efficacious than the pharmaceutical acarbose (IC50 of 112.0 and 137.8 μg/mL, respectively). The activity of F. distichus fractions was associated with phlorotannin oligomers. Normal-phase liquid chromatography-mass spectrometry (NPLC-MS) was employed to characterize individual oligomers. Accurate masses and fragmentation patterns confirmed the presence of fucophloroethol structures with degrees of polymerization from 3 to 18 monomer units. These findings suggest that coastal Alaskan seaweeds are sources of α-glucosidase and α-amylase inhibitory phlorotannins, and thus have potential to limit the release of sugar from carbohydrates and thus alleviate postprandial hyperglycemia. PMID:25341030

  16. Bacterial communities and enzyme activities of PAHs polluted soils.

    PubMed

    Andreoni, V; Cavalca, L; Rao, M A; Nocerino, G; Bernasconi, S; Dell'Amico, E; Colombo, M; Gianfreda, L

    2004-11-01

    Three soils (i.e. a Belgian soil, B-BT, a German soil, G, and an Italian agricultural soil, I-BT) with different properties and hydrocarbon-pollution history with regard to their potential to degrade phenanthrene were investigated. A chemical and microbiological evaluation of soils was done using measurements of routine chemical properties, bacterial counts and several enzyme activities. The three soils showed different levels of polycyclic aromatic hydrocarbons (PAHs), being their contamination strictly associated to their pollution history. High values of enzyme activities and culturable heterotrophic bacteria were detected in the soil with no or negligible presence of organic pollutants. Genetic diversity of soil samples and enrichment cultures was measured as bands on denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the soil and enrichment community DNAs. When analysed by Shannon index (H'), the highest genetic biodiversity (H'=2.87) was found in the Belgian soil B-BT with a medium-term exposition to PAHs and the poorest biodiversity (H'=0.85) in the German soil with a long-term exposition to alkanes and PAHs and where absence, or lower levels of enzyme activities were measured. For the Italian agricultural soil I-BT, containing negligible amounts of organic pollutants but the highest Cu content, a Shannon index=2.13 was found. The enrichment of four mixed cultures capable of degrading solid phenanthrene in batch liquid systems was also studied. Phenanthrene degradation rates in batch systems were culture-dependent, and simple (one-slope) and complex (two-slope) kinetic behaviours were observed. The presence of common bands of microbial species in the cultures and in the native soil DNA indicated that those strains could be potential in situ phenanthrene degraders. Consistent with this assumption are the decrease of PAH and phenanthrene contents of Belgian soil B-BT and the isolation of phenanthrene-degrading bacteria. From

  17. The structure of an enzyme-activating fragment of human telomerase RNA.

    PubMed

    Leeper, Thomas C; Varani, Gabriele

    2005-04-01

    The ribonucleoprotein enzyme telomerase ensures the stability and fidelity of linear chromosome ends by elongating the telomeric DNA that is lost during each round of DNA replication. All telomerases contain a catalytic protein component homologous to viral reverse transcriptases (TERT) and an RNA (TR) that provides the template sequence, acts as the scaffold for ribonucleoprotein assembly, and activates the enzyme for catalysis. Vertebrate telomerase RNAs contain three highly conserved structural and functional domains: the template domain, the "CR4-CR5" or "activation" domain essential for activation of the enzymatic activity, and a 3'-terminal "box H/ACA"-homology domain responsible for ribonucleprotein assembly and maturation. Here we report the NMR structure of a functionally essential RNA structural element derived from the human telomerase RNA CR4-CR5 domain. This RNA, referred to as hTR J6, forms a stable hairpin interrupted by a single nucleotide bulge and an asymmetric internal loop. Previous work on telomerase has shown that deletion of the hTR J6 asymmetric internal loop results in an RNA incapable of binding the enzymatic protein component of the RNP and therefore an inactive RNP without telomerase activity. We demonstrate here that the J6 internal loop introduces a twist in the RNA structure that may position the entire domain into the catalytic site of the enzyme. PMID:15703438

  18. Effect of clofibrate on the enzyme activity of rat liver plasma membranes.

    PubMed

    Renaud, G; Foliot, A; Marais, J; Infante, R

    1980-03-15

    The activity of 3 plasma membranes marker enzymes (5'-nucleotidase, Mg++-ATPase and alkaline phosphodiesterase-I) was determined in plasma membranes isolated from liver of control and of clofibrate-treated rats. A complete indentity of plasma membranes enzyme activity in the 2 groups of experimental animals was observed for the 3 enzymes studied. PMID:6102923

