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Sample records for activating enzyme e1

  1. Largazole and Its Derivatives Selectively Inhibit Ubiquitin Activating Enzyme (E1)

    PubMed Central

    Nasveschuk, Christopher G.; Wang, Wei; Quade, Bettina; Zhang, Gan; Kuchta, Robert D.; Phillips, Andrew J.; Liu, Xuedong

    2012-01-01

    Protein ubiquitination plays an important role in the regulation of almost every aspect of eukaryotic cellular function; therefore, its destabilization is often observed in most human diseases and cancers. Consequently, developing inhibitors of the ubiquitination system for the treatment of cancer has been a recent area of interest. Currently, only a few classes of compounds have been discovered to inhibit the ubiquitin-activating enzyme (E1) and only one class is relatively selective in E1 inhibition in cells. We now report that Largazole and its ester and ketone analogs selectively inhibit ubiquitin conjugation to p27Kip1 and TRF1 in vitro. The inhibitory activity of these small molecules on ubiquitin conjugation has been traced to their inhibition of the ubiquitin E1 enzyme. To further dissect the mechanism of E1 inhibition, we analyzed the effects of these inhibitors on each of the two steps of E1 activation. We show that Largazole and its derivatives specifically inhibit the adenylation step of the E1 reaction while having no effect on thioester bond formation between ubiquitin and E1. E1 inhibition appears to be specific to human E1 as Largazole ketone fails to inhibit the activation of Uba1p, a homolog of E1 in Schizosaccharomyces pombe. Moreover, Largazole analogs do not significantly inhibit SUMO E1. Thus, Largazole and select analogs are a novel class of ubiquitin E1 inhibitors and valuable tools for studying ubiquitination in vitro. This class of compounds could be further developed and potentially be a useful tool in cells. PMID:22279528

  2. Mutation in E1, the ubiquitin activating enzyme, reduces Drosophila lifespan and results in motor impairment.

    PubMed

    Liu, Hsiu-Yu; Pfleger, Cathie M

    2013-01-01

    Neurodegenerative diseases cause tremendous suffering for those afflicted and their families. Many of these diseases involve accumulation of mis-folded or aggregated proteins thought to play a causal role in disease pathology. Ubiquitinated proteins are often found in these protein aggregates, and the aggregates themselves have been shown to inhibit the activity of the proteasome. These and other alterations in the Ubiquitin Pathway observed in neurodegenerative diseases have led to the question of whether impairment of the Ubiquitin Pathway on its own can increase mortality or if ongoing neurodegeneration alters Ubiquitin Pathway function as a side-effect. To address the role of the Ubiquitin Pathway in vivo, we studied loss-of-function mutations in the Drosophila Ubiquitin Activating Enzyme, Uba1 or E1, the most upstream enzyme in the Ubiquitin Pathway. Loss of only one functional copy of E1 caused a significant reduction in adult lifespan. Rare homozygous hypomorphic E1 mutants reached adulthood. These mutants exhibited further reduced lifespan and showed inappropriate Ras activation in the brain. Removing just one functional copy of Ras restored the lifespan of heterozygous E1 mutants to that of wild-type flies and increased the survival of homozygous E1 mutants. E1 homozygous mutants also showed severe motor impairment. Our findings suggest that processes that impair the Ubiquitin Pathway are sufficient to cause early mortality. Reduced lifespan and motor impairment are seen in the human disease X-linked Infantile Spinal Muscular Atrophy, which is associated with mutation in human E1 warranting further analysis of these mutants as a potential animal model for study of this disease.

  3. A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

    PubMed

    Mulder, Monique P C; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C O; Schulman, Brenda A; Komander, David; Neefjes, Jacques; El Oualid, Farid; Ovaa, Huib

    2016-07-01

    Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades. PMID:27182664

  4. Crystal Structure of the Human Ubiquitin-activating Enzyme 5 (UBA5) Bound to ATP Mechanistic Insights into a Minimalistic E1 Enzyme

    SciTech Connect

    Bacik, John-Paul; Walker, John R.; Ali, Mohsin; Schimmer, Aaron D.; Dhe-Paganon, Sirano

    2010-08-30

    E1 ubiquitin-activating enzymes (UBAs) are large multidomain proteins that catalyze formation of a thioester bond between the terminal carboxylate of a ubiquitin or ubiquitin-like modifier (UBL) and a conserved cysteine in an E2 protein, producing reactive ubiquityl units for subsequent ligation to substrate lysines. Two important E1 reaction intermediates have been identified: a ubiquityl-adenylate phosphoester and a ubiquityl-enzyme thioester. However, the mechanism of thioester bond formation and its subsequent transfer to an E2 enzyme remains poorly understood. We have determined the crystal structure of the human UFM1 (ubiquitin-fold modifier 1) E1-activating enzyme UBA5, bound to ATP, revealing a structure that shares similarities with both large canonical E1 enzymes and smaller ancestral E1-like enzymes. In contrast to other E1 active site cysteines, which are in a variably sized domain that is separate and flexible relative to the adenylation domain, the catalytic cysteine of UBA5 (Cys{sup 250}) is part of the adenylation domain in an {alpha}-helical motif. The novel position of the UBA5 catalytic cysteine and conformational changes associated with ATP binding provides insight into the possible mechanisms through which the ubiquityl-enzyme thioester is formed. These studies reveal structural features that further our understanding of the UBA5 enzyme reaction mechanism and provide insight into the evolution of ubiquitin activation.

  5. Immunoelectron microscopic localization of the ubiquitin-activating enzyme E1 in HepG2 cells.

    PubMed Central

    Schwartz, A L; Trausch, J S; Ciechanover, A; Slot, J W; Geuze, H

    1992-01-01

    As the first enzyme in the ubiquitin system the ubiquitin-activating enzyme E1 plays a pivotal role in all pathways of protein ubiquitination. In an effort to learn more about the cell biology of this pathway, we have purified the 110-kDa enzyme to homogeneity and generated a panel of distinct monoclonal antibodies to it. Using quantitative electron microscopic immunolocalization with these anti-E1 monoclonal antibodies, we find that E1 is abundant both within the cytoplasm and nucleus. Within the cytoplasm, E1 was found throughout the cytoplasmic volume as well as enriched along the cytoplasmic face of the rough endoplasmic reticulum and associated with the dense material along the desmosomal junctions. E1 was also found associated with the cytoplasmic surface of endosomal/lysosomal vacuoles. Interestingly, E1 was also found within the mitochondria. The lumen of rough endoplasmic reticulum, Golgi complex, endosomes, and lysosomes were negative. The specific localization of E1 to distinct subcellular organelles suggests that E1 may play multiple physiological roles within the cell. Images PMID:1376922

  6. The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autonomously and tissue growth non-autonomously

    PubMed Central

    Lee, Tom V.; Ding, Tian; Chen, Zhihong; Rajendran, Vani; Scherr, Heather; Lackey, Melinda; Bolduc, Clare; Bergmann, Andreas

    2008-01-01

    Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene. PMID:18045837

  7. Human ubiquitin-activating enzyme, E1. Indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cDNA constructs.

    PubMed

    Handley-Gearhart, P M; Stephen, A G; Trausch-Azar, J S; Ciechanover, A; Schwartz, A L

    1994-12-30

    The ubiquitin-activating enzyme E1 catalyzes the first step in the ubiquitin conjugation pathway. Previously, we have cloned and sequenced the cDNA for human E1. Expression of the E1 cDNA in the ts20 cell line, which harbors a thermolabile E1, abrogated the phenotypic defects associated with this line. However, little is known of the cell biology of the E1 protein or the nature of the E1 doublet. Thus, we constructed epitope-tagged E1 cDNAs in which the HA monoclonal antibody epitope tag sequence (from influenza hemagglutinin and recognized by the 12CA5 monoclonal antibody) was fused to the amino terminus of E1. Because the amino-terminal amino acid sequence of E1 is unknown, three constructs were made in which the HA tag was placed at each of the first three ATGs in the open reading frame (HA-1E1, HA-2E1, and HA-3E1). Western analysis of HeLa cells transfected with the constructs revealed that HA-1E1 closely comigrated with the upper band of the E1 doublet, and HA-2E1 comigrated with the lower band of the E1 doublet; HA-3E1 appeared smaller than either of the E1 bands. Metabolic labeling with 32P and immunoprecipitation with anti-HA antibody revealed that only the HA-1E1 protein product is phosphorylated; polyclonal anti-E1 antibody showed that only the upper band of the endogenous E1 doublet is phosphorylated. Each of the constructs was able to rescue the mutant phenotype of the ts20 cell line. Immunofluorescence studies showed that HA-2E1 and HA-3E1 were distributed in the cytoplasm with both negative and positive nuclei. This pattern of distribution has also been observed when immunostaining with a monoclonal antibody to E1 (1C5). However, the staining pattern associated with a polyclonal anti-E1 antibody (JJJ) is characterized by positive staining cytoplasm and nuclei in all cells. The HA-1E1 construct exhibited apparently exclusive nuclear distribution in HeLa cells. The difference between the staining patterns of the polyclonal and monoclonal anti-E1

  8. Substrate-Assisted Inhibition of Ubiquitin-like Protein-Activating Enzymes: The NEDD8 E1 Inhibitor MLN4924 Forms a NEDD8-AMP Mimetic In Situ

    SciTech Connect

    Brownell, James E.; Sintchak, Michael D.; Gavin, James M.; Liao, Hua; Bruzzese, Frank J.; Bump, Nancy J.; Soucy, Teresa A.; Milhollen, Michael A.; Yang, Xiaofeng; Burkhardt, Anne L.; Ma, Jingya; Loke, Huay-Keng; Lingaraj, Trupti; Wu, Dongyun; Hamman, Kristin B.; Spelman, James J.; Cullis, Courtney A.; Langston, Steven P.; Vyskocil, Stepan; Sells, Todd B.; Mallender, William D.; Visiers, Irache; Li, Ping; Claiborne, Christopher F.; Rolfe, Mark; Bolen, Joseph B.; Dick, Lawrence R.

    2010-11-15

    The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.

  9. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors.

    PubMed

    Qiu, Jiazhang; Sheedlo, Michael J; Yu, Kaiwen; Tan, Yunhao; Nakayasu, Ernesto S; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2016-05-01

    Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP.

  10. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors.

    PubMed

    Qiu, Jiazhang; Sheedlo, Michael J; Yu, Kaiwen; Tan, Yunhao; Nakayasu, Ernesto S; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2016-05-01

    Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP. PMID:27049943

  11. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

    PubMed Central

    Qiu, Jiazhang; Sheedlo, Michael J.; Yu, Kaiwen; Tan, Yunhao; Nakayasu, Ernesto S.; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2016-01-01

    Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or as enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalyzed by these enzymes is energized by NAD which activates ubiquitin by the formation of ADP-ribosylated ubiquitin (ADPR-Ub). These results establish that ubiquitination can be catalyzed by a single enzyme whose activity does not require ATP. PMID:27049943

  12. Ubiquitination Accomplished: E1 and E2 Enzymes Were Not Necessary.

    PubMed

    Nakasone, Mark A; Huang, Danny T

    2016-06-16

    Qiu et al. (2016) show that a mono-ADP-ribosyltransferase, SdeA, from Legionella pneumophila catalyzes ADP-ribosylation of ubiquitin, allowing SdeA to modify substrate with ubiquitin in the absence of E1 and E2 enzymes. PMID:27315555

  13. Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme.

    PubMed

    Naselli, Flores; Catanzaro, Irene; Bellavia, Daniele; Perez, Alessandro; Sposito, Laura; Caradonna, Fabio

    2014-02-15

    Different individuals possess slightly different genetic information and show genetically-determined differences in several enzyme activities due to genetic variability. Following an integrated approach, we studied the polymorphisms and methylation of sites contained in the 5' flanking region of the metabolizing enzyme CYP2E1 in correlation to its expression in both tumor and non-neoplastic liver cell lines, since to date little is known about the influence of these (epi)genetic elements in basal conditions and under induction by the specific inductor and a demethylating agent. In treated cells, reduced DNA methylation, assessed both at genomic and gene level, was not consistently associated with the increase of enzyme expression. Interestingly, the Rsa/Pst haplotype differentially influenced CYP2E1 enzyme expression. In addition, regarding the Variable Number of Tandem Repeats polymorphism, cells with A4/A4 genotype showed a greater expression inhibition (ranging from 20% to 30%) compared with others carrying the A2/A2 one, while those cells bringing A2/A3 genotype showed an increase of expression (of 25%, about). Finally, we demonstrated for the first time that the A2 and A3 CYP2E1 alleles play a more important role in the expression of the enzyme, compared with other (epi)genetic factors, since they are binding sites for trans-acting proteins. PMID:24333271

  14. Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme.

    PubMed

    Naselli, Flores; Catanzaro, Irene; Bellavia, Daniele; Perez, Alessandro; Sposito, Laura; Caradonna, Fabio

    2014-02-15

    Different individuals possess slightly different genetic information and show genetically-determined differences in several enzyme activities due to genetic variability. Following an integrated approach, we studied the polymorphisms and methylation of sites contained in the 5' flanking region of the metabolizing enzyme CYP2E1 in correlation to its expression in both tumor and non-neoplastic liver cell lines, since to date little is known about the influence of these (epi)genetic elements in basal conditions and under induction by the specific inductor and a demethylating agent. In treated cells, reduced DNA methylation, assessed both at genomic and gene level, was not consistently associated with the increase of enzyme expression. Interestingly, the Rsa/Pst haplotype differentially influenced CYP2E1 enzyme expression. In addition, regarding the Variable Number of Tandem Repeats polymorphism, cells with A4/A4 genotype showed a greater expression inhibition (ranging from 20% to 30%) compared with others carrying the A2/A2 one, while those cells bringing A2/A3 genotype showed an increase of expression (of 25%, about). Finally, we demonstrated for the first time that the A2 and A3 CYP2E1 alleles play a more important role in the expression of the enzyme, compared with other (epi)genetic factors, since they are binding sites for trans-acting proteins.

  15. Involvement of CYP 2E1 enzyme in ovotoxicity caused by 4-vinylcyclohexene and its metabolites

    SciTech Connect

    Rajapaksa, Kathila S.; Cannady, Ellen A.; Sipes, I. Glenn; Hoyer, Patricia B. . E-mail: hoyer@u.arizona.edu

    2007-06-01

    4-Vinylcyclohexene (VCH) is bioactivated by hepatic CYP 2A and 2B to a monoepoxide (VCM) and subsequently to an ovotoxic diepoxide metabolite (VCD). Studies suggest that the ovary can directly bioactivate VCH via CYP 2E1. The current study was designed to evaluate the role of ovarian CYP 2E1 in VCM-induced ovotoxicity. Postnatal day 4 B6C3F{sub 1} and CYP 2E1 wild-type (+/+) and null (-/-) mouse ovaries were cultured (15 days) with VCD (30 {mu}M), 1,2-VCM (125-1000 {mu}M), or vehicle. Twenty-eight days female CYP 2E1 +/+ and -/- mice were dosed daily (15 days; ip) with VCH, 1,2-VCM, VCD or vehicle. Following culture or in vivo dosing, ovaries were histologically evaluated. In culture, VCD decreased (p < 0.05) primordial and primary follicles in ovaries from all three groups of mice. 1,2-VCM decreased (p < 0.05) primordial follicles in B6C3F{sub 1} and CYP 2E1 +/+ ovaries, but not in CYP 2E1 -/- ovaries in culture. 1,2-VCM did not affect primary follicles in any group of mouse ovaries. Conversely, following in vivo dosing, primordial and primary follicles were reduced (p < 0.05) by VCD and VCM in CYP2E1 +/+ and -/-, and by VCH in +/+ mice. The data demonstrate that, whereas in vitro ovarian bioactivation of VCM requires CYP 2E1 enzyme, in vivo CYP 2E1 plays a minimal role. Thus, the findings support that hepatic metabolism dominates the contribution made by the ovary in bioactivation of VCM to its ovotoxic metabolite, VCD. This study also demonstrates the use of a novel ovarian culture system to evaluate ovary-specific metabolism of xenobiotics.

  16. Methodology to assay CYP2E1 mixed function oxidase catalytic activity and its induction

    PubMed Central

    Cederbaum, Arthur I.

    2014-01-01

    The cytochrome P450 mixed function oxidase enzymes are the major catalysts involved in drug metabolism. There are many forms of P450. CYP2E1 metabolizes many toxicologically important compounds including ethanol and is active in generating reactive oxygen species. Since several of the contributions in the common theme series “Role of CYP2E1 and Oxidative/Nitrosative Stress in the Hepatotoxic Actions of Alcohol” discuss CYP2E1, this methodology review describes assays on how CYP2E1 catalytic activity and its induction by ethanol and other inducers can be measured using substrate probes such as the oxidation of para-nitrophenol to para-nitrocatechol and the oxidation of ethanol to acetaldehyde. Approaches to validate that a particular reaction e.g. oxidation of a drug or toxin is catalyzed by CYP2E1 or that induction of that reaction is due to induction of CYP2E1 are important and specific examples using inhibitors of CYP2E1, anti-CYP2E1 IgG or CYP2E1 knockout and knockin mice will be discussed. PMID:25454746

  17. Activation of NF-κB by Human Papillomavirus 16 E1 Limits E1-Dependent Viral Replication through Degradation of E1

    PubMed Central

    Nakahara, Tomomi; Tanaka, Katsuyuki; Ohno, Shin-ichi; Egawa, Nagayasu; Yugawa, Takashi

    2015-01-01

    ABSTRACT NF-κB is a family of transcription factors that regulate gene expression involved in many processes, such as the inflammatory response and cancer progression. Little is known about associations of NF-κB with the human papillomavirus (HPV) life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of the human papillomavirus 16 (HPV16) genome in human cervical keratinocytes and found that expression of HPV16 E1, a viral helicase, results in reduction of IκBα and subsequent activation of NF-κB in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IκBα, which inhibits the activation of NF-κB, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and, to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NF-κB and augmented E1-dependent replication of the HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of wortmannin as well as the IκBα mutant. Collectively, we propose that expression of E1 induces NF-κB activation at least in part through the ATR-dependent DNA damage response and that NF-κB in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NF-κB may constitute a negative feedback loop. IMPORTANCE A major risk factor in human papillomavirus (HPV)-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that human papillomavirus 16 (HPV16) E1, a viral DNA helicase essential for amplification of the viral genomes, induces NF-κB activation and that this limits E1-dependent

  18. Effects of Mutations in the Rubella Virus E1 Glycoprotein on E1-E2 Interaction and Membrane Fusion Activity

    PubMed Central

    Yang, Decheng; Hwang, Dorothy; Qiu, Zhiyong; Gillam, Shirley

    1998-01-01

    Rubella virus (RV) virions contain two glycosylated membrane proteins, E1 and E2, that exist as a heterodimer and form the viral spike complexes on the virion surface. Formation of an E1-E2 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi apparatus and plasma membrane. To investigate the nature of the E1-E2 interaction, we have introduced mutations in the internal hydrophobic region (residues 81 to 109) of E1. Substitution of serine at Cys82 (mutant C82S) or deletion of this hydrophobic domain (mutant dt) of E1 resulted in a disruption of the E1 conformation that ultimately affected E1-E2 heterodimer formation and cell surface expression of both E1 and E2. Substitution of either aspartic acid at Gly93 (G93D) or glycine at Pro104 (P104G) was found to impair neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. Fusion of RV-infected cells is induced by a brief treatment at a pH below 6.0. To test whether this internal hydrophobic domain is involved in the membrane fusion activity of RV, transformed BHK cell lines expressing either wild-type or mutant spike proteins were exposed to an acidic pH and polykaryon formation was measured. No fusion activity was observed in the C82S, dt, and G93D mutants; however, the wild type and the P104G mutant exhibited fusogenic activities, with greater than 60% and 20 to 40% of the cells being fused, respectively, at pH 4.8. These results suggest that it is likely that the region of E1 between amino acids 81 and 109 is involved in the membrane fusion activity of RV and that it may be important for the interaction of that protein with E2 to form the E1-E2 heterodimer. PMID:9765418

  19. Measurement of enzyme activity.

    PubMed

    Harris, T K; Keshwani, M M

    2009-01-01

    To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

  20. 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

  1. Facile synthesis of covalent probes to capture enzymatic intermediates during E1 enzyme catalysis.

    PubMed

    An, Heeseon; Statsyuk, Alexander V

    2016-02-11

    We report a facile synthetic strategy to prepare UBL-AMP electrophilic probes that form a covalent bond with the catalytic cysteine of cognate E1s, mimicking the tetrahedral intermediate of the E1-UBL-AMP complex. These probes enable the structural and biochemical study of both canonical- and non-canonical E1s.

  2. Active site remodeling accompanies thioester bond formation in the SUMO E1

    PubMed Central

    Olsen, Shaun K.; Capili, Allan D.; Lu, Xuequan; Tan, Derek S.; Lima, Christopher D.

    2009-01-01

    E1 enzymes activate ubiquitin (Ub) and ubiquitin-like (Ubl) proteins in two steps by carboxy-terminal adenylation and thioester bond formation to a conserved catalytic cysteine in the E1 Cys domain. The structural basis for these intermediates remains unknown. Here we report crystal structures for human SUMO E1 in complex with SUMO adenylate and tetrahedral intermediate analogs at 2.45 Å and 2.6 Å, respectively. These structures show that side chain contacts to ATP·Mg are released after adenylation to facilitate a 130 degree rotation of the Cys domain during thioester bond formation that is accompanied by remodeling of key structural elements including the helix that contains the E1 catalytic cysteine, the cross-over and re-entry loops, and refolding of two helices that are required for adenylation. These changes displace side chains required for adenylation with side chains required for thioester bond formation. Mutational and biochemical analyses suggest these mechanisms are conserved in other E1s. PMID:20164921

  3. Active site remodelling accompanies thioester bond formation in the SUMO E1

    SciTech Connect

    Olsen, Shaun K.; Capili, Allan D.; Lu, Xuequan; Tan, Derek S.; Lima, Christopher D.

    2010-03-30

    E1 enzymes activate ubiquitin (Ub) and ubiquitin-like (Ubl) proteins in two steps by carboxy-terminal adenylation and thioester bond formation to a conserved catalytic cysteine in the E1 Cys domain. The structural basis for these intermediates remains unknown. Here we report crystal structures for human SUMO E1 in complex with SUMO adenylate and tetrahedral intermediate analogues at 2.45 and 2.6 {angstrom}, respectively. These structures show that side chain contacts to ATP-Mg are released after adenylation to facilitate a 130 degree rotation of the Cys domain during thioester bond formation that is accompanied by remodelling of key structural elements including the helix that contains the E1 catalytic cysteine, the crossover and re-entry loops, and refolding of two helices that are required for adenylation. These changes displace side chains required for adenylation with side chains required for thioester bond formation. Mutational and biochemical analyses indicate these mechanisms are conserved in other E1s.

  4. Effects of Ca++ and Prostaglandin E1 on Vasopressin Activation of Renal Adenyl Cyclase

    PubMed Central

    Marumo, Fumiaki; Edelman, Isidore S.

    1971-01-01

    Adenyl cyclase activity was assayed in crude homogenates of the renal cortex, medulla, and papilla of the golden hamster. The specific activity (moles C-AMP/unit of time per mg protein of tissue) of the enzyme under basal conditions, was greatest in papilla, somewhat lower in medulla, and least in cortex. On an absolute scale, the sensitivity to vasopressin was greater in the medullary and papillary than in the cortical homogenates. In addition, at concentrations of 0.1-1.0 mm, CaCl2 inhibited the enzyme in the order papilla > medulla > cortex. These results imply the existence of distinct differences in the composition of the adenyl cyclase-receptor complex in various parts of the kidney. We proposed that Ca++ inhibits the core enzyme directly since at the minimally inhibitory concentration (0.1 mm), CaCl2 reduced to an equivalent extent (a) basal activity, (b) the response to graded doses of vasopressin (0.5 to 50.0 mU/ml) and (c) the response to maximal stimulatory concentrations of NaF (10 mm). Prostaglandin E1 (PGE1 = 10−7m) had no effect on either basal adenyl-cyclase activity or the response to 10 mm NaF in medullary and papillary homogenates. 7-Oxa-13-prostynoic acid (10−4m) similarly had no effect under basal conditions or on stimulation with NaF in medullary homogenates. Both fatty acids, however, inhibited the enzymic response to vasopressin, particularly at low concentrations of the peptide. The straight-chain fatty acid, 11-eicosanoic acid (10−7m), was inactive on basal activity or on the response to vasopressin. The possibility that PGE1 modifies the coupling mechanism between the core enzyme and the hormone-specific receptor is discussed. PMID:4329002

  5. Conserved tyrosine 182 residue in hyperthermophilic esterase EstE1 plays a critical role in stabilizing the active site.

    PubMed

    Truongvan, Ngoc; Chung, Hye-Shin; Jang, Sei-Heon; Lee, ChangWoo

    2016-03-01

    An aromatic amino acid, Tyr or Trp, located in the esterase active site wall, is highly conserved, with hyperthermophilic esterases showing preference for Tyr and lower temperature esterases showing preference for Trp. In this study, we investigated the role of Tyr(182) in the active site wall of hyperthermophilic esterase EstE1. Mutation of Tyr to Phe or Ala had a moderate effect on EstE1 thermal stability. However, a small-to-large mutation such as Tyr to His or Trp had a devastating effect on thermal stability. All mutant EstE1 enzymes showed reduced catalytic rates and enhanced substrate affinities as compared with wild-type EstE1. Hydrogen bond formation involving Tyr(182) was unimportant for maintaining EstE1 thermal stability, as the EstE1 structure is already adapted to high temperatures via increased intramolecular interactions. However, removal of hydrogen bond from Tyr(182) significantly decreased EstE1 catalytic activity, suggesting its role in stabilization of the active site. These results suggest that Tyr is preferred over a similarly sized Phe residue or bulky His or Trp residue in the active site walls of hyperthermophilic esterases for stabilizing the active site and regulating catalytic activity at high temperatures. PMID:26838013

  6. Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

    PubMed

    Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William

    2004-03-01

    Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis. PMID:14992577

  7. Chronic administration of caderofloxacin, a new fluoroquinolone, increases hepatic CYP2E1 expression and activity in rats

    PubMed Central

    Liu, Li; Miao, Ming-xing; Zhong, Ze-yu; Xu, Ping; Chen, Yang; Liu, Xiao-dong

    2016-01-01

    Aim: Caderofloxacin is a new fluoroquinolone that is under phase III clinical trials in China. Here we examined the effects of caderofloxacin on rat hepatic cytochrome P450 (CYP450) isoforms as well as the potential of caderofloxacin interacting with co-administered drugs. Methods: Male rats were treated with caderofloxacin (9 mg/kg, ig) once or twice daily for 14 consecutive days. The effects of caderofloxacin on CYP3A, 2D6, 2C19, 1A2, 2E1 and 2C9 were evaluated using a “cocktail” of 6 probes (midazolam, dextromethorphan, omeprazole, theophylline, chlorzoxazone and diclofenac) injected on d 0 (prior to caderofloxacin exposure) and d 15 (after caderofloxacin exposure). Hepatic microsomes from the caderofloxacin-treated rats were used to assess CYP2E1 activity and chlorzoxazone metabolism. The expression of CYP2E1 mRNA and protein in hepatic microsomes was analyzed with RT-PCR and Western blotting, respectively. Results: Fourteen-day administration of caderofloxacin significantly increased the activity of hepatic CYP2E1, leading to enhanced metabolism of chlorzoxazone. In vitro microsomal study confirmed that CYP2E1 was a major metabolic enzyme involved in chlorzoxazone metabolism, and the 14-d administration of caderofloxacin significantly increased the activity of CYP2E1 in hepatic microsomes, resulting in increased formation of 6-hydroxychlorzoxazone. Furthermore, the 14-d administration of caderofloxacin significantly increased the expression of CYP2E1 mRNA and protein in liver microsomes, which was consistent with the pharmacokinetic results. Conclusion: Fourteen-day administration of caderofloxacin can induce the expression and activity of hepatic CYP2E1 in rats. When caderofloxacin is administered, a potential drug-drug interaction mediated by CYP2E1 induction should be considered. PMID:26838075

  8. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers

    PubMed Central

    Nemeria, Natalia S.; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-01-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4′-aminopyrimidine N1′ atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu571, Glu235, and Glu237) and Arg606 resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu235 makes no direct contact with the cofactor. The role of the conserved Glu571 residue in both catalysis and cofactor orientation is revealed by the combined results for the first time. PMID:20106967

  9. Determination of lipolytic enzyme activities.

    PubMed

    Jaeger, Karl-Erich; Kovacic, Filip

    2014-01-01

    Pseudomonas aeruginosa is a versatile human opportunistic pathogen that produces and secretes an arsenal of enzymes, proteins and small molecules many of which serve as virulence factors. Notably, about 40 % of P. aeruginosa genes code for proteins of unknown function, among them more than 80 encoding putative, but still unknown lipolytic enzymes. This group of hydrolases (EC 3.1.1) is known already for decades, but only recently, several of these enzymes have attracted attention as potential virulence factors. Reliable and reproducible enzymatic activity assays are crucial to determine their physiological function and particularly assess their contribution to pathogenicity. As a consequence of the unique biochemical properties of lipids resulting in the formation of micellar structures in water, the reproducible preparation of substrate emulsions is strongly dependent on the method used. Furthermore, the physicochemical properties of the respective substrate emulsion may drastically affect the activities of the tested lipolytic enzymes. Here, we describe common methods for the activity determination of lipase, esterase, phospholipase, and lysophospholipase. These methods cover lipolytic activity assays carried out in vitro, with cell extracts or separated subcellular compartments and with purified enzymes. We have attempted to describe standardized protocols, allowing the determination and comparison of enzymatic activities of lipolytic enzymes from different sources. These methods should also encourage the Pseudomonas community to address the wealth of still unexplored lipolytic enzymes encoded and produced by P. aeruginosa.

  10. The dual effect of adenovirus type 5 E1A 13S protein on NF-kappaB activation is antagonized by E1B 19K.

    PubMed Central

    Schmitz, M L; Indorf, A; Limbourg, F P; Städtler, H; Traenckner, E B; Baeuerle, P A

    1996-01-01

    The genomes of human adenoviruses encode several regulatory proteins, including the two differentially spliced gene products E1A and E1B. Here, we show that the 13S but not the 12S splice variant of E1A of adenovirus type 5 can activate the human transcription factor NF-kappaB in a bimodal fashion. One mode is the activation of NF-kappaB containing the p65 subunit from the cytoplasmic NF-kappaB-IkappaB complex. This activation required reactive oxygen intermediates and the phosphorylation of IkappaBalpha at serines 32 and 36, followed by IkappaBalpha degradation and the nuclear uptake of NF-kappaB. In addition, 13S E1A stimulated the transcriptional activity of the C-terminal 80 amino acids of p65 at a core promoter with either a TATA box or an initiator (INR) element. The C-terminal 80 amino acids of p65 were found to associate with E1A in vitro. The activation of NF-kappaB-dependent reporter gene transcription by E1A was potently suppressed upon coexpression of the E1B 19-kDa protein (19K). E1B 19K prevented both the activation of NF-kappaB and the E1A-mediated transcriptional enhancement of p65. These inhibitory effects were not found for the 55-kDa splice variant of the E1B protein. We suggest that the inductive effect of E1A 13S on the host factor NF-kappaB, whose activation is important for the transcription of various adenovirus genes, must be counteracted by the suppressive effect of E1B 19K so that the adenovirus-infected cell can escape the immune-stimulatory and apoptotic effects of NF-kappaB. PMID:8754803

  11. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new function for

  12. Enzyme activities in activated sludge flocs.

    PubMed

    Yu, Guang-Hui; He, Pin-Jing; Shao, Li-Ming; Lee, Duu-Jong

    2007-12-01

    This study quantified the activities of enzymes in extracellular polymeric substances (EPS) and in pellets. Seven commonly adopted extraction schemes were utilized to extract from aerobic flocs the contained EPS, which were further categorized into loosely bound (LB) and tightly bound (TB) fractions. Ultrasonication effectively extracted the EPS from sludge flocs. Enzyme assay tests showed that the protease activity was localized mainly on the pellets, alpha-amylase and alpha-glucosidase activities were largely bound with LB-EPS, and few protease, alpha-amylase, or alpha-glucosidase activities were associated with the TB-EPS fraction. There exists no correlation between the biochemical compositions of EPS and the distribution of enzyme activities in the sludge matrix. The 44-65% of alpha-amylase and 59-100% of alpha-glucosidase activities noted with the LB-EPS indicate heterogeneous hydrolysis patterns in the sludge flocs with proteins and carbohydrates.

  13. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

    PubMed

    Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

    2016-01-01

    Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

  14. Inhibition of human cytochrome P450 2E1 and 2A6 by aldehydes: structure and activity relationships.

    PubMed

    Kandagatla, Suneel K; Mack, Todd; Simpson, Sean; Sollenberger, Jill; Helton, Eric; Raner, Gregory M

    2014-08-01

    The purpose of this study was to probe active site structure and dynamics of human cytochrome P4502E1 and P4502A6 using a series of related short chain fatty aldehydes. Binding efficiency of the aldehydes was monitored via their ability to inhibit the binding and activation of the probe substrates p-nitrophenol (2E1) and coumarin (2A6). Oxidation of the aldehydes was observed in reactions with individually expressed 2E1, but not 2A6, suggesting alternate binding modes. For saturated aldehydes the optimum chain length for inhibition of 2E1 was 9 carbons (KI=7.8 ± 0.3 μM), whereas for 2A6 heptanal was most potent (KI=15.8 ± 1.1 μM). A double bond in the 2-position of the aldehyde significantly decreased the observed KI relative to the corresponding saturated compound in most cases. A clear difference in the effect of the double bond was observed between the two isoforms. With 2E1, the double bond appeared to remove steric constraints on aldehyde binding with KI values for the 5-12 carbon compounds ranging between 2.6 ± 0.1 μM and 12.8 ± 0.5 μM, whereas steric effects remained the dominant factor in the binding of the unsaturated aldehydes to 2A6 (observed KI values between 7.0 ± 0.5 μM and >1000 μM). The aldehyde function was essential for effective inhibition, as the corresponding carboxylic acids had very little effect on enzyme activity over the same range of concentrations, and branching at the 3-position of the aldehydes increased the corresponding KI value in all cases examined. The results suggest that a conjugated π-system may be a key structural determinant in the binding of these compounds to both enzymes, and may also be an important feature for the expansion of the active site volume in 2E1.

  15. Inhibition of human Cytochrome P450 2E1 and 2A6 by aldehydes: Structure and activity relationships

    PubMed Central

    Kandagatla, Suneel K.; Mack, Todd; Simpson, Sean; Sollenberger, Jill; Helton, Eric; Raner, Gregory M.