  19. Effects of Fertilization on Tomato Growth and Soil Enzyme Activity

    NASA Astrophysics Data System (ADS)

    Mu, Zhen; Hu, Xue-Feng; Cheng, Chang; Luo, Zhi-qing

    2015-04-01

    To study the effects of different fertilizer applications on soil enzyme activity, tomato plant growth and tomato yield and quality, a field experiment on tomato cultivation was carried out in the suburb of Shanghai. Three fertilizer treatments, chemical fertilizer (CF) (N, 260 g/kg; P, 25.71g/kg; K, 83.00g/kg), rapeseed cake manure (CM) (N, 37.4 g/kg; P, 9.0 g/kg; K, 8.46 g/kg), crop-leaf fermenting manure (FM) (N, 23.67 g/kg; P, 6.39 g/kg; K 44.32 g/kg), and a control without using any fertilizers (CK), were designed. The total amounts of fertilizer application to each plot for the CF, CM, FM and CK were 0.6 kg, 1.35 kg, 3.75 kg and 0 kg, respectively, 50% of which were applied as base fertilizer, and another 50% were applied after the first fruit picking as top dressing. Each experimental plot was 9 m2 (1 m × 9 m) in area. Each treatment was replicated for three times. No any pesticides and herbicides were applied during the entire period of tomato growth to prevent their disturbance to soil microbial activities. Soil enzyme activities at each plot were constantly tested during the growing period; the tomato fruit quality was also constantly analyzed and the tomato yield was calculated after the final harvesting. The results were as follows: (1) Urease activity in the soils treated with the CF, CM and FM increased quickly after applying base fertilizer. That with the CF reached the highest level. Sucrase activity was inhibited by the CF and CM to some extent, which was 32.4% and 11.2% lower than that with the CK, respectively; while that with the FM was 15.7% higher than that with the CK. Likewise, catalase activity with the CF increased by 12.3% - 28.6%; that with the CM increased by 87.8% - 95.1%; that with the FM increased by 86.4% - 93.0%. Phosphatase activity with the CF increased rapidly and reached a maximum 44 days after base fertilizer application, and then declined quickly. In comparison, that with the CM and FM increased slowly and reached a maximum

  20. Polymer-based protein engineering can rationally tune enzyme activity, pH-dependence, and stability.

    PubMed

    Murata, Hironobu; Cummings, Chad S; Koepsel, Richard R; Russell, Alan J

    2013-06-10

    The attachment of inert polymers, such as polyethylene glycol, to proteins has driven the emergence of a multibillion dollar biotechnology industry. In all cases, proteins have been stabilized or altered by covalently coupling the pre-existing polymer to the surface of the protein. This approach is inherently limited by a lack of exquisite control of polymer architecture, site and density of attachment. Using a novel water-soluble atom transfer radical polymerization initiator, we have grown temperature- and pH-responsive polymers from the surface of a model protein, the enzyme chymotrypsin. Poly(2-(dimethylamino)ethyl methacrylate) changes in conformation with altered temperature and pH. Growing the polymer from the surface of chymotrypsin we were able to demonstrate that changes in temperature or pH can change predictably the conformation of the polymer surrounding the enzyme, which in turn enabled the rational tailoring of enzyme activity and stability. Using what we now term "Polymer-Based Protein Engineering", we have increased the activity and stability of chymotrypsin by an order of magnitude at pHs where the enzyme is usually inactive or unstable. PMID:23600667

  1. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation.

    PubMed

    Dzamukova, Maria R; Naumenko, Ekaterina A; Lvov, Yuri M; Fakhrullin, Rawil F

    2015-01-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment. PMID:25976444

  2. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

    PubMed Central

    Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2015-01-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (А549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment. PMID:25976444

  3. Microbial enzyme activities of peatland soils in south central Alaska lowlands

    EPA Science Inventory

    Microbial enzyme activities related to carbon and nutrient acquisition were measured on Alaskan peatland soils as indicators of nutrient limitation and biochemical sustainability. Peat decomposition is mediated by microorganisms and enzymes that in turn are limited by various ph...