    2014-01-01

    The purpose of this study was to probe active site structure and dynamics of human cytochrome P4502E1 and P4502A6 using a series of related short chain fatty aldehydes. Binding efficiency of the aldehydes was monitored via their ability to inhibit the binding and activation of the probe substrates p-nitrophenol (2E1) and coumarin (2A6). Oxidation of the aldehydes was observed in reactions with individually expressed 2E1, but not 2A6, suggesting alternate binding modes. For saturated aldehydes the optimum chain length for inhibition of 2E1 was 9 carbons (KI=7.8 ±0.3 μM), whereas for 2A6 heptanal was most potent (KI=15.8 ±1.1 μM). A double bond in the 2-position of the aldehyde significantly decreased the observed KI relative to the corresponding saturated compound in most cases. A clear difference in the effect of the double bond was observed between the two isoforms. With 2E1, the double bond appeared to remove steric constraints on aldehyde binding with KI values for the 5–12 carbon compounds ranging between 2.6 ± 0.1 μM and 12.8± 0.5 μM, whereas steric effects remained the dominant factor in the binding of the unsaturated aldehydes to 2A6 (observed KI values between 7.0± 0.5 μM and >1000 μM). The aldehyde function was essential for effective inhibition, as the corresponding carboxylic acids had very little effect on enzyme activity over the same range of concentrations, and branching at the 3-position of the aldehydes increased the corresponding KI value in all cases examined. The results suggest that a conjugated π-system may be a key structural determinant in the binding of these compounds to both enzymes, and may also be an important feature for the expansion of the active site volume in 2E1. PMID:24924949

  16. The E1A transcriptional control region is efficiently activated in proliferating tissues of transgenic mice.

    PubMed

    Dieckmann, A; Krippl, B

    1994-08-01

    To study the in vivo regulation of the adenovirus E1A transcriptional regulatory region in transgenic mice, we have constructed two hybrid genes in which the viral control element regulates the expression of the CAT and the lacZ reporter gene. The fusion constructs were introduced into the mouse germline. The expression of the transgenes were monitored during embryogenesis and during postnatal development as well as in adult organs. We show that the E1A regulatory region is recognized and activated in undifferentiated cells during early embryonic cleavage, in the morula, in the inner cell mass and in the trophectoderm of the blastocyst. Transcription initiation at the E1A promoter leads to higher marker gene expression in proliferative centers in postimplantation embryos at the beginning of the neural tube closure. Analysing marker gene expression during postnatal development, a correlation of transcriptional activity of the E1A regulatory region and cell proliferation could be demonstrated. The expression profile of the transgene in different adult organs parallels with DNA synthesis. Marker gene expression was high in cells of organs known to have a high mitotic rate, such as the intestine, the stomach, the skin and the bone marrow, whereas little activity of the E1A control region was observed in the post-proliferative brain. These results are consistent with the finding that activation of the viral cis-regulating elements dramatically increased in the kidney after mitotic stimulation by folic acid. These observations strongly suggests a cell cycle regulated expression from the E1A enhancer/promoter in the absence of the E1A autoregulatory proteins in the living animal. PMID:8036008

  17. Serum enzyme activities after cardioversion

    PubMed Central

    Mandecki, Tadeusz; Giec, Leszek; Kargul, Włodzimierz

    1970-01-01

    Serum aspartate aminotransferase (SGOT), alanine aminotransferase (SGPT), creatinine phosphokinase (CPK), and butyric acid dehydrogenase (BDH) were determined in 94 patients before, 1½ hours, and 24 hours after cardioversion. An increase in SGOT and CPK activity was observed 24 hours after cardioversion in the group of patients treated with two or more DC shocks. The importance of this enzyme activity increase is discussed. It originates in the skeletal muscles and probably has no clinical significance, as no other signs of myocardial damage were observed simultaneously in a large group of patients. PMID:5470040

  18. Leinamycin E1 acting as an anticancer prodrug activated by reactive oxygen species

    PubMed Central

    Huang, Sheng-Xiong; Yun, Bong-Sik; Ma, Ming; Basu, Hirak S.; Church, Dawn R.; Ingenhorst, Gudrun; Huang, Yong; Yang, Dong; Lohman, Jeremy R.; Tang, Gong-Li; Ju, Jianhua; Liu, Tao; Wilding, George; Shen, Ben

    2015-01-01

    Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140, featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. Upon reductive activation in the presence of cellular thiols, LNM exerts its antitumor activity by an episulfonium ion-mediated DNA alkylation. Previously, we have cloned the lnm gene cluster from S. atroolivaceus S-140 and characterized the biosynthetic machinery responsible for the 18-membered lactam backbone and the alkyl branch at C3 of LNM. We now report the isolation and characterization of leinamycin E1 (LNM E1) from S. atroolivacues SB3033, a ΔlnmE mutant strain of S. atroolivaceus S-140. Complementary to the reductive activation of LNM by cellular thiols, LNM E1 can be oxidatively activated by cellular reactive oxygen species (ROS) to generate a similar episulfonium ion intermediate, thereby alkylating DNA and leading to eventual cell death. The feasibility of exploiting LNM E1 as an anticancer prodrug activated by ROS was demonstrated in two prostate cancer cell lines, LNCaP and DU-145. Because many cancer cells are under higher cellular oxidative stress with increased levels of ROS than normal cells, these findings support the idea of exploiting ROS as a means to target cancer cells and highlight LNM E1 as a novel lead for the development of anticancer prodrugs activated by ROS. The structure of LNM E1 also reveals critical new insights into LNM biosynthesis, setting the stage to investigate sulfur incorporation, as well as the tailoring steps that convert the nascent hybrid peptide–polyketide biosynthetic intermediate into LNM. PMID:26056295

  19. Alcohol metabolism's damaging effects on the cell: a focus on reactive oxygen generation by the enzyme cytochrome P450 2E1.

    PubMed

    Koop, Dennis R

    2006-01-01

    Alcohol metabolism's various processes create harmful compounds that contribute to cell and tissue damage. In particular, the enzyme cytochrome P450 2E1 (CYP2E1) plays a role in creating a harmful condition known as oxidative stress. This condition is related to oxygen's ability to accept electrons and the subsequent highly reactive and harmful byproducts created by these chemical reactions. CYP2E1's use of oxygen in alcohol metabolism generates reactive oxygen species, ultimately leading to oxidative stress and tissue damage.

  20. Rb function is required for E1A-induced S-phase checkpoint activation.

    PubMed

    Nemajerova, A; Talos, F; Moll, U M; Petrenko, O

    2008-09-01

    It is widely accepted that adenoviral E1A exerts its influence on recipient cells through binding to the retinoblastoma (Rb) family proteins, followed by a global release of E2F factors from pocket-protein control. Our study challenges this simple paradigm by demonstrating previously unappreciated complexity. We show that E1A-expressing primary and transformed cells are characterized by the persistence of Rb-E2F1 complexes. We provide evidence that E1A causes Rb stabilization by interfering with its proteasomal degradation. Functional experiments supported by biochemical data reveal not only a dramatic increase in Rb and E2F1 protein levels in E1A-expressing cells but also demonstrate their activation throughout the cell cycle. We further show that E1A activates an Rb- and E2F1-dependent S-phase checkpoint that attenuates the growth of cells that became hyperploid through errors in mitosis and supports the fidelity DNA replication even in the absence of E2F complexes with other Rb family proteins, thereby functionally substituting for the loss of p53. Our results support the essential role of Rb and E2F1 in the regulation of genomic stability and DNA damage checkpoints.

  1. Characterization of papillomavirus E1 helicase mutants defective for interaction with the SUMO-conjugating enzyme Ubc9

    SciTech Connect

    Fradet-Turcotte, Amelie; Brault, Karine; Titolo, Steve; Howley, Peter M.; Archambault, Jacques

    2009-12-20

    The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and binding to DNA and ATP.

  2. Inhibitory Potency of 4-Carbon Alkanes and Alkenes toward CYP2E1 Activity

    PubMed Central

    Hartman, Jessica H.; Miller, Grover P.; Boysen, Gunnar

    2016-01-01

    CYP2E1 has been implicated in the bioactivation of many small molecules into reactive metabolites which form adducts with proteins and DNA, and thus a better understanding of the molecular determinants of its selectivity are critical for accurate toxicological predictions. In this study, we determined the potency of inhibition of human CYP2E1 for various 4-carbon alkanes, alkenes and alcohols. In addition, known CYP2E1 substrates and inhibitors including 4-methylpyrazole, aniline, and dimethylnitrosamine were included to determine their relative potencies. Of the 1,3-butadiene-derived metabolites studied, 3,4-epoxy-1-butene was the strongest inhibitor with an IC50 of 110 μM compared to 1700 μM and 6600 μM for 1,2-butenediol and 1,2:3,4-diepoxybutane, respectively. Compared to known inhibitors, inhibitory potency of 3,4-epoxy-1-butene is between 4-methylpyrazole (IC50 = 1.8 μM) and dimethylnitrosamine (IC50 = 230 μM). All three butadiene metabolites inhibit CYP2E1 activity through a simple competitive mechanism. Among the 4-carbon compounds studied, the presence and location of polar groups seems to influence inhibitory potency. To further examine this notion, the investigation was extended to include structurally and chemically similar analogs, including propylene oxide and various butane alcohols. Those results demonstrated preferential recognition of CYP2E1 toward the type and location of polar and hydrophobic structural elements. Taken together, CYP2E1 metabolism may be modified in vivo by exposure to 4-carbon compounds, such as drugs, and nutritional constituents, a finding that highlights the complexity of exposure to mixtures. PMID:24561005

  3. 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)…

  4. Combined System of Activated Sludge and Ozonation for the Treatment of Kraft E1 Effluent

    PubMed Central

    Assalin, Marcia Regina; dos Santos Almeida, Edna; Durán, Nelson

    2009-01-01

    The treatment of paper mill effluent for COD, TOC, total phenols and color removal was investigated using combined activated sludge-ozonation processes and single processes. The combined activated sludge-O3/pH 10 treatment was able to remove around 80% of COD, TOC and color from Kraft E1 effluent. For the total phenols, the efficiency removal was around 70%. The ozonation post treatment carried out at pH 8.3 also showed better results than the single process. The COD, TOC, color and total phenols removal efficiency obtained were 75.5, 59.1, 77 and 52.3%, respectively. The difference in the concentrations of free radical produced by activated sludge-O3/pH 10 and activated sludge-O3/pH 8.3 affected mainly the TOC and total phenol removal values. PMID:19440438

  5. The E1 proteins

    SciTech Connect

    Bergvall, Monika; Melendy, Thomas; Archambault, Jacques

    2013-10-15

    E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA using a spiral escalator mechanism. E1 is tightly regulated in vivo, in particular by post-translational modifications that restrict its accumulation in the nucleus. Here we review how different functional domains of E1 orchestrate viral DNA replication, with an emphasis on their interactions with substrate DNA, host DNA replication factors and modifying enzymes. These studies have made E1 one of the best characterized helicases and provided unique insights on how PVs usurp different host-cell machineries to replicate and amplify their genome in a tightly controlled manner. - Highlights: • The papillomavirus E1 helicase orchestrates replication of the viral DNA genome. • E1 assembles into a double-hexamer at the viral origin with the help of E2. • E1 interacts with cellular DNA replication factors. • E1 unwinds DNA using a spiral escalator mechanism. • Nuclear accumulation of E1 is regulated by post-translational modifications.

  6. 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)

  7. A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.

    PubMed

    Arjunan, Palaniappa; Sax, Martin; Brunskill, Andrew; Chandrasekhar, Krishnamoorthy; Nemeria, Natalia; Zhang, Sheng; Jordan, Frank; Furey, William

    2006-06-01

    The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important. PMID:16531404

  8. Enzyme activity down to -100 degrees C.

    PubMed

    Bragger, J M; Dunn, R V; Daniel, R M

    2000-07-14

    The activities of two enzymes, beef liver catalase (EC 1.11.1.6) and calf intestine alkaline phosphatase (EC 3.1.3.1), have been measured down to -97 degrees C and -100 degrees C, respectively. Enzyme activity has not previously been measured at such low temperatures. For catalase, the cryosolvents used were methanol:ethylene glycol:water (70:10:20) and DMSO:ethylene glycol:water (60:20:20). For alkaline phosphatase, methanol:ethylene glycol:water (70:10:20) was used. All of the Arrhenius plots were linear over the whole of the temperature range examined. Since the lowest temperatures at which activity was measured are well below the dynamic transition observed for proteins, the results indicate that the motions which cease below the dynamic transition are not essential for enzyme activity. In all cases the use of cryosolvent led to substantial increases in Arrhenius activation energies, and this imposed practical limitations on the measurement of enzyme activity below -100 degrees C. At even lower temperatures, enzyme activity may be limited by the effect of solvent fluidity on substrate/product diffusion, but overall there is no evidence that any intrinsic enzyme property imposes a lower temperature limit for enzyme activity. PMID:10899628

  9. 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)

  10. Glutamate 636 of the Escherichia coli pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity.

    PubMed

    Nemeria, Natalia; Tittmann, Kai; Joseph, Ebenezer; Zhou, Leon; Vazquez-Coll, Michelle B; Arjunan, Palaniappa; Hübner, Gerhard; Furey, William; Jordan, Frank

    2005-06-01

    The residue Glu636 is located near the thiamine diphosphate (ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a K(d,ThDP) = 4.34 microM and K(m,ThDP) = 11 microM were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1',4'-iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)-acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp28-substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity (re or si) vis à vis pyruvate. The tryptic peptide map (mass spectral analysis) revealed that the Glu636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu636 has important functions both in active center communication and in protecting the active center from undesirable "carboligase" side reactions.

  11. 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.

  12. Histidine 407, a phantom residue in the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex, activates reductive acetylation of lipoamide on the E2 subunit. An explanation for conservation of active sites between the E1 subunit and transketolase.

    PubMed

    Nemeria, Natalia; Arjunan, Palaniappa; Brunskill, Andrew; Sheibani, Farzad; Wei, Wen; Yan, Yan; Zhang, Sheng; Jordan, Frank; Furey, William

    2002-12-31

    Least squares alignment of the E. coli pyruvate dehydrogenase multienzyme complex E1 subunit and yeast transketolase crystal structures indicates a general structural similarity between the two enzymes and provides a plausible location for a short-loop region in the E1 structure that was unobserved due to disorder. The residue H407, located in this region, is shown to be able to penetrate the active site. Suggested by this comparison, the H407A E1 variant was created, and H407 was shown to participate in the reductive acetylation of both an independently expressed lipoyl domain and the intact 1-lipoyl E2 subunit. While the H407A substitution only modestly affected the reaction through pyruvate decarboxylation (ca. 14% activity compared to parental E1), the overall complex has a much impaired activity, at most 0.15% compared to parental E1. Isothermal titration calorimetry measurements show that the binding of the lipoyl domain to the H407A E1 variant is much weaker than that to parental E1. At the same time, mass spectrometric measurements clearly demonstrate much impaired reductive acetylation of the independently expressed lipoyl domain and of the intact 1-lipoyl E2 by the H407A variant compared to the parental E1. A proposal is presented to explain the remarkable conservation of the three-dimensional structure at the active centers of the E. coli E1 subunit and transketolase on the basis of the parallels in the ligation-type reactions carried out and the need to protonate a very weak acid, a dithiolane sulfur atom in the former, and a carbonyl oxygen atom in the latter. PMID:12501174

  13. 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.

  14. 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.

  15. Regulation of hepatic sulfotransferase (SULT) 1E1 expression and effects on estrogenic activity in cystic fibrosis (CF)☆

    PubMed Central

    Falany, Charles N.; He, Dongning; Li, Li; Falany, Josie L.; Wilborn, Teresa W.; Kocarek, Thomas A.; Runge-Morris, Melissa

    2013-01-01

    Cystic fibrosis (CF) is a major genetic disease in Caucasians affecting 1 in 2500 newborns. Hepatobiliary pathology is a major cause of morbidity and mortality in CF second only to pulmonary disease. SULT1E1 activity is significantly elevated, generally 20–30-fold, in hepatocytes of mouse models of CF. SULT1E1 is responsible for the inactivation of β-estradiol (E2) at physiological concentrations via conjugation with sulfonate. The increase in SULT1E1 activity results in the alteration of E2-regulated protein expression in CF mouse liver. To investigate the mechanism by which the absence of CFTR in human cholangiocytes induces SULT1E1 expression in hepatocytes, a membrane-separated human MMNK-1 cholangiocyte and human HepG2 hepatocyte co-culture system was developed. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in bile duct cholangiocytes but not hepatocytes, whereas SULT1E1 is expressed in hepatocytes but not cholangiocytes. CFTR expression in MMNK-1 cells was inhibited with siRNA by >90% as determined by immunoblot and immunohistochemical analysis. Control and CFTR-siRNA-MMNK-1 cells were co-cultured with HepG2 cells in a Transwell membrane-separated system. After 8 h of co-culture, HepG2 cells were removed from exposure to MMNK-1 cells and placed in fresh medium. After 24–48 h, expression of SULT1E1 and selected E2-regulated proteins was analyzed in the HepG2 cells. Results demonstrated that SULT1E1 message and activity were selectively induced in HepG2 cells co-cultured with CFTR-deficient MMNK-1 cells. The expression of E2-regulated proteins (IGF-1, GST-P1 and carbonic anhydrase II) was also altered in response to decreased E2 levels. Thus, the loss of CFTR activity in cholangiocytes stimulates the expression of SULT1E1 in hepatocytes by a paracrine mechanism. SULT1E1 expression in HepG2 cells is inducible by sterol mediated liver-X-receptor (LXR) activation although not by progestins that induce SULT1E1 in the endometrium

  16. Effects of sex, weight, diet and hCG administration on levels of skatole and indole in the liver and hepatic activities of cytochromes P4502E1 and P4502A6 in pigs.

    PubMed

    Zamaratskaia, G; Chen, G; Lundström, K

    2006-02-01

    Cytochromes P4502E1 (CYP2E1) and P4502A6 (CYP2A6) catalyse metabolic reactions of skatole and indole metabolism. The objectives of this study were as follows: to evaluate whether activities of CYP2E1 and CYP2A6 in pigs of two live weights (LW) differ between males and females; to investigate whether activities of CYP2E1 and CYP2A6 are affected by hCG stimulation; and to investigate whether the levels of skatole and indole in the liver and the activities of CYP2E1 and CYP2A6 are affected by raw potato starch (RPS). Female pigs expressed higher CYP2A6 activity at 90kg LW, and higher CYP2E1 activity at 115kg LW compared to male pigs. Skatole levels in the liver were higher in male pigs than in female pigs at both LW, whereas indole levels were higher in males only at 115 kg LW. Neither levels of indolic compounds in the liver nor enzyme activities were affected by hCG stimulation. The inclusion of RPS in the diet reduced skatole levels in the liver in both sexes and increased CYP2A6 activity in female pigs. It was concluded that the incidence of boar taint may depend on both skatole amount, which reach the liver, and the activities of enzymes involved in skatole metabolism, which may vary depending on sex, live weight, and diet. PMID:22061562

  17. Activity assessment of microbial fibrinolytic enzymes.

    PubMed

    Kotb, Essam

    2013-08-01

    Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

  18. Ubiquitin enzymes, ubiquitin and proteasome activity in blood mononuclear cells of MCI, Alzheimer and Parkinson patients.

    PubMed

    Ullrich, C; Mlekusch, R; Kuschnig, A; Marksteiner, J; Humpel, C

    2010-09-01

    Alzheimer's disease (AD) is a severe chronic neurodegenerative disease. During aging and neurodegeneration, misfolded proteins accumulate and activate the ubiquitin-proteasome system. The aim of the present study is to explore whether ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin or proteasome activity are affected in peripheral blood mononuclear cells (PBMC) of AD, mild cognitive impairment (MCI) and Parkinson's disease (PD) patients compared to healthy subjects. PBMCs were isolated from EDTA blood samples and extracts were analyzed by Western Blot. Proteasome activity was measured with fluorogenic substrates. When compared to healthy subjects, the concentration of enzyme E1 was increased in PBMCs of AD patients, whereas the concentration of the enzyme E2 was decreased in these same patients. Ubiquitin levels and proteasome activity were unchanged in AD patients. No changes in enzyme expression or proteasome activity was observed in MCI patients compared to healthy and AD subjects. In PD patients E2 levels and proteasomal activity were significantly reduced, while ubiquitin and E1 levels were unchanged. The present investigation demonstrates the differences in enzyme and proteasome activity patterns of AD and PD patients. These results suggest that different mechanisms are involved in regulating the ubiquitin-proteasomal system in different neurodegenerative diseases.

  19. NOVEL ASSAY TO ASSESS CYP-2E1-LIKE ACTIVITY IN THE JAPANESE MEDAKA (ORYZIAS LATIPES).

    EPA Science Inventory

    Liver microsomes and S-9 fraction of Japanese medaka (Oryzias latipes) metabolized the CYP2E1 specific substrate, p-nitrophenol (PNP), to a single hydroxylated product, 4-nitrocatechol. The use of liver S-9 fraction proved to be a viable alternative to liver microsomes and allowe...

  20. 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)

  1. Arabinogalactan proteins: focus on carbohydrate active enzymes

    PubMed Central

    Knoch, Eva; Dilokpimol, Adiphol; Geshi, Naomi

    2014-01-01

    Arabinogalactan proteins (AGPs) are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/) involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development. PMID:24966860

  2. Concentration profiles near an activated enzyme.

    PubMed

    Park, Soohyung; Agmon, Noam

    2008-09-25

    When a resting enzyme is activated, substrate concentration profile evolves in its vicinity, ultimately tending to steady state. We use modern theories for many-body effects on diffusion-influenced reactions to derive approximate analytical expressions for the steady-state profile and the Laplace transform of the transient concentration profiles. These show excellent agreement with accurate many-particle Brownian-dynamics simulations for the Michaelis-Menten kinetics. The steady-state profile has a hyperbolic dependence on the distance of the substrate from the enzyme, albeit with a prefactor containing the complexity of the many-body effects. These are most conspicuous for the substrate concentration at the surface of the enzyme. It shows an interesting transition as a function of the enzyme turnover rate. When it is high, the contact concentration decays monotonically to steady state. However, for slow turnover it is nonmonotonic, showing a minimum due to reversible substrate binding, then a maximum due to diffusion of new substrate toward the enzyme, and finally decay to steady state. Under certain conditions one can obtain a good estimate for the critical value of the turnover rate constant at the transition.

  3. Heterogeneity of adenovirus type 5 E1A proteins: multiple serine phosphorylations induce slow-migrating electrophoretic variants but do not affect E1A-induced transcriptional activation or transformation.

    PubMed Central

    Richter, J D; Slavicek, J M; Schneider, J F; Jones, N C

    1988-01-01

    The 289-amino-acid product encoded by the adenovirus E1A 13S mRNA has several pleiotropic activities, including transcriptional activation, transcriptional repression, and when acting in concert with certain oncogene products, cell transformation. In all cell types in which E1A has been introduced (except bacteria), E1A protein is extensively posttranslationally modified to yield several isoelectric and molecular weight variants. The most striking variant is one that has a retarded mobility, by about Mr = 2,000, in sodium dodecyl sulfate gels. We have investigated the nature of this modification and have assessed its importance for E1A activity. Phosphorylation is responsible for the altered mobility of E1A, since acid phosphatase treatment eliminates the higher apparent molecular weight products. By using several E1A deletion mutants, we show that at least two seryl residues, residing between residues 86 and 120 and 224 and 289, are the sites of phosphorylation and that each phosphorylation can independently induce the mobility shift. However, E1A mutants lacking these seryl residues transcriptionally activate the adenovirus E3 and E2A promoters and transform baby rat kidney cells to near wild-type levels. Images PMID:2835499

  4. 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

  5. Local encoding of computationally designed enzyme activity

    PubMed Central

    Allert, Malin; Dwyer, Mary A.; Hellinga, Homme W.

    2007-01-01

    One aim of computational protein design is to introduce novel enzyme activity into proteins of known structure by predicting mutations that stabilize transition states. Previously we have shown that it is possible to introduce triose phosphate isomerase activity into the ribose-binding protein of Escherichia coli by constructing 17 mutations in the first two layers of residues that surround the wild-type ligand-binding site. Here we report that these mutations can be “transplanted” into a homologous ribose-binding protein, isolated from the hyperthermophilic bacterium Thermoanaerobacter tengcongensis, with retention of catalytic activity, substrate affinity, and reaction pH dependence. The observed 105–106-fold rate enhancement corresponds to 70% of the maximally known transition-state binding energy. The wild-type sequences in these two homologues are almost perfectly conserved in the vicinity of their ribose-binding sites, but diverge significantly at increasing distance from these sites. The results demonstrate that the computationally designed mutations are sufficient to encode the observed enzyme activity, that all the observed activity is locally encoded within the layer of residues directly in contact with the substrate, and that in this case at least 70% of transition state stabilization energy can be achieved using straightforward considerations of stereochemical complementarity between enzyme and reactants. PMID:17196220

  6. Identification of a region within the ubiquitin-activating enzyme required for nuclear targeting and phosphorylation.

    PubMed

    Stephen, A G; Trausch-Azar, J S; Handley-Gearhart, P M; Ciechanover, A; Schwartz, A L

    1997-04-18

    The ubiquitin-activating enzyme exists as two isoforms: E1a, localized predominantly in the nucleus, and E1b, localized in the cytoplasm. Previously we generated hemagglutinin (HA) epitope-tagged cDNA constructs, HA1-E1 (epitope tag placed after the first methionine) and HA2-E1 (epitope tag placed after the second methionine) (Handley-Gearhart, P. M., Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A., and Schwartz, A. L. (1994) J. Biol. Chem. 269, 33171-33178), which represent the native isoforms. HA1-E1 is exclusively nuclear, whereas HA2-E1 is found predominantly in the cytoplasm. Using high resolution isoelectric focusing and SDS-polyacrylamide gel electrophoresis, we confirm that these epitope-tagged constructs HA1-E1 and HA2-E1 represent the two isoforms E1a and E1b. HA1-E1/E1a exists as one non-phosphorylated and four phosphorylated forms, and HA2-E1/E1b exists as one predominant non-phosphorylated form and two minor phosphorylated forms. We demonstrate that the first 11 amino acids are essential for phosphorylation and exclusive nuclear localization of HA1-E1. Within this region are four serine residues and a putative nuclear localization sequence (NLS; 5PLSKKRR). Removal of these four serine residues reduced phosphorylation levels by 60% but had no effect on nuclear localization of HA1-E1. Each serine residue was independently mutated to an alanine and analyzed by two-dimensional electrophoresis; only serine 4 was phosphorylated. Disruption of the basic amino acids within the NLS resulted in loss of exclusive nuclear localization and a 90-95% decrease in the phosphorylation of HA1-E1. This putative NLS was able to confer nuclear import on a non-nuclear protein in digitonin-permeabilized cells in a temperature- and ATP-dependent manner. Thus the predominant requirement for efficient phosphorylation of HA1-E1/E1a is a functional NLS, suggesting that E1a may be phosphorylated within the nucleus. PMID:9099746

  7. Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock.

    PubMed

    Li, Guang-Qi; Zang, Xiao-Nan; Zhang, Xue-Cheng; Lu, Ning; Ding, Yan; Gong, Le; Chen, Wen-Chao

    2014-03-15

    To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes - ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) - of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis-wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis.

  8. Effects of herbal products and their constituents on human cytochrome P450(2E1) activity.

    PubMed

    Raner, Gregory M; Cornelious, Sean; Moulick, Kamalika; Wang, Yingqing; Mortenson, Ashley; Cech, Nadja B

    2007-12-01

    Ethanolic extracts from fresh Echinacea purpurea and Spilanthes acmella and dried Hydrastis canadensis were examined with regard to their ability to inhibit cytochrome P450(2E1) mediated oxidation of p-nitrophenol in vitro. In addition, individual constituents of these extracts, including alkylamides from E. purpurea and S. acmella, caffeic acid derivatives from E. purpurea, and several of the major alkaloids from H. canadensis, were tested for inhibition using the same assay. H. canadensis (goldenseal) was a strong inhibitor of the P450(2E1), and the inhibition appeared to be related to the presence of the alkaloids berberine, hydrastine and canadine in the extract. These compounds inhibited 2E1 with K(I) values ranging from 2.8 microM for hydrastine to 18 microM for berberine. The alkylamides present in E. purpurea and S. acmella also showed significant inhibition at concentrations as low as 25 microM, whereas the caffeic acid derivatives had no effect. Commercial green tea preparations, along with four of the individual tea catechins, were also examined and were found to have no effect on the activity of P450(2E1). PMID:17658211

  9. Microwave-assisted Synthesis and antifungal activity of coumarin[8,7-e][1,3]oxazine derivatives.

    PubMed

    Zhang, Ming-Zhi; Zhang, Rong-Rong; Yin, Wen-Zheng; Yu, Xiang; Zhang, Ya-Ling; Liu, Pin; Gu, Yu-Cheng; Zhang, Wei-Hua

    2016-08-01

    The synthesis of novel coumarin[8,7-e][1,3]oxazine derivatives through a microwave-assisted three-component one-pot Mannich reaction is described in this study. All the target compounds were evaluated in vitro for their antifungal activity against Botrytis cinerea, Colletotrichum capsici, Alternaria solani, Gibberella zeae, Rhizoctonia solani, and Alternaria mali. The preliminary bioassays showed that 5e, 5m, and 5s exhibited good antifungal activity and the most active compound was 5m with an [Formula: see text] value as low as 2.1 nM against Botrytis cinerea. PMID:26880591

  10. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    PubMed

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  11. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Lu, Guoxin

    2007-01-01

    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

  12. Synthesis and kinase inhibitory activity of new sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazines.

    PubMed

    Mojzych, Mariusz; Šubertová, Veronika; Bielawska, Anna; Bielawski, Krzysztof; Bazgier, Václav; Berka, Karel; Gucký, Tomáš; Fornal, Emilia; Kryštof, Vladimír

    2014-05-01

    A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine has been synthesized and characterized. Their anticancer activity was tested in vitro against multiple human cancer cell lines and were found to have dose-dependent antiproliferative effects. Furthermore, some of the new compounds inhibited the Abl protein kinase with low micromolar IC50 values and exhibited selective activity against the Bcr-Abl positive K562 and BV173 cell lines, providing starting points for the further development of this new kinase inhibitor scaffold.

  13. 26 CFR 1.924(e)-1 - Activities relating to the disposition of export property.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... that advertising activity is a foreign cost. The FSC may rely upon the distribution statistics of the.... If the distribution statistics show that 85 percent or more of the readership, radio listeners, or... supplier to United States distributors for resale to Canadian retail customers. The related supplier,...

  14. 26 CFR 1.924(e)-1 - Activities relating to the disposition of export property.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... account as advertising costs as long as the activity is not taken into account for purposes of the sales... costs of sales promotion. Direct costs of sales promotion include costs such as rental of space at trade... costs for travel, meals, and lodging for direct sales people attending the event if these costs are...

  15. [Detection of enzyme activity in decontaminated spices in industrial use].

    PubMed

    Müller, R; Theobald, R

    1995-03-01

    A range of decontaminated species of industrial use have been examined for their enzymes (catalase, peroxidase, amylase, lipase activity). The genuine enzymes remain fully active in irradiated spices, whereas the microbial load is clearly reduced. In contrast steam treated spices no longer demonstrate enzyme activities. Steam treatment offers e.g. black pepper without lipase activity, which can no longer cause fat deterioration. Low microbial load in combination with clearly detectable enzyme activity in spices is an indication for irradiation, whereas, reduced microbial contamination combined with enzyme inactivation indicate steam treatment of raw material.

  16. Communication between thiamin cofactors in the Escherichia coli pyruvate dehydrogenase complex E1 component active centers: evidence for a "direct pathway" between the 4'-aminopyrimidine N1' atoms.

    PubMed

    Nemeria, Natalia S; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-04-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4'-aminopyrimidine N1' atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu(571), Glu(235), and Glu(237)) and Arg(606) resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu(235) makes no direct contact with the cofactor. The role of the conserved Glu(571) residue in both catalysis and cofactor orientation is revealed by the combined results for the first time.

  17. Communication between thiamin cofactors in the Escherichia coli pyruvate dehydrogenase complex E1 component active centers: evidence for a "direct pathway" between the 4'-aminopyrimidine N1' atoms.

    PubMed

    Nemeria, Natalia S; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-04-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4'-aminopyrimidine N1' atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu(571), Glu(235), and Glu(237)) and Arg(606) resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu(235) makes no direct contact with the cofactor. The role of the conserved Glu(571) residue in both catalysis and cofactor orientation is revealed by the combined results for the first time. PMID:20106967

  18. The Impact of Enzyme Orientation and Electrode Topology on the Catalytic Activity of Adsorbed Redox Enzymes

    PubMed Central

    McMillan, Duncan G. G.; Marritt, Sophie J.; Kemp, Gemma L.; Gordon-Brown, Piers; Butt, Julea N.; Jeuken, Lars J. C.

    2014-01-01

    It is well established that the structural details of electrodes and their interaction with adsorbed enzyme influences the interfacial electron transfer rate. However, for nanostructured electrodes, it is likely that the structure also impacts on substrate flux near the adsorbed enzymes and thus catalytic activity. Furthermore, for enzymes converting macro-molecular substrates it is possible that the enzyme orientation determines the nature of interactions between the adsorbed enzyme and substrate and therefore catalytic rates. In essence the electrode may impede substrate access to the active site of the enzyme. We have tested these possibilities through studies of the catalytic performance of two enzymes adsorbed on topologically distinct electrode materials. Escherichia coli NrfA, a nitrite reductase, was adsorbed on mesoporous, nanocrystalline SnO2 electrodes. CymA from Shewanella oneidensis MR-1 reduces menaquinone-7 within 200 nm sized liposomes and this reaction was studied with the enzyme adsorbed on SAM modified ultra-flat gold electrodes. PMID:24634538

  19. The Impact of Enzyme Orientation and Electrode Topology on the Catalytic Activity of Adsorbed Redox Enzymes.

    PubMed

    McMillan, Duncan G G; Marritt, Sophie J; Kemp, Gemma L; Gordon-Brown, Piers; Butt, Julea N; Jeuken, Lars J C

    2013-11-01

    It is well established that the structural details of electrodes and their interaction with adsorbed enzyme influences the interfacial electron transfer rate. However, for nanostructured electrodes, it is likely that the structure also impacts on substrate flux near the adsorbed enzymes and thus catalytic activity. Furthermore, for enzymes converting macro-molecular substrates it is possible that the enzyme orientation determines the nature of interactions between the adsorbed enzyme and substrate and therefore catalytic rates. In essence the electrode may impede substrate access to the active site of the enzyme. We have tested these possibilities through studies of the catalytic performance of two enzymes adsorbed on topologically distinct electrode materials. Escherichia coli NrfA, a nitrite reductase, was adsorbed on mesoporous, nanocrystalline SnO2 electrodes. CymA from Shewanella oneidensis MR-1 reduces menaquinone-7 within 200 nm sized liposomes and this reaction was studied with the enzyme adsorbed on SAM modified ultra-flat gold electrodes.

  20. Exploration of the spontaneous fluctuating activity of single enzyme molecules.

    PubMed

    Schwabe, Anne; Maarleveld, Timo R; Bruggeman, Frank J

    2013-09-01

    Single enzyme molecules display inevitable, stochastic fluctuations in their catalytic activity. In metabolism, for instance, the stochastic activity of individual enzymes is averaged out due to their high copy numbers per single cell. However, many processes inside cells rely on single enzyme activity, such as transcription, replication, translation, and histone modifications. Here we introduce the main theoretical concepts of stochastic single-enzyme activity starting from the Michaelis-Menten enzyme mechanism. Next, we discuss stochasticity of multi-substrate enzymes, of enzymes and receptors with multiple conformational states and finally, how fluctuations in receptor activity arise from fluctuations in signal concentration. This paper aims to introduce the exciting field of single-molecule enzyme kinetics and stochasticity to a wider audience of biochemists and systems biologists.

  1. Identification of quinazolinyloxy biaryl urea as a new class of SUMO activating enzyme 1 inhibitors.

    PubMed

    Kumar, Ashutosh; Ito, Akihiro; Hirohama, Mikako; Yoshida, Minoru; Zhang, Kam Y J

    2013-09-15

    SUMO activating enzyme 1 (SUMO E1) is the first enzyme in sumoylation pathway and an important cancer drug target. However, only a few inhibitors were reported up to now that includes three natural products, semi-synthetic protein inhibitors and one AMP mimic. Here, we report the identification of quinazolinyloxy biaryl urea as a new class of SUMO E1 inhibitors. The most active compound of this class inhibited the in vitro sumoylation with an IC50 of 13.4 μM. This compound inhibits sumoylation by blocking the formation of SUMOE1-SUMO thioester intermediate. The biological activity of the most active compound is comparable to previously reported inhibitors with properties suitable for medicinal chemistry optimization for potency and druggability.

  2. Glycyl radical activating enzymes: structure, mechanism, and substrate interactions.

    PubMed

    Shisler, Krista A; Broderick, Joan B

    2014-03-15

    The glycyl radical enzyme activating enzymes (GRE-AEs) are a group of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily and utilize a [4Fe-4S] cluster and SAM to catalyze H-atom abstraction from their substrate proteins. GRE-AEs activate homodimeric proteins known as glycyl radical enzymes (GREs) through the production of a glycyl radical. After activation, these GREs catalyze diverse reactions through the production of their own substrate radicals. The GRE-AE pyruvate formate lyase activating enzyme (PFL-AE) is extensively characterized and has provided insights into the active site structure of radical SAM enzymes including GRE-AEs, illustrating the nature of the interactions with their corresponding substrate GREs and external electron donors. This review will highlight research on PFL-AE and will also discuss a few GREs and their respective activating enzymes.

  3. 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

  4. Gene coding for the E1 endoglucanase

    DOEpatents

    Thomas, Steven R.; Laymon, Robert A.; Himmel, Michael E.

    1996-01-01

    The gene encoding Acidothermus cellulolyticus E1 endoglucanase is cloned and expressed in heterologous microorganisms. A new modified E1 endoglucanase enzyme is produced along with variants of the gene and enzyme. The E1 endoglucanase is useful for hydrolyzing cellulose to sugars for simultaneous or later fermentation into alcohol.

  5. Gene coding for the E1 endoglucanase

    DOEpatents

    Thomas, S.R.; Laymon, R.A.; Himmel, M.E.

    1996-07-16

    The gene encoding Acidothermus cellulolyticus E1 endoglucanase is cloned and expressed in heterologous microorganisms. A new modified E1 endoglucanase enzyme is produced along with variants of the gene and enzyme. The E1 endoglucanase is useful for hydrolyzing cellulose to sugars for simultaneous or later fermentation into alcohol. 6 figs.

  6. Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

    PubMed Central

    Kanazawa, Ippei; Yamaguchi, Toru; Yano, Shozo; Yamauchi, Mika; Yamamoto, Masahiro; Sugimoto, Toshitsugu

    2007-01-01

    Background Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results Adiponectin receptor type 1 (AdipoR1) mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase) was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR), in the cells. AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM) in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml) also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusion Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions. PMID:18047638

  7. Observing single enzyme molecules interconvert between activity states upon heating.

    PubMed

    Rojek, Marcin J; Walt, David R

    2014-01-01

    In this paper, we demonstrate that single enzyme molecules of β-galactosidase interconvert between different activity states upon exposure to short pulses of heat. We show that these changes in activity are the result of different enzyme conformations. Hundreds of single β-galactosidase molecules are trapped in femtoliter reaction chambers and the individual enzymes are subjected to short heating pulses. When heating pulses are introduced into the system, the enzyme molecules switch between different activity states. Furthermore, we observe that the changes in activity are random and do not correlate with the enzyme's original activity. This study demonstrates that different stable conformations play an important role in the static heterogeneity reported previously, resulting in distinct long-lived activity states of enzyme molecules in a population.

  8. Human Papillomavirus Type 16 E1∧E4-Induced G2 Arrest Is Associated with Cytoplasmic Retention of Active Cdk1/Cyclin B1 Complexes

    PubMed Central

    Davy, Clare E.; Jackson, Deborah J.; Raj, Kenneth; Peh, Woei Ling; Southern, Shirley A.; Das, Papia; Sorathia, Rina; Laskey, Peter; Middleton, Kate; Nakahara, Tomomi; Wang, Qian; Masterson, Phillip J.; Lambert, Paul F.; Cuthill, Scott; Millar, Jonathan B. A.; Doorbar, John

    2005-01-01

    Human papillomavirus type 16 (HPV16) can cause cervical cancer. Expression of the viral E1∧E4 protein is lost during malignant progression, but in premalignant lesions, E1∧E4 is abundant in cells supporting viral DNA amplification. Expression of 16E1∧E4 in cell culture causes G2 cell cycle arrest. Here we show that unlike many other G2 arrest mechanisms, 16E1∧E4 does not inhibit the kinase activity of the Cdk1/cyclin B1 complex. Instead, 16E1∧E4 uses a novel mechanism in which it sequesters Cdk1/cyclin B1 onto the cytokeratin network. This prevents the accumulation of active Cdk1/cyclin B1 complexes in the nucleus and hence prevents mitosis. A mutant 16E1∧E4 (T22A, T23A) which does not bind cyclin B1 or alter its intracellular location fails to induce G2 arrest. The significance of these results is highlighted by the observation that in lesions induced by HPV16, there is evidence for Cdk1/cyclin B1 activity on the keratins of 16E1∧E4-expressing cells. We hypothesize that E1∧E4-induced G2 arrest may play a role in creating an environment optimal for viral DNA replication and that loss of E1∧E4 expression may contribute to malignant progression. PMID:15767402

  9. Why do crown ethers activate enzymes in organic solvents?

    PubMed

    van Unen, Dirk-Jan; Engbersen, Johan F J; Reinhoudt, David N

    2002-02-01

    One of the major drawbacks of enzymes in nonaqueous solvents is that their activity is often dramatically low compared to that in water. This limitation can be largely overcome by crown ether treatment of enzymes. In this paper, we describe a number of carefully designed new experiments that have improved the insights into the mechanisms that are operative in the crown ether activation of enzymes in organic solvents. The enhancement of enzyme activity upon addition of 18-crown-6 to the organic solvent can be reconciled with a mechanism in which macrocyclic interactions of 18-crown-6 with the enzyme play an important role. Macrocyclic interactions (e.g., complexation with lysine ammonium groups of the enzyme) can lead to a reduced formation of inter- and intramolecular salt bridges and, consequently, to lowering of the kinetic conformational barriers, enabling the enzyme to refold into thermodynamically stable, catalytically (more) active conformations. This assumption is supported by the observation that the crown-ether-enhanced enzyme activity is retained after removal of the crown by washing with a dry organic solvent. A much stronger crown ether activation is observed when 18-crown-6 is added prior to lyophilization, and this can be explained by a combination of two effects: the before-mentioned macrocyclic complexation effect, and a less specific, nonmacrocyclic, lyoprotecting effect. The magnitude of the total crown ether effect depends on the polarity and thermodynamic water activity of the solvent, the activation being highest in dry and apolar media, where kinetic conformational barriers are highest. By determination of the specific activity of crown-ether-lyophilized enzyme as a function of the enzyme concentration, the macrocyclic crown ether (linearly dependent on the enzyme concentration) and the nonmacrocyclic lyoprotection effect (not dependent on the enzyme concentration) could be separated. These measurements reveal that the contribution of the

  10. 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

  11. 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

  12. 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

  13. Microbial Enzyme Activity and Carbon Cycling in Grassland Soil Fractions

    NASA Astrophysics Data System (ADS)

    Allison, S. D.; Jastrow, J. D.

    2004-12-01

    Extracellular enzymes are necessary to degrade complex organic compounds present in soils. Using physical fractionation procedures, we tested whether old soil carbon is spatially isolated from degradative enzymes across a prairie restoration chronosequence in Illinois, USA. We found that carbon-degrading enzymes were abundant in all soil fractions, including macroaggregates, microaggregates, and the clay fraction, which contains carbon with a mean residence time of ~200 years. The activities of two cellulose-degrading enzymes and a chitin-degrading enzyme were 2-10 times greater in organic matter fractions than in bulk soil, consistent with the rapid turnover of these fractions. Polyphenol oxidase activity was 3 times greater in the clay fraction than in the bulk soil, despite very slow carbon turnover in this fraction. Changes in enzyme activity across the restoration chronosequence were small once adjusted for increases in soil carbon concentration, although polyphenol oxidase activity per unit carbon declined by 50% in native prairie versus cultivated soil. These results are consistent with a `two-pool' model of enzyme and carbon turnover in grassland soils. In light organic matter fractions, enzyme production and carbon turnover both occur rapidly. However, in mineral-dominated fractions, both enzymes and their carbon substrates are immobilized on mineral surfaces, leading to slow turnover. Soil carbon accumulation in the clay fraction and across the prairie restoration chronosequence probably reflects increasing physical isolation of enzymes and substrates on the molecular scale, rather than the micron to millimeter scale.

  14. 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,…

  15. Ultrasound in Enzyme Activation and Inactivation

    NASA Astrophysics Data System (ADS)

    Mawson, Raymond; Gamage, Mala; Terefe, Netsanet Shiferaw; Knoerzer, Kai

    As discussed in previous chapters, most effects due to ultrasound arise from cavitation events, in particular, collapsing cavitation bubbles. These collapsing bubbles generate very high localized temperatures and pressure shockwaves along with micro-streaming that is associated with high shear forces. These effects can be used to accelerate the transport of substrates and reaction products to and from enzymes, and to enhance mass transfer in enzyme reactor systems, and thus improve efficiency. However, the high velocity streaming, together with the formation of hydroxy radicals and heat generation during collapsing of bubbles, may also potentially affect the biocatalyst stability, and this can be a limiting factor in combined ultrasound/enzymatic applications. Typically, enzymes can be readily denatured by slight changes in environmental conditions, including temperature, pressure, shear stress, pH and ionic strength.

  16. Manganese enzymes with binuclear active sites

    SciTech Connect

    Dismukes, G.C.

    1996-11-01

    The purpose of this article is twofold. First, to review the recent literature dealing with the mechanisms of catalysis by binuclear manganese enzymes. Second, to summarize and illustrate the general principles of catalysis which distinguish binuclear metalloenzymes from monometallic centers. This review covers primarily the published literature from 1991 up to May 1996. A summary of the major structurally characterized dimanganese enzymes is given. These perform various reaction types including several redox reactions, (de)hydrations, isomerizations, (de)phosphorylation, and phosphoryl transfer. 114 refs.

  17. Determination of concentration and activity of immobilized enzymes.

    PubMed

    Singh, Priyanka; Morris, Holly; Tivanski, Alexei V; Kohen, Amnon

    2015-09-01

    Methods that directly measure the concentration of surface-immobilized biomolecules are scarce. More commonly, the concentration of the soluble molecule is measured before and after immobilization, and the bound concentration is assessed by elimination, assuming that all bound molecules are active. An assay was developed for measuring the active site concentration, activity, and thereby the catalytic turnover rate (kcat) of an immobilized dihydrofolate reductase as a model system. The new method yielded a similar first-order rate constant, kcat, to that of the same enzyme in solution. The findings indicate that the activity of the immobilized enzyme, when separated from the surface by the DNA spacers, has not been altered. In addition, a new immobilization method that leads to solution-like activity of the enzyme on the surface is described. The approaches developed here for immobilization and for determining the concentration of an immobilized enzyme are general and can be extended to other enzymes, receptors, and antibodies.

  18. Arg-Gly-Asp (RGD)-Modified E1A/E1B Double Mutant Adenovirus Enhances Antitumor Activity in Prostate Cancer Cells In Vitro and in Mice.

    PubMed

    Shen, Yue-Hong; Yang, Fei; Wang, Hua; Cai, Zhi-Jian; Xu, Yi-Peng; Zhao, An; Su, Ying; Zhang, Gu; Zhu, Shao-Xing

    2016-01-01

    CAR is a transmembrane protein that is expressed in various epithelial and endothelial cells. CAR mediates adenoviral infection, as well as adenovirus-mediated oncolysis of AxdAdB-3, an E1A/E1B double-restricted oncolytic adenovirus, in prostate cancer cells. This study further assessed the therapeutic efficacy of AxdAdB-3 with Arg-Gly-Asp (RGD)-fiber modification (AxdAdB3-F/RGD), which enables integrin-dependent infection, in prostate cancer. Susceptibility of prostate cancer cells LNCaP, PC3, and DU145 to adenovirus infection was associated with CAR expression. All of the prostate cancer cell lines expressed integrin αvβ3 and αvβ5. AxdAdB-3 was more cytopathic in CAR-positive prostate cancer cells than in CAR-negative cells, whereas AxdAdB3-F/RGD caused potent oncolysis in both CAR-positive and CAR-negative prostate cancer cells. In contrast, AxdAdB3-F/RGD was not cytopathic against normal prostate epithelial cells, RWPE-1. Intratumoral injection of AxdAdB3-F/RGD into CAR-negative prostate cancer cell xenografts in nude mice inhibited tumor growth. The current study demonstrates that E1A/E1B double-restricted oncolytic adenovirus with an RGD-fiber modification enhances infection efficiency and anti-tumor activity in CAR-deficient prostate cancer cells, while sparing normal cells. Future studies will evaluate the therapeutic potential of AxdAdB3-F/RGD in prostate cancer. PMID:26799485

  19. Enzyme catalysis in an aqueous/organic segment flow microreactor: ways to stabilize enzyme activity.

    PubMed

    Karande, Rohan; Schmid, Andreas; Buehler, Katja

    2010-06-01

    Multiphase flow microreactors benefit from rapid mixing and high mass transfer rates, yet their application in enzymatic catalysis is limited due to the fast inactivation of enzymes used as biocatalysts. Enzyme inactivation during segment flow is due to the large interfacial area between aqueous and organic phases. The Peclet number of the system points to strong convective forces within the segments, and this results in rapid deactivation of the enzyme depending on segment length and flow rate. Addition of surfactant to the aqueous phase or enzyme immobilization prevents the biocatalyst from direct contact with the interface and thus stabilizes the enzyme activity. Almost 100% enzyme activity can be recovered compared to 45% without any enzyme or medium modification. Drop tensiometry measurements point to a mixed enzyme-surfactant interfacial adsorption, and above a certain concentration, the surfactant forms a protective layer between the interface and the biocatalyst in the aqueous compartments. Theoretical models were used to compare adsorption kinetics of the protein to the interface in the segment flow microreactor and in the drop tensiometry measurements. This study is the basis for the development of segment flow microreactors as a tool to perform productive enzymatic catalysis. PMID:20201570

  20. The 68-kilodalton E1 protein of bovine papillomavirus is a DNA binding phosphoprotein which associates with the E2 transcriptional activator in vitro.

    PubMed Central

    Blitz, I L; Laimins, L A

    1991-01-01

    The E1 open reading frame of bovine papillomavirus type 1 encodes factors necessary for extrachromosomal maintenance of the viral genome in transformed cells. To facilitate biochemical characterization of the gene products encoded by this open reading frame, we have expressed the full-length E1 protein in a baculovirus-insect cell system. This protein was found to be phosphorylated and localized to the nucleus of infected cells. The E1 protein alone has affinity for DNA but appears to lack specificity for viral sequences. In addition, we present evidence that the E1 protein interacts with the virally encoded transcriptional activator E2 in vitro. These results are consistent with a model in which the E1 protein, as part of a complex with E2, interacts with specific DNA sequences in the viral genome. Images PMID:1846189

  1. Human papillomavirus 16 E2 stability and transcriptional activation is enhanced by E1 via a direct protein-protein interaction

    SciTech Connect

    King, Lauren E.; Dornan, Edward S.; Donaldson, Mary M.; Morgan, Iain M.

    2011-05-25

    Human papillomavirus 16 E1 and E2 interact with cellular factors to replicate the viral genome. E2 forms homodimers and binds to 12 bp palindromic sequences adjacent to the viral origin and recruits E1 to the origin. E1 forms a di-hexameric helicase complex that replicates the viral genome. This manuscript demonstrates that E1 stabilises the E2 protein, increasing the half life in both C33a and 293 T cells respectively. This stabilisation requires a direct protein--protein interaction. In addition, the E1 protein enhances E2 transcription function in a manner that suggests the E1 protein itself can contribute to transcriptional regulation not simply by E2 stabilisation but by direct stimulation of transcription. This activation of E2 transcription is again dependent upon an interaction with E1. Overall the results suggest that in the viral life cycle, co-expression of E1 with E2 can increase E2 stability and enhance E2 function.

  2. Phosphorylation at the carboxy terminus of the 55-kilodalton adenovirus type 5 E1B protein regulates transforming activity.

    PubMed Central

    Teodoro, J G; Halliday, T; Whalen, S G; Takayesu, D; Graham, F L; Branton, P E

    1994-01-01

    The 55-kDa product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation through complex formation with and inactivation of the cellular tumor suppressor p53. We have used both biochemical and genetic approaches to show that this 496-residue (496R) protein of adenovirus type 5 is phosphorylated at serine and threonine residues near the carboxy terminus within sequences characteristic of substrates of casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased viral replication and greatly reduced the efficiency of transformation of primary baby rat kidney cells. Such mutant 496R proteins interacted with p53 at efficiencies similar to those of wild-type 496R but only partially inhibited p53 transactivation activity. These results indicated that phosphorylation at these carboxy-terminal sites either regulates the inhibition of p53 or regulates some other 496R function required for cell transformation. Images PMID:8289381

  3. 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

  4. 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

  5. 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

  6. Serodiagnosis of Lyme disease by kinetic enzyme-linked immunosorbent assay using recombinant VlsE1 or peptide antigens of Borrelia burgdorferi compared with 2-tiered testing using whole-cell lysates.

    PubMed

    Bacon, Rendi Murphree; Biggerstaff, Brad J; Schriefer, Martin E; Gilmore, Robert D; Philipp, Mario T; Steere, Allen C; Wormser, Gary P; Marques, Adriana R; Johnson, Barbara J B

    2003-04-15

    In a study of US patients with Lyme disease, immunoglobulin (Ig) G and IgM antibody responses to recombinant Borrelia burgdorferi antigen VlsE1 (rVlsE1), IgG responses to a synthetic peptide homologous to a conserved internal sequence of VlsE (C6), and IgM responses to a synthetic peptide comprising the C-terminal 10 amino acid residues of a B. burgdorferi outer-surface protein C (pepC10) were evaluated by kinetic enzyme-linked immunoassay. At 99% specificity, the overall sensitivities for detecting IgG antibody to rVlsE1 or C6 in samples from patients with diverse manifestations of Lyme disease were equivalent to that of 2-tiered testing. When data were considered in parallel, 2 combinations (IgG responses to either rVlsE1 or C6 in parallel with IgM responses to pepC10) maintained high specificity (98%) and were significantly more sensitive than 2-tiered analysis in detecting antibodies to B. burgdorferi in patients with acute erythema migrans. In later stages of Lyme disease, the sensitivities of the in parallel tests and 2-tiered testing were high and statistically equivalent.

  7. Elevated glutathione level does not protect against chronic alcohol mediated apoptosis in recombinant human hepatoma cell line VL-17A over-expressing alcohol metabolizing enzymes--alcohol dehydrogenase and Cytochrome P450 2E1.

    PubMed

    Chandrasekaran, Karthikeyan; Swaminathan, Kavitha; Kumar, S Mathan; Chatterjee, Suvro; Clemens, Dahn L; Dey, Aparajita

    2011-06-01

    Chronic consumption of alcohol leads to liver injury. Ethanol-inducible Cytochrome P450 2E1 (CYP2E1) plays a critical role in alcohol mediated oxidative stress due to its ability to metabolize ethanol. In the present study, using the recombinant human hepatoma cell line VL-17A that over-expresses the alcohol metabolizing enzymes-alcohol dehydrogenase (ADH) and CYP2E1; and control HepG2 cells, the mechanism and mode of cell death due to chronic ethanol exposure were studied. Untreated VL-17A cells exhibited apoptosis and oxidative stress when compared with untreated HepG2 cells. Chronic alcohol exposure, i.e., 100 mM ethanol treatment for 72 h caused a significant decrease in viability (47%) in VL-17A cells but not in HepG2 cells. Chronic ethanol mediated cell death in VL-17A cells was predominantly apoptotic, with increased oxidative stress as the underlying mechanism. Chronic ethanol exposure of VL-17A cells resulted in 1.1- to 2.5-fold increased levels of ADH and CYP2E1. Interestingly, the level of the antioxidant GSH was found to be 3-fold upregulated in VL-17A cells treated with ethanol, which may be a metabolic adaptation to the persistent and overwhelming oxidative stress. In conclusion, the increased GSH level may not be sufficient enough to protect VL-17A cells from chronic alcohol mediated oxidative stress and resultant apoptosis. PMID:21414402

  8. 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...

  9. 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 ...

  10. 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.

  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. TISSUE ENZYME ACTIVITIES IN KEMP'S RIDLEY TURTLES (LEPIDOCHELYS KEMPII).

    PubMed

    Petrosky, Keiko Y; Knoll, Joyce S; Innis, Charles

    2015-09-01

    This study determined the tissue distribution and activities of eight enzymes in 13 juvenile Kemp's ridley turtles (Lepidochelys kempii) that died after stranding. Samples from the liver, kidney, skeletal muscle, cardiac muscle, pancreas, lung, small intestine, and spleen were evaluated for activities of alanine aminotransferase (ALT), alkaline phosphatase (ALP), amylase, aspartate aminotransferase (AST), creatine kinase (CK), γ-glutamyl transferase (GGT), lactate dehydrogenase (LDH), and lipase. AST, CK, and LDH activities were highest in cardiac and skeletal muscle but were also found in all other tissues. Amylase and lipase activities were highest in the pancreas and low in all other tissues. ALP activity was highest in the lung. ALT activity was highest in liver, kidney, and cardiac muscle, and GGT activity was highest in the kidney, but activities of these enzymes were low in all tissues. These data may assist clinicians in interpretation of plasma enzyme activities of Kemp's ridley turtles.

  13. ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA

    PubMed Central

    Stine, G. J.

    1968-01-01

    Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged. PMID:4384627

  14. 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

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

    PubMed

    Karan, Ram; Capes, Melinda D; Dassarma, Shiladitya

    2012-02-02

    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.

  16. 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.

  17. State-dependent electrostatic interactions of S4 arginines with E1 in S2 during Kv7.1 activation.

    PubMed

    Wu, Dick; Delaloye, Kelli; Zaydman, Mark A; Nekouzadeh, Ali; Rudy, Yoram; Cui, Jianmin

    2010-06-01

    The voltage-sensing domain of voltage-gated channels is comprised of four transmembrane helices (S1-S4), with conserved positively charged residues in S4 moving across the membrane in response to changes in transmembrane voltage. Although it has been shown that positive charges in S4 interact with negative countercharges in S2 and S3 to facilitate protein maturation, how these electrostatic interactions participate in channel gating remains unclear. We studied a mutation in Kv7.1 (also known as KCNQ1 or KvLQT1) channels associated with long QT syndrome (E1K in S2) and found that reversal of the charge at E1 eliminates macroscopic current without inhibiting protein trafficking to the membrane. Pairing E1R with individual charge reversal mutations of arginines in S4 (R1-R4) can restore current, demonstrating that R1-R4 interact with E1. After mutating E1 to cysteine, we probed E1C with charged methanethiosulfonate (MTS) reagents. MTS reagents could not modify E1C in the absence of KCNE1. With KCNE1, (2-sulfonatoethyl) MTS (MTSES)(-) could modify E1C, but [2-(trimethylammonium)ethyl] MTS (MTSET)(+) could not, confirming the presence of a positively charged environment around E1C that allows approach by MTSES(-) but repels MTSET(+). We could change the local electrostatic environment of E1C by making charge reversal and/or neutralization mutations of R1 and R4, such that MTSET(+) modified these constructs depending on activation states of the voltage sensor. Our results confirm the interaction between E1 and the fourth arginine in S4 (R4) predicted from open-state crystal structures of Kv channels and reveal an E1-R1 interaction in the resting state. Thus, E1 engages in electrostatic interactions with arginines in S4 sequentially during the gating movement of S4. These electrostatic interactions contribute energetically to voltage-dependent gating and are important in setting the limits for S4 movement.

  18. Activation volumes of enzymes adsorbed on silica particles.

    PubMed

    Schuabb, Vitor; Czeslik, Claus

    2014-12-30

    The immobilization of enzymes on carrier particles is useful in many biotechnological processes. In this way, enzymes can be separated from the reaction solution by filtering and can be reused in several cycles. On the other hand, there is a series of examples of free enzymes in solution that can be activated by the application of pressure. Thus, a potential loss of enzymatic activity upon immobilization on carrier particles might be compensated by pressure. In this study, we have determined the activation volumes of two enzymes, α-chymotrypsin (α-CT) and horseradish peroxidase (HRP), when they are adsorbed on silica particles and free in solution. The experiments have been carried out using fluorescence assays under pressures up to 2000 bar. In all cases, activation volumes were found to depend on the applied pressure, suggesting different compressions of the enzyme-substrate complex and the transition state. The volume profiles of free and adsorbed HRP are similar. For α-CT, larger activation volumes are found in the adsorbed state. However, up to about 500 bar, the enzymatic reaction of α-CT, which is adsorbed on silica particles, is characterized by a negative activation volume. This observation suggests that application of pressure might indeed be useful to enhance the activity of enzymes on carrier particles.

  19. Induction of sensitivity to the cytotoxic action of tumor necrosis factor alpha by adenovirus E1A is independent of transformation and transcriptional activation.

    PubMed

    Ames, R S; Holskin, B; Mitcho, M; Shalloway, D; Chen, M J

    1990-09-01

    We have previously shown that expression of the adenovirus E1A 12S or 13S products in NIH 3T3 fibroblasts induces susceptibility to the cytotoxic actions of tumor necrosis factor alpha (TNF alpha). A large number of studies have mapped the multiple biological functions of the 12S and 13S products to three highly conserved regions (CR) within the E1A sequence. Here we used plasmids coding for E1A deletion and point mutants in these regions to generate target cell lines for TNF alpha cytotoxicity assays to determine which regions and functions are necessary for the induction of TNF alpha sensitivity. Expression of CR1 was required for the induction of TNF alpha sensitivity. This finding did not reflect a requirement for transforming or transcriptional repression activity, since some mutants that were defective in both of these properties were able to induce TNF alpha sensitivity. CR2 transformation-defective point mutants, but not a CR2/3 region deletion mutant, were also able to induce sensitivity. In addition, NIH 3T3 cells expressing the retroviral transcription activators tat from human immunodeficiency virus type 1 and tax from human T-lymphotropic virus type I were not sensitive to TNF alpha. However, the possibility that E1A-mediated transcriptional activation can augment the induction of TNF alpha sensitivity is not excluded. Comparison of data from previous biological studies with the TNF alpha cytotoxicity assays presented here suggested that the mechanism by which E1A induces sensitivity to TNF alpha in NIH 3T3 cells is independent of many of the known E1A biological functions, including transformation in cooperation with ras, immortalization, induction of DNA synthesis in quiescent cells, and transcriptional repression. A novel E1A-mediated effect may be involved, although our data do not exclude the possibility that sensitization to TNF alpha is mediated through E1A binding to cellular proteins. PMID:2143540

  20. Induction of sensitivity to the cytotoxic action of tumor necrosis factor alpha by adenovirus E1A is independent of transformation and transcriptional activation.

    PubMed Central

    Ames, R S; Holskin, B; Mitcho, M; Shalloway, D; Chen, M J

    1990-01-01

    We have previously shown that expression of the adenovirus E1A 12S or 13S products in NIH 3T3 fibroblasts induces susceptibility to the cytotoxic actions of tumor necrosis factor alpha (TNF alpha). A large number of studies have mapped the multiple biological functions of the 12S and 13S products to three highly conserved regions (CR) within the E1A sequence. Here we used plasmids coding for E1A deletion and point mutants in these regions to generate target cell lines for TNF alpha cytotoxicity assays to determine which regions and functions are necessary for the induction of TNF alpha sensitivity. Expression of CR1 was required for the induction of TNF alpha sensitivity. This finding did not reflect a requirement for transforming or transcriptional repression activity, since some mutants that were defective in both of these properties were able to induce TNF alpha sensitivity. CR2 transformation-defective point mutants, but not a CR2/3 region deletion mutant, were also able to induce sensitivity. In addition, NIH 3T3 cells expressing the retroviral transcription activators tat from human immunodeficiency virus type 1 and tax from human T-lymphotropic virus type I were not sensitive to TNF alpha. However, the possibility that E1A-mediated transcriptional activation can augment the induction of TNF alpha sensitivity is not excluded. Comparison of data from previous biological studies with the TNF alpha cytotoxicity assays presented here suggested that the mechanism by which E1A induces sensitivity to TNF alpha in NIH 3T3 cells is independent of many of the known E1A biological functions, including transformation in cooperation with ras, immortalization, induction of DNA synthesis in quiescent cells, and transcriptional repression. A novel E1A-mediated effect may be involved, although our data do not exclude the possibility that sensitization to TNF alpha is mediated through E1A binding to cellular proteins. Images PMID:2143540

  1. Microbial hydrolytic enzyme activities in deep-sea sediments

    NASA Astrophysics Data System (ADS)

    Boetius, A.

    1995-03-01

    The potential hydrolysis rates of five different hydrolytic enzymes were determined in deep-sea sediments from the northeast Atlantic (BIOTRANS area) in March 1992. Fluorogenic substrates were used to assay extracellular α- and β-glucosidase, chitobiase, lipase and aminopeptidase. The potential activity of most of the enzymes investigated decreased to a minimum within the upper two centimetre range, whereas aminopeptidase was high over the upper five centimetre range. Exceptions were found when macrofaunal burrows occurred in the cores, always increasing the activities of some hydrolases, and therefore indicating the impact of bioturbation on degradation rates. The most striking feature of the investigated enzyme spectrum was the 50 2000 times higher specific activity of the aminopeptidase, compared with the other hydrolases. The activity of hydrolytic enzymes most likely reflects the availability of their respective substrates and is not a function of bacterial biomass.

  2. Silk Microgels Formed by Proteolytic Enzyme Activity

    PubMed Central

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

    2013-01-01

    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 (SMG) at physiological temperature. These microgels consist of lamellar crystals in the micrometer scale, in contrast to the nanometer scaled crystals in native silkworm fibers. SDS-PAGE and zeta potential results demonstrated that α-chymotrypsin utilized only the nonamorphous 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 potential and that the prepared SMGS have useful features for studies related to biomaterials and pharmaceutical needs. This process is also an easy approach to obtain the amorphous peptide chains for further study. PMID:23756227

  3. 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

  4. Diced electrophoresis gel assay for screening enzymes with specified activities.

    PubMed

    Komatsu, Toru; Hanaoka, Kenjiro; Adibekian, Alexander; Yoshioka, Kentaro; Terai, Takuya; Ueno, Tasuku; Kawaguchi, Mitsuyasu; Cravatt, Benjamin F; Nagano, Tetsuo

    2013-04-24

    We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants.

  5. Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

    PubMed Central

    Du, Li; Li, Yi-Jia; Fakih, Marwan; Wiatrek, Rebecca L.; Duldulao, Marjun; Chen, Zhenbin; Chu, Peiguo; Garcia-Aguilar, Julio; Chen, Yuan

    2016-01-01

    Cancer stem cells (CSCs) have key roles in treatment resistance, tumour metastasis and relapse. Using colorectal cancer (CC) cell lines, patient-derived xenograft (PDX) tissues and patient tissues, here we report that CC CSCs, which resist chemoradiation, have higher SUMO activating enzyme (E1) and global SUMOylation levels than non-CSCs. Knockdown of SUMO E1 or SUMO conjugating enzyme (E2) inhibits CC CSC maintenance and self-renewal, while overexpression of SUMO E1 or E2 increases CC cell stemness. We found that SUMOylation regulates CSCs through Oct-1, a transcription factor for aldehyde dehydrogenases (ALDHs). ALDH activity is not only a marker for CSCs but also important in CSC biology. SUMO does not modify Oct-1 directly, but regulates the expression of TRIM21 that enhances Oct-1 ubiquitination and, consequently, reducing Oct-1 stability. In summary, our findings suggest that SUMOylation could be a target to inhibit CSCs and ultimately to reduce treatment resistance, tumour metastasis and relapse. PMID:27465491

  6. Compounds from Silicones Alter Enzyme Activity in Curing Barnacle Glue and Model Enzymes

    PubMed Central

    Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H.

    2011-01-01

    Background Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. Methodology/Principal Findings GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Conclusions/Significance Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management. PMID:21379573

  7. A novel approach to predict active sites of enzyme molecules.

    PubMed

    Chou, Kuo-Chen; Cai, Yu-dong

    2004-04-01

    Enzymes are critical in many cellular signaling cascades. With many enzyme structures being solved, there is an increasing need to develop an automated method for identifying their active sites. However, given the atomic coordinates of an enzyme molecule, how can we predict its active site? This is a vitally important problem because the core of an enzyme molecule is its active site from the viewpoints of both pure scientific research and industrial application. In this article, a topological entity was introduced to characterize the enzymatic active site. Based on such a concept, the covariant discriminant algorithm was formulated for identifying the active site. As a paradigm, the serine hydrolase family was demonstrated. The overall success rate by jackknife test for a data set of 88 enzyme molecules was 99.92%, and that for a data set of 50 independent enzyme molecules was 99.91%. Meanwhile, it was shown through an example that the prediction algorithm can also be used to find any typographic error of a PDB file in annotating the constituent amino acids of catalytic triad and to suggest a possible correction. The very high success rates are due to the introduction of a covariance matrix in the prediction algorithm that makes allowance for taking into account the coupling effects among the key constituent atoms of active site. It is anticipated that the novel approach is quite promising and may become a useful high throughput tool in enzymology, proteomics, and structural bioinformatics. PMID:14997541

  8. In Vitro Antibody-Enzyme Conjugates with Specific Bactericidal Activity

    PubMed Central

    Knowles, Daniel M.; Sullivan, Timothy J.; Parker, Charles W.; Williams, Ralph C.

    1973-01-01

    IgG with antibacterial antibody opsonic activity was isolated from rabbit antisera produced by intravenous hyperimmunization with several test strains of pneumococci, Group A β-hemolytic streptococci, Staphylococcus aureus, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli. Antibody-enzyme conjugates were prepared, using diethylmalonimidate to couple glucose oxidase to IgG antibacterial antibody preparations. Opsonic human IgG obtained from serum of patients with subacute bacterial endocarditis was also conjugated to glucose oxidase. Antibody-enzyme conjugates retained combining specificity for test bacteria as demonstrated by indirect immunofluorescence. In vitro test for bactericidal activity of antibody-enzyme conjugates utilized potassium iodide, lactoperoxidase, and glucose as cofactors. Under these conditions glucose oxidase conjugated to antibody generates hydrogen peroxide, and lactoperoxidase enzyme catalyzes the reduction of hydrogen peroxide with simultaneous oxidation of I- and halogenation and killing of test bacteria. Potent in vitro bactericidal activity of this system was repeatedly demonstrated for antibody-enzyme conjugates against pneumococci, streptococci, S. aureus, P. mirabilis, and E. coli. However, no bactericidal effect was demonstrable with antibody-enzyme conjugates and two test strains of P. aeruginosa. Bactericidal activity of antibody-enzyme conjugates appeared to parallel original opsonic potency of unconjugated IgG preparations. Antibody-enzyme conjugates at concentrations as low as 0.01 mg/ml were capable of intense bactericidal activity producing substantial drops in surviving bacterial counts within 30-60 min after initiation of assay. These in vitro bactericidal systems indicate that the concept of antibacterial antibody-enzyme conjugates may possibly be adaptable as a mechanism for treatment of patients with leukocyte dysfunction or fulminant bacteremia. PMID:4145026

  9. Activities of virE1 and the VirE1 secretion chaperone in export of the multifunctional VirE2 effector via an Agrobacterium type IV secretion pathway.