  4. A Review on the Effects of Supercritical Carbon Dioxide on Enzyme Activity

    PubMed Central

    Wimmer, Zdeněk; Zarevúcka, Marie

    2010-01-01

    Different types of enzymes such as lipases, several phosphatases, dehydrogenases, oxidases, amylases and others are well suited for the reactions in SC-CO2. The stability and the activity of enzymes exposed to carbon dioxide under high pressure depend on enzyme species, water content in the solution and on the pressure and temperature of the reaction system. The three-dimensional structure of enzymes may be significantly altered under extreme conditions, causing their denaturation and consequent loss of activity. If the conditions are less adverse, the protein structure may be largely retained. Minor structural changes may induce an alternative active protein state with altered enzyme activity, specificity and stability. PMID:20162013

  5. Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

    PubMed Central

    Brust, Belinda; Lecoufle, Mélanie; Tuaillon, Edouard; Dedieu, Luc; Canaan, Stéphane; Valverde, Viviane; Kremer, Laurent

    2011-01-01

    Background New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. Methods Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. Results A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. Conclusion These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high

  6. Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance.

    PubMed

    Sun, Baodong; Bird, Andrew; Young, Sarah P; Kishnani, Priya S; Chen, Y-T; Koeberl, Dwight D

    2007-11-01

    Pompe disease, which results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid alpha -glucosidase (GAA) (also called "acid maltase"), causes death in early childhood related to glycogen accumulation in striated muscle and an accompanying infantile-onset cardiomyopathy. The efficacy of enzyme replacement therapy (ERT) with recombinant human GAA was demonstrated during clinical trials that prolonged subjects' overall survival, prolonged ventilator-free survival, and also improved cardiomyopathy, which led to broad-label approval by the U.S. Food and Drug Administration. Patients who lack any residual GAA expression and are deemed negative for cross-reacting immunologic material (CRIM) have a poor response to ERT. We previously showed that gene therapy with an adeno-associated virus (AAV) vector containing a liver-specific promoter elevated the GAA activity in plasma and prevented anti-GAA antibody formation in immunocompetent GAA-knockout mice for 18 wk, predicting that liver-specific expression of human GAA with the AAV vector would induce immune tolerance and enhance the efficacy of ERT. In this study, a very low number of AAV vector particles was administered before initiation of ERT, to prevent the antibody response in GAA-knockout mice. A robust antibody response was provoked in naive GAA-knockout mice by 6 wk after a challenge with human GAA and Freund's adjuvant; in contrast, administration of the AAV vector before the GAA challenge prevented the antibody response. Most compellingly, the antibody response was prevented by AAV vector administration during the 12 wk of ERT, and the efficacy of ERT was thereby enhanced. Thus, AAV vector-mediated gene therapy induced a tolerance to introduced GAA, and this strategy could enhance the efficacy of ERT in CRIM-negative patients with Pompe disease and in patients with other lysosomal storage diseases. PMID:17924344

  7. Enhanced Response to Enzyme Replacement Therapy in Pompe Disease after the Induction of Immune Tolerance

    PubMed Central

    Sun, Baodong ; Bird, Andrew ; Young, Sarah P. ; Kishnani, Priya S. ; Chen, Y.-T. ; Koeberl, Dwight D. 

    2007-01-01

    Pompe disease, which results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid α-glucosidase (GAA) (also called “acid maltase”), causes death in early childhood related to glycogen accumulation in striated muscle and an accompanying infantile-onset cardiomyopathy. The efficacy of enzyme replacement therapy (ERT) with recombinant human GAA was demonstrated during clinical trials that prolonged subjects’ overall survival, prolonged ventilator-free survival, and also improved cardiomyopathy, which led to broad-label approval by the U.S. Food and Drug Administration. Patients who lack any residual GAA expression and are deemed negative for cross-reacting immunologic material (CRIM) have a poor response to ERT. We previously showed that gene therapy with an adeno-associated virus (AAV) vector containing a liver-specific promoter elevated the GAA activity in plasma and prevented anti-GAA antibody formation in immunocompetent GAA-knockout mice for 18 wk, predicting that liver-specific expression of human GAA with the AAV vector would induce immune tolerance and enhance the efficacy of ERT. In this study, a very low number of AAV vector particles was administered before initiation of ERT, to prevent the antibody response in GAA-knockout mice. A robust antibody response was provoked in naive GAA-knockout mice by 6 wk after a challenge with human GAA and Freund’s adjuvant; in contrast, administration of the AAV vector before the GAA challenge prevented the antibody response. Most compellingly, the antibody response was prevented by AAV vector administration during the 12 wk of ERT, and the efficacy of ERT was thereby enhanced. Thus, AAV vector–mediated gene therapy induced a tolerance to introduced GAA, and this strategy could enhance the efficacy of ERT in CRIM-negative patients with Pompe disease and in patients with other lysosomal storage diseases. PMID:17924344

  8. Fall armyworm sensitivity to flavone: Limited role of constitutive and induced detoxifying enzyme activity.

    PubMed

    Wheeler, G S; Slansky, F; Yu, S J

    1993-04-01

    responses to inhibitor treatment and the relatively small benefit of enzyme induction suggest that these enzyme systems have minimal impact on the detoxification of flavone inS. frugiperda, even though this allelochemical induces enzyme activity and has been reported to be metabolized in vitro. PMID:24249008

  9. Mutant α-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin

    PubMed Central

    Ishii, Satoshi; Chang, Hui-Hwa; Kawasaki, Kunito; Yasuda, Kayo; Wu, Hui-Li; Garman, Scott C.; Fan, Jian-Qiang

    2007-01-01

    Fabry disease is a lysosomal storage disorder caused by the deficiency of α-Gal A (α-galactosidase A) activity. In order to understand the molecular mechanism underlying α-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal Km and Vmax values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) α-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q α-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant α-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant α-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations. PMID:17555407

  10. Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs)

    PubMed Central

    Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

    2016-01-01

    Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘Vitis vinifera cv. Pinot Noir’ and ‘Vitis vinifera cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs. PMID:27551866

  11. Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs).

    PubMed

    Tang, Yujin; Wang, Ruipu; Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

    2016-01-01

    Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the 'Vitis vinifera cv. Pinot Noir' and 'Vitis vinifera cv. Thompson Seedless' varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs. PMID:27551866

  12. Endocannabinoid Catabolic Enzymes Play Differential Roles in Thermal Homeostasis in Response to Environmental or Immune Challenge.

    PubMed

    Nass, Sara R; Long, Jonathan Z; Schlosburg, Joel E; Cravatt, Benjamin F; Lichtman, Aron H; Kinsey, Steven G

    2015-06-01

    Cannabinoid receptor agonists, such as Δ(9)-THC, the primary active constituent of Cannabis sativa, have anti-pyrogenic effects in a variety of assays. Recently, attention has turned to the endogenous cannabinoid system and how endocannabinoids, including 2-arachidonoylglycerol (2-AG) and anandamide, regulate multiple homeostatic processes, including thermoregulation. Inhibiting endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH), elevates levels of 2-AG or anandamide in vivo, respectively. The purpose of this experiment was to test the hypothesis that endocannabinoid catabolic enzymes function to maintain thermal homeostasis in response to hypothermic challenge. In separate experiments, male C57BL/6J mice were administered a MAGL or FAAH inhibitor, and then challenged with the bacterial endotoxin lipopolysaccharide (LPS; 2 mg/kg ip) or a cold (4 °C) ambient environment. Systemic LPS administration caused a significant decrease in core body temperature after 6 h, and this hypothermia persisted for at least 12 h. Similarly, cold environment induced mild hypothermia that resolved within 30 min. JZL184 exacerbated hypothermia induced by either LPS or cold challenge, both of which effects were blocked by rimonabant, but not SR144528, indicating a CB1 cannabinoid receptor mechanism of action. In contrast, the FAAH inhibitor, PF-3845, had no effect on either LPS-induced or cold-induced hypothermia. These data indicate that unlike direct acting cannabinoid receptor agonists, which elicit profound hypothermic responses on their own, neither MAGL nor FAAH inhibitors affect normal body temperature. However, these endocannabinoid catabolic enzymes play distinct roles in thermoregulation following hypothermic challenges. PMID:25715681

  13. Optimal response of key enzymes and uncoupling protein to cold in BAT depends on local T/sub 3/ generation

    SciTech Connect

    Bianco, A.C.; Silva, J.E.