    PubMed

    Zhao, Z; Sagulenko, E; Ding, Z; Christie, P J

    2001-07-01

    Agrobacterium tumefaciens uses a type IV secretion system to deliver oncogenic nucleoprotein particles and effector proteins, such as the multifunctional VirE2 protein, to plant cells. In this study, we examined the function of virE1 and its product, the VirE1 secretion chaperone, in mediating VirE2 export. A nonpolar virE1 null mutant accumulated low levels of VirE2, and trans expression of virE1 in this mutant only partially restored VirE2 abundance. Deletion of virE1 did not affect transcription but decreased translation of virE2, as shown by analysis of lacZ transcriptional and translational fusions. VirE2 was stable for a prolonged period, more than 6 h, when it was expressed in cis with virE1, and it exhibited half-lives of about 2 h when it was expressed in trans with virE1 and less than 10 min when it was expressed in the absence of virE1, as shown by pulse-chase experiments. VirE1 stabilized VirE2 via an interaction with a domain near the N terminus of VirE2, as shown by analyses of VirE2 truncation and insertion mutants synthesized in A. tumefaciens. VirE1 self-association was demonstrated by using bacteriophage lambda cI repressor fusion and pull-down assays, and evidence of VirE1 homomultimerization in vivo was obtained by native polyacrylamide gel electrophoresis and gel filtration chromatography. A putative VirE1-VirE2 complex with a molecular mass of about 70 to 80 kDa was detected by gel filtration chromatography of extracts from wild-type cells, whereas higher-order VirE2 complexes or aggregates were detected in extracts from a virE1 mutant. Taken together, our findings show that virE1 contributes in several ways to VirE2 export:(i) virE1 regulates efficient virE2 translation in the context of expression from the native P(virE) promoter; (ii) the VirE1 secretion chaperone stabilizes VirE2, most probably via an interaction with an N-terminal domain; and (iii) VirE1 forms a VirE1-VirE2 complex with a predicted 2:1 stoichiometry that inhibits assembly

  10. 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.

  11. Effects of cadium, zinc and lead on soil enzyme activities.

    PubMed

    Yang, Zhi-xin; Liu, Shu-qing; Zheng, Da-wei; Feng, Sheng-dong

    2006-01-01

    Heavy metal (HM) is a major hazard to the soil-plant system. This study investigated the combined effects of cadium (Cd), zinc (Zn) and lead (Pb) on activities of four enzymes in soil, including calatase, urease, invertase and alkalin phosphatase. HM content in tops of canola and four enzymes activities in soil were analyzed at two months after the metal additions to the soil. Pb was not significantly inhibitory than the other heavy metals for the four enzyme activities and was shown to have a protective role on calatase activity in the combined presence of Cd, Zn and Pb; whereas Cd significantly inhibited the four enzyme activities, and Zn only inhibited urease and calatase activities. The inhibiting effect of Cd and Zn on urease and calatase activities can be intensified significantly by the additions of Zn and Cd. There was a negative synergistic inhibitory effect of Cd and Zn on the two enzymes in the presence of Cd, Zn and Pb. The urease activity was inhibited more by the HM combinations than by the metals alone and reduced approximately 20%-40% of urease activity. The intertase and alkaline phosphatase activities significantly decreased only with the increase of Cd concentration in the soil. It was shown that urease was much more sensitive to HM than the other enzymes. There was a obvious negative correlation between the ionic impulsion of HM in soil, the ionic impulsion of HM in canola plants tops and urease activity. It is concluded that the soil urease activity may be a sensitive tool for assessing additive toxic combination effect on soil biochemical parameters.

  12. Enzyme:nanoparticle bioconjugates with two sequential enzymes: stoichiometry and activity of malate dehydrogenase and citrate synthase on Au nanoparticles.

    PubMed

    Keighron, Jacqueline D; Keating, Christine D

    2010-12-21

    We report the synthesis and characterization of bioconjugates in which the enzymes malate dehydrogenase (MDH) and/or citrate synthase (CS) were adsorbed to 30 nm diameter Au nanoparticles. Enzyme:Au stoichiometry and kinetic parameters (specific activity, k(cat), K(M), and activity per particle) were determined for MDH:Au, CS:Au, and three types of dual-activity MDH/CS:Au bioconjugates. For single-activity bioconjugates (MDH:Au and CS:Au), the number of enzyme molecules adsorbed per particle was dependent upon the enzyme concentration in solution, with multilayers forming at high enzyme:Au solution ratios. The specific activity of adsorbed enzyme increased with increasing number adsorbed per particle for CS:Au, but was less sensitive to stoichiometry for MDH:Au. Dual activity bioconjugates were prepared in three ways: (1) by adsorption of MDH followed by CS, (2) by adsorption of CS followed by MDH, and (3) by coadsorption of both enzymes from the same solution. The resulting bioconjugates differed substantially in the number of enzyme molecules adsorbed per particle, the specific activity of the adsorbed enzymes, and also the enzymatic activity per particle. Bioconjugates formed by adding CS to the Au nanoparticles before MDH was added exhibited higher specific activities for both enzymes than those formed by adding the enzymes in the reverse order. These bioconjugates also had 3-fold higher per-particle sequential activity for conversion of malate to citrate, despite substantially fewer copies of both enzymes present.

  13. 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.

  14. 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

  15. Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites.

    PubMed Central

    Montell, C; Fisher, E F; Caruthers, M H; Berk, A J

    1984-01-01

    The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells. Images PMID:6727875

  16. 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

  17. Catalytically active nanomaterials: a promising candidate for artificial enzymes.

    PubMed

    Lin, Youhui; Ren, Jinsong; Qu, Xiaogang

    2014-04-15

    Natural enzymes, exquisite biocatalysts mediating every biological process in living organisms, are able to accelerate the rate of chemical reactions up to 10(19) times for specific substrates and reactions. However, the practical application of enzymes is often hampered by their intrinsic drawbacks, such as low operational stability, sensitivity of catalytic activity to environmental conditions, and high costs in preparation and purification. Therefore, the discovery and development of artificial enzymes is highly desired. Recently, the merging of nanotechnology with biology has ignited extensive research efforts for designing functional nanomaterials that exhibit various properties intrinsic to enzymes. As a promising candidate for artificial enzymes, catalytically active nanomaterials (nanozymes) show several advantages over natural enzymes, such as controlled synthesis in low cost, tunability in catalytic activities, as well as high stability against stringent conditions. In this Account, we focus on our recent progress in exploring and constructing such nanoparticulate artificial enzymes, including graphene oxide, graphene-hemin nanocomposites, carbon nanotubes, carbon nanodots, mesoporous silica-encapsulated gold nanoparticles, gold nanoclusters, and nanoceria. According to their structural characteristics, these enzyme mimics are categorized into three classes: carbon-, metal-, and metal-oxide-based nanomaterials. We aim to highlight the important role of catalytic nanomaterials in the fields of biomimetics. First, we provide a practical introduction to the identification of these nanozymes, the source of the enzyme-like activities, and the enhancement of activities via rational design and engineering. Then we briefly describe new or enhanced applications of certain nanozymes in biomedical diagnosis, environmental monitoring, and therapeutics. For instance, we have successfully used these biomimetic catalysts as colorimetric probes for the detection of

  18. Functionalized graphene oxide in enzyme engineering: a selective modulator for enzyme activity and thermostability.

    PubMed

    Jin, Liling; Yang, Kai; Yao, Kai; Zhang, Shuai; Tao, Huiquan; Lee, Shuit-Tong; Liu, Zhuang; Peng, Rui

    2012-06-26

    The understanding of interactions between nanomaterials and biomolecules is of fundamental importance to the area of nanobiotechnology. Graphene and its derivative, graphene oxide (GO), are two-dimensional (2-D) nanomaterials with interesting physical and chemical properties and have been widely explored in various directions of biomedicine in recent years. However, how functionalized GO interacts with bioactive proteins such as enzymes and its potential in enzyme engineering have been rarely explored. In this study, we carefully investigated the interactions between serine proteases and GO functionalized with different amine-terminated polyethylene glycol (PEG). Three well-characterized serine proteases (trypsin, chymotrypsin, and proteinase K) with important biomedical and industrial applications were analyzed. It is found that these PEGylated GOs could selectively improve trypsin activity and thermostability (60-70% retained activity at 80 °C), while exhibiting barely any effect on chymotrypsin or proteinase K. Detailed investigation illustrates that the PEGylated GO-induced acceleration is substrate-dependent, affecting only phosphorylated protein substrates, and that at least up to 43-fold increase could be achieved depending on the substrate concentration. This unique phenomenon, interestingly, is found to be attributed to both the terminal amino groups on polymer coatings and the 2-D structure of GO. Moreover, an enzyme-based bioassay system is further demonstrated utilizing our GO-based enzyme modulator in a proof-of-concept experiment. To our best knowledge, this work is the first success of using functionalized GO as an efficient enzyme positive modulator with great selectivity, exhibiting a novel potential of GO, when appropriately functionalized, in enzyme engineering as well as enzyme-based biosensing and detection.

  19. Optimization to Low Temperature Activity in Psychrophilic Enzymes

    PubMed Central

    Struvay, Caroline; Feller, Georges

    2012-01-01

    Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. Considering the subtle structural adjustments required for low temperature activity, directed evolution appears to be the most suitable methodology to engineer cold activity in biological catalysts. PMID:23109875

  20. Angiotensin I converting enzyme activity in rabbit corneal endothelial cells.

    PubMed

    Neels, H M; Vanden Berghe, D A; Neetens, A J; Delgadillo, R A; Scharpe, S L

    1983-01-01

    Angiotensin I converting enzyme (ACE) was studied in Vero cells, rabbit corneal fibroblasts, and rabbit corneal endothelial cells. The enzyme activity was determined by means of an assay employing hippuryl-glycyl-glycine as a substrate. The hippuric acid end product was separated from the substrate by reversed phase liquid chromatography and measured spectrophotometrically at 228 nm. The enzyme was further characterized by a captopril inhibition study. Significant ACE activity was found in rabbit corneal endothelial cells but not in other types of cells tested. This is the first report of the presence of this enzyme in a specific ocular cell type and suggests that angiotensin II may play a role in normal ocular physiology.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. Improving activity of salt-lyophilized enzymes in organic media

    SciTech Connect

    Borole, Abhijeet P; Davison, Brian H

    2008-01-01

    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.

  6. trans-dominant mutants of E1A provide genetic evidence that the zinc finger of the trans-activating domain binds a transcription factor.

    PubMed Central

    Webster, L C; Ricciardi, R P

    1991-01-01

    The 289R E1A protein of adenovirus stimulates transcription of early viral and certain cellular genes. trans-Activation requires residues 140 to 188, which encompass a zinc finger. Several studies have indicated that trans-activation by E1A is mediated through cellular transcription factors. In particular, the ability of the trans-dominant E1A point mutant hr5 (Ser-185 to Asn) to inhibit wild-type E1A trans-activation was proposed to result from the sequestration of a cellular factor. Using site-directed mutagenesis, we individually replaced every residue within and flanking the trans-activating domain with a conservative amino acid, revealing 16 critical residues. Six of the individual substitutions lying in a contiguous stretch C terminal to the zinc finger (carboxyl region183-188) imparted a trans-dominant phenotype. trans-Dominance was even produced by deletion of the entire carboxyl region183-188. Conversely, an intact finger region147-177 was absolutely required for trans-dominance, since second-site substitution of every critical residue in this region abrogated the trans-dominant phenotype of the hr5 protein. These data indicate that the finger region147-177 bind a limiting cellular transcription factor and that the carboxyl region183-188 provides a separate and essential function. In addition, we show that four negatively charged residues within the trans-activating domain do not comprise a distinct acidic activating region. We present a model in which the trans-activating domain of E1A binds to two different cellular protein targets through the finger and carboxyl regions. Images PMID:1831535

  7. 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

  8. Enzyme activities of lung lavage in silicosis.

    PubMed

    Larivée, P; Cantin, A; Dufresne, A; Bégin, R

    1990-01-01

    The cytotoxic effect of quartz on lung cells has been well documented by in vitro and animal studies, but the pertinence of these findings to humans has not yet been documented. We measured lactate dehydrogenase (LDH) activities in the lung lavage of 24 long-term workers in the Québec granite industry and 25 control subjects. We found significant increases in LDH activities in the workers' lung lavage, even in the absence of established silicosis (9 subjects). We looked at a similar observation in the sheep model of early silicosis, measured quartz content of lung lavage, and found significant correlation with LDH levels (R = 0.64, p less than 0.001). All of the quartz particles in human and sheep lung lavage were in the alveolar macrophages. To test further the relationship of macrophage damage (cytotoxicity of quartz) we measured the release of LDH by sheep alveolar macrophage in 24 h cell culture under control conditions, exposure to inert dust, titanium, minusil-5 quartz, or aluminum-treated quartz. The LDH release was at control levels during titanium exposure and showed a significantly dose-related increase during quartz exposure. The latter cytotoxic effect was largely attenuated by aluminum treatment of quartz. These in vitro data agreed with previous reports. This study presents evidence of a cytotoxic effect of quartz inhalation in humans. The effect is related to the intensity of quartz retention in the lung macrophages; it is not a nonspecific dust exposure effect and can be attenuated by surface modification of the quartz.

  9. Structure activity optimization of 6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazines as Jak1 kinase inhibitors.

    PubMed

    Friedman, Michael; Frank, Kristine E; Aguirre, Ana; Argiriadi, Maria A; Davis, Heather; Edmunds, Jeremy J; George, Dawn M; George, Jonathan S; Goedken, Eric; Fiamengo, Bryan; Hyland, Deborah; Li, Bin; Murtaza, Anwar; Morytko, Michael; Somal, Gagandeep; Stewart, Kent; Tarcsa, Edit; Van Epps, Stacy; Voss, Jeffrey; Wang, Lu; Woller, Kevin; Wishart, Neil

    2015-10-15

    Previous work investigating tricyclic pyrrolopyrazines as kinase cores led to the discovery that 1-cyclohexyl-6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazine (12) had Jak inhibitory activity. Herein we describe our initial efforts to develop orally bioavailable analogs of 12 with improved selectivity of Jak1 over Jak2. PMID:26372653

  10. 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

  11. 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

  12. 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

  13. Water modulation of stratum corneum chymotryptic enzyme activity and desquamation.

    PubMed

    Watkinson, A; Harding, C; Moore, A; Coan, P

    2001-09-01

    Exposure to a dry environment leads to depletion of water from the peripheral stratum corneum layers in a process dependent on the relative humidity (RH) and the intrinsic properties of the tissue. We hypothesized that by modulating the water content of the stratum corneum in the surface layers, RH effects the rate of desquamation by modulating the activity of the desquamatory enzymes, and specifically stratum corneum chymotryptic enzyme (SCCE). Using a novel air interface in vitro desquamatory model, we demonstrated RH-dependent corneocyte release with desquamatory rates decreasing below 80% RH. Application of 10% glycerol or a glycerol-containing moisturizing lotion further increased desquamation, even in humid conditions, demonstrating that water was the rate-limiting factor in the final stages of desquamation. Furthermore, even in humid conditions desquamation was sub-maximal. In situ stratum corneum SCCE activity showed a dependence on RH: activity was significantly higher at 100% than at 44% RH. Further increases in SCCE activity were induced by applying a 10% glycerol solution. Since SCCE, a water-requiring enzyme, must function in the water-depleted outer stratum corneum, we sought to determine whether this enzyme has a tolerance to lowered water activity. Using concentrated sucrose solutions to lower water activity, we analysed the activity of recombinant SCCE and compared it to that of trypsin and chymotrypsin. SCCE activity demonstrated a tolerance to water restriction, and this may be an adaptation to maintain enzyme activity even within the water-depleted stratum corneum intercellular space. Overall these findings support the concept that in the upper stratum corneum, RH modulates desquamation by its effect upon SCCE activity, and possibly other desquamatory hydrolases. In addition, SCCE may be adapted to function in the water-restricted stratum corneum intercellular space.

  14. 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.

  15. 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.

  16. CHIP−/−-Mouse Liver: Adiponectin-AMPK-FOXO-Activation Overrides CYP2E1-Elicited JNK1-Activation, Delaying Onset of NASH: Therapeutic Implications

    PubMed Central

    Kim, Sung-Mi; Grenert, James P.; Patterson, Cam; Correia, Maria Almira

    2016-01-01

    Genetic ablation of C-terminus of Hsc70-interacting protein (CHIP) E3 ubiquitin-ligase impairs hepatic cytochrome P450 CYP2E1 degradation. Consequent CYP2E1 gain of function accelerates reactive O2 species (ROS) production, triggering oxidative/proteotoxic stress associated with sustained activation of c-Jun NH2-terminal kinase (JNK)-signaling cascades, pro-inflammatory effectors/cytokines, insulin resistance, progressive hepatocellular ballooning and microvesicular steatosis. Despite this, little evidence of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) was found in CHIP−/−-mice over the first 8–9-months of life. We herein document that this lack of tissue injury is largely due to the concurrent up-regulation and/or activation of the adiponectin-5′-AMP-activated protein kinase (AMPK)-forkhead box O (FOXO)-signaling axis stemming from at the least three synergistic features: Up-regulated expression of adipose tissue adiponectin and its hepatic adipoR1/adipoR2 receptors, stabilization of hepatic AMPKα1-isoform, identified herein for the first time as a CHIP-ubiquitination substrate (unlike its AMPKα2-isoform), as well as nuclear stabilization of FOXOs, well-known CHIP-ubiquitination targets. Such beneficial predominance of the adiponectin-AMPK-FOXO-signaling axis over the sustained JNK-elevation and injurious insulin resistance in CHIP−/−-livers apparently counteracts/delays rapid progression of the hepatic microvesicular steatosis to the characteristic macrovesicular steatosis observed in clinical NASH and/or rodent NASH-models. PMID:27406999

  17. 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

  18. Melatonin promotes osteoblast differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions through activation of PKD/p38 pathways.

    PubMed

    Son, Jang-Ho; Cho, Yeong-Cheol; Sung, Iel-Yong; Kim, In-Ryoung; Park, Bong-Soo; Kim, Yong-Deok

    2014-11-01

    Osteoblastic differentiation and bone-forming capacity are known to be suppressed under hypoxic conditions. Melatonin has been shown to influence cell differentiation. A number of in vitro and in vivo studies have suggested that melatonin also has an anabolic effect on bone, by promoting osteoblastic differentiation. However, the precise mechanisms and the signaling pathways involved in this process, particularly under hypoxic conditions, are unknown. This study investigated whether melatonin could promote osteoblastic differentiation and mineralization of preosteoblastic MC3T3-E1 cells under hypoxic conditions. Additionally, we examined the molecular signaling pathways by which melatonin mediates this process. We found that melatonin is capable of promoting differentiation and mineralization of MC3T3-E1 cells cultured under hypoxic conditions. Melatonin upregulated ALP activity and mRNA levels of Alp, Osx, Col1, and Ocn in a time- and concentration-dependent manner. Alizarin red S staining showed that the mineralized matrix in hypoxic MC3T3-E1 cells formed in a manner that was dependent on melatonin concentration. Moreover, melatonin stimulated phosphorylation of p38 Mapk and Prkd1 in these MC3T3-E1 cells. We concluded that melatonin promotes osteoblastic differentiation of MC3T3-E1 cells under hypoxic conditions via the p38 Mapk and Prkd1 signaling pathways. PMID:25250639

  19. Phosphorylation within the transactivation domain of adenovirus E1A protein by mitogen-activated protein kinase regulates expression of early region 4.

    PubMed Central

    Whalen, S G; Marcellus, R C; Whalen, A; Ahn, N G; Ricciardi, R P; Branton, P E

    1997-01-01

    A critical role of the 289-residue (289R) E1A protein of human adenovirus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to conserved region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zinc binding motif in CR3 and in some cases a region at the carboxy-terminal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously unidentified sites at Ser-185 and Ser-188 are the targets for decreased phosphorylation in response to cAMP. At least one of these sites, presumably Ser-185, is phosphorylated in vitro by purified mitogen-activated protein kinase (MAPK), and both are hyperphosphorylated in cells which express a constitutively active form of MAPK kinase. Analysis of E1A-mediated transactivation activity indicated that elevated phosphorylation at these sites increased expression of the E4 promoter but not that of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter. PMID:9094626

  20. Prilocaine- and lidocaine-induced methemoglobinemia is caused by human carboxylesterase-, CYP2E1-, and CYP3A4-mediated metabolic activation.

    PubMed

    Higuchi, Ryota; Fukami, Tatsuki; Nakajima, Miki; Yokoi, Tsuyoshi

    2013-06-01

    Prilocaine and lidocaine are classified as amide-type local anesthetics for which serious adverse effects include methemoglobinemia. Although the hydrolyzed metabolites of prilocaine (o-toluidine) and lidocaine (2,6-xylidine) have been suspected to induce methemoglobinemia, the metabolic enzymes that are involved remain uncharacterized. In the present study, we aimed to identify the human enzymes that are responsible for prilocaine- and lidocaine-induced methemoglobinemia. Our experiments revealed that prilocaine was hydrolyzed by recombinant human carboxylesterase (CES) 1A and CES2, whereas lidocaine was hydrolyzed by only human CES1A. When the parent compounds (prilocaine and lidocaine) were incubated with human liver microsomes (HLM), methemoglobin (Met-Hb) formation was lower than when the hydrolyzed metabolites were incubated with HLM. In addition, Met-Hb formation when prilocaine and o-toluidine were incubated with HLM was higher than that when lidocaine and 2,6-xylidine were incubated with HLM. Incubation with diisopropyl fluorophosphate and bis-(4-nitrophenyl) phosphate, which are general inhibitors of CES, significantly decreased Met-Hb formation when prilocaine and lidocaine were incubated with HLM. An anti-CYP3A4 antibody further decreased the residual formation of Met-Hb. Met-Hb formation after the incubation of o-toluidine and 2,6-xylidine with HLM was only markedly decreased by incubation with an anti-CYP2E1 antibody. o-Toluidine and 2,6-xylidine were further metabolized by CYP2E1 to 4- and 6-hydroxy-o-toluidine and 4-hydroxy-2,6-xylidine, respectively, and these metabolites were shown to more efficiently induce Met-Hb formation than the parent compounds. Collectively, we found that the metabolites produced by human CES-, CYP2E1-, and CYP3A4-mediated metabolism were involved in prilocaine- and lidocaine-induced methemoglobinemia.

  1. 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

  2. Lipid peroxidation and antioxidant enzymes activity in avian semen.

    PubMed

    Partyka, Agnieszka; Lukaszewicz, Ewa; Niżański, Wojciech

    2012-10-01

    The present study compared the antioxidant system and lipid peroxidation in semen of two avian species: chicken and goose. The experiment was conducted on Greenleg Partridge roosters and White Koluda(®) ganders, each represented by 10 mature males. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma. In gander spermatozoa, the amount of MDA was 10 times greater (P<0.01) than in rooster spermatozoa. Each of the investigated antioxidant enzymes had greater (P<0.01) activity in goose than chicken sperm. Catalase activity was detected in seminal plasma and spermatozoa from both studied species for the first time. In seminal plasma, the activity of GPx was two times greater (P<0.01) in the White Koluda(®) than in chickens, whereas SOD activity was less (P<0.01) than in chickens. This is the first study describing the presence of CAT in avian semen and the occurrence of indicator of lipid peroxidation (LPO) in geese. Data from the present study clearly show the species-specific differences in the activity of antioxidant defense and LPO. The greater amount of lipid peroxidation and greater activity of antioxidant enzymes in goose semen might suggest that spermatozoa were under greater oxidative stress and the enzymes were not utilized for the protection of functionally and structurally impaired cells. In turn, in fresh chicken semen a lesser activity of antioxidant enzymes accompanied with a lesser lipid peroxidation amount and good semen quality could indicate that fowl spermatozoa were under oxidative stress, but the enzymes were employed to protect and maintain sperm quality.

  3. [Enzyme activity of an actinomycete producer of carotenes and macrotetrolides].

    PubMed

    Nefelova, M V; Sverdlova, A N

    1982-01-01

    The activity of pyruvate dehydrogenase and dehydrogenases of the tricarboxylic acid cycle was assayed in the mycelium of Streptomyces chrysomallus var. Carotenoides growing under different conditions of the medium. The activity of the enzymes increased when acetic, citric and succinic acids were added at different periods of the growth. Moreover, addition of the acids increased the time of intensive functioning of the dehydrogenases whose activity abruptly decreased after 60 h of the growth under the control conditions.

  4. 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

  5. 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

  6. 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

  7. [Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

    PubMed

    Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

    2015-09-01

    Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

  8. 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...

  9. 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

  10. 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

  11. Construction of chimeric enzymes out of maize endosperm branching enzymes I and II: activity and properties.

    PubMed

    Kuriki, T; Stewart, D C; Preiss, J

    1997-11-14

    Branching enzyme I and II isoforms from maize endosperm (mBE I and mBE II, respectively) have quite different properties, and to elucidate the domain(s) that determines the differences, chimeric genes consisting of part mBE I and part mBE II were constructed. When expressed under the control of the T7 promoter in Escherichia coli, several of the chimeric enzymes were inactive. The only fully active chimeric enzyme was mBE II-I BspHI, in which the carboxyl-terminal part of mBE II was exchanged for that of mBE I at a BspHI restriction site and was purified to homogeneity and characterized. Another chimeric enzyme, mBE I-II HindIII, in which the amino-terminal end of mBE II was replaced with that of mBE I, had very little activity and was only partially characterized. The purified mBE II-I BspHI exhibited higher activity than wild-type mBE I and mBE II when assayed by the phosphorylase a stimulation assay. mBE II-I BspHI had substrate specificity (preference for amylose rather than amylopectin) and catalytic capacity similar to mBE I, despite the fact that only the carboxyl terminus was from mBE I, suggesting that the carboxyl terminus may be involved in determining substrate specificity and catalytic capacity. In chain transfer experiments, mBE II-I BspHI transferred more short chains (with a degree of polymerization of around 6) in a fashion similar to mBE II. In contrast, mBE I-II HindIII transferred more long chains (with a degree of polymerization of around 11-12), similar to mBE I, suggesting that the amino terminus of mBEs may play a role in the size of oligosaccharide chain transferred. This study challenges the notion that the catalytic centers for branching enzymes are exclusively located in the central portion of the enzyme; it suggests instead that the amino and carboxyl termini may also be involved in determining substrate preference, catalytic capacity, and chain length transfer.

  12. Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

    PubMed

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

    Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry.

  13. Improved Plant-based Production of E1 endoglucanase Using Potato: Expression Optimization and Tissue Targeting

    SciTech Connect

    Dai, Ziyu; Hooker, Brian S.; Anderson, Daniel B.; Thomas, Steven R.

    2000-06-01

    Optimization of Acidothermus cellulolyticus endoglucanase (E1) gene expression in transgenic potato (Solanum tuberosum L.) was examined in this study, where the E1 coding sequence was transcribed under control of a leaf specific promoter (tomato RbcS-3C) or the Mac promoter (a hybrid promoter of mannopine synthase promoter and cauliflower mosaic virus 35S promoter enhancer region). Average E1 activity in leaf extracts of potato transformants, in which E1 protein was targeted by a chloroplast signal peptide and an apoplast signal peptide were much higher than those by an E1 native signal peptide and a vacuole signal peptide. E1 protein accumulated up to 2.6% of total leaf soluble protein, where E1 gene was under control of the RbcS-3C promoter, alfalfa mosaic virus 5-untranslated leader, and RbcS-2A signal peptide. E1 protein production, based on average E1 activity and E1 protein accumulation in leaf extracts, is higher in potato than those measured previously in transgenic tobacco bearing the same transgene constructs. Comparisons of E1 activity, protein accumulation, and relative mRNA levels showed that E1 expression under control of tomato RbcS-3C promoter was specifically localized in leaf tissues, while E1 gene was expressed in both leaf and tuber tissues under control of Mac promoter. This suggests dual-crop applications in which potato vines serve as enzyme production `bioreactors' while tubers are preserved for culinary applications.

  14. Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS.

    PubMed

    Taylor-Harding, Barbie; Aspuria, Paul-Joseph; Agadjanian, Hasmik; Cheon, Dong-Joo; Mizuno, Takako; Greenberg, Danielle; Allen, Jenieke R; Spurka, Lindsay; Funari, Vincent; Spiteri, Elizabeth; Wang, Qiang; Orsulic, Sandra; Walsh, Christine; Karlan, Beth Y; Wiedemeyer, W Ruprecht

    2015-01-20

    High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC. PMID:25557169

  15. Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

    PubMed Central

    Taylor-Harding, Barbie; Aspuria, Paul-Joseph; Agadjanian, Hasmik; Cheon, Dong-Joo; Mizuno, Takako; Greenberg, Danielle; Allen, Jenieke R.; Spurka, Lindsay; Funari, Vincent; Spiteri, Elizabeth; Wang, Qiang; Orsulic, Sandra; Walsh, Christine; Karlan, Beth Y.; Wiedemeyer, W. Ruprecht

    2015-01-01

    High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC. PMID:25557169

  16. Stimulation of hERG1 channel activity promotes a calcium-dependent degradation of cyclin E2, but not cyclin E1, in breast cancer cells.

    PubMed

    Perez-Neut, Mathew; Shum, Andrew; Cuevas, Bruce D; Miller, Richard; Gentile, Saverio

    2015-01-30

    Cyclin E2 gene amplification, but not cyclin E1, has been recently defined as marker for poor prognosis in breast cancer, and appears to play a major role in proliferation and therapeutic resistance in several breast cancer cells. Our laboratory has previously reported that stimulation of the hERG1 potassium channel with selective activators led to down-regulation of cyclin E2 in breast cancer cells. In this work, we demonstrate that stimulation of hERG1 promotes an ubiquitin-proteasome-dependent degradation of cyclin E2 in multiple breast cancer cell lines representing Luminal A, HER2+ and Trastuzumab-resistant breast cancer cells. In addition we have also reveal that hERG1 stimulation induces an increase in intracellular calcium that is required for cyclin E2 degradation. This novel function for hERG1 activity was specific for cyclin E2, as cyclins A, B, D E1 were unaltered by the treatment. Our results reveal a novel mechanism by which hERG1 activation impacts the tumor marker cyclin E2 that is independent of cyclin E1, and suggest a potential therapeutic use for hERG1 channel activators.

  17. Lysophosphatidic Acid-induced ERK Activation and Chemotaxis in MC3T3-E1 Preosteoblasts are Independent of EGF Receptor Transactivation

    SciTech Connect

    Karagiosis, Sue A.; Chrisler, William B.; Bollinger, Nikki; Karin, Norman J.

    2009-06-01

    Growing evidence indicates that bone-forming osteoblasts and their progenitors are target cells for the lipid growth factor lysophosphatidic acid (LPA) which is produced by degranulating platelets at sites of injury. LPA is a potent inducer of bone cell migration, proliferation and survival in vitro and an attractive candidate to facilitate preosteoblast chemotaxis during skeletal regeneration in vivo, but the intracellular signaling pathways mediating the effects of this lipid on bone cells are not defined. In this study we measured the ability of LPA to stimulate extracellular signal-related kinase (ERK1/2) in MC3T3-E1 preosteoblastic cells and determined the contribution of this pathway to LPA-stimulated chemotaxis. LPA-treated cells exhibited a bimodal activation of ERK1/2 with maximal phosphorylation at 5 and 60 minutes. The kinetics of ERK1/2 phosphorylation were not coupled to Ras activation or LPA-induced elevations in cytosolic Ca2+. While LPA is coupled to the transactivation of the EGF receptor in many cell types, LPA-stimulated ERK1/2 activation in MC3T3-E1 cells was unaffected by inhibition of EGF receptor function. ERK isoforms rapidly accumulated at nuclear sites in LPA-treated cells, a process that was blocked if ERK1/2 phosphorylation was prevented with the MEK1 inhibitor U0126. Blocking ERK1/2 phosphorylation with U0126 also diminished MC3T3-E1 cell migration and altered the normal disassembly of LPA-induced stress fibers, while the inhibition of EGF receptor function had no effect on LPA-coupled preosteoblast motility. Our results identify ERK1/2 activation as a mediatora mediator of LPA-stimulated MC3T3-E1 cell migration that may be relevant to preosteoblast motility during bone repair in vivo.

  18. β₂ adrenergic receptor activation suppresses bone morphogenetic protein (BMP)-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

    PubMed

    Yamada, Takayuki; Ezura, Yoichi; Hayata, Tadayoshi; Moriya, Shuichi; Shirakawa, Jumpei; Notomi, Takuya; Arayal, Smriti; Kawasaki, Makiri; Izu, Yayoi; Harada, Kiyoshi; Noda, Masaki

    2015-06-01

    β adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of β2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

  19. 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

  20. 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

  1. Molecular imaging of macrophage enzyme activity in cardiac inflammation

    PubMed Central

    Ali, Muhammad; Pulli, Benjamin; Chen, John W.

    2014-01-01

    Molecular imaging is highly advantageous as various insidious inflammatory events can be imaged in a serial and quantitative fashion. Combined with the conventional imaging modalities like computed tomography (CT), magnetic resonance (MR) and nuclear imaging, it helps us resolve the extent of ongoing pathology, quantify inflammation and predict outcome. Macrophages are increasingly gaining importance as an imaging biomarker in inflammatory cardiovascular diseases. Macrophages, recruited to the site of injury, internalize necrotic or foreign material. Along with phagocytosis, activated macrophages release proteolytic enzymes like matrix metalloproteinases (MMPs) and cathepsins into the extracellular environment. Pro-inflammatory monocytes and macrophages also induce tissue oxidative damage through the inflammatory enzyme myeloperoxidase (MPO). In this review we will highlight recent advances in molecular macrophage imaging. Particular stress will be given to macrophage functional and enzymatic activity imaging which targets phagocytosis, proteolysis and myeloperoxidase activity imaging. PMID:24729833

  2. [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

  3. 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.

  4. Resolvins AT-D1 and E1 differentially impact functional outcome, post-traumatic sleep, and microglial activation following diffuse brain injury in the mouse.