    1987-09-01

    The authors have examined the activity of three lipogenic enzymes (malic enzyme (ME), glucose-6-phosphate dehydrogenase (G-6-PD), and acetyl coenzyme A (CoA) carboxylase), the activity of the mitochondrial FAD-dependent ..cap alpha..-glycerolphosphate dehydrogenase (..cap alpha..-GPD), and the mitochondrial concentration of uncoupling protein (UCP) in brown adipose tissue (BAT) of euthyroid and hypothyroid rats, both at room temperature and in response to acute cold stress. These enzymes and UCP are important for the thermogenic response of BAT in adaptation to cold. The basal level of the lipogenic enzymes was normal or slightly elevated in hypothyroid rats maintained at 23/sup 0/C, but the levels of ..cap alpha..-GPD and UCP were markedly reduced. Forty-eight hours at 4/sup 0/C resulted in an increase in the activity of G-6-PD, acetyl-CoA carboxylase, and ..cap alpha..-GPD and in the concentration of UCP both in euthyroid and hypothyroid animals, but the levels reached were invariably less in hypothyroid animals, indicating that thyroid hormone is necessary for a full metabolic response of BAT under maximal demands. Of all variables measured, the most affected was UCP followed by ..cap alpha..-GDP. Dose-response relationship analysis of the UCP response to T/sub 3/ indicated that the normalization of the response to cold requires saturation of the nuclear T/sub 3/ receptors. They concluded, therefore, that the activation of the BAT 5'-deiodinase induced by cold exposure is essential to provide the high levels of nuclear T/sub 3/ required for the full expression of BAT thermogenic potential.

  14. Application of Activity-Based Protein Profiling to Study Enzyme Function in Adipocytes

    PubMed Central

    Galmozzi, Andrea; Dominguez, Eduardo; Cravatt, Benjamin F.; Saez, Enrique

    2014-01-01

    Activity-Based Protein Profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes, and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways. PMID:24529438

  15. Microbial enzyme and biomass responses: Deciphering the effects of earthworms and seasonal variation on treating excess sludge.

    PubMed

    Ma, Xiaojie; Xing, Meiyan; Wang, Yin; Xu, Zhe; Yang, Jian

    2016-04-01

    This paper reports on a seasonal pattern comparison of microbial enzymatic activities and biomass responses based on a conventional biofilter (BF, without earthworm) and a vermifilter (VF, with earthworm, Eisenia fetida) for excess sludge treatment. The volatile suspended solids (VSS) reduction, viable cell number and enzyme activities were assayed to probe what made the VF operate stably. The results indicated that the earthworm activities can polish the VSS reduction with 27.17% more than the BF. Though the VF had a lower level in the viable cell number compared with the BF, the earthworm strongly improved the microbial enzymatic activities such as INT-dehydrogenase, protease, β-glucosidase and amylase, which can explain the excellent performance of VSS reduction. The correlation analysis documented that the VSS reduction was positively correlated with microbial enzyme activities. More importantly, the earthworm enabled the VF to avoid the detrimental influence of temperature, which guaranteed a stable performance during seasonal variations. PMID:26840985

  16. Correlation Among Soil Enzyme Activities, Root Enzyme Activities, and Contaminant Removal in Two-Stage In Situ Constructed Wetlands Purifying Domestic Wastewater.

    PubMed

    Ni, Lixiao; Xu, Jiajun; Chu, Xianglin; Li, Shiyin; Wang, Peifang; Li, Yiping; Li, Yong; Zhu, Liang; Wang, Chao

    2016-07-01

    Two-stage in situ wetlands (two vertical flow constructed wetlands in parallel and a horizontal flow constructed wetland) were constructed for studying domestic wastewater purification and the correlations between contaminant removal and plant and soil enzyme activities. Results indicated the removal efficiency of NH4 (+) and NO3 (-) were significantly correlated with both urease and protease activity, and the removal of total phosphorus was significantly correlated with phosphatase activity. Chemical oxygen demand removal was not correlated with enzyme activity in constructed wetlands. Plant root enzyme (urease, phosphatase, protease and cellulose) activity correlation was apparent with all contaminant removal in the two vertical flow constructed wetlands. However, the correlation between the plant root enzyme activity and contaminant removal was poor in horizontal flow constructed wetlands. Results indicated that plant roots clearly played a role in the removal of contaminants. PMID:27230025

  17. E2 potentializes benzo(a)pyrene-induced hepatic cytochrome P450 enzyme activities in Nile tilapia at high concentrations.

    PubMed

    Rodrigues, Aline Cristina Ferreira; Moneró, Tatiana de Oliveira; Frighetto, Rosa Toyoko Shiraishi; de Almeida, Eduardo Alves

    2015-11-01

    In the aquatic environment, biotransformation enzymes are established biomarkers for assessing PAH exposure in fish, but little is known about the effect of 17β-estradiol (E2) on these enzymes during exposure to benzo(a)pyrene (BaP). In this study, Nile tilapia (Oreochromis niloticus) were exposed for 3, 5, and 10 days to BaP (300 μg L(-1)) and E2 (5 μg L(-1)). These substances were applied isolated or mixed. In the mixture experiment, fish were analyzed pre- and postexposure in order to better understand whether preexposure to the hormone masks the responses activated by PAH or vice versa. Phase I enzymes ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-depenthylase (PROD), and benzyloxyresorufin-O-debenzylase (BROD) activities as well as the phase II enzyme glutathione S-transferase (GST) were analyzed. Isolated E2 treatment decreased EROD activity after 3 days, but this enzyme activity returned to control values after 5 and 10 days of exposure. Isolated BaP treatment significantly induced EROD activity after 3 and 5 days, and the activity returned to control levels after ten exposure days. Combined treatment (E2 + Bap) significantly increased EROD activity, both in the pre- and postexposure. This increase was even higher than in the isolated BaP treatment, suggesting a synergism between these two compounds. When E2 and BaP were used singly, they did not change BROD and PROD activities. However, combined treatment (E2 + Bap) significantly increased PROD activity. Isolated BaP treatment increased GST activity after 10 days. However, this response was not observed in the mixture treatment, suggesting that E2 suppressed the GST induction modulated by BaP. The results put together indicated that E2 altered the biotransformation pathway regarding enzymes activated by BaP in Nile tilapia. PMID:25280508

  18. Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model

    PubMed Central

    2013-01-01

    Background For screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity. Results During uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity. Conclusion Specific activity calculated from the level of

  19. Ultrasonic Monitoring of Enzyme Catalysis; Enzyme Activity in Formulations for Lactose-Intolerant Infants.

    PubMed

    Altas, Margarida C; Kudryashov, Evgeny; Buckin, Vitaly

    2016-05-01

    The paper introduces ultrasonic technology for real-time, nondestructive, precision monitoring of enzyme-catalyzed reactions in solutions and in complex opaque media. The capabilities of the technology are examined in a comprehensive analysis of the effects of a variety of diverse factors on the performance of enzyme β-galactosidase in formulations for reduction of levels of lactose in infant milks. These formulations are added to infant's milk bottles prior to feeding to overcome the frequently observed intolerance to lactose (a milk sugar), a serious issue in healthy development of infants. The results highlight important impediments in the development of these formulations and also illustrate the capability of the described ultrasonic tools in the assessment of the performance of enzymes in complex reaction media and in various environmental conditions. PMID:27018312

  20. Electrical stimulation affects metabolic enzyme phosphorylation, protease activation, and meat tenderization in beef.

    PubMed

    Li, C B; Li, J; Zhou, G H; Lametsch, R; Ertbjerg, P; Brüggemann, D A; Huang, H G; Karlsson, A H; Hviid, M; Lundström, K

    2012-05-01

    The objective of this study was to investigate the response of sarcoplasmic proteins in bovine LM to low-voltage electrical stimulation (ES; 80 V, 35 s) after dressing and its contribution to meat tenderization at an early postmortem time. Proteome analysis showed that ES resulted in decreased (P < 0.05) phosphorylation of creatine kinase M chain, fructose bisphosphate aldolase C-A, β-enolase, and pyruvate kinase at 3 h postmortem. Zymography indicated an earlier (P < 0.05) activation of μ-calpain in ES muscles. Free lysosomal cathepsin B and L activity increased faster (P < 0.05) in ES muscles up to 24 h. Immunohistochemistry and transmission electron microscopy further indicated that lysosomal enzymes were released at an early postmortem time. Electrical stimulation also induced ultrastructural disruption of sarcomeres. In addition, ES accelerated (P < 0.05) the depletion of ATP, creatine phosphate, and glycogen, as well as a pH decline and the more preferred pH/temperature decline mode. Finally, ES accelerated meat tenderization, resulting in lesser (P < 0.05) shear force values than the control over the testing time. A possible relationship was suggested between a change in the phosphorylation of energy metabolic enzymes and the postmortem tenderization of beef. Our results suggested the possible importance of the activation of μ-calpain, phosphorylation of sarcoplasmic proteins, and release of lysosomal enzymes for ES-induced tenderization of beef muscle. PMID:22147478