    PubMed

    Harrison, Jordan L; Rowe, Rachel K; Ellis, Timothy W; Yee, Nicole S; O'Hara, Bruce F; Adelson, P David; Lifshitz, Jonathan

    2015-07-01

    Traumatic brain injury (TBI) is induced by mechanical forces which initiate a cascade of secondary injury processes, including inflammation. Therapies which resolve the inflammatory response may promote neural repair without exacerbating the primary injury. Specific derivatives of omega-3 fatty acids loosely grouped as specialized pro-resolving lipid mediators (SPMs) and termed resolvins promote the active resolution of inflammation. In the current study, we investigate the effect of two resolvin molecules, RvE1 and AT-RvD1, on post-traumatic sleep and functional outcome following diffuse TBI through modulation of the inflammatory response. Adult, male C57BL/6 mice were injured using a midline fluid percussion injury (mFPI) model (6-10min righting reflex time for brain-injured mice). Experimental groups included mFPI administered RvE1 (100ng daily), AT-RvD1 (100ng daily), or vehicle (sterile saline) and counterbalanced with uninjured sham mice. Resolvins or saline were administered daily for seven consecutive days beginning 3days prior to TBI to evaluate proof-of-principle to improve outcome. Immediately following diffuse TBI, post-traumatic sleep was recorded for 24h post-injury. For days 1-7 post-injury, motor outcome was assessed by rotarod. Cognitive function was measured at 6days post-injury using novel object recognition (NOR). At 7days post-injury, microglial activation was quantified using immunohistochemistry for Iba-1. In the diffuse brain-injured mouse, AT-RvD1 treatment, but not RvE1, mitigated motor and cognitive deficits. RvE1 treatment significantly increased post-traumatic sleep in brain-injured mice compared to all other groups. RvE1 treated mice displayed a higher proportion of ramified microglia and lower proportion of activated rod microglia in the cortex compared to saline or AT-RvD1 treated brain-injured mice. Thus, RvE1 treatment modulated post-traumatic sleep and the inflammatory response to TBI, albeit independently of improvement in motor

  5. Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii.

    PubMed

    Shtaida, Nastassia; Khozin-Goldberg, Inna; Solovchenko, Alexei; Chekanov, Konstantin; Didi-Cohen, Shoshana; Leu, Stefan; Cohen, Zvi; Boussiba, Sammy

    2014-12-01

    The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga.

  6. Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii.

    PubMed

    Shtaida, Nastassia; Khozin-Goldberg, Inna; Solovchenko, Alexei; Chekanov, Konstantin; Didi-Cohen, Shoshana; Leu, Stefan; Cohen, Zvi; Boussiba, Sammy

    2014-12-01

    The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga. PMID:25210079

  7. Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii

    PubMed Central

    Shtaida, Nastassia; Khozin-Goldberg, Inna; Solovchenko, Alexei; Chekanov, Konstantin; Didi-Cohen, Shoshana; Leu, Stefan; Cohen, Zvi; Boussiba, Sammy

    2014-01-01

    The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga. PMID:25210079

  8. Extracellular enzyme activity and biogeochemical cycling in restored prairies

    NASA Astrophysics Data System (ADS)

    Lynch, L.; Hernandez, D.; Schade, J. D.

    2011-12-01

    Winter microbial activity in mid-latitude prairie ecosystems is thermally sensitive and significantly influenced by snow depth. Snow insulates the soil column facilitating microbial processing of complex organic substrates. Previous studies in forests and tundra ecosystems suggest patterns of substrate utilization and limitation are seasonal; above freezing, soil microbes access fresh litter inputs and sugar exudates from plant roots, while under frozen condition they recycle nutrients incorporated in microbial biomass. In order to liberate nutrients required for carbon degradation, soil microbes invest energy in the production of extracellular enzymes that cleave monomers from polymer bonds. The inverse relationship between relative enzyme abundance and substrate availability makes enzyme assays a useful proxy to assess changes in resources over time. Our objective in this study was to assess patterns in microbial biomass, nutrient availability, and extracellular enzyme activity in four snow exclosure sites over a seven-month period. Over the past three years, we have maintained a snow removal experiment on two restored prairies in central Minnesota. In each prairie, snow was continuously removed annually from two 4 x 4 m plots by shoveling after each snow event. Extractable C, N and P, and microbial C, N and P in soil samples were measured in samples collected from these snow removal plots, as well as in adjacent unmanipulated prairie control plots. Pools of C, N, and P were estimated using standard extraction protocols, and microbial pools were estimated using chloroform fumigation direct extraction (CFDE). We conducted fluorometric extracellular enzyme assays (EEA) to assess how the degradation potential of cellulose (cellobiohydrolase, CBH), protein (leucine aminopeptidase, LAP), and phosphate esters (phosphatase, PHOS) changed seasonally. Microbial C and N declined between October and June, while microbial P declined during the fall and winter, but increased

  9. Extracellular enzyme activity in a willow sewage treatment system.

    PubMed

    Brzezinska, Maria Swiontek; Lalke-Porczyk, Elżbieta; Kalwasińska, Agnieszka

    2012-12-01

    This paper presents the results of studies on the activity of extra-cellular enzymes in soil-willow vegetation filter soil which is used in the post-treatment of household sewage in an onsite wastewater treatment system located in central Poland. Wastewater is discharged from the detached house by gravity into the onsite wastewater treatment system. It flows through a connecting pipe into a single-chamber septic tank and is directed by the connecting pipe to a control well to be further channelled in the soil-willow filter by means of a subsurface leaching system. Soil samples for the studies were collected from two depths of 5 cm and 1 m from three plots: close to the wastewater inflow, at mid-length of the plot and close to its terminal part. Soil samples were collected from May to October 2009. The activity of the extra-cellular enzymes was assayed by the fluorometric method using 4-methylumbelliferyl and 7-amido-4-methylcoumarin substrate. The ranking of potential activity of the assayed enzymes was the same at 5 cm and 1 m soil depths, i.e. esterase > phosphmomoesterase > leucine-aminopeptidase > β-glucosidase > α-glucosidase. The highest values of enzymatic activity were recorded in the surface layer of the soil at the wastewater inflow and decreased with increasing distance from that point.

  10. Enzyme-like activities of algal polysaccharide - cerium complexes

    NASA Astrophysics Data System (ADS)

    Wang, Dongfeng; Sun, Jipeng; Du, Dehong; Ye, Shen; Wang, Changhong; Zhou, Xiaoling; Xue, Changhu

    2005-01-01

    Water-soluble algal polysaccharides (APS) (alginic acid, fucoidan and laminaran) possess many pharmacological activities. The results of this study showed that the APS-Ce4+ complexes have some enzyme-like activities. Fucoidan and its complex with Ce4+ have activities similar to those of SOD. The activities of laminaran, alginic acid and their complexes are not measurable. The APS do not show measurable activities in the digestion of plasmid DNA. In contrast, the APS - Ce4+ complexes show these measurable activities under the comparable condition when APS bind Ce4+ and form homogenous solutions. The laminaran - Ce4+ complex shows the most obvious activity in the digestion of plasmid DNA, pNPP and chloropy-rifos under neutral conditions.

  11. Polyphosphate-degrading enzymes in Acinetobacter spp. and activated sludge.

    PubMed Central

    van Groenestijn, J W; Bentvelsen, M M; Deinema, M H; Zehnder, A J

    1989-01-01

    Polyphosphate-degrading enzymes were studied in Acinetobacter spp. and activated sludge. Polyphosphate: AMP phosphotransferase activity in Acinetobacter strain 210A decreased with increasing growth rates. The activity of this enzyme in cell extracts of Acinetobacter strain 210A was maximal at a pH of 8.5 and a temperature of 40 degrees C and was stimulated by (NH4)2SO4. The Km for AMP was 0.6 mM, and the Vmax was 60 nmol/min per mg of protein. Cell extracts of this strain also contained polyphosphatase, which was able to degrade native polyphosphate and synthetic magnesium polyphosphate and was strongly stimulated by 300 to 400 mM NH4Cl. A positive correlation was found between polyphosphate:AMP phosphotransferase activity, adenylate kinase activity, and phosphorus accumulation in six Acinetobacter strains. Significant activities of polyphosphate kinase were detected only in strain P, which contained no polyphosphate:AMP phosphotransferase. In samples of activated sludge from different plants, the activity of adenylate kinase correlated well with the ability of the sludge to remove phosphate biologically from wastewater. PMID:2539774

  12. 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.

  13. Suppression of Myc, but not E1a, transformation activity by Max-associated proteins, Mad and Mxi1.

    PubMed Central

    Lahoz, E G; Xu, L; Schreiber-Agus, N; DePinho, R A

    1994-01-01

    Mad and Mxi1, two members of the Myc-related basic-region helix-loop-helix/leucine-zipper family of proteins, associate directly with Max to form sequence-specific DNA binding heterodimers that are transactivation-incompetent. Mad-Max complexes have been shown to exert a strong repressive effect on Myc-induced transactivation, perhaps through the competitive occupation of common promoter binding sites also recognized by active Myc-Max heterodimers. To place these recent biochemical observations in a biological context, mad and mxi1 expression vectors were tested for their ability to influence Myc transformation activity in the rat embryo fibroblast cooperation assay. Addition of an equimolar amount of mad or mxi1 expression vector to mouse c-myc/ras cotransfections resulted in a dramatic reduction in both the number of foci generated and the severity of the malignant phenotype. Myc-specific suppression by Mad and Mxi1 was demonstrated by their ability to affect c- and N-myc-, but not ela-, induced transformation. In contrast, mad and mxi1 expression constructs bearing deletions in the basic region exerted only mild repressive effects on Myc transformation activity, suggesting that occupation of common DNA binding sites by transactivation-incompetent Mad-Max or Mxi1-Max complexes appears to play a more dominant role in this suppression than titration of limited intracellular pools of Max away from active Myc-Max complexes. Thus, these biological data support a current model for regulation of Myc function in which relative intracellular levels of Mad and Mxi1 in comparison to those of Myc may determine the degree of activation of Myc-responsive growth pathways. Images PMID:8202517

  14. 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

  15. Sample storage for soil enzyme activity and bacterial community profiles.

    PubMed

    Wallenius, K; Rita, H; Simpanen, S; Mikkonen, A; Niemi, R M

    2010-04-01

    Storage of samples is often an unavoidable step in environmental data collection, since available analytical capacity seldom permits immediate processing of large sample sets needed for representative data. In microbiological soil studies, sample pretreatments may have a strong influence on measurement results, and thus careful consideration is required in the selection of storage conditions. The aim of this study was to investigate the suitability of prolonged (up to 16 weeks) frozen or air-dried storage for divergent soil materials. The samples selected to this study were mineral soil (clay loam) from an agricultural field, humus from a pine forest and compost from a municipal sewage sludge composting field. The measured microbiological parameters included functional profiling with ten different hydrolysing enzyme activities determined by artificial fluorogenic substrates, and structural profiling with bacterial 16S rDNA community fingerprints by amplicon length heterogeneity analysis (LH-PCR). Storage of samples affected the observed fluorescence intensity of the enzyme assay's fluorophor standards dissolved in soil suspension. The impact was highly dependent on the soil matrix and storage method, making it important to use separate standardisation for each combination of matrix type, storage method and time. Freezing proved to be a better storage method than air-drying for all the matrices and enzyme activities studied. The effect of freezing on the enzyme activities was small (<20%) in clay loam and forest humus and moderate (generally 20-30%) in compost. The most dramatic decreases (>50%) in activity were observed in compost after air-drying. The bacterial LH-PCR community fingerprints were unaffected by frozen storage in all matrices. The effect of storage treatments was tested using a new statistical method based on showing similarity rather than difference of results.

  16. Angiotensin-converting enzyme 2 activation improves endothelial function.

    PubMed

    Fraga-Silva, Rodrigo A; Costa-Fraga, Fabiana P; Murça, Tatiane M; Moraes, Patrícia L; Martins Lima, Augusto; Lautner, Roberto Q; Castro, Carlos H; Soares, Célia Maria A; Borges, Clayton L; Nadu, Ana Paula; Oliveira, Marilene L; Shenoy, Vinayak; Katovich, Michael J; Santos, Robson A S; Raizada, Mohan K; Ferreira, Anderson J

    2013-06-01

    Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

  17. Metabolic inactivation of resolvin E1 and stabilization of its anti-inflammatory actions.

    PubMed

    Arita, Makoto; Oh, Sungwhan F; Chonan, Tomomichi; Hong, Song; Elangovan, Siva; Sun, Yee-Ping; Uddin, Jasim; Petasis, Nicos A; Serhan, Charles N

    2006-08-11

    The resolvins (Rv) are lipid mediators derived from omega-3 polyunsaturated fatty acids that act within a local inflammatory milieu to stop leukocyte recruitment and promote resolution. Resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is an oxygenase product derived from omega-3 eicosapentaenoic acid that displays potent anti-inflammation/pro-resolution actions in vivo. Here, we determined whether oxidoreductase enzymes catalyze the conversion of RvE1 and assessed the biological activity of the RvE1 metabolite. With NAD+ as a cofactor, recombinant 15-hydroxyprostaglandin dehydrogenase acted as an 18-hydroxyl dehydrogenase to form 18-oxo-RvE1. In the murine lung, dehydrogenation of the hydroxyl group at carbon 18 position to form 18-oxo-RvE1 represented the major initial metabolic route for RvE1. At a concentration where RvE1 potently reduced polymorphonuclear leukocyte (PMN) recruitment in zymosan-induced peritonitis, 18-oxo-RvE1 was devoid of activity. In human neutrophils, carbon 20 hydroxylation of RvE1 was the main route of conversion. An RvE1 analog, i.e. 19-(p-fluorophenoxy)-RvE1, was synthesized that resisted rapid metabolic inactivation and proved to retain biological activity reducing PMN infiltration and pro-inflammatory cytokine/chemokine production in vivo. These results established the structure of a novel RvE1 initial metabolite, indicating that conversion of RvE1 to the oxo product represents a mode of RvE1 inactivation. Moreover, the designed RvE1 analog, which resisted further metabolism/inactivation, could be a useful tool to evaluate the actions of RvE1 in complex disease models.

  18. Metabolic inactivation of resolvin E1 and stabilization of its anti-inflammatory actions.

    PubMed

    Arita, Makoto; Oh, Sungwhan F; Chonan, Tomomichi; Hong, Song; Elangovan, Siva; Sun, Yee-Ping; Uddin, Jasim; Petasis, Nicos A; Serhan, Charles N

    2006-08-11

    The resolvins (Rv) are lipid mediators derived from omega-3 polyunsaturated fatty acids that act within a local inflammatory milieu to stop leukocyte recruitment and promote resolution. Resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is an oxygenase product derived from omega-3 eicosapentaenoic acid that displays potent anti-inflammation/pro-resolution actions in vivo. Here, we determined whether oxidoreductase enzymes catalyze the conversion of RvE1 and assessed the biological activity of the RvE1 metabolite. With NAD+ as a cofactor, recombinant 15-hydroxyprostaglandin dehydrogenase acted as an 18-hydroxyl dehydrogenase to form 18-oxo-RvE1. In the murine lung, dehydrogenation of the hydroxyl group at carbon 18 position to form 18-oxo-RvE1 represented the major initial metabolic route for RvE1. At a concentration where RvE1 potently reduced polymorphonuclear leukocyte (PMN) recruitment in zymosan-induced peritonitis, 18-oxo-RvE1 was devoid of activity. In human neutrophils, carbon 20 hydroxylation of RvE1 was the main route of conversion. An RvE1 analog, i.e. 19-(p-fluorophenoxy)-RvE1, was synthesized that resisted rapid metabolic inactivation and proved to retain biological activity reducing PMN infiltration and pro-inflammatory cytokine/chemokine production in vivo. These results established the structure of a novel RvE1 initial metabolite, indicating that conversion of RvE1 to the oxo product represents a mode of RvE1 inactivation. Moreover, the designed RvE1 analog, which resisted further metabolism/inactivation, could be a useful tool to evaluate the actions of RvE1 in complex disease models. PMID:16757471

  19. 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

  20. 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.

  1. 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

  2. Polymer nanocarriers protecting active enzyme cargo against proteolysis.

    PubMed

    Dziubla, Thomas D; Karim, Adnan; Muzykantov, Vladimir R

    2005-02-01

    Polymeric nanocarriers (PNCs), proposed as an attractive vehicle for vascular drug delivery, remain an orphan technology for enzyme therapies due to poor loading and inactivation of protein cargoes. To unite enzyme delivery by PNC with a clinically relevant goal of containment of vascular oxidative stress, a novel freeze-thaw encapsulation strategy was designed and provides approximately 20% efficiency loading of an active large antioxidant enzyme, catalase, into PNC (200-300 nm) composed of biodegradable block copolymers poly(ethylene glycol)-b-poly(lactic-glycolic acid). Catalase's substrate, H(2)O(2), was freely diffusible in the PNC polymer. Furthermore, PNC-loaded catalase stably retained 25-30% of H(2)O(2)-degrading activity for at least 18 h in a proteolytic environment, while free catalase lost activity within 1 h. Delivery and protection of catalase from lysosomal degradation afforded by PNC nanotechnology may advance effectiveness and duration of treatment of diverse disease conditions associated with vascular oxidative stress. PMID:15653162

  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. 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

  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. A dynamic loop at the active center of the Escherichia coli pyruvate dehydrogenase complex E1 component modulates substrate utilization and chemical communication with the E2 component.

    PubMed

    Kale, Sachin; Arjunan, Palaniappa; Furey, William; Jordan, Frank

    2007-09-21

    Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.

  7. Expression and Characterization of Acidothermus celluloyticus E1 Endoglucanase in Transgenic Duckweed Lemna minor 8627

    SciTech Connect

    Sun, Y.; Cheng, J. J.; Himmel, M. E.; Skory, C. D.; Adney, W. S.; Thomas, S. R.; Tisserat, B.; Nishimura, Y.; Yamamoto, Y. T.

    2007-01-01

    Endoglucanase E1 from Acidothermus cellulolyticus was expressed cytosolically under control of the cauliflower mosaic virus 35S promoter in transgenic duckweed, Lemna minor 8627 without any obvious observable phenotypic effects on morphology or rate of growth. The recombinant enzyme co-migrated with the purified catalytic domain fraction of the native E1 protein on western blot analysis, revealing that the cellulose-binding domain was cleaved near or in the linker region. The duckweed-expressed enzyme was biologically active and the expression level was up to 0.24% of total soluble protein. The endoglucanase activity with carboxymethylcellulose averaged 0.2 units mg protein{sup -1} extracted from fresh duckweed. The optimal temperature and pH for E1 enzyme activity were about 80 C and pH 5, respectively. While extraction with HEPES (N-[2-hydroxyethyl]piperazine-N{prime}-[2-ethanesulfonic acid]) buffer (pH 8) resulted in the highest recovery of total soluble proteins and E1 enzyme, extraction with citrate buffer (pH 4.8) at 65 C enriched relative amounts of E1 enzyme in the extract. This study demonstrates that duckweed may offer new options for the expression of cellulolytic enzymes in transgenic plants.

  8. Resveratrol reduces prostaglandin E1-stimulated osteoprotegerin synthesis in osteoblasts: suppression of stress-activated protein kinase/c-Jun N-terminal kinase.

    PubMed

    Yamamoto, Naohiro; Otsuka, Takanobu; Kuroyanagi, Gen; Kondo, Akira; Kainuma, Shingo; Nakakami, Akira; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Tokuda, Haruhiko

    2015-01-01

    Resveratrol, a natural polyphenol mainly existing in red grapes and berries, possesses beneficial effects on human being. We have previously reported that prostaglandin E1 (PGE1) stimulates vascular endothelial growth factor synthesis via activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the PGE1-effect on osteoprotegerin (OPG) synthesis and the effect of resveratrol on the synthesis in MC3T3-E1 cells. PGE1 induced the expression levels of OPG mRNA and stimulated the OPG release. Resveratrol significantly reduced the PGE1-induced OPG release and the mRNA expression. SRT1720, an activator of SIRT1, suppressed the release of OPG. The protein levels of SIRT1 were not up-regulated by resveratrol with or without PGE1. Both SB203580 and SP600125, a specific p38 MAP kinase inhibitor and a specific SAPK/JNK inhibitor, respectively, but not PD98059, a specific MEK inhibitor, reduced the PGE1-stimulated OPG release. Resveratrol or SRT1720 failed to affect the phosphorylation of p38 MAP kinase. On the contrary, PGE1-induced phosphorylation of SAPK/JNK was significantly attenuated by both resveratrol and SRT1720. Our results strongly suggest that resveratrol inhibits PGE1-stimulated OPG synthesis via suppressing SAPK/JNK but not p38 MAP kinase in osteoblasts.

  9. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers EVIDENCE FOR A DIRECT PATHWAY BETWEEN THE 4′-AMINOPYRIMIDINE N1′ ATOMS

    SciTech Connect

    Nemeria, Natalia S; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-11-03

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4{prime}-aminopyrimidine N1{prime} atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu{sup 571}, Glu{sup 235}, and Glu{sup 237}) and Arg{sup 606} resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. (1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. (2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. (3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. (4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu{sup 235} makes no direct contact with the cofactor. The role of the conserved Glu{sup 571} residue in both catalysis and cofactor orientation is revealed by the combined results for the first time.

  10. Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

    PubMed

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

    2015-09-01

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical 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 four-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

  11. Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

    PubMed

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

    2015-09-01

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical 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 four-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

  12. Hydrostatic Pressure Increases the Catalytic Activity of Amyloid Fibril Enzymes.

    PubMed

    Luong, Trung Quan; Erwin, Nelli; Neumann, Matthias; Schmidt, Andreas; Loos, Cornelia; Schmidt, Volker; Fändrich, Marcus; Winter, Roland

    2016-09-26

    We studied the combined effects of pressure (0.1-200 MPa) and temperature (22, 30, and 38 °C) on the catalytic activity of designed amyloid fibrils using a high-pressure stopped-flow system with rapid UV/Vis absorption detection. Complementary FT-IR spectroscopic data revealed a remarkably high pressure and temperature stability of the fibrillar systems. High pressure enhances the esterase activity as a consequence of a negative activation volume at all temperatures (about -14 cm(3)  mol(-1) ). The enhancement is sustained in the whole temperature range covered, which allows a further acceleration of the enzymatic activity at high temperatures (activation energy 45-60 kJ mol(-1) ). Our data reveal the great potential of using both pressure and temperature modulation to optimize the enzyme efficiency of catalytic amyloid fibrils.

  13. Hydrostatic Pressure Increases the Catalytic Activity of Amyloid Fibril Enzymes.

    PubMed

    Luong, Trung Quan; Erwin, Nelli; Neumann, Matthias; Schmidt, Andreas; Loos, Cornelia; Schmidt, Volker; Fändrich, Marcus; Winter, Roland

    2016-09-26

    We studied the combined effects of pressure (0.1-200 MPa) and temperature (22, 30, and 38 °C) on the catalytic activity of designed amyloid fibrils using a high-pressure stopped-flow system with rapid UV/Vis absorption detection. Complementary FT-IR spectroscopic data revealed a remarkably high pressure and temperature stability of the fibrillar systems. High pressure enhances the esterase activity as a consequence of a negative activation volume at all temperatures (about -14 cm(3)  mol(-1) ). The enhancement is sustained in the whole temperature range covered, which allows a further acceleration of the enzymatic activity at high temperatures (activation energy 45-60 kJ mol(-1) ). Our data reveal the great potential of using both pressure and temperature modulation to optimize the enzyme efficiency of catalytic amyloid fibrils. PMID:27573584

  14. 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.

  15. Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme activity.

    PubMed

    Jadhav, Swati B; Bankar, Sandip B; Granström, Tom; Ojamo, Heikki; Singhal, Rekha S; Survase, Shrikant A

    2015-09-01

    Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.

  16. 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

  17. PAPSS2 Promotes Alkaline Phosphates Activity and Mineralization of Osteoblastic MC3T3-E1 Cells by Crosstalk and Smads Signal Pathways

    PubMed Central

    Wang, Weizhuo; Li, Fang; Wang, Kunzheng; Cheng, Bin; Guo, Xiong

    2012-01-01

    Several studies have indicated that PAPSS2 (3′-phosphoadenosine-5′-phosphosulfate synthetase 2) activity is important to normal skeletal development. Mouse PAPSS2 is predominantly expressed during the formation of the skeleton and cartilaginous elements of the mouse embryo and in newborn mice. However, the role and mechanism of PAPSS2 in bone formation remains largely unidentified. By analyzing the expression pattern of the PAPSS2 gene, we have found that PAPSS2 is expressed in bone tissue and bone formation. PAPSS2 transcripts increase during osteoblast differentiation and are in less level in RANKL-induced osteoclast like cells. By using lentivirus-mediated RNA interference (RNAi) technology, we knocked down PAPSS2 expression in MC3T3-E1 osteoblast. Silencing of PAPSS2 expression significantly decreases ALP activity and cell mineralization, inhibits expression of osteoblast marker osteopontin (OPN) and collagen I. Conversely, overexpression of PAPSS2 promotes the MC3T3-E1 to differentiate into osteoblast and mineralization. Moreover, compared to that in the control cells, the mRNA level and protein expression of phosphorylated Smad 2/3, which is a key transcriptional factor in the Smad osteoblast differentiation pathway, showed significant decreases in PAPSS2-silenced cells and increases in PAPSS2-overexpression cells. These results suggest that PAPSS2 might regulate osteoblast ALP activity and cell mineralization, probably through Smads signal pathways. PMID:22916269

  18. Protoplast fusion enhances lignocellulolytic enzyme activities in Trichoderma reesei.

    PubMed

    Cui, Yu-xiao; Liu, Jia-jing; Liu, Yan; Cheng, Qi-yue; Yu, Qun; Chen, Xin; Ren, Xiao-dong

    2014-12-01

    Protoplast fusion was used to obtain a higher production of lignocellulolytic enzymes with protoplast fusion in Trichoderma reesei. The fusant strain T. reesei JL6 was obtained from protoplast fusion from T. reesei strains QM9414, MCG77, and Rut C-30. Filter paper activity of T. reesei JL6 increased by 18% compared with that of Rut C-30. β-Glucosidase, hemicellulase and pectinase activities of T. reesei JL6 were also higher. The former activity was 0.39 Uml(-1), while those of QM9414, MCG77, and Rut C-30 were 0.13, 0.11, and 0.16 Uml(-1), respectively. Pectinase and hemicellulase activities of JL6 were 5.4 and 15.6 Uml(-1), respectively, which were slightly higher than those of the parents. The effects of corn stover and wheat bran carbon sources on the cellulase production and growth curve of T. reesei JL6 were also investigated.

  19. Auto-ubiquitination of ubiquitin-activating enzymes from chicken breast muscle.

    PubMed Central

    Arnold, J E; Gevers, W

    1990-01-01

    A soluble ubiquitin-depleted fraction from chicken skeletal muscle (fraction II), when incubated at neutral pH for several hours with 125I-ubiquitin and ATP, formed small amounts of a ubiquitin derivative (Mr 115,000) of the ubiquitin-activating enzyme E1 as well as certain similarly modified E2 species (Mr 37,000, 34,000 and 24,000). Treatment of such mixtures with NaOH during the incubations, even at early times, greatly enhanced the appearance of these entities; up to two-thirds of the thiolesters of ubiquitin bound to these proteins before alkali treatment were thus converted. The bonds involved had properties compatible with their being peptidic in nature, suggesting that auto-ubiquitination had occurred in each case. The protease inhibitor and alkylating agent tosyl-lysylchloromethane ('TLCK'), when preincubated at 50 microM with fraction II for 2 h at 37 degrees C before the addition of 125I-ubiquitin and ATP, promoted the subsequent auto-ubiquitination of E1 and inhibited its adenylate-forming and thiolester-transferring activities. The findings have a bearing on the physiological substrate- and site-specificity of ubiquitin-conjugating reactions. Images Fig. 1. Fig. 3. Fig. 5. PMID:2160236

  20. 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

  1. 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

  2. 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.

  3. 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

  4. Application of capillary enzyme micro-reactor in enzyme activity and inhibitors studies of glucose-6-phosphate dehydrogenase.

    PubMed

    Camara, Mohamed Amara; Tian, Miaomiao; Guo, Liping; Yang, Li

    2015-05-15

    In this study, we present an on-line measurement of enzyme activity and inhibition of Glucose-6-phosphate dehydrogenase (G6PDH) enzyme using capillary electrophoresis based immobilized enzyme micro-reactor (CE-based IMER). The IMER was prepared using a two-step protocol based on electrostatic assembly. The micro-reactor exhibited good stability and reproducibility for on-line assay of G6PDH enzyme. Both the activity as well as the inhibition of the G6PDH enzyme by six inhibitors, including three metals (Cu(2+), Pb(2+), Cd(2+)), vancomycin, urea and KMnO4, were investigated using on-line assay of the CE-based IMERs. The enzyme activity and inhibition kinetic constants were measured using the IMERs which were found to be consistent with those using traditional off-line enzyme assays. The kinetic mechanism of each inhibitor was also determined. The present study demonstrates the feasibility of using CE-based IMERs for rapid and efficient on-line assay of G6PDH, an important enzyme in the pentosephosphate pathway of human metabolism.

  5. 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.

  6. 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.

  7. CYP2E1 hydroxylation of aniline involves negative cooperativity.

    PubMed

    Hartman, Jessica H; Knott, Katie; Miller, Grover P

    2014-02-01

    CYP2E1 plays a role in the metabolic activation and elimination of aniline, yet there are conflicting reports on its mechanism of action, and hence relevance, in aniline metabolism. Based on our work with similar compounds, we hypothesized that aniline binds two CYP2E1 sites during metabolism resulting in cooperative reaction kinetics and tested this hypothesis through rigorous in vitro studies. The kinetic profile for recombinant CYP2E1 demonstrated significant negative cooperativity based on a fit of data to the Hill equation (n=0.56). Mechanistically, the data were best explained through a two-binding site cooperative model in which aniline binds with high affinity (K(s)=30 μM) followed by a second weaker binding event (K(ss)=1100 uM) resulting in a threefold increase in the oxidation rate. Binding sites for aniline were confirmed by inhibition studies with 4-methylpyrazole. Inhibitor phenotyping experiments with human liver microsomes validated the central role for CYP2E1 in aniline hydroxylation and indicated minor roles for CYP2A6 and CYP2C9. Importantly, inhibition of minor metabolic pathways resulted in a kinetic profile for microsomal CYP2E1 that replicated the preferred mechanism and parameters observed with the recombinant enzyme. Scaled modeling of in vitro CYP2E1 metabolism of aniline to in vivo clearance, especially at low aniline levels, led to significant deviations from the traditional model based on non-cooperative, Michaelis-Menten kinetics. These findings provide a critical mechanistic perspective on the potential importance of CYP2E1 in the metabolic activation and elimination of aniline as well as the first experimental evidence of a negatively cooperative metabolic reaction catalyzed by CYP2E1.

  8. Growth characteristics and enzyme activity in Batrachochytrium dendrobatidis isolates.

    PubMed

    Symonds, E Pearl; Trott, Darren J; Bird, Philip S; Mills, Paul

    2008-09-01

    Batrachochytrium dendrobatidis is a member of the phylum Chytridiomycota and the causative organism chytridiomycosis, a disease of amphibians associated with global population declines and mass mortality events. The organism targets keratin-forming epithelium in adult and larval amphibians, which suggests that keratinolytic activity may be required to infect amphibian hosts. To investigate this hypothesis, we tested 10 isolates of B. dendrobatidis for their ability to grow on a range of keratin-supplemented agars and measured keratolytic enzyme activity using a commercially available kit (bioMerieux API ZYM). The most dense and fastest growth of isolates were recorded on tryptone agar, followed by growth on frog skin agar and the slowest growth recorded on feather meal and boiled snake skin agar. Growth patterns were distinctive for each nutrient source. All 10 isolates were strongly positive for a range of proteolytic enzymes which may be keratinolytic, including trypsin and chymotrypsin. These findings support the predilection of B. dendrobatidis for amphibian skin. PMID:18568420

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. Puromycin-Sensitive Aminopeptidase: An Antiviral Prodrug Activating Enzyme

    PubMed Central

    Tehler, Ulrika; Nelson, Cara H.; Peterson, Larryn W.; Provoda, Chester J.; Hilfinger, John M.; Lee, Kyung-Dall; McKenna, Charles E.; Amidon, Gordon L.

    2010-01-01

    Cidofovir (HPMPC) is a broad-spectrum antiviral agent, currently used to treat AIDS-related human cytomegalovirus retinitis. Cidofovir has recognized therapeutic potential for orthopox virus infections, although its use is hampered by its inherent low oral bioavailability. Val-Ser-cyclic HPMPC (Val-Ser-cHPMPC) is a promising peptide prodrug which has previously been shown by us to improve the permeability and bioavailability of the parent compound in rodent models (Eriksson et al. Molecular Pharmaceutics, 2008 vol 5 598-609). Puromycin-sensitive aminopeptidase was partially purified from Caco-2 cell homogenates and identified as a prodrug activating enzyme for Val-Ser-cHPMPC. The prodrug activation process initially involves an enzymatic step where the l-Valine residue is removed by puromycin-sensitive aminopeptidase, a step that is bestatin-sensitive. Subsequent chemical hydrolysis results in the generation of cHPMPC. A recombinant puromycin-sensitive aminopeptidase was generated and its substrate specificity investigated. The kcat for Val-pNA was significantly lower than that for Ala-pNA, suggesting that some amino acids are preferred over others. Furthermore, the three-fold higher kcat for Val-Ser-cHPMPC as compared to Val-pNA suggests that the leaving group may play an important role in determining hydrolytic activity. In addition to its ability to hydrolyze a variety of substrates, these observations strongly suggest that puromycin-sensitive aminopeptidase is an important enzyme for activating Val-Ser-cHPMPC in vivo. Taken together, our data suggest that puromycin-sensitive aminopeptidase makes an attractive target for future prodrug design. PMID:19969024

  15. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

    CONSPECTUS: The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes' enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme-substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C-H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed.

  16. 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

  17. County-scale spatial distribution of soil enzyme activities and enzyme activity indices in agricultural land: implications for soil quality assessment.

    PubMed

    Tan, Xiangping; 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 km(2)) 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

  18. CYP2E1 Sensitizes the Liver to LPS- and TNF α-Induced Toxicity via Elevated Oxidative and Nitrosative Stress and Activation of ASK-1 and JNK Mitogen-Activated Kinases

    PubMed Central

    Cederbaum, Arthur I.; Yang, Lili; Wang, Xiaodong; Wu, Defeng

    2012-01-01

    The mechanisms by which alcohol causes cell injury are not clear. A major mechanism is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a role in how alcohol induces oxidative stress. Considerable attention has been given to alcohol elevated production of lipopolysaccharide (LPS) and TNFα and to alcohol induction of CYP2E1. These two pathways are not exclusive of each other; however, interactions between them, have not been extensively evaluated. Increased oxidative stress from induction of CYP2E1 sensitizes hepatocytes to LPS and TNFα toxicity and oxidants, activation of inducible nitric oxide synthase and p38 and JNK MAP kinases, and mitochondrial dysfunction are downstream mediators of this CYP2E1-LPS/TNFα-potentiated hepatotoxicity. This paper will summarize studies showing potentiated interactions between these two risk factors in promoting liver injury and the mechanisms involved including activation of the mitogen-activated kinase kinase kinase ASK-1. Decreasing either cytosolic or mitochondrial thioredoxin in HepG2 cells expressing CYP2E1 causes loss of cell viability and elevated oxidative stress via an ASK-1/JNK-dependent mechanism. We hypothesize that similar interactions occur as a result of ethanol induction of CYP2E1 and TNFα. PMID:22028977

  19. The frequency of cytochrome P450 2E1 polymorphisms in Black South Africans.

    PubMed

    Chelule, Paul K; Pegoraro, Rosemary J; Gqaleni, Nceba; Dutton, Michael F

    2006-01-01

    Polymorphisms in the promoter region of the Cytochrome P4502E1 (CYP2E1) gene reportedly modify the metabolic activity of CYP2E1 enzyme, and have been associated with increased susceptibility to squamous cell carcinoma (SCC) of the oesophagus in high prevalence areas such as China. To assess the frequency of these polymorphisms in Black South Africans, a population with a high incidence of oesophageal SCC, this study examined genomic DNA from 331 subjects for restriction fragment length polymorphisms in the CYP2E1 (RsaI and PstI digestion). The frequency of the CYP2E1 c1/c1 and c1/c3 genotypes was 95% and 5% respectively. The frequency of the CYP2E1 allele distribution was found to be markedly different between Chinese and South African populations; hence it is important to place racial differences into consideration when proposing allelic variants as genetic markers for cancer. PMID:17264406

  20. Modelling thermal stability and activity of free and immobilized enzymes as a novel tool for enzyme reactor design.

    PubMed

    Santos, A M P; Oliveira, M G; Maugeri, F

    2007-11-01

    In this work, a novel method is proposed to establish the most suitable operational temperature for an enzyme reactor. The method was based on mathematical modelling of the thermal stability and activity of the enzyme and was developed using thermodynamic concepts and experimental data from free and immobilized inulinases (2,1-beta-D fructan frutanohydrolase, EC 3.2.1.7) from Kluyveromyces marxianus, which were used as examples. The model was, therefore, designed to predict the enzyme activity with respect to the temperature and time course of the enzymatic process, as well as its half-life, in a broad temperature range. The knowledge and information provided by the model could be used to design the operational temperature conditions, leading to higher enzyme activities, while preserving acceptable stability levels, which represent the link between higher productivity and lower process costs. For the inulinase used in this study, the optimum temperature conditions leading to higher enzyme activities were shown to be 63 degrees C and 57.5 degrees C for the free and immobilized inulinases, respectively. However, according to the novel method of approach used here, the more appropriate operating temperatures would be 52 degrees C for free and 42 degrees C for immobilized inulinases, showing that the working temperature is not necessarily the same as the maximum reaction rate temperature, but preferably a lower temperature where the enzyme is much more stable.

  1. Phlorotannins from Alaskan seaweed inhibit carbolytic enzyme activity.

    PubMed

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

    2014-10-22

    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.

  2. Multiple Activities of the Plant Pathogen Type III Effector Proteins WtsE and AvrE1 require WxxxE Motifs

    PubMed Central

    Ham, Jong Hyun; Majerczak, Doris R.; Nomura, Kinya; Mecey, Christy; Uribe, Francisco; He, Sheng-Yang; Mackey, David; Coplin, David L.

    2009-01-01

    The broadly conserved AvrE-family of type III effectors from Gram-negative plant pathogenic bacteria includes important virulence factors, yet little is known about the mechanisms by which these effectors function inside plant cells to promote disease. We have identified two conserved motifs in AvrE-family effectors: a WxxxE motif and a putative C-terminal endoplasmic reticulum membrane retention/retrieval signal (ERMRS). The WxxxE and ERMRS motifs are both required for the virulence activities of WtsE and AvrE1, which are major virulence factors of the corn pathogen Pantoea stewartii subsp. stewartii and the tomato/Arabidopsis pathogen Pseudomonas syringae pv. tomato, respectively. The WxxxE and the predicted ERMRS motifs are also required for other biological activities of WtsE, including elicitation of the hypersensitive response in nonhost plants and suppression of defense responses in Arabidopsis. A family of type III effectors from mammalian bacterial pathogens requires WxxxE and sub-cellular targeting motifs for virulence functions that involve their ability to mimic activated G-proteins. The conservation of related motifs and their necessity for the function of type III effectors from plant pathogens indicates that disturbing host pathways by mimicking activated host G-proteins may be a virulence mechanism employed by plant pathogens as well. PMID:19445595

  3. Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese.

    PubMed

    Torres-Llanez, M J; González-Córdova, A F; Hernandez-Mendoza, A; Garcia, H S; Vallejo-Cordoba, B

    2011-08-01

    The objective of this study was to evaluate if Mexican Fresco cheese manufactured with specific lactic acid bacteria (LAB) presented angiotensin I-converting enzyme inhibitory (ACEI) activity. Water-soluble extracts (3 kDa) obtained from Mexican Fresco cheese prepared with specific LAB (Lactococcus, Lactobacillus, Enterococcus, and mixtures: Lactococcus-Lactobacillus and Lactococcus-Enterococcus) were evaluated for ACEI activity. Specific peptide fractions with high ACEI were analyzed using reverse phase-HPLC coupled to mass spectrometry for determination of amino acid sequence. Cheese containing Enterococcus faecium or a Lactococcus lactis ssp. lactis-Enterococcus faecium mixture showed the largest number of fractions with ACEI activity and the lowest half-maximal inhibitory concentration (IC(50); <10 μg/mL). Various ACEI peptides derived from β-casein [(f(193-205), f(193-207), and f(193-209)] and α(S1)-casein [f(1-15), f(1-22), f(14-23), and f(24-34)] were found. The Mexican Fresco cheese manufactured with specific LAB strains produced peptides with potential antihypertensive activity.

  4. 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

  5. Enzyme architecture: deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5'-monophosphate decarboxylase.

    PubMed

    Goldman, Lawrence M; Amyes, Tina L; Goryanova, Bogdana; Gerlt, John A; Richard, John P

    2014-07-16

    The mechanism for activation of orotidine 5'-monophosphate decarboxylase (OMPDC) by interactions of side chains from Gln215 and Try217 at a gripper loop and R235, adjacent to this loop, with the phosphodianion of OMP was probed by determining the kinetic parameters k(cat) and K(m) for all combinations of single, double, and triple Q215A, Y217F, and R235A mutations. The 12 kcal/mol intrinsic binding energy of the phosphodianion is shown to be equal to the sum of the binding energies of the side chains of R235 (6 kcal/mol), Q215 (2 kcal/mol), Y217 (2 kcal/mol), and hydrogen bonds to the G234 and R235 backbone amides (2 kcal/mol). Analysis of a triple mutant cube shows small (ca. 1 kcal/mol) interactions between phosphodianion gripper side chains, which are consistent with steric crowding of the side chains around the phosphodianion at wild-type OMPDC. These mutations result in the same change in the activation barrier to the OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces (1-β-D-erythrofuranosyl)orotic acid (EO) and phosphite dianion. This shows that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The 12 kcal/mol intrinsic phosphodianion binding energy of OMP is divided between the 8 kcal/mol of binding energy, which is utilized to drive a thermodynamically unfavorable conformational change of the free enzyme, resulting in an increase in (k(cat))(obs) for OMPDC-catalyzed decarboxylation of OMP, and the 4 kcal/mol of binding energy, which is utilized to stabilize the Michaelis complex, resulting in a decrease in (K(m))(obs).

  6. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

    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 (A549) 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.

  8. Rat oocyte tissue plasminogen activator is a catalytically efficient enzyme in the absence of fibrin. Endogenous potentiation of enzyme activity.

    PubMed

    Bicsak, T A; Hsueh, A J

    1989-01-01

    Rat oocytes synthesize tissue plasminogen activator (tPA) in response to stimuli which initiate meiotic maturation. Purified tPA exhibits optimal activity only in the presence of fibrin or fibrin substitutes. Because oocytes are not exposed to fibrin in situ, we investigated the possible stimulation of rat oocyte tPA activity by other endogenous factor(s). Oocytes were obtained from immature female rats which were induced to ovulate with gonadotropins. tPA activity was measured by the plasminogen-dependent cleavage of a chromogenic substrate. Measurements of kinetic parameters with Glu- or Lys-plasminogen revealed a Km for the rat oocyte enzyme of 1.3-2.1 microM compared with 23-24 microM for purified human tPA. Inclusion of the soluble fibrin substitute polylysine lowered the Km of human tPA by 30-fold (0.8 microM) but had no effect on the oocyte tPA Km. Polylysine had no significant effect on the Vmax values. The rate of plasminogen activation catalyzed by oocyte tPA was increased only 4.3-fold by fibrin while fibrin stimulated purified human tPA activity by 15.2-fold. After fractionation of oocyte extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polylysine enhanced oocyte tPA activity as seen by casein zymography. tPA activity in the conditioned medium of a rat insulinoma cell line was also not stimulated with polylysine prior to fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that extravascular cells which elaborate tPA may produce stimulatory factor(s) which allow for full tPA activity at physiological concentrations of plasminogen in the absence of fibrin. PMID:2491854

  9. 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...

  10. 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

  11. Development of radiometric assays for quantification of enzyme activities of the key enzymes of thyroid hormones metabolism.

    PubMed

    Pavelka, S

    2014-01-01

    We newly elaborated and adapted several radiometric enzyme assays for the determination of activities of the key enzymes engaged in the biosynthesis (thyroid peroxidase, TPO) and metabolic transformations (conjugating enzymes and iodothyronine deiodinases, IDs) of thyroid hormones (THs) in the thyroid gland and in peripheral tissues, especially in white adipose tissue (WAT). We also elaborated novel, reliable radiometric methods for extremely sensitive determination of enzyme activities of IDs of types 1, 2 and 3 in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. The use of optimized TLC separation of radioactive products from the unconsumed substrates and film-less autoradiography of radiochromatograms, taking advantage of storage phosphor screens, enabled us to determine IDs enzyme activities as low as 10(-18) katals. In studies of the interaction of fluoxetine (Fluox) with the metabolism of THs, we applied adapted radiometric enzyme assays for iodothyronine sulfotransferases (ST) and uridine 5'-diphospho-glucuronyltransferase (UDP-GT). Fluox is the most frequently used representative of a new group of non-tricyclic antidepressant drugs--selective serotonin re-uptake inhibitors. We used the elaborated assays for quantification the effects of Fluox and for the assessment of the degree of potential induction of rat liver ST and/or UDP-GT enzyme activities by Fluox alone or in combination with T(3). Furthermore, we studied possible changes in IDs activities in murine adipose tissue under the conditions that promoted either tissue hypertrophy (obesogenic treatment) or involution (caloric restriction), and in response to leptin, using our newly developed radiometric enzyme assays for IDs. Our results suggest that deiodinase D1 has a functional role in WAT, with D1 possibly being involved in the control of adipose tissue metabolism and/or accumulation of the tissue. Significant positive correlation between

  12. Effect of age and diet composition on activity of pancreatic enzymes in birds.

    PubMed

    Brzęk, Paweł; Ciminari, M Eugenia; Kohl, Kevin D; Lessner, Krista; Karasov, William H; Caviedes-Vidal, Enrique

    2013-07-01

    Digestive enzymes produced by the pancreas and intestinal epithelium cooperate closely during food hydrolysis. Therefore, activities of pancreatic and intestinal enzymes processing the same substrate can be hypothesized to change together in unison, as well as to be adjusted to the concentration of their substrate in the diet. However, our knowledge of ontogenetic and diet-related changes in the digestive enzymes of birds is limited mainly to intestinal enzymes; it is largely unknown whether they are accompanied by changes in activities of enzymes produced by the pancreas. Here, we analyzed age- and diet-related changes in activities of pancreatic enzymes in five passerine and galloanserine species, and compared them with simultaneous changes in activities of intestinal enzymes. Mass-specific activity of pancreatic amylase increased with age in young house sparrows but not in zebra finches, in agreement with changes in typical dietary starch content and activity of intestinal maltase. However, we found little evidence for the presence of adaptive, diet-related modulation of pancreatic enzymes in both passerine and galloanserine species, even though in several cases the same birds adaptively modulated activities of their intestinal enzymes. In general, diet-related changes in mass-specific activities of pancreatic and intestinal enzymes were not correlated. We conclude that activity of pancreatic enzymes in birds is under strong genetic control, which enables evolutionary adjustment to typical diet composition but is less adept for short term, diet-related flexibility.

  13. Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6.

    PubMed Central

    Panning, B; Smiley, J R

    1993-01-01

    We found that transcription of endogenous human Alu elements by RNA polymerase III was strongly stimulated following infection of HeLa cells with adenovirus type 5, leading to the accumulation of high levels of Alu transcripts initiated from Alu polymerase III promoters. In contrast to previously reported cases of adenovirus-induced activation of polymerase III transcription, induction required the E1b 58-kDa protein and the products of E4 open reading frames 3 and 6 in addition to the 289-residue E1a protein. In addition, E1a function was not required at high multiplicities of infection, suggesting that E1a plays an indirect role in Alu activation. These results suggest previously unsuspected regulatory properties of the adenovirus E1b and E4 gene products and provide a novel approach to the study of the biology of the most abundant class of dispersed repetitive DNA in the human genome. Images PMID:7684492

  14. 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

  15. 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.

  16. 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

  17. Mechanism of porphobilinogen synthase. Requirement of Zn2+ for enzyme activity.

    PubMed

    Bevan, D R; Bodlaender, P; Shemin, D

    1980-03-10

    The role of metal ions in the mechanism of action of bovine liver porphobilinogen synthase was investigated. Studies with chelating agents were consistent with a requirement of metal ions for enzyme activity, and the use of 8-hydroxyquinoline-5-sulfonic acid suggested that Zn2+ was present in the enzyme. The low activity detected in metal-free apoporphobilinogen synthase was attributed to adventitious metal ions. Addition of Zn2+ to the apoenzyme completely restored enzyme activity if the essential sulfhydryl groups on the enzyme were first reduced with sulfhydryl reagents. It does not follow necessarily from this observation that Zn2+ forms a bond with a sulfhydryl group in the enzyme. However, we also observed that Zn2+ did not bind to the enzyme unless the essential cysteinyl residues were reduced. We have concluded that the octameric enzyme contains 4 g atoms of Zn2+/mol from our enzyme activity measurements and binding studies. Alkylation of the enzyme resulted in a marked reduction in the binding of Zn2+ to the enzyme. These observations are consistent with the suggestion that the interaction of the Zn2+ ions with the enzyme occurs with sulfhydryl groups at the active site. It appears that Zn2+ does not participate in substrate binding nor in the maintenance of the quaternary structure of the enzyme. Possible mechanistic roles for Zn2+ in porphobilinogen synthase are discussed. It should be noted that Cd2+ was the only other element found which restored activity to the apoenzyme.

  18. Application of activity-based protein profiling to study enzyme function in adipocytes.

    PubMed

    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.

  19. Microbial extracellular enzyme activities in HUMEX Lake Skjervatjern

    SciTech Connect

    Muenster, U. )

    1992-01-01

    Two microbial extracellular enzyme activities (MEEA) were studied in HUMEX Lake Skjervatjern: acid phosphatase (APHA) and leucine aminopeptidase (LeuAMPA). Both enzyme activities varied in the vertical and horizontal scale in both lake sites. APHA varied in the acidfied Basin A between 945-1706 nmol L[sup [minus]1] h[sup [minus]1] and LeuAMPA between 3.7-25 nmol L[sup [minus]1] h[sup [minus]1]. Both MEEA reached maxima in 0.5 m depth. In the control site (Basin B), APHA was lower by a factor of two, and varied between 156-669 nmol L[sup [minus]1] h[sup [minus]1]. LeuAMPA reached similar values as in Basin A and varied between 7.8-34.8 nmol L[sup [minus]1] h[sup [minus]1]. Maxima of APHA were found in the upper layer (0-2 m), while LeuAMPA had only one distinct maxima at 2-2.5 m depth. The number of bacteria (AFDC) varied between 4.4-8.8 10[sup 6] cells mL[sup [minus]1] and was not significantly different in either side, but both had maxima in the thermocline. Highest specific LeuAMPA activities were found in the thermocline (3.2-4.5 fmol L[sup [minus]1] h[sup [minus]1] cell[sup [minus]1]) in both sides and varied between 0.4-4.5 fmol L[sup [minus]1] h[sup [minus]1] cell[sup [minus]1] in both water columns. The main contributor (60-70%) to LeuAMPA was found in the microplankton fraction, retained on Nuclepore filters with pore sizes between 2.0-0.2 [mu]m. APHA was retained less even on a filter with pore size smaller than 0.2 [mu]m. About 50-70% of APHA passed through 0.2 [mu]m-0.1 [mu]m Nuclepore filters and could be found in the dissolved organic matter (DOM) fraction. APHA and bacteria counts (AFDC) showed a distinct gradient from the littoral zone to the pelagial in the surface water samples (0.2 m depth). APHA and LeuAMPA are regarded as important regulators for nutrient availabilty to microplankton. 40 refs., 6 figs.

  20. 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

  1. 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.

  2. Role of Metabolic Enzymes P450 (CYP) on Activating Procarcinogen and their Polymorphisms on the Risk of Cancers.

    PubMed

    He, Xin; Feng, Shan

    2015-01-01

    Cytochrome P450 (CYP450) enzymes are the most important metabolizing enzyme family exists among all organs. Apart from their role in the deactivation of most endogenous compounds and xenobiotics, they also mediate most procarcinogens oxidation to ultimate carcinogens. There are several modes of CYP450s activation of procarcinogens. 1) Formation of epoxide and diol-epoxides intermediates, such as CYP1A1 and CYP1B1 mediates PAHs oxidation to epoxide intermediates; 2) Formation of diazonium ions, such as CYP2A6, CYP2A13 and CYP2E1 mediates activation of most nitrosamines to unstable metabolites, which can rearrange to give diazonium ions. 3) Formation of reactive semiquinones and quinines, such as CYP1A1 and CYP1B1 transformation of estradiol to catechol estrogens, subsequently formation semiquinones; 4) Formation of toxic O-esterification, such as CYP1A1 and CYP1A2 metabolizes PhIP to N(2)-acetoxy-PhIP and N(2)-sulfonyloxy-PhIP, which are carcinogenic metabolites. 5) Formation of free radical, such as CYP2E1 is involved in activation tetrachloromethane to free radicals. While for CYP2B6 and CYP2D6, only a minor role has been found in procarcinogens activation. In addition, as the gene polymorphisms reflected, the polymorphisms of CYP1A1 (-3801T/C and -4889A/G), CYP1A2 (- 163C/A and -2467T/delT), CYP1B1 (-48G/C, -119G/T and -432G/C), CYP2E1 (-1293G/C and -1053 C/T) have been associated with an increased risk of lung cancer. The polymorphisms CYP1A1 (-3801T/C and -4889A/G), and CYP2E1 (PstI/Rsa and 9-bp insertion) have an association with higher risk colon cancers, whereas CYP1A2 (-163C/A and -3860G/A) polymorphism is found to be among the protective factors. The polymorphisms CYP1A1 (-3801T/C and -4889A/G), CYP1B1 -432G/C, CYP2B6 (-516G/T and -785A/G) may increase the risk of breast cancer. In conclusion, CYP1A1, CYP1A2, CYP1B1, CYP2A6, and CYP2E1 are responsible for most of the procarcinogens activation, and their gene polymorphisms are associated with the risk of

  3. Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

    PubMed Central

    Meneely, Kathleen M.; Luo, Qianyi; Lamb, Audrey L.

    2013-01-01

    The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the “interchange” hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, “permute” hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities. PMID:24055536

  4. Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities.

    PubMed

    Meneely, Kathleen M; Luo, Qianyi; Lamb, Audrey L

    2013-11-01

    The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the "interchange" hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, "permute" hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.

  5. 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

  6. Erectile Dysfunction Drugs Changed the Protein Expressions and Activities of Drug-Metabolising Enzymes in the Liver of Male Rats

    PubMed Central

    Hassan, Mostafa

    2016-01-01

    Erectile dysfunction (ED) is a major health problem and is mainly associated with the persistent inability of men to maintain sufficient erection for satisfactory sexual performance. Millions of men are using sildenafil, vardenafil, and/or tadalafil for ED treatment. Cytochrome P450s (CYPs) play a central role in the metabolism of a wide range of xenobiotics as well as endogenous compounds. Susceptibility of individuals to the adverse effects of different drugs is mainly dependent on the expression of CYPs proteins. Therefore, changes in activities of phase I drug-metabolising enzymes [arylhydrocarbon hydroxylase (AHH), dimethylnitrosamine N-demethylase (DMN-dI), 7-ethoxycoumarin-O-deethylase (ECOD), and ethoxyresorufin-O-deethylase ((EROD)] and the protein expression of different CYPs isozymes (CYP1A2, CYP2E1, CYP2B1/2, CYP3A4, CYP2C23, and CYP2C6) were determined after treatment of male rats with either low or high doses of sildenafil (Viagra), tadalafil (Cialis), and/or vardenafil (Levitra) for 3 weeks. The present study showed that low doses of tadalafil and vardenafil increased DMN-dI activity by 32 and 23%, respectively. On the other hand, high doses of tadalafil, vardenafil, and sildenafil decreased such activity by 50, 56, and 52%, respectively. In addition, low doses of tadalafil and vardenafil induced the protein expression of CYP2E1. On the other hand, high doses of either tadalafil or sildenafil were more potent inhibitors to CYP2E1 expression than vardenafil. Moreover, low doses of both vardenafil and sildenafil markedly increased AHH activity by 162 and 247%, respectively, whereas high doses of tadalafil, vardenafil, and sildenafil inhibited such activity by 36, 49, and 57% and inhibited the EROD activity by 39, 49, and 33%, respectively. Low and high doses of tadalafil, vardenafil, and sildenafil inhibited the activity of NADPH-cytochrome c reductase as well as its protein expression. In addition, such drugs inhibited the expression of CYP B1/2 along

  7. 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

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

    PubMed

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

    2016-02-10

    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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. TEMPERATURE ACTIVATION OF CERTAIN RESPIRATORY ENZYMES OF STENOTHERMOPHILIC BACTERIA

    PubMed Central

    Gaughran, Eugene R. L.

    1949-01-01

    The results of this study of the effect of temperature on the respiratory mechanism of five stenothermophilic bacteria may be summarized as follows:— 1. The respiratory mechanism and its various components of the stenothermophilic bacteria were found to function at temperatures below the minimum temperature for growth of these organisms. In every case the rates of the individual reactions involved in the respiratory chain increased exponentially with temperature until the temperature at which inactivation became apparent was reached. 2. The mean activation energies, calculated from the "best" value for the slope of the straight lines resulting from a plot of log rate against the reciprocal of the absolute temperature were: Dehydrogenases: 28,000 to 28,500 calories per gram molecule. Glucose, fructose, galactose, mannose, xylose, arabinose, maltose, lactose, sucrose, glycine, β-alanine, monosodium glutamate, (asparagine). 19,500 to 20,500 calories per gram molecule. Ethyl alcohol, succinate, pyruvate, lactate, acetate. 19,500 to 20,500 calories per gram molecule. Ethyl alcohol, succinate, pyruvate, lactate, acetate. 15,000 calories per gram molecule. Formate. Cytochrome oxidase and cytochrome b and c (substrate: p-phenylenediamine): 16,800 calories per gram molecule. Cytochrome oxidase and cytochrome c (substrate: hydroquinone): 20,200 calories per gram molecule. Catalase: 4,100 calories per gram molecule. Complete aerobic respiratory system (plus added glucose): 29,500 calories per gram molecule. 3. The identity of the energies of activation of the respiratory system and its enzymic components at temperatures above and below the minimum temperature for growth of the stenothermophilic bacteria was demonstrated. 4. An attempt has been made to indicate a relationship between the nature of the substrate and the activation energy by grouping substrates on the basis of common µ values obtained for their dehydrogenation by resting cell preparations of

  14. 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

  15. Activities of Tricarboxylic Acid Cycle Enzymes, Glyoxylate Cycle Enzymes, and Fructose Diphosphatase in Bakers' Yeast During Adaptation to Acetate Oxidation

    PubMed Central

    Gosling, J. P.; Duggan, P. F.

    1971-01-01

    Bakers' yeast oxidizes acetate at a high rate only after an adaptation period during which the capacity of the glyoxylate cycle is found to increase. There was apparently no necessity for the activity of acetyl-coenzyme A synthetase, the capacity of the tricarboxylic acid cycle, or the concentrations of the cytochromes to increase for this adaptation to occur. Elevation of fructose 1,6 diphosphatase occurred only when acetate oxidation was nearly maximal. Cycloheximide almost completely inhibited adaptation as well as increases in the activities of isocitrate lyase and aconitate hydratase, the only enzymes assayed. p-Fluorophenylalanine was partially effective and chloramphenicol did not inhibit at all. The presence of ammonium, which considerably delayed adaptation of the yeast to acetate oxidation, inhibited the increases in the activities of the glyoxylate cycle enzymes to different degrees, demonstrating noncoordinate control of these enzymes. Under the various conditions, the only enzyme activity increase consistently related to the rising oxygen uptake rate was that of isocitrate lyase which apparently limited the activity of the cycle. PMID:5557595

  16. 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.

  17. Chaperone-like activities of {alpha}-synuclein: {alpha}-Synuclein assists enzyme activities of esterases

    SciTech Connect

    Ahn, Misun; Kim, SeungBum; Kang, Mira; Ryu, Yeonwoo . E-mail: ywryu@ajou.ac.kr; Doohun Kim, T. . E-mail: doohunkim@ajou.ac.kr

    2006-08-11

    {alpha}-Synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of {alpha}-synuclein has not yet been known. Here we have shown that {alpha}-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of {alpha}-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with {alpha}-synuclein. Our results indicate that {alpha}-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo.

  18. Controlled exogenous enzyme imbibition and activation in whole chickpea seed enzyme reactor (SER).

    PubMed

    Kliger, Eynav; Fischer, Lutz; Lutz-Wahl, Sabine; Saguy, I Sam

    2011-05-01

    Chickpeas are of excellent quality (protein, vitamins, minerals, unsaturated fatty acids) and very low in phytoestrogen, making them a potentially promising source for vegetarian-based infant formula (VBIF). However, their high starch and fiber concentration could hinder their utilization for infants. To overcome this natural shortcoming, a solid-state "enzymation" (SSE) process was developed in which imbibition of exogenous enzyme facilitates hydrolysis within the intact chickpea seed. The process was termed seed enzyme reactor (SER). Liquid imbibition data of dry chickpeas during soaking were fitted with the Weibull distribution model. The derived Weibull shape parameter, β, value (0.77 ± 0.11) indicated that the imbibition mechanism followed Fickian diffusion. Imbibition occurred through the coat and external layers. The process was tested using green fluorescent protein (GFP) as an exogenous marker, and involved soaking, thermal treatment, peeling, microwave partial drying, rehydration in enzyme solution, and SSE at an adjusted pH, time, and temperature. Amylases, or a combination of amylases and cellulases, resulted in significant carbohydrate hydrolysis (23% and 47% of the available starch, respectively). In addition, chickpea initial raffinose and stachyose concentration was significantly reduced (91% and 92%, respectively). The process could serve as a proof of concept, requiring additional development and optimization to become a full industrial application.

  19. Carbohydrate-active enzymes exemplify entropic principles in metabolism

    PubMed Central

    Kartal, Önder; Mahlow, Sebastian; Skupin, Alexander; Ebenhöh, Oliver

    2011-01-01

    Glycans comprise ubiquitous and essential biopolymers, which usually occur as highly diverse mixtures. The myriad different structures are generated by a limited number of carbohydrate-active enzymes (CAZymes), which are unusual in that they catalyze multiple reactions by being relatively unspecific with respect to substrate size. Existing experimental and theoretical descriptions of CAZyme-mediated reaction systems neither comprehensively explain observed action patterns nor suggest biological functions of polydisperse pools in metabolism. Here, we overcome these limitations with a novel theoretical description of this important class of biological systems in which the mixing entropy of polydisperse pools emerges as an important system variable. In vitro assays of three CAZymes essential for central carbon metabolism confirm the power of our approach to predict equilibrium distributions and non-equilibrium dynamics. A computational study of the turnover of the soluble heteroglycan pool exemplifies how entropy-driven reactions establish a metabolic buffer in vivo that attenuates fluctuations in carbohydrate availability. We argue that this interplay between energy- and entropy-driven processes represents an important regulatory design principle of metabolic systems. PMID:22027553

  20. Detection of Sulfatase Enzyme Activity with a CatalyCEST MRI Contrast Agent.

    PubMed

    Sinharay, Sanhita; Fernández-Cuervo, Gabriela; Acfalle, Jasmine P; Pagel, Mark D

    2016-05-01

    A chemical exchange saturation transfer (CEST) MRI contrast agent has been developed that detects sulfatase enzyme activity. The agent produces a CEST signal at δ=5.0 ppm before enzyme activity, and a second CEST signal appears at δ=9.0 ppm after the enzyme cleaves a sulfate group from the agent. The comparison of the two signals improved detection of sulfatase activity.

  1. Effects of capsaicin and dihydrocapsaicin on human and rat liver microsomal CYP450 enzyme activities in vitro and in vivo.

    PubMed

    Zhang, Qing-Hao; Hu, Jin-Ping; Wang, Bao-Lian; Li, Yan

    2012-01-01

    Capsaicin and dihydrocapsaicin, the two most abundant members of capsaicinoids in chili peppers, are widely used as food additives and for other purposes. In this study, we examined the inhibitory potentials of capsaicin and dihydrocapsaicin against CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 activities in human liver microsomes. The effects of these two capsaicinoids on CYP450 enzymes were also evaluated in vivo in rats. The results demonstrated that capsaicin and dihydrocapsaicin moderately inhibited five isozymes (IC₅₀) values ranging from 4.4 to 61.8 μM), with the exception of CYP2E1 (IC₅₀ > 200 μM). Both capsaicinoids exhibited competitive, mixed, and noncompetitive inhibition on these isozymes (K (i) = 3.1 ± 0.5 - 78.6 ± 8.4 μM). Time-dependent inhibition of CYP3A4/5 by capsaicin was found. After multiple administrations of capsaicin and dihydrocapsaicin (1, 4, and 10 mg/kg) to rats, chlorzoxazone 6-hydroxylase activity and the expression of CYP2E1 were increased in liver microsomes. Our findings indicated that the possibility of food-drug interactions mediated by capsaicin and dihydrocapsaicin could not be excluded, and provided the useful information for evaluating the anticarcinogenic potentials of these two capsaicinoids. PMID:22375877

  2. Cross-linked enzyme aggregates (CLEAs) of Pencilluim notatum lipase enzyme with improved activity, stability and reusability characteristics.

    PubMed

    Rehman, Saima; Bhatti, Haq Nawaz; Bilal, Muhammad; Asgher, Muhammad

    2016-10-01

    Cross-linked enzyme aggregates (CLEAs) are considered as an effective tool for the immobilization of enzyme. In this study, Pencillium notatum lipase (PNL) was immobilized as carrier free cross-linked enzyme aggregates using glutaraldehyde (GLA) and Ethylene glycol-bis [succinic acid N-hydroxysuccinimide] (EG-NHS) as cross-linking agents. The optimal conditions for the synthesis of an efficient lipase CLEAs such as precipitant type, the nature and amount of cross-linking reagent, and cross-linking time were optimized. The recovered activities of CLEAs were considerably dependent on the concentration of GLA; however, the activity recovery was not severely affected by EG-NHS as a mild cross-linker. The EG-NHS aggregates displayed superior hydrolytic (52.08±2.52%) and esterification (64.42%) activities as compared to GLA aggregates which showed 23.8±1.86 and 34.54% of hydrolytic and esterification activity, respectively. Morphological analysis by fluorescence and scanning electron microscope revealed that EG-NHS aggregates were smaller in size with larger surface area compared to GLA aggregates. The pH optima of both types of CLEAs were displaced to slightly alkaline region and higher temperature as compared to native enzyme. Highest enzyme activity of CLEAs was achieved at the pH of 9.0 and 42°C temperature. Moreover, a significant improvement in the thermal resistance was also recorded after immobilization. After ten reusability cycles in aqueous medium, GLA and EG-NHS cross-linked lipase CLEAs preserved 63.62% and 70.9% of their original activities, respectively. The results suggest that this novel CLEA-lipase is potentially usable in many industrial applications.

  3. Cross-linked enzyme aggregates (CLEAs) of Pencilluim notatum lipase enzyme with improved activity, stability and reusability characteristics.

    PubMed

    Rehman, Saima; Bhatti, Haq Nawaz; Bilal, Muhammad; Asgher, Muhammad

    2016-10-01

    Cross-linked enzyme aggregates (CLEAs) are considered as an effective tool for the immobilization of enzyme. In this study, Pencillium notatum lipase (PNL) was immobilized as carrier free cross-linked enzyme aggregates using glutaraldehyde (GLA) and Ethylene glycol-bis [succinic acid N-hydroxysuccinimide] (EG-NHS) as cross-linking agents. The optimal conditions for the synthesis of an efficient lipase CLEAs such as precipitant type, the nature and amount of cross-linking reagent, and cross-linking time were optimized. The recovered activities of CLEAs were considerably dependent on the concentration of GLA; however, the activity recovery was not severely affected by EG-NHS as a mild cross-linker. The EG-NHS aggregates displayed superior hydrolytic (52.08±2.52%) and esterification (64.42%) activities as compared to GLA aggregates which showed 23.8±1.86 and 34.54% of hydrolytic and esterification activity, respectively. Morphological analysis by fluorescence and scanning electron microscope revealed that EG-NHS aggregates were smaller in size with larger surface area compared to GLA aggregates. The pH optima of both types of CLEAs were displaced to slightly alkaline region and higher temperature as compared to native enzyme. Highest enzyme activity of CLEAs was achieved at the pH of 9.0 and 42°C temperature. Moreover, a significant improvement in the thermal resistance was also recorded after immobilization. After ten reusability cycles in aqueous medium, GLA and EG-NHS cross-linked lipase CLEAs preserved 63.62% and 70.9% of their original activities, respectively. The results suggest that this novel CLEA-lipase is potentially usable in many industrial applications. PMID:27365121

  4. Modulation of insulin degrading enzyme activity and liver cell proliferation.

    PubMed

    Pivovarova, Olga; von Loeffelholz, Christian; Ilkavets, Iryna; Sticht, Carsten; Zhuk, Sergei; Murahovschi, Veronica; Lukowski, Sonja; Döcke, Stephanie; Kriebel, Jennifer; de las Heras Gala, Tonia; Malashicheva, Anna; Kostareva, Anna; Lock, Johan F; Stockmann, Martin; Grallert, Harald; Gretz, Norbert; Dooley, Steven; Pfeiffer, Andreas F H; Rudovich, Natalia

    2015-01-01

    Diabetes mellitus type 2 (T2DM), insulin therapy, and hyperinsulinemia are independent risk factors of liver cancer. Recently, the use of a novel inhibitor of insulin degrading enzyme (IDE) was proposed as a new therapeutic strategy in T2DM. However, IDE inhibition might stimulate liver cell proliferation via increased intracellular insulin concentration. The aim of this study was to characterize effects of inhibition of IDE activity in HepG2 hepatoma cells and to analyze liver specific expression of IDE in subjects with T2DM. HepG2 cells were treated with 10 nM insulin for 24 h with or without inhibition of IDE activity using IDE RNAi, and cell transcriptome and proliferation rate were analyzed. Human liver samples (n = 22) were used for the gene expression profiling by microarrays. In HepG2 cells, IDE knockdown changed expression of genes involved in cell cycle and apoptosis pathways. Proliferation rate was lower in IDE knockdown cells than in controls. Microarray analysis revealed the decrease of hepatic IDE expression in subjects with T2DM accompanied by the downregulation of the p53-dependent genes FAS and CCNG2, but not by the upregulation of proliferation markers MKI67, MCM2 and PCNA. Similar results were found in the liver microarray dataset from GEO Profiles database. In conclusion, IDE expression is decreased in liver of subjects with T2DM which is accompanied by the dysregulation of p53 pathway. Prolonged use of IDE inhibitors for T2DM treatment should be carefully tested in animal studies regarding its potential effect on hepatic tumorigenesis.