  1. Dual Enzyme-Responsive Capsules of Hyaluronic Acid-block-Poly(Lactic Acid) for Sensing Bacterial Enzymes.

    PubMed

    Tücking, Katrin-Stephanie; Grützner, Verena; Unger, Ronald E; Schönherr, Holger

    2015-07-01

    The synthesis of novel amphiphilic hyaluronic acid (HYA) and poly(lactic acid) (PLA) block copolymers is reported as the key element of a strategy to detect the presence of pathogenic bacterial enzymes. In addition to the formation of defined HYA-block-PLA assemblies, the encapsulation of fluorescent reporter dyes and the selective enzymatic degradation of the capsules by hyaluronidase and proteinase K are studied. The synthesis of the dual enzyme-responsive HYA-b-PLA is carried out by copper-catalyzed Huisgen 1,3-dipolar cycloaddition. The resulting copolymers are assembled in water to form vesicular structures, which are characterized by scanning electron microscopy, transmission electron microscopy, dynamic light scattering (DLS), and fluorescence lifetime imaging microscopy (FLIM). DLS measurements show that both enzymes cause a rapid decrease in the hydrodynamic diameter of the nanocapsules. Fluorescence spectroscopy data confirm the liberation of encapsulated dye, which indicates the disintegration of the capsules and validates the concept of enzymatically triggered payload release. Finally, cytotoxicity assays confirm that the HYA-b-PLA nanocapsules are biocompatible with primary human dermal microvascular endothelial cells. PMID:25940300

  2. Engineering a hyper-catalytic enzyme by photo-activated conformation modulation

    SciTech Connect

    Agarwal, Pratul K

    2012-01-01

    Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted that mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8-52 fold better catalytic activity than the unmodulated enzyme.

  3. Activity, life time and effect of hydrolytic enzymes for enhanced biogas production from sludge anaerobic digestion.

    PubMed

    Odnell, Anna; Recktenwald, Michael; Stensén, Katarina; Jonsson, Bengt-Harald; Karlsson, Martin

    2016-10-15

    As an alternative to energy intensive physical methods, enzymatic treatment of sludge produced at wastewater treatment plants for increased hydrolysis and biogas production was investigated. Several hydrolytic enzymes were assessed with a focus on how enzyme activity and life time was influenced by sludge environments. It could be concluded that the activity life time of added enzymes was limited (<24 h) in both waste activated sludge and anaerobic digester sludge environments and that this was, for the majority of enzymes, due to endogenous protease activity. In biogas in situ experiments, subtilisin at a 1% mixture on basis of volatile solids, was the only enzyme providing a significantly increased biomethane production of 37%. However, even at this high concentration, subtilisin could not hydrolyze all available substrate within the life time of the enzyme. Thus, for large scale implementation, enzymes better suited to the sludge environments are needed. PMID:27498254

  4. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    PubMed Central

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-01-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

  5. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    NASA Astrophysics Data System (ADS)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-02-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

  6. Monocyte Tumor Necrosis Factor-α–Converting Enzyme Catalytic Activity and Substrate Shedding in Sepsis and Noninfectious Systemic Inflammation*

    PubMed Central

    O’Callaghan, David J. P.; O’Dea, Kieran P.; Scott, Alasdair J.; Takata, Masao

    2015-01-01

    Objectives: To determine the effect of severe sepsis on monocyte tumor necrosis factor-α–converting enzyme baseline and inducible activity profiles. Design: Observational clinical study. Setting: Mixed surgical/medical teaching hospital ICU. Patients: Sixteen patients with severe sepsis, 15 healthy volunteers, and eight critically ill patients with noninfectious systemic inflammatory response syndrome. Interventions: None. Measurements and Main Results: Monocyte expression of human leukocyte antigen-D-related peptide, sol-tumor necrosis factor production, tumor necrosis factor-α–converting enzyme expression and catalytic activity, tumor necrosis factor receptor 1 and 2 expression, and shedding at 48-hour intervals from day 0 to day 4, as well as p38-mitogen activated protein kinase expression. Compared with healthy volunteers, both sepsis and systemic inflammatory response syndrome patients’ monocytes expressed reduced levels of human leukocyte antigen-D-related peptide and released less sol-tumor necrosis factor on in vitro lipopolysaccharide stimulation, consistent with the term monocyte deactivation. However, patients with sepsis had substantially elevated levels of basal tumor necrosis factor-α–converting enzyme activity that were refractory to lipopolysaccharide stimulation and this was accompanied by similar changes in p38-mitogen activated protein kinase signaling. In patients with systemic inflammatory response syndrome, monocyte basal tumor necrosis factor-α–converting enzyme, and its induction by lipopolysaccharide, appeared similar to healthy controls. Changes in basal tumor necrosis factor-α–converting enzyme activity at day 0 for sepsis patients correlated with Acute Physiology and Chronic Health Evaluation II score and the attenuated tumor necrosis factor-α–converting enzyme response to lipopolysaccharide was associated with increased mortality. Similar changes in monocyte tumor necrosis factor-α–converting enzyme activity could