  5. A microsystem to assay lysosomal enzyme activities in cultured retinal pigment epithelial cells.

    PubMed

    Cabral, L; Unger, W; Boulton, M; Marshall, J

    1988-11-01

    A microsystem to assay the activity of lysosomal enzymes in a small number of cultured RPE cells is described. The activities of acid phosphatase, a-mannosidase, B-glucuronidase and N-acetyl-B-glucosaminidase were estimated in different human RPE cultures of varying passages. Some biochemical characteristics for each of the enzyme assays were studied including the effect of pH, the saturating concentrations of the appropriate substrates and the relationship between the enzyme activity and the number of cells assayed. The method presented is straightforward, avoids complicated tissue fractionation procedures and is able to estimate enzyme activities in as few as 10(4) cells. PMID:3243083

  6. Enzyme activities in plasma, kidney, liver, and muscle of five avian species

    USGS Publications Warehouse

    Franson, J.C.; Murray, H.C.; Bunck, C.

    1985-01-01

    Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine phosphokinase (CPK), and lactate dehydrogenase (LDH) were determined in plasma, kidney, liver, and muscle from five species of captive birds. Few differences occurred in plasma activities between sexes but considerable differences occurred between species. All five enzymes were detected in each of the tissues sampled. Relative enzyme activities in liver, kidney, and muscle were similar for each species. CPK activity was much higher in muscle than in liver or kidney and, of the five enzymes studied, may be the best indicator of muscle damage. Most of the other enzymes were more evenly distributed among the three tissues, and no organ-specific enzyme could be identified for liver or kidney. Because of interspecific variations in plasma enzyme activities, it is important to establish baseline values for each species to ensure accurate interpretation of results.

  7. Annexation of a high-activity enzyme in a synthetic three-enzyme complex greatly decreases the degree of substrate channeling.

    PubMed

    You, Chun; Zhang, Y-H Percival

    2014-06-20

    The self-assembled three-enzyme complex containing triosephosphate isomerase (TIM), aldolase (ALD), and fructose 1,6-biphosphatase (FBP) was constructed via a mini-scaffoldin containing three different cohesins and the three dockerin-containing enzymes. This enzyme complex exhibited 1 order of magnitude higher initial reaction rates than the mixture of noncomplexed three enzymes. In this enzyme cascade reactions, the reaction mediated by ALD was the rate-limiting step. To understand the in-depth role of the rate-limiting enzyme ALD in influencing the substrate channeling effect of synthetic enzyme complexes, low-activity ALD from Thermotoga maritima was replaced with a similar-size ALD isolated from Thermus thermophilus, where the latter had more than 5 times specific activity of the former. The synthetic three-enzyme complexes annexed with either low-activity or high-activity ALDs exhibited higher initial reaction rates than the mixtures of the two-enzyme complex (TIM-FBP) and the nonbound low-activity or high activity ALD at the same enzyme concentration. It was also found that the annexation of more high-activity ALD in the synthetic enzyme complexes drastically decreased the degree of substrate channeling from 7.5 to 1.5. These results suggested that the degree of substrate channeling in synthetic enzyme complexes depended on the enzyme choice. This study implied that the construction of synthetic enzyme enzymes in synthetic cascade pathways could be a very important tool to accrelerate rate-limiting steps controlled by low-activity enzymes.

  8. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    PubMed

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

  9. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    PubMed

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor. PMID:26802007

  10. Guanidinylated neomycin mediates heparan sulfate-dependent transport of active enzymes to lysosomes.

    PubMed

    Sarrazin, Stéphane; Wilson, Beth; Sly, William S; Tor, Yitzhak; Esko, Jeffrey D

    2010-07-01

    Guanidinylated neomycin (GNeo) can transport bioactive, high molecular weight cargo into the interior of cells in a process that depends on cell surface heparan sulfate proteoglycans. In this report, we show that GNeo-modified quantum dots bind to cell surface heparan sulfate, undergo endocytosis and eventually reach the lysosomal compartment. An N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS) was prepared and conjugated to two lysosomal enzymes, beta-D-glucuronidase (GUS) and alpha-L-iduronidase. Conjugation did not interfere with enzyme activity and enabled binding of the enzymes to heparin-Sepharose and heparan sulfate on primary human fibroblasts. Cells lacking the corresponding lysosomal enzyme took up sufficient amounts of the conjugated enzymes to restore normal turnover of glycosaminoglycans. The high capacity of proteoglycan-mediated uptake suggests that this method of delivery might be used for enzyme replacement or introduction of foreign enzymes into cells.

  11. Activity-based proteomics probes for carbohydrate-processing enzymes: current trends and future outlook.

    PubMed

    Stubbs, Keith A

    2014-05-22

    Carbohydrate-processing enzymes are gaining more attention due to their roles in health and disease as these enzymes are involved in the construction and deconstruction of vast arrays of glycan structures. As a result, the development of methods to identify these enzymes in complex biological systems is of increasing interest. Activity-based proteomics probes (ABPPs) are increasingly being used in glycobiology to detect and identify functionally related proteins (and homologues) within a biological system. This review will describe the design of activity-based proteomics probes, provide examples of compounds that have been used to profile activity in the area of carbohydrate-processing enzymes, and give some future perspectives.

  12. Preparation of biocatalytic nanofibers with high activity and stability via enzyme aggregate coating on polymer nanofibers

    SciTech Connect

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

    2005-04-01

    We have developed a unique approach for the fabrication of enzyme coating on the surface of electrospun polymer nanofibers. This approach employs covalent attachment of seed enzymes onto nanofibers, followed by the glutaraldehyde treatment that crosslinks additional enzymes onto the seed enzyme molecules. These crosslinked enzyme aggregates, covalently attached to the nanofibers via seed enzyme linker, would improve not only the enzyme activity due to increased enzyme loading, but also the enzyme stability. To demonstrate the principle of concept, we fabricated the coating of alpha-chymotrypsin (CT) on the nanofibers electrospun from a mixture of polystyrene and poly(styrene-co-maleic anhydride). The addition of poly(styrene-co-maleic anhydride) makes it much easier to attach the seed enzyme molecules onto electrospun nanofibers without any rigorous functionalization of nanofibers for the attachment of enzymes. The initial activity of final CT coating was 17 and 9 times higher than those of simply-adsorbed CT and covalently-attached CT, respectively. While adsorbed and covalently-attached CT resulted in a serious enzyme leaching during initial incubation in a shaking condition, the CT coating did not show any leaching from the beginning of incubation in the same condition. As a result, the enzyme stability of CT coating was impressively improved with a half-life of 686 days under rigorous shaking while the half-life of covalently-attached CT was only 21 hours. This new approach of enzyme coating with high stability and activity will make a great impact in various applications of enzymes such as bioconversion, bioremediation, and biosensors.

  13. Molecular architectures and functions of radical enzymes and their (re)activating proteins.

    PubMed

    Shibata, Naoki; Toraya, Tetsuo

    2015-10-01

    Certain proteins utilize the high reactivity of radicals for catalysing chemically challenging reactions. These proteins contain or form a radical and therefore named 'radical enzymes'. Radicals are introduced by enzymes themselves or by (re)activating proteins called (re)activases. The X-ray structures of radical enzymes and their (re)activases revealed some structural features of these molecular apparatuses which solved common enigmas of radical enzymes—i.e. how the enzymes form or introduce radicals at the active sites, how they use the high reactivity of radicals for catalysis, how they suppress undesired side reactions of highly reactive radicals and how they are (re)activated when inactivated by extinction of radicals. This review highlights molecular architectures of radical B12 enzymes, radical SAM enzymes, tyrosyl radical enzymes, glycyl radical enzymes and their (re)activating proteins that support their functions. For generalization, comparisons of the recently reported structures of radical enzymes with those of canonical radical enzymes are summarized here.

  14. Endothelin converting enzyme (ECE) activity in human vascular smooth muscle

    PubMed Central

    Maguire, Janet J; Johnson, Christopher M; Mockridge, James W; Davenport, Anthony P

    1997-01-01

    of a phosphoramidon-sensitive ECE on the smooth muscle layer of the human umbilical vein which can convert big ET-1, big ET-2(1-37), big ET-2(1-38) and big ET-3 to their mature biologically active forms. The precise subcellular localization of this enzyme and its physiological relevance remains to be determined. PMID:9422810

  15. 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

  16. RING E3-Catalyzed E2 Self-Ubiquitination Attenuates the Activity of Ube2E Ubiquitin-Conjugating Enzymes.

    PubMed

    Banka, Prerana Agarwal; Behera, Adaitya Prasad; Sarkar, Sayani; Datta, Ajit B

    2015-07-01

    Ubiquitination of a target protein is accomplished through sequential actions of the E1, E2s, and the E3s. E2s dictate the modification topology while E3 ligases confer substrate specificity and recruit the cognate E2. Human genome codes for ~35 different E2 proteins; all of which contain the characteristic ubiquitin-conjugating UBC core domain sufficient for catalysis. Many of these E2 enzymes also have N- or C-terminal extensions; roles of which are not very well understood. We show that the N-terminal extension of Ube2E1 undergoes intramolecular auto-ubiquitination. This self-ubiquitination activity is enhanced in the presence of interacting RING E3 ligases and results in a progressive attenuation of the E2 activity toward substrate/E3 modification. We also find that the N-terminal ubiquitination sites are conserved in all the three Ube2Es and replacing them with arginine renders all three full-length Ube2Es equally active as their core UBC domains. Based on these results, we propose that E3-catalyzed self-ubiquitination acts as a key regulatory mechanism that controls the activity of Ube2E class of ubiquitin E2s.

  17. Enhanced diffusion, chemotaxis, and pumping by active enzymes: progress toward an organizing principle of molecular machines.

    PubMed

    Astumian, R Dean

    2014-12-23

    Active enzymes diffuse more rapidly than inactive enzymes. This phenomenon may be due to catalysis-driven conformational changes that result in "swimming" through the aqueous solution. Recent additional work has demonstrated that active enzymes can undergo chemotaxis toward regions of high substrate concentration, whereas inactive enzymes do not, and, further, that active enzymes immobilized at surfaces can directionally pump liquids. In this Perspective, I will discuss these phenomena in light of Purcell's work on directed motion at low Reynold's number and in the context of microscopic reversibility. The conclusions suggest that a deep understanding of catalytically driven enhanced diffusion of enzymes and related phenomena can lead toward a general organizing principle for the design, characterization, and operation of molecular machines.

  18. Antibacterial, Antifungal, Cytotoxic, Phytotoxic, Insecticidal, and Enzyme Inhibitory Activities of Geranium wallichianum

    PubMed Central

    Ismail, Muhammad; Hussain, Javid; Khan, Arif-ullah; Khan, Abdul Latif; Ali, Liaqat; Khan, Farman-ullah; Khan, Amir Zada; Niaz, Uzma; Lee, In-Jung

    2012-01-01

    The present study describes the phytochemical investigations of the crude extracts of rhizomes and leaves of Geranium wallichianum. The crude extracts were fractionated to obtain n-hexane, ethyl acetate, and n-butanol fractions, which were subjected to different biological activities and enzyme inhibition assays to explore the therapeutic potential of this medicinally important herb. The results indicated that the crude extracts and different fractions of rhizomes and leaves showed varied degree of antimicrobial activities and enzyme inhibitions in different assays. Overall, the rhizome extract and its different fractions showed comparatively better activities in various assays. Furthermore, the purified constituents from the repeated chromatographic separations were also subjected to enzyme inhibition studies against three different enzymes. The results of these studies showed that lipoxygenase enzyme was significantly inhibited as compared to urease. In case of chemical constituents, the sterols (2–4) showed no inhibition, while ursolic acid (1) and benzoic ester (6) showed significant inhibition of urease enzymes. PMID:23049606

  19. [Glycolytic activity of enzyme preparation from the red king crab (Paralithodes camtschaticus) hepatopancreas].

    PubMed

    Rysakova, K S; Novikov, V Iu; Mukhin, V A; Serafimchik, E M

    2008-01-01

    Enzyme preparation exhibiting glycolytic activity yielding chitooligosaccharides along with N-acetyl-D-glucosamine was obtained from the red king crab (Paralithodes camtschaticus) hepatopancreas. The results of the analysis confirmed the presence of endo- and exochitinase activities in the preparation. HPLC showed that the hydrolysis products of chitin and chitosan did not contain D(+)-glucosamine, which is indicative of the absence of deacetylase and, apparently, exochitosanase activities. A comparison of the dependence of the enzyme preparation activity on temperature and pH of the incubation medium suggests that chitinase and protease activities are exhibited by different enzymes.

  20. 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

  1. Spatial distribution of enzyme activities along the root and in the rhizosphere of different plants

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

    Extracellular enzymes are important for decomposition of many biological macromolecules abundant in soil such as cellulose, hemicelluloses and proteins. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. So far acquisition of in situ data about local activity of different enzymes in soil has been challenged. That is why there is an urgent need in spatially explicit methods such as 2-D zymography to determine the variation of enzymes along the roots in different plants. Here, we developed further the zymography technique in order to quantitatively visualize the enzyme activities (Spohn and Kuzyakov, 2013), with a better spatial resolution We grew Maize (Zea mays L.) and Lentil (Lens culinaris) in rhizoboxes under optimum conditions for 21 days to study spatial distribution of enzyme activity in soil and along roots. We visualized the 2D distribution of the activity of three enzymes:β-glucosidase, leucine amino peptidase and phosphatase, 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 shows different pattern of spatial distribution of enzyme activity along roots and soil of different plants. We observed a uniform distribution of enzyme activities along the root system of Lentil. However, root system of Maize demonstrated inhomogeneity of enzyme activities. The apical part of an individual root (root tip) in maize showed the highest activity. The activity of all enzymes was the highest at vicinity of the roots and it 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

  2. A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes*

    PubMed Central

    Vidal-Melgosa, Silvia; Pedersen, Henriette L.; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B.; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G. T.

    2015-01-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

  3. A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

    PubMed

    Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T

    2015-04-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths.

  4. Temperature adaptation of enzymes: roles of the free energy, the enthalpy, and the entropy of activation.

    PubMed

    Low, P S; Bada, J L; Somero, G N

    1973-02-01

    The enzymic reactions of ectothermic (cold-blooded) species differ from those of avian and mammalian species in terms of the magnitudes of the three thermodynamic activation parameters, the free energy of activation (DeltaG()), the enthalpy of activation (DeltaH()), and the entropy of activation (DeltaS()). Ectothermic enzymes are more efficient than the homologous enzymes of birds and mammals in reducing the DeltaG() "energy barrier" to a chemical reaction. Moreover, the relative importance of the enthalpic and entropic contributions to DeltaG() differs between these two broad classes of organisms.

  5. [Relationship between soil enzyme activities and trace element contents in Eucalyptus plantation soil].

    PubMed

    Li, Yuelin; Peng, Shaolin; Li, Zhihui; Ren, Hai; Li, Zhi'an

    2003-03-01

    Canonical correlation analysis on soil enzyme activities and trace element contents in Eucalyptus plantation soil showed that among the test elements, only Zn and Mn affected enzyme activity. Both Zn and Mn increased soil proteinase activity. Zn decreased the activities of soil urease and peroxidase, while Mn promoted them. "Integral soil enzyme factor" could be used as an index of soil fertility. Together with other growth factors, this index should be considered when evaluating soil fertility of Eucalyptus forest sites. It also had a definite significance on the division of Eucalyptus soil families. PMID:12836538

  6. 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

  7. Secretion of an articular cartilage proteoglycan-degrading enzyme activity by murine T lymphocytes in vitro.

    PubMed Central

    Kammer, G M; Sapolsky, A I; Malemud, C J

    1985-01-01

    Destruction of articular cartilage is the hallmark of inflammatory arthritides. Enzymes elaborated by mononuclear cells infiltrating the synovium mediate, in part, the degradation of the cartilage extracellular matrix. Since mononuclear cells are the dominant cell type found in chronic inflammatory synovitis, we investigated whether interaction of immune mononuclear cells with antigen initiated the synthesis and secretion of a proteoglycan-degrading enzyme activity. Proteoglycan-degrading enzyme activity was monitored by the capacity of murine spleen cell conditioned medium to release [3H]serine/35SO4 incorporated into rabbit cartilage proteoglycan monomer fraction (A1D1), and by the relative change in specific viscosity of bovine nasal cartilage proteoglycan monomer. The results demonstrated that both virgin and immune mononuclear cells spontaneously generated proteoglycan-degrading enzyme activity and that cellular activation and proliferation induced by the antigen keyhole limpet hemocyanin or the mitogen phytohemagglutinin was not required. Kinetic studies demonstrated stable release of the enzyme activity over 72 h. Cell separation studies showed that T lymphocytes, a thymoma line, and macrophages separately produced proteoglycan-degrading enzyme activity. The enzyme activity has been partially characterized and appears to belong to a class of neutral pH metal-dependent proteinases. These observations, the first to demonstrate that T lymphocytes secrete an enzyme capable of degrading cartilage proteoglycan, raise the possibility that this enzyme activity contributes to cartilage extracellular matrix destruction in vivo. Moreover, these data support the conclusion that production of this enzyme by T lymphocytes is independent of an antigen-specific stimulus. PMID:3897284

  8. 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

  9. Redox Regulation of Human Estrogen Sulfotransferase (hSULT1E1)†

    PubMed Central

    Maiti, Smarajit; Zhang, Jimei; Chen, Guangping

    2007-01-01

    Sulfotransferases (SULTs) are enzymes that catalyze the sulfation of hydroxyl-containing compounds. Sulfation regulates hormone activities and detoxifies xenobiotics. Human estrogen sulfotransferase (hSULT1E1) catalyzes the sulfation of estrogens and regulates estrogen bioactivities. Oxidative regulation provides a biological mechanism for regulating enzyme activities in vivo. The oxidative regulation of human SULTs has not been reported. In this study, we used amino acid modification, manipulation of intracellular redox state, and site-directed mutagenesis to study the redox regulation of human SULTs and specifically the mechanism of hSULT1E1 inhibitory regulation by oxidized glutathione (GSSG). Of the four major human SULTs, hSULT1A1, hSULT1A3, and hSULT2A1 do not undergo redox regulation; hSULT1E1, on the other hand, can be redox regulated. GSSG inactivated hSULT1E1 activity in an efficient, time- and concentration-dependant manner. The co-enzyme adenosine 3′-phosphate 5′-phosphosulfate protected hSULT1E1 from GSSG-associated inactivation. A reduced glutathione (GSH) inducer (N-acetyl cysteine) significantly increased while a GSH depletor (buthionine sulfoxamine) significantly decreased hSULT1E1 activity, but both failed to affect the amount of hSULT1E1 protein in human hepatocyte carcinoma Hep G2 cells. Crystal structure suggested that no Cys residues exist near the active sites of hSULT1A1, hSULT1A3, and hSULT2A1, but Cys residues do exist within the active site of hSULT1E1. Site-directed mutagenesis demonstrated that Cys83 is critical for the redox regulation of hSULT1E1. This first report on the redox regulation of human SULTs suggests that the redox regulation of hSULT1E1 may interrupt the regulation and function of estrogens under various physiological and pathological conditions. PMID:17266938

  10. Determination of Diamine Oxidase in Lentil Seedlings by Enzymic Activity and Immunoreactivity

    PubMed Central

    Federico, Rodolfo; Angelini, Riccardo; Cesta, Alberinda; Pini, Carlo

    1985-01-01

    A competitive radioimmunoassay for the quantitation of diamine oxidase (EC 1.4.3.6) from Lens culinaris is reported. Specific antibodies raised in rabbits immunized with a homogeneous preparation of the enzyme were incubated with purified 125I-enzyme and with either unlabeled diamine oxidase or plant material. Antigen-antibody complexes were isolated from the mixture by incubation with Staphylococcus protein A. The sensitivity of the test was about 5 nanograms in terms of enzyme protein. This assay was applied to the determination of the enzyme in extracts from lentil shoots grown either in the dark or in the light. Diamine oxidase activity and enzyme protein (as determined by radioimmunoassay) were measured during 7 days after germination. Both enzymic activity and enzyme protein declined slowly in the dark and rapidly in the light. These results indicate that fluctuation of the enzymic activity in this organ, both in the light and in the dark, are mediated via changes in the amount of the enzyme protein and not via the action of an inhibitor. PMID:16664402

  11. Structure of the pyruvate dehydrogenase multienzyme complex E1 component from Escherichia coli at 1.85 A resolution.

    PubMed

    Arjunan, Palaniappa; Nemeria, Natalia; Brunskill, Andrew; Chandrasekhar, Krishnamoorthy; Sax, Martin; Yan, Yan; Jordan, Frank; Guest, John R; Furey, William

    2002-04-23

    The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined at a resolution of 1.85 A. The E. coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit. Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having alpha/beta folds. The functional dimer contains two catalytic centers located at the interface between subunits. The ThDP cofactors are bound in the "V" conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold. The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts. Significant structural differences are observed between individual domains of E1p relative to heterotetrameric multienzyme complex E1 components operating on branched chain substrates. These differences may be responsible for reported alternative E1p binding modes to E2 components within the respective complexes. This paper represents the first structural example of a functional pyruvate dehydrogenase E1p component from any species. It also provides the first representative example for the entire family of homodimeric (alpha2) E1 multienzyme complex components, and should serve as a model for this class of enzymes. PMID:11955070

  12. Inhibition of the hepatitis C virus helicase-associated ATPase activity by the combination of ADP, NaF, MgCl2, and poly(rU). Two ADP binding sites on the enzyme-nucleic acid complex.

    PubMed

    Porter, D J

    1998-03-27

    Hepatitis C virus (HCV) helicase has an intrinsic ATPase activity and a nucleic acid (poly(rU))-stimulated ATPase activity. The poly(rU)-stimulated ATPase activity was inhibited by F- in a time-dependent manner during ATP hydrolysis. Inhibition was the result of trapping an enzyme-bound ADP-poly(rU) ternary complex generated during the catalytic cycle and was not the result of generating enzyme-free ADP that subsequently inhibited the enzyme. However, catalysis was not required for efficient inhibition by F-. The stimulated and the intrinsic ATPase activities were also inhibited by treatment of the enzyme with F-, ADP, and poly(rU). The inhibited enzyme slowly recovered (t1/2 = 23 min) ATPase activity after a 2000-fold dilution into assay buffer. The onset of inhibition by 500 microM ADP and 15 mM F- in the absence of nucleic acid was very slow (t1/2 > 40 min). However, the sequence of addition of poly(rU) to a diluted solution of ADP/NaF-treated enzyme had a profound effect on the extent of inhibition. If the ADP/NaF-treated enzyme was diluted into an assay that lacked poly(rU) and the assay was subsequently initiated with poly(rU), the treated enzyme was not inhibited. Alternatively, if the treated enzyme was diluted into an assay containing poly(rU), the enzyme was inhibited. ATP protected the enzyme from inhibition by ADP/NaF. The stoichiometry between ADP and enzyme monomer in the inhibited enzyme complex was 2, as determined from titration of the ATPase activity ([ADP]/[E] = 2.2) and from the number of radiolabeled ADP bound to the inhibited enzyme ([ADP]/[E] = 1.7) in the presence of excess NaF, MgCl2, and poly(rU). The Hill coefficient for titration of ATPase activity with F- (n = 2.8) or MgCl2 (n = 2.1) in the presence of excess ADP and poly(rU) suggested that multiple F- and Mg2+ were involved in forming the inhibited enzyme complex. The stoichiometry between (dU)18, a defined oligomeric nucleic acid substituting for poly(rU), and enzyme monomer in the

  13. High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

    PubMed Central

    Bell, Colin W.; Fricks, Barbara E.; Rocca, Jennifer D.; Steinweg, Jessica M.; McMahon, Shawna K.; Wallenstein, Matthew D.

    2013-01-01

    Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample). Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil

  14. Catechins Variously Affect Activities of Conjugation Enzymes in Proliferating and Differentiated Caco-2 Cells.

    PubMed

    Lněničková, Kateřina; Procházková, Eliška; Skálová, Lenka; Matoušková, Petra; Bártíková, Hana; Souček, Pavel; Szotáková, Barbora

    2016-01-01

    The knowledge of processes in intestinal cells is essential, as most xenobiotics come into contact with the small intestine first. Caco-2 cells are human colorectal adenocarcinoma that once differentiated, exhibit enterocyte-like characteristics. Our study compares activities and expressions of important conjugation enzymes and their modulation by green tea extract (GTE) and epigallocatechin gallate (EGCG) using both proliferating (P) and differentiated (D) caco-2 cells. The mRNA levels of the main conjugation enzymes were significantly elevated after the differentiation of Caco-2 cells. However, no increase in conjugation enzymes' activities in differentiated cells was detected in comparison to proliferating ones. GTE/EGCG treatment did not affect the mRNA levels of any of the conjugation enzymes tested in either type of cells. Concerning conjugation enzymes activities, GTE/EGCG treatment elevated glutathione S-transferase (GST) activity by approx. 30% and inhibited catechol-O-methyltransferase (COMT) activity by approx. 20% in differentiated cells. On the other hand, GTE as well as EGCG treatment did not significantly affect the activities of conjugation enzymes in proliferating cells. Administration of GTE/EGCG mediated only mild changes of GST and COMT activities in enterocyte-like cells, indicating a low risk of GTE/EGCG interactions with concomitantly administered drugs. However, a considerable chemo-protective effect of GTE via the pronounced induction of detoxifying enzymes cannot be expected as well. PMID:27617982

  15. Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line

    PubMed Central

    Zhuge, Jian; Luo, Ye; Yu, Ying-Nian

    2003-01-01

    AIM: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many small-molecule substrates and volatile anesthetics. CYP2E1 is involved in chemical activation of many carcinogens, procarcinogens, and toxicants. To assess the metabolic and toxicological characteristics of CYP2E1, we cloned CYP2E1 cDNA and established a HepG2 cell line stably expressing recombinant CYP 2E1. METHODS: Human CYP2E1 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2E1 to HepG2 cells. The expression of CYP2E1 mRNA was validated by RT-PCR. The enzyme activity of CYP2E1 catalyzing oxidation of 4-nitrophenol in postmitochondrial supernate (S9) fraction of the cells was determined by spectrophotometry. The metabolic activation of HepG2-CYP2E1 cells was assayed by N-nitrosodiethylamine (NDEA) cytotoxicity and micronucleus test. RESULTS: The cloned CYP2E1 cDNA segment was identical to that reported by Umeno et al (GenBank access No. J02843). HepG2-CYP2E1 cells expressed CYP2E1 mRNA and had 4-nitrophenol hydroxylase activity (0.162 ± 0.025 nmol·min-1·mg-1 S9 protein), which were undetectable in parent HepG2 cells. HepG2-CYP2E1 cells increased the cytotoxicity and micronucleus rate of NDEA in comparison with those of HepG2 cells. CONCLUSION: The cDNA of human CYP2E1 can be successfully cloned, and a cell line, HepG2-CYP2E1, which can efficiently express mRNA and has CYP2E1 activity, is established. The cell line is useful for testing the cytotoxicity, mutagenicity and metabolism of xenobiotics, which may possibly be activated or metabolized by CYP2E1. PMID:14669323

  16. Quantum dot-based nanosensors for diagnosis via enzyme activity measurement.

    PubMed

    Knudsen, Birgitta R; Jepsen, Morten Leth; Ho, Yi-Ping

    2013-05-01

    Enzymes are essential in the human body, and the disorder of enzymatic activities has been associated with many different diseases and stages of disease. Luminescent semiconductor nanocrystals, also known as quantum dots (QDs), have garnered great attention in molecular diagnostics. Owing to their superior optical properties, tunable and narrow emissions, stable brightness and long lifetime, QD-based enzyme activity measurement has demonstrated improved detection sensitivity, which is considered particularly valuable for early disease diagnosis. Recent studies have also shown that QD-based nanosensors are capable of probing multiple enzyme activities simultaneously. This review highlights the current development of QD-based nanosensors for enzyme detection. The enzyme-QD hybrid system, equipped with unique electronic, optical and catalytic properties, is envisioned as a potential solution in addressing challenges in diagnostics and therapeutics.

  17. Retaining and recovering enzyme activity during degradation of TCE by methanotrophs.

    PubMed

    Palumbo, A V; Strong-Gunderson, J M; Carroll, S

    1997-01-01

    To determine if compounds added during trichloroethylene (TCE) degradation could reduce the loss of enzyme activity or increase enzyme recovery, different compounds serving as energy and carbon sources, pH buffers, or free radical scavengers were tested. Formate and formic acid (reducing power and a carbon source), as well as ascorbic acid and citric acid (free radical scavengers) were added during TCE degradation at a concentration of 2 mM. A saturated solution of calcium carbonate was also tested to address pH concerns. In the presence of formate and methane, only calcium carbonate and formic acid had a beneficial effect on enzyme recovery. The calcium carbonate and formic acid both reduced the loss of enzyme activity and resulted in the highest levels of enzyme activity after recovery. PMID:18576132

  18. Retaining and recovering enzyme activity during degradation of TCE by methanotrophs

    SciTech Connect

    Palumbo, A.V.; Strong-Gunderson, J.M.; Carroll, S.

    1997-12-31

    To determine if compounds added during trichloroethylene (TCE) degradation could reduce the loss of enzyme activity or increase enzyme recovery, different compounds serving as energy and carbon sources, pH buffers, or free radical scavengers were tested. Formate and formic acid (reducing power and a carbon source), as well as ascorbic acid and citric acid (free radical scavengers) were added during TCE degradation at a concentration of 2 mM. A saturated solution of calcium carbonate was also tested to address pH concerns. In the presence of formate and methane, only calcium carbonate and formic acid had a beneficial effect on enzyme recovery. The calcium carbonate and formic acid both reduced the loss of enzyme activity and resulted in the highest levels of enzyme activity after recovery. 19 refs., 3 figs.

  19. [Relationship among soil enzyme activities, vegetation state, and soil chemical properties of coal cinder yard].

    PubMed

    Wang, Youbao; Zhang, Li; Liu, Dengyi

    2003-01-01

    From field investigation and laboratory analysis, the relationships among soil enzyme activities, vegetation state and soil chemical properties of coal cinder yard in thermal power station were studied. The results showed that vegetation on coal cinder yard was distributed in scattered patch mainly with single species of plant, and herbs were the dominant species. At the same time, the activity of three soil enzymes had a stronger relativity to environment conditions, such as vegetation state and soil chemical properties. The sensitivity of three soil enzymes to environmental stress was in order of urease > sucrase > catalase. The relativity of three soil enzymes to environmental factor was in order of sucrase > urease > catalase. Because of urease being the most susceptible enzyme to environmental conditions, and it was marked or utmost marked interrelated with vegetation state and soil chemical properties, urease activity could be used as an indicator for the reclamation of wasteland.

  20. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  1. Inhibitory activity of Plantago major L. on angiotensin I-converting enzyme.

    PubMed

    Nhiem, Nguyen Xuan; Tai, Bui Huu; Van Kiem, Phan; Van Minh, Chau; Cuong, Nguyen Xuan; Tung, Nguyen Huu; Thu, Vu Kim; Trung, Trinh Nam; Anh, Hoang Le Tuan; Jo, Sung-Hoon; Jang, Hae-Dong; Kwon, Young-In; Kim, Young Ho

    2011-03-01

    Eight compounds were isolated from methanol extract of Plantago major L. leaves and investigated for their ability to inhibit angiotensin I-converting enzyme activity. Among them, compound 1 showed the most potent inhibition with rate of 28.06 ± 0.21% at a concentration of 100 μM. Compounds 2 and 8 exhibited weak activities. These results suggest that compound 1 might contribute to the ability of P. major to inhibit the activity of angiotensin I- converting enzyme.

  2. Soil Enzyme Activities as Affected by Manure Types, Application Rates and Management Practices

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The application of manure can restore soil ecosystem services related to nutrient cycling and soil organic matter (SOM) dynamics through biochemical transformations mediated by soil enzymes. Enzyme activities are very crucial in soil metabolic functioning as they drive the decomposition of organic r...

  3. Illustrating the Effect of pH on Enzyme Activity Using Gibbs Energy Profiles

    ERIC Educational Resources Information Center

    Bearne, Stephen L.

    2014-01-01

    Gibbs energy profiles provide students with a visual representation of the energy changes that occur during enzyme catalysis, making such profiles useful as teaching and learning tools. Traditional kinetic topics, such as the effect of pH on enzyme activity, are often not discussed in terms of Gibbs energy profiles. Herein, the symbolism of Gibbs…

  4. Reconciling apparent variability in effects of biochar amendment on soil enzyme activities by assay optimization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We studied the effects of a biochar made from switchgrass on four soil enzymes (ß- glucosidase, ß-N-acetylglucosaminidase, lipase, and leucine aminopeptidase) to determine if biochar would consistently modify soil biological activities. Inconsistent results from enzyme assays of char-amended soils s...

  5. Sediment Microbial Enzyme Activity as an Indicator of Nutrient Limitation in Great Lakes Coastal Wetlands

    EPA Science Inventory

    This study, the first to link microbial enzyme activities to regional-scale anthropogenic stressors, suggests that microbial enzyme regulation of carbon and nutrient dynamics may be sensitive indicators of nutrient dynamics in aquatic ecosystems, but further work is needed to elu...

  6. Enzyme activity in terrestrial soil in relation to exploration of the Martian surface

    NASA Technical Reports Server (NTRS)

    Mclaren, A. D.

    1974-01-01

    Sensitive tests for the detection of extracellular enzyme activity in Martian soil was investigated using simulated Martian soil. Enzyme action at solid-liquid water interfaces and at low humidity were studied, and a kinetic scheme was devised and tested based on the growth of microorganisms and the oxidation of ammonium nitrite.

  7. Measuring potential denitrification enzyme activity rates using the membrane inlet mass spectrometer

    EPA Science Inventory

    The denitrification enzyme activity (DEA) assay, provides a quantitative assessment of the multi enzyme, biological process of reactive nitrogen removal via the reduction of N03 to N2. Measured in soil, usually under non limiting carbon and nitrate concentrations, this short ter...

  8. Enzymatic immuno-assembly of gold nanoparticles for visualized activity screening of histone-modifying enzymes.

    PubMed

    Zhen, Zhen; Tang, Li-Juan; Long, Haoxu; Jiang, Jian-Hui

    2012-04-17

    Activity screening of histone-modifying enzymes is of paramount importance for epigenetic research as well as clinical diagnostics and therapeutics. A novel biosensing strategy has been developed for sensitive and selective detection of histone-modifying enzymes as well as their inhibitors. This strategy relies on the antibody-mediated assembly of gold nanoparticles (AuNPs) decorated with substrate peptides that are subjected to enzymatic modifications by the histone-modifying enzymes. This design allows a visual and homogeneous assay of the enzyme activity using antibodies without any labels, which circumvents the requirements to prefunctionalize the antibody and affords improved assay simplicity and throughput. Additionally, the use of antibody-based recognition of modified peptides could offer improved specificity as compared with existing techniques based on the enzyme coupled assay. We have demonstrated this strategy using a histone methyltransferase acting on histone H3 (Lys 4) and a histone acetyltransferase acting on histone H3 (Lys 14). The results reveal that the absorption peak characteristic for AuNPs decreases dynamically with increasing activity of the enzymes with concomitant visualizable color attenuation, and subnanomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a robust and convenient visualized platform for screening the enzyme activities and their inhibitors with high sensitivity and selectivity.

  9. Quantitation of Lipase Activity from a Bee: An Introductory Enzyme Experiment.

    ERIC Educational Resources Information Center

    Farley, Kathleen A.; Jones, Marjorie A.

    1989-01-01

    This four-hour experiment uses a bee as a source of the enzyme which is reacted with a radioactive substrate to determine the specific activity of the enzyme. Uses thin layer chromatography, visible spectrophotometry, and liquid scintillation spectrometry (if not available a Geiger-Muller counter can be substituted). (MVL)

  10. Inhibitors of enzymes catalyzing modifications to histone lysine residues: structure, function and activity.

    PubMed

    Lillico, Ryan; Stesco, Nicholas; Khorshid Amhad, Tina; Cortes, Claudia; Namaka, Mike P; Lakowski, Ted M

    2016-05-01

    Gene expression is partly controlled by epigenetic mechanisms including histone-modifying enzymes. Some diseases are caused by changes in gene expression that can be mitigated by inhibiting histone-modifying enzymes. This review covers the enzyme inhibitors targeting histone lysine modifications. We summarize the enzymatic mechanisms of histone lysine acetylation, deacetylation, methylation and demethylation and discuss the biochemical roles of these modifications in gene expression and in disease. We discuss inhibitors of lysine acetylation, deacetylation, methylation and demethylation defining their structure-activity relationships and their potential mechanisms. We show that there are potentially indiscriminant off-target effects on gene expression even with the use of selective epigenetic enzyme inhibitors.

  11. Activity of an enzyme immobilized on superparamagnetic particles in a rotational magnetic field

    SciTech Connect

    Mizuki, Toru; Watanabe, Noriyuki; Nagaoka, Yutaka; Fukushima, Tadamasa; Morimoto, Hisao; Usami, Ron; Maekawa, Toru

    2010-03-19

    We immobilize {alpha}-amylase extracted from Bacillus Iicheniformis on the surfaces of superparamagnetic particles and investigate the effect of a rotational magnetic field on the enzyme's activity. We find that the activity of the enzyme molecules immobilized on superparamagnetic particles increases in the rotational magnetic field and reaches maximum at a certain frequency. We clarify the effect of the cluster structures formed by the superparamagnetic particles on the activity. Enzyme reactions are enhanced even in a tiny volume of solution using the present method, which is very important for the development of efficient micro reactors and micro total analysis systems ({mu}-TAS).

  12. The effect of aspartame on the activity of rat liver xenobiotic-metabolizing enzymes.

    PubMed

    Tutelyan, V A; Kravchenko, L V; Kuzmina, E E

    1990-01-01

    Male, Wistar rats were administered aspartame (40 or 4000 mg/kg body weight) in their diet for 90 days. By 45 days, the activities of three microsomal enzymes, epoxide hydrolase, carboxylesterase, and p-nitrophenyl-UDP-glucuronosyltransferase, were significantly increased in rats consuming 4000 mg/kg of aspartame. By 90 days, however, the activity of the xenobiotic-metabolizing enzymes of the rats given aspartame did not differ significantly from the activity of control animals. From these results, we conclude that the consumption of aspartame does not substantially alter the function of the hepatic microsomal enzymes which protect the organism from foreign compounds found in its environment and food.

  13. Antioxidative capacity and enzyme activity in Haematococcus pluvialis cells exposed to superoxide free radicals

    NASA Astrophysics Data System (ADS)

    Liu, Jianguo; Zhang, Xiaoli; Sun, Yanhong; Lin, Wei

    2010-01-01

    The antioxidative capacity of astaxanthin and enzyme activity of reactive oxygen eliminating enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were studied in three cell types of Haematococcus pluvialis exposed to high concentrations of a superoxide anion radical (O{2/-}). The results show that defensive enzymes and astaxanthin-related mechanisms were both active in H. pluvialis during exposure to reactive oxygen species (ROS) such as O{2/-}. Astaxanthin reacted with ROS much faster than did the protective enzymes, and had the strongest antioxidative capacity to protect against lipid peroxidation. The defensive mechanisms varied significantly between the three cell types and were related to the level of astaxanthin that had accumulated in those cells. Astaxanthin-enriched red cells had the strongest antioxidative capacity, followed by brown cells, and astaxanthin-deficient green cells. Although there was no significant increase in expression of protective enzymes, the malondialdehyde (MDA) content in red cells was sustained at a low level because of the antioxidative effect of astaxanthin, which quenched O{2/-} before the protective enzymes could act. In green cells, astaxanthin is very low or absent; therefore, scavenging of ROS is inevitably reliant on antioxidative enzymes. Accordingly, in green cells, these enzymes play the leading role in scavenging ROS, and the expression of these enzymes is rapidly increased to reduce excessive ROS. However, because ROS were constantly increased in this study, the enhance enzyme activity in the green cells was not able to repair the ROS damage, leading to elevated MDA content. Of the four defensive enzymes measured in astaxanthin-deficient green cells, SOD eliminates O{2/-}, POD eliminates H2O2, which is a by-product of SOD activity, and APX and CAT are then initiated to scavenge excessive ROS.

  14. Experimental strategy to discover microbes with gluten-degrading enzyme activities

    NASA Astrophysics Data System (ADS)

    Helmerhorst, Eva J.; Wei, Guoxian

    2014-06-01

    Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

  15. Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities

    PubMed Central

    Helmerhorst, Eva J.; Wei, Guoxian

    2015-01-01

    Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement. PMID:26113763

  16. Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain.

    PubMed

    Liu, C C; Chung, C H; Lee, M L

    1973-10-01

    l-Tryptophan-activating enzyme [l-tryptophan-tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different K(m) and V(max.) values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg(2+), ATP, in any combination.

  17. DprE1--from the discovery to the promising tuberculosis drug target.

    PubMed

    Mikusová, Katarína; Makarov, Vadim; Neres, João

    2014-01-01

    Several groups working in the field of the development of new antituberculosis drugs have recently reported active compounds targeting mycobacterial enzyme DprE1. Along with its counterpart, DprE2, it catalyses a unique epimerization reaction resulting in the synthesis of decaprenylphosphoryl arabinose, the single donor of arabinosyl residues for the build-up of arabinans, fundamental components of the mycobacterial cell wall. This review presents the historical background leading to the discovery of DprE1, focusing on the biochemical and structural characterization of this important emerging target and introducing the molecules acting on DprE1 including the development of the most successful series--the benzothiazinones, currently in late pre-clinical development, which turned to be suicide inhibitors of DprE1.

  18. Microbial dynamics and enzyme activities in tropical Andosols depending on land use and nutrient inputs

    NASA Astrophysics Data System (ADS)

    Mganga, Kevin; Razavi, Bahar; Kuzyakov, Yakov

    2015-04-01

    Microbial decomposition of soil organic matter is mediated by enzymes and is a key source of terrestrial CO2 emissions. Microbial and enzyme activities are necessary to understand soil biochemical functioning and identify changes in soil quality. However, little is known about land use and nutrients availability effects on enzyme activities and microbial processes, especially in tropical soils of Africa. This study was conducted to examine how microbial and enzyme activities differ between different land uses and nutrient availability. As Andosols of Mt. Kilimanjaro are limited by nutrient concentrations, we hypothesize that N and P additions will stimulate enzyme activity. N and P were added to soil samples (0-20 cm) representing common land use types in East Africa: (1) savannah, (2) maize fields, (3) lower montane forest, (4) coffee plantation, (5) grasslands and (6) traditional Chagga homegardens. Total CO2 efflux from soil, microbial biomass and activities of β-glucosidase, cellobiohydrolase, chitinase and phosphatase involved in C, N and P cycling, respectively was monitored for 60 days. Total CO2 production, microbial biomass and enzyme activities varied in the order forest soils > grassland soils > arable soils. Increased β-glucosidase and cellobiohydrolase activities after N addition of grassland soils suggest that microorganisms increased N uptake and utilization to produce C-acquiring enzymes. Low N concentration in all soils inhibited chitinase activity. Depending on land use, N and P addition had an inhibitory or neutral effect on phosphatase activity. We attribute this to the high P retention of Andosols and low impact of N and P on the labile P fractions. Enhanced CO2 production after P addition suggests that increased P availability could stimulate soil organic matter biodegradation in Andosols. In conclusion, land use and nutrients influenced soil enzyme activities and microbial dynamics and demonstrated the decline in soil quality after landuse

  19. Heterologous Acidothermus cellulolyticus 1,4-β-Endoglucanase E1 Produced Within the Corn Biomass Converts Corn Stover Into Glucose

    NASA Astrophysics Data System (ADS)

    Ransom, Callista; Balan, Venkatesh; Biswas, Gadab; Dale, Bruce; Crockett, Elaine; Sticklen, Mariam

    Commercial conversion of lignocellulosic biomass to fermentable sugars requires inexpensive bulk production of biologically active cellulase enzymes, which might be achieved through direct production of these enzymes within the biomass crops. Transgenic corn plants containing the catalytic domain of Acidothermus cellulolyticus E1 endo-1,4-β glucanase and the bar bialaphos resistance coding sequences were generated after Biolistic® (BioRad Hercules, CA) bombardment of immature embryo-derived cells. E1 sequences were regulated under the control of the cauliflower mosaic virus 35S promoter and tobacco mosaic virus translational enhancer, and E1 protein was targeted to the apoplast using the signal peptide of tobacco pathogenesis-related protein to achieve accumulation of this enzyme. The integration, expression, and segregation of E1 and bar transgenes were demonstrated, respectively, through Southern and Western blotting, and progeny analyses. Accumulation of up to 1.13% of transgenic plant total soluble proteins was detected as biologically active E1 by enzymatic activity assay. The corn-produced, heterologous E1 could successfully convert ammonia fiber explosion-pretreated corn stover polysaccharides into glucose as a fermentable sugar for ethanol production, confirming that the E1 enzyme is produced in its active from.

  20. Physics-based enzyme design: predicting binding affinity and catalytic activity.

    PubMed

    Sirin, Sarah; Pearlman, David A; Sherman, Woody

    2014-12-01

    Computational enzyme design is an emerging field that has yielded promising success stories, but where numerous challenges remain. Accurate methods to rapidly evaluate possible enzyme design variants could provide significant value when combined with experimental efforts by reducing the number of variants needed to be synthesized and speeding the time to reach the desired endpoint of the design. To that end, extending our computational methods to model the fundamental physical-chemical principles that regulate activity in a protocol that is automated and accessible to a broad population of enzyme design researchers is essential. Here, we apply a physics-based implicit solvent MM-GBSA scoring approach to enzyme design and benchmark the computational predictions against experimentally determined activities. Specifically, we evaluate the ability of MM-GBSA to predict changes in affinity for a steroid binder protein, catalytic turnover for a Kemp eliminase, and catalytic activity for α-Gliadin peptidase variants. Using the enzyme design framework developed here, we accurately rank the most experimentally active enzyme variants, suggesting that this approach could provide enrichment of active variants in real-world enzyme design applications.

  1. A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

    PubMed

    Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

    2015-11-10

    In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. PMID:26257292

  2. A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

    PubMed

    Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

    2015-11-10

    In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device.

  3. Chemical modification of an alpha 3-fucosyltransferase; definition of amino acid residues essential for enzyme activity.

    PubMed

    Britten, C J; Bird, M I

    1997-02-11

    The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe(x)) is dependent on the activity of an alpha 3-fucosyltransferase (EC 2.4.1.152, GDP-fucose:Gal beta (1-4)GlcNAc-R alpha (1-3)fucosyltransferase). This enzyme catalyses the transfer of fucose from GDP-beta-fucose to the 3-OH of N-acetylglucosamine present in lactosamine acceptors. In this report, we have investigated the amino acids essential for the activity of a recombinant alpha 3-fucosyltransferase (FucT-VI) through chemical modification of the enzyme with group-selective reagents. FucT-VI activity was found to be particularly sensitive to the histidine-selective reagent diethylpyrocarbonate and the cysteine reagent N-ethylmaleimide, with IC50 values of less than 200 microM. Reagents selective for arginine and lysine had no effect on enzyme activity. The inclusion of GDP-beta-fucose during preincubation with NEM reduces the rate of inactivation whereas inclusion of an acceptor saccharide for the enzyme, Gal beta (1-4)GlcNAc, had no effect. No protective effect with either GDP-beta-fucose or Gal beta (1-4)GlcNAc was observed on treatment of the enzyme with diethylpyrocarbonate. These data suggest that in addition to an NEM-reactive cysteine in, or adjacent to, the substrate-binding site of the enzyme, FucT-VI possesses histidine residue(s) that are essential for enzyme activity.

  4. Studies on the activating enzyme for iron protein of nitrogenase from Rhodospirillum rubrum.

    PubMed

    Saari, L L; Pope, M R; Murrell, S A; Ludden, P W

    1986-04-15

    Removal of ADP-ribose from the iron protein of nitrogenase by activating enzyme resulted in the activation of the inactive iron protein. A radioassay that directly measured the initial velocity of the activation was developed using iron protein radiolabeled with either [8-3H]- or [G-32P]ADP-ribose. The release of radiolabeled ADP-ribose by activating enzyme was linearly correlated with the increase in the specific activity of the iron protein as measured by acetylene reduction. Both ATP and MnCl2 were required for the activation of inactive iron protein. The optimal ratio of [MnCl2]/[ATP] in the radioassay was 2:1, and the optimal concentrations were 4 mM and 2 mM for [MnCl2] and [ATP], respectively. The Km for inactive iron protein was 74 microM and the Vmax was 628 pmol of [32P] ADP-ribose released min-1 microgram of activating enzyme-1. Adenosine, cytidine, guanosine, or uridine mono-, di-, or triphosphates did not substitute for ATP in the activation of native iron protein. Activating enzyme removed ADP-ribose from oxygen-denatured iron protein in the absence of ATP. ADP, ADP-ribose, pyrophosphate, and high concentrations of NaCl inhibited activating enzyme activity. PMID:3082874

  5. Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes.

    PubMed Central

    Khan, A. R.; James, M. N.

    1998-01-01

    Proteolytic enzymes are synthesized as inactive precursors, or "zymogens," to prevent unwanted protein degradation, and to enable spatial and temporal regulation of proteolytic activity. Upon sorting or appropriate compartmentalization, zymogen conversion to the active enzyme typically involves limited proteolysis and removal of an "activation segment." The sizes of activation segments range from dipeptide units to independently folding domains comprising more than 100 residues. A common form of the activation segment is an N-terminal extension of the mature enzyme, or "prosegment," that sterically blocks the active site, and thereby prevents binding of substrates. In addition to their inhibitory role, prosegments are frequently important for the folding, stability, and/or intracellular sorting of the zymogen. The mechanisms of conversion to active enzymes are diverse in nature, ranging from enzymatic or nonenzymatic cofactors that trigger activation, to a simple change in pH that results in conversion by an autocatalytic mechanism. Recent X-ray crystallographic studies of zymogens and comparisons with their active counterparts have identified the structural changes that accompany conversion. This review will focus upon the structural basis for inhibition by activation segments, as well as the molecular events that lead to the conversion of zymogens to active enzymes. PMID:9568890

  6. Investigations on the activity of poly(2-oxazoline) enzyme conjugates dissolved in organic solvents.

    PubMed

    Konieczny, Stefan; Krumm, Christian; Doert, Dominik; Neufeld, Katharina; Tiller, Joerg C

    2014-07-10

    The use of enzymes in organic solvents offers a great opportunity for the highly selective synthesis of complex organic compounds. In this study we investigate the POXylation of several enzymes with different polyoxazolines ranging from the hydrophilic poly(2-methyl-oxazoline) (PMOx) to the hydrophobic poly(2-heptyl-oxazoline) (PHeptOx). As reported previously on the examples of model enzymes POXylation mediated by pyromellitic acid dianhydride results in highly modified, organosoluble protein conjugates. This procedure is here extended to a larger number of proteins and optimized for the different polyoxazolines. The resulting polymer-enzyme conjugates (PEC) became soluble in different organic solvents ranging from hydrophilic DMF to even toluene. These conjugates were characterized regarding their solubility and especially their activity in organic solvents and in some cases the PECs showed significantly (up to 153,000 fold) higher activities than the respective native enzymes. PMID:24709400

  7. 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

  8. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  9. Modeling in situ soil enzyme activity using continuous field soil moisture and temperature data

    NASA Astrophysics Data System (ADS)

    Steinweg, J. M.; Wallenstein, M. D.

    2010-12-01

    Moisture and temperature are key drivers of soil organic matter decomposition, but there is little consensus on how climate change will affect the degradation of specific soil compounds under field conditions. Soil enzyme activities are a useful metric of soil community microbial function because they are they are the direct agents of decomposition for specific substrates in soil. However, current standard enzyme assays are conducted under optimized conditions in the laboratory and do not accurately reflect in situ enzyme activity, where diffusion and substrate availability may limit reaction rates. The Arrhenius equation, k= A*e(-Ea/RT), can be used to predict enzyme activity (k), collision frequency (A) or activation energy (Ea), but is difficult to parameterize when activities are measured under artificial conditions without diffusion or substrate limitation. We developed a modifed equation to estimate collision frequency and activation energy based on soil moisture to model in-situ enzyme activites. Our model was parameterized using data we collected from the Boston Area Climate Experiment (BACE) in Massachusetts; a multi-factor climate change experiment that provides an opportunity to assess how changes in moisture availability and temperature may impact enzyme activity. Soils were collected from three precipitation treatments and four temperature treatments arranged in a full-factorial design at the BACE site in June 2008, August 2008, January 2009 and June 2009. Enzyme assays were performed at four temperatures (4, 15, 25 and 35°C) to calculate temperature sensitivity and activation energy over the different treatments and seasons. Enzymes activities were measured for six common enzymes involved in carbon (β-glucosidase, cellobiohydrolase, xylosidase), phosphorus (phosphatase) and nitrogen cycling (N-acetyl glucosaminidase, and leucine amino peptidase). Potential enzyme activity was not significantly affected by precipitation, warming or the interaction of

  10. Extracellular enzyme activities during cassava fermentation for 'fufu' production.

    PubMed

    Oyewole, O B; Odunfa, S A

    1992-01-01

    Amylase and pectin methyl esterase activities increased rapidly during the early period of the fermentation of cassava for 'fufu' production, attaining their peak activities after 12 and 24h, respectively. Cellulase activity was lower and approximately constant for most of the fermentation period.

  11. Androgen-activating enzymes in the central nervous system.

    PubMed

    Poletti, A; Martini, L

    1999-01-01

    In the rat brain, several steroids can be converted by specific enzymes to either more potent compounds or to derivatives showing new biological effects. One of the most studied enzyme is the 5alpha-reductase (5alpha-R), which acts on 3keto-delta4 steroids. In males, testosterone is the main substrate and gives rise to the most potent natural androgen dihydrotestosterone. In females, progesterone is reduced to dihydroprogesterone, a precursor of allopregnanolone, a natural anxiolytic/anesthetic steroid. Other substrates are some gluco- and minero-corticoids. Two isoforms of the 5alpha-R, with limited degree of homology, have been cloned: 5alpha-R type 1 and type 2. The 5alpha-R type 1 possesses low affinity for the various substrates and is widely distributed in the body, with the highest levels in the liver; in the brain, this isoform is expressed throughout life and does not appear to be controlled by androgens. 5Alpha-R type 1 in the rat brain is mainly concentrated in myelin membranes, where it might be involved in the catabolism of potentially neurotoxic steroids. The 5alpha-R type 2 shows high affinity for the various substrates, a peculiar pH optimum at acidic values and is localized in androgen-dependent structures. In the rat brain, the type 2 isoform is expressed at high levels only in the perinatal period and is controlled by androgens, at least in males. In adulthood, the type 2 gene appears to be specifically expressed in localised brain regions, like the hypothalamus and the hippocampus. The 5alpha-R type 2 is present in the GT1 cells, a model of LHRH-secreting neurons. These cells also contain the androgen receptor, which is probably involved in the central negative feedback effect exerted by androgens on the hypothalamic-pituitary-gonadal axis. The physiological significance of these and additional data will be discussed.

  12. Studies on antioxidant activity of teasaponins after hydrolyzed by enzyme

    NASA Astrophysics Data System (ADS)

    Tian, Jing; Zhao, Sen; Xu, Longquan; Fei, Xu; Wang, Xiuying; Wang, Yi

    The biological activity of teasaponins and their molecular structure are closely related, and the activity of saponins may be increased with the change of their molecular structure. In this report, teasaponins were hydrolyzed by Aspergillus niger for increasing the antioxidant activity. The antioxidant activity of teasaponins before and after hydrolyzed was tested by DPPH, and the result showed four new teasaponins were produced after hydrolysis, and their antioxidant activity was increased significantly than the original teasaponins before hydrolysis, the radical scavenging capacity (RSC) was partly up to 95 %.

  13. Function-based classification of carbohydrate-active enzymes by recognition of short, conserved peptide motifs.

    PubMed

    Busk, Peter Kamp; Lange, Lene

    2013-06-01

    Functional prediction of carbohydrate-active enzymes is difficult due to low sequence identity. However, similar enzymes often share a few short motifs, e.g., around the active site, even when the overall sequences are very different. To exploit this notion for functional prediction of carbohydrate-active enzymes, we developed a simple algorithm, peptide pattern recognition (PPR), that can divide proteins into groups of sequences that share a set of short conserved sequences. When this method was used on 118 glycoside hydrolase 5 proteins with 9% average pairwise identity and representing four characterized enzymatic functions, 97% of the proteins were sorted into groups correlating with their enzymatic activity. Furthermore, we analyzed 8,138 glycoside hydrolase 13 proteins including 204 experimentally characterized enzymes with 28 different functions. There was a 91% correlation between group and enzyme activity. These results indicate that the function of carbohydrate-active enzymes can be predicted with high precision by finding short, conserved motifs in their sequences. The glycoside hydrolase 61 family is important for fungal biomass conversion, but only a few proteins of this family have been functionally characterized. Interestingly, PPR divided 743 glycoside hydrolase 61 proteins into 16 subfamilies useful for targeted investigation of the function of these proteins and pinpointed three conserved motifs with putative importance for enzyme activity. Furthermore, the conserved sequences were useful for cloning of new, subfamily-specific glycoside hydrolase 61 proteins from 14 fungi. In conclusion, identification of conserved sequence motifs is a new approach to sequence analysis that can predict carbohydrate-active enzyme functions with high precision. PMID:23524681

  14. Comparative ontogenetic changes in enzyme activity during embryonic development of calyptraeid gastropods.

    PubMed

    Collin, Rachel; Starr, Matthew J

    2013-09-01

    A modification of a semi-quantitative color-based enzyme assay was used to quantify the activity of 19 enzymes (5 peptidases, 3 lipases, 3 phosphotases, and 8 carbohydrases) during five stages of development in eight species of calyptraeid gastropods. Sixteen of the 19 enzymes showed a significant effect of mode of development on the concentration of the reaction product after incubation of homogenates standardized for protein content. The overall pattern was that planktotrophs showed the highest activities, followed by adelphophages, and nonfeeding embryos, which had the lowest enzyme activities. Thirteen enzymes showed significant differences across developmental stages. Of these, eight showed a clear increase during development. Only one of the enzymes showed a sudden jump in activity between the unfed, pre-hatching stage and post-hatching stages that were fed Isochrysis galbana. In three cases, ANOVA identified two exclusive, significantly different groups of species. In naphthol-AS-BI-phosphohydrolase, the measured absorbance of Crucibulum spinosum samples was significantly higher than in all of the other species. The activity of α-fucosidase in Crepipatella occulta was significantly greater than in the other seven species. Finally, the activity of β-galactosidase was significantly higher in C. occulta, Crucibulum spinosum, and Bostrycapulus calyptraeformis than in the four Crepidula species. This is the only enzyme for which there is an indication of a phylogenetic effect. Relative enzyme activities were similar to those reported for other herbivorous gastropods, with the three phosphohydrolases, four carbohydrases (β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, and α-fucosidase), and leucine arylamidase showing high activities.

  15. Function-based classification of carbohydrate-active enzymes by recognition of short, conserved peptide motifs.

    PubMed

    Busk, Peter Kamp; Lange, Lene

    2013-06-01

    Functional prediction of carbohydrate-active enzymes is difficult due to low sequence identity. However, similar enzymes often share a few short motifs, e.g., around the active site, even when the overall sequences are very different. To exploit this notion for functional prediction of carbohydrate-active enzymes, we developed a simple algorithm, peptide pattern recognition (PPR), that can divide proteins into groups of sequences that share a set of short conserved sequences. When this method was used on 118 glycoside hydrolase 5 proteins with 9% average pairwise identity and representing four characterized enzymatic functions, 97% of the proteins were sorted into groups correlating with their enzymatic activity. Furthermore, we analyzed 8,138 glycoside hydrolase 13 proteins including 204 experimentally characterized enzymes with 28 different functions. There was a 91% correlation between group and enzyme activity. These results indicate that the function of carbohydrate-active enzymes can be predicted with high precision by finding short, conserved motifs in their sequences. The glycoside hydrolase 61 family is important for fungal biomass conversion, but only a few proteins of this family have been functionally characterized. Interestingly, PPR divided 743 glycoside hydrolase 61 proteins into 16 subfamilies useful for targeted investigation of the function of these proteins and pinpointed three conserved motifs with putative importance for enzyme activity. Furthermore, the conserved sequences were useful for cloning of new, subfamily-specific glycoside hydrolase 61 proteins from 14 fungi. In conclusion, identification of conserved sequence motifs is a new approach to sequence analysis that can predict carbohydrate-active enzyme functions with high precision.

  16. Quantum dot based enzyme activity sensors present deviations from Michaelis-Menten kinetic model

    NASA Astrophysics Data System (ADS)

    Díaz, Sebastián. A.; Brown, Carl W.; Malanoski, Anthony P.; Oh, Eunkeu; Susumu, Kimihiro; Medintz, Igor L.

    2016-03-01

    Nanosensors employing quantum dots (QDs) and enzyme substrates with fluorescent moieties offer tremendous promise for disease surveillance/diagnostics and as high-throughput co-factor assays. Advantages of QDs over other nanoscaffolds include their small size and inherent photochemical properties such as size tunable fluorescence, ease in attaching functional moieties, and resistance to photobleaching. These properties make QDs excellent Förster Resonance Energy Transfer (FRET) donors; well-suited for rapid, optical measurement applications. We report enzyme sensors designed with a single FRET donor, the QD donor acting as a scaffold to multiple substrates or acceptors. The QD-sensor follows the concrete activity of the enzyme, as compared to the most common methodologies that quantify the enzyme amount or its mRNA precursor. As the sensor reports on the enzyme activity in real-time we can actively follow the kinetics of the enzyme. Though classic Michaelis-Menten (MM) parameters can be obtained to describe the activity. In the course of these experiments deviations, both decreasing and increasing the kinetics, from the common MM model were observed upon close examinations. From these observations additional experiments were undertaken to understand the varying mechanisms. Different enzymes can present different deviations depending on the chosen target, e.g. trypsin appears to present a positive hopping mechanism while collagenase demonstrates a QD caused reversible inhibition.

  17. Improved complementary polymer pair system: switching for enzyme activity by PEGylated polymers.

    PubMed

    Kurinomaru, Takaaki; Tomita, Shunsuke; Kudo, Shinpei; Ganguli, Sumon; Nagasaki, Yukio; Shiraki, Kentaro

    2012-03-01

    The development of technology for on/off switching of enzyme activity is expected to expand the applications of enzyme in a wide range of research fields. We have previously developed a complementary polymer pair system (CPPS) that enables the activity of several enzymes to be controlled by a pair of oppositely charged polymers. However, it failed to control the activity of large and unstable α-amylase because the aggregation of the complex between anionic α-amylase and cationic poly(allylamine) (PAA) induced irreversible denaturation of the enzyme. To address this issue, we herein designed and synthesized a cationic copolymer with a poly(ethylene glycol) backbone, poly(N,N-diethylaminoethyl methacrylate)-block-poly(ethylene glycol) (PEAMA-b-PEG). In contrast to PAA, α-amylase and β-galactosidase were inactivated by PEAMA-b-PEG with the formation of soluble complexes. The enzyme/PEAMA-b-PEG complexes were then successfully recovered from the complex by the addition of anionic poly(acrylic acid) (PAAc). Thus, dispersion of the complex by PEG segment in PEAMA-b-PEG clearly plays a crucial role for regulating the activities of these enzymes, suggesting that PEGylated charged polymer is a new candidate for CPPS for large and unstable enzymes.

  18. Development of Activity-based Cost Functions for Cellulase, Invertase, and Other Enzymes

    NASA Astrophysics Data System (ADS)

    Stowers, Chris C.; Ferguson, Elizabeth M.; Tanner, Robert D.

    As enzyme chemistry plays an increasingly important role in the chemical industry, cost analysis of these enzymes becomes a necessity. In this paper, we examine the aspects that affect the cost of enzymes based upon enzyme activity. The basis for this study stems from a previously developed objective function that quantifies the tradeoffs in enzyme purification via the foam fractionation process (Cherry et al., Braz J Chem Eng 17:233-238, 2000). A generalized cost function is developed from our results that could be used to aid in both industrial and lab scale chemical processing. The generalized cost function shows several nonobvious results that could lead to significant savings. Additionally, the parameters involved in the operation and scaling up of enzyme processing could be optimized to minimize costs. We show that there are typically three regimes in the enzyme cost analysis function: the low activity prelinear region, the moderate activity linear region, and high activity power-law region. The overall form of the cost analysis function appears to robustly fit the power law form.

  19. High Inorganic Triphosphatase Activities in Bacteria and Mammalian Cells: Identification of the Enzymes Involved

    PubMed Central

    Lakaye, Bernard; Servais, Anne-Catherine; Scholer, Georges; Fillet, Marianne; Elias, Benjamin; Derochette, Jean-Michel; Crommen, Jacques; Wins, Pierre; Bettendorff, Lucien

    2012-01-01

    Background We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. Methodology/Principal Findings Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. Conclusions and General Significance We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling. PMID:22984449

  20. Extracellular enzyme activities during regulated hydrolysis of high-solid organic wastes.

    PubMed

    Zhang, Bo; He, Pin-Jing; Lü, Fan; Shao, Li-Ming; Wang, Pei

    2007-11-01

    The hydrolysis process, where the complex insoluble organic materials are hydrolyzed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic solid wastes. Recirculating the leachate from hydrolysis reactor and recycling the effluent from methanogenic reactor to hydrolysis reactor in the two-stage solid-liquid anaerobic digestion process could accelerate degradation of organic solid wastes. To justify the influencing mechanism of recirculation and recycling on hydrolysis, the relationship of hydrolysis to the synthesis and locations of extracellular enzymes was evaluated by regulating the dilution rate of the methanogenic effluent recycle. The results showed that the hydrolysis could be enhanced by increasing the dilution rate, resulting from improved total extracellular enzyme activities. About 15%, 25%, 37%, 56% and 92% of carbon, and about 9%, 18%, 27%, 45% and 80% of nitrogen were converted from the solid phase to the liquid phase at dilution rates of 0.09, 0.25, 0.5, 0.9 and 1.8d(-1), respectively. The hydrolysis of organic wastes was mainly attributable to cell-free enzyme, followed by biofilm-associated enzyme. Increasing the dilution rate afforded cell-free extracellular enzymes with more opportunity to access the surface of organic solid waste, which ensured a faster renewal of niche where extracellular enzymes functioned actively. Meanwhile, the increment of biofilm-associated enzyme was promoted concomitantly, and therefore improved the hydrolysis of organic solid wastes.

  1. Effects of deep tillage and straw returning on soil microorganism and enzyme activities.

    PubMed

    Ji, Baoyi; Hu, Hao; Zhao, Yali; Mu, Xinyuan; Liu, Kui; Li, Chaohai

    2014-01-01

    Two field experiments were conducted for two years with the aim of studying the effects of deep tillage and straw returning on soil microorganism and enzyme activity in clay and loam soil. Three treatments, (1) conventional tillage (CT), shallow tillage and straw returning; (2) deep tillage (DT), deep tillage and straw returning; and (3) deep tillage with no straw returning (DNT), were carried out in clay and loam soil. The results showed that deep tillage and straw returning increased the abundance of soil microorganism and most enzyme activities. Deep tillage was more effective for increasing enzyme activities in clay, while straw returning was more effective in loam. Soil microorganism abundance and most enzyme activities decreased with the increase of soil depth. Deep tillage mainly affected soil enzyme activities in loam at the soil depth of 20-30 cm and in clay at the depth of 0-40 cm. Straw returning mainly affected soil microorganism and enzyme activities at the depths of 0-30 cm and 0-40 cm, respectively.

  2. Common regulatory control of CTP synthase enzyme activity and filament formation

    PubMed Central

    Noree, Chalongrat; Monfort, Elena; Shiau, Andrew K.; Wilhelm, James E.

    2014-01-01

    The ability of enzymes to assemble into visible supramolecular complexes is a widespread phenomenon. Such complexes have been hypothesized to play a number of roles; however, little is known about how the regulation of enzyme activity is coupled to the assembly/disassembly of these cellular structures. CTP synthase is an ideal model system for addressing this question because its activity is regulated via multiple mechanisms and its filament-forming ability is evolutionarily conserved. Our structure–function studies of CTP synthase in Saccharomyces cerevisiae reveal that destabilization of the active tetrameric form of the enzyme increases filament formation, suggesting that the filaments comprise inactive CTP synthase dimers. Furthermore, the sites responsible for feedback inhibition and allosteric activation control filament length, implying that multiple regions of the enzyme can influence filament structure. In contrast, blocking catalysis without disrupting the regulatory sites of the enzyme does not affect filament formation or length. Together our results argue that the regulatory sites that control CTP synthase function, but not enzymatic activity per se, are critical for controlling filament assembly. We predict that the ability of enzymes to form supramolecular structures in general is closely coupled to the mechanisms that regulate their activity. PMID:24920825

  3. Induction of antioxidant enzyme activity and lipid peroxidation level in ion-beam-bombarded rice seeds

    NASA Astrophysics Data System (ADS)

    Semsang, Nuananong; Yu, LiangDeng

    2013-07-01

    Low-energy ion beam bombardment has been used to mutate a wide variety of plant species. To explore the indirect effects of low-energy ion beam on biological damage due to the free radical production in plant cells, the increase in antioxidant enzyme activities and lipid peroxidation level was investigated in ion-bombarded rice seeds. Local rice seeds were bombarded with nitrogen or argon ion beams at energies of 29-60 keV and ion fluences of 1 × 1016 ions cm-2. The activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST) and lipid peroxidation level were assayed in the germinated rice seeds after ion bombardment. The results showed most of the enzyme activities and lipid peroxidation levels in both the argon and nitrogen bombarded samples were higher than those in the natural control. N-ion bombardment could induce higher levels of antioxidant enzyme activities in the rice samples than the Ar-ion bombardment. Additional effects due to the vacuum condition were found to affect activities of some antioxidant enzymes and lipid peroxidation level. This study demonstrates that ion beam bombardment and vacuum condition could induce the antioxidant enzyme activity and lipid peroxidation level which might be due to free radical production in the bombarded rice seeds.

  4. Seasonal effects on intestinal enzyme activity in the Australian agamid lizard, Lophognathus temporalis.

    PubMed

    Iglesias, Sebastian; Tracy, Christopher R; Bedford, Gavin S; McWhorter, Todd J; Christian, Keith