  7. Preparation of biocatalytic nanofibres with high activity and stability via enzyme aggregate coating on polymer nanofibres

    NASA Astrophysics Data System (ADS)

    Kim, Byoung Chan; Nair, Sujith; Kim, Jungbae; Kwak, Ja Hun; Grate, Jay W.; Kim, Seong H.; Gu, Man Bock

    2005-07-01

    We have developed a unique approach for the fabrication of enzyme aggregate coatings on the surfaces of electrospun polymer nanofibres. This approach employs covalent attachment of seed enzymes onto nanofibres consisting of a mixture of polystyrene and poly(styrene-co-maleic anhydride), followed by a glutaraldehyde (GA) treatment that cross-links additional enzyme molecules and aggregates from the solution onto the covalently attached seed enzyme molecules. These cross-linked enzyme aggregates, covalently attached to the nanofibres via the linkers of seed enzyme molecules, are expected to improve the enzyme activity due to increased enzyme loading, and also the enzyme stability. To demonstrate the principle, we coated α-chymotrypsin (CT) on nanofibres electrospun from a mixture of polystyrene and poly(styrene-co-maleic anhydride). The initial activity of CT-aggregate-coated nanofibres was nine times higher than nanofibres with just a layer of covalently attached CT molecules. The enzyme stability of CT-aggregate-coated nanofibres was greatly improved with essentially no measurable loss of activity over a month of observation under rigorous shaking conditions. This new approach of enzyme coating on nanofibres, yielding high activity and stability, creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, and biosensors.

  8. Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects cardiomyocytes against oxidant stress.

    PubMed

    Reuland, Danielle J; Khademi, Shadi; Castle, Christopher J; Irwin, David C; McCord, Joe M; Miller, Benjamin F; Hamilton, Karyn L

    2013-03-01

    Increased production of reactive oxygen species has been implicated in the pathogenesis of cardiovascular disease (CVD), and enhanced endogenous antioxidants have been proposed as a mechanism for regulating redox balance. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcriptional regulator of phase II antioxidant enzymes, and activation of Nrf2 has been suggested to be an important step in attenuating oxidative stress associated with CVD. A well-defined combination of five widely studied medicinal plants derived from botanical sources (Bacopa monniera, Silybum marianum (milk thistle), Withania somnifera (Ashwagandha), Camellia sinensis (green tea), and Curcuma longa (turmeric)) has been shown to activate Nrf2 and induce phase II enzymes through the antioxidant response element. The purpose of these experiments was to determine if treatment of cardiomyocytes with this phytochemical composition, marketed as Protandim, activates Nrf2, induces phase II detoxification enzymes, and protects cardiomyocytes from oxidant-induced apoptosis in a Nrf2-dependent manner. In cultured HL-1 cardiomyocytes, phytochemical treatment was associated with nuclear accumulation of Nrf2, significant induction of phase II enzymes, and concomitant protection against hydrogen peroxide-induced apoptosis. The protection against oxidant stress was abolished when Nrf2 was silenced by shRNA, suggesting that our phytochemical treatment worked through the Nrf2 pathway. Interestingly, phytochemical treatment was found to be a more robust activator of Nrf2 than oxidant treatment, supporting the use of the phytochemicals as a potential treatment to increase antioxidant defenses and protect heart cells against an oxidative challenge. PMID:23201694

  9. Enzyme catalysis: C-H activation is a Reiske business

    NASA Astrophysics Data System (ADS)

    Bruner, Steven D.

    2011-05-01

    Enzymes that selectively oxidize unactivated C-H bonds are capable of constructing complex molecules with high efficiency. A new member of this enzyme family is RedG, a Reiske-type oxygenase that catalyses chemically challenging cyclizations in the biosynthesis of prodiginine natural products.

  10. Light/dark modulation of enzyme activity in photosynthesis

    SciTech Connect

    Anderson, L.E.; Ashton, A.R.; Mohamed, A.H.; Scheibe, R.

    1982-02-01

    In photosynthetic species ranging from cyanobacteria to higher plants, many enzymes are light modulated. Most of the known light modulated enzymes are chloroplastic. Four mechanisms of modulation have been proposed. One function of light modulation is probably the autocatalytic build-up of reductive pentose phosphate cycle intermediates during the induction period of photosynthetic CO/sub 2/ fixation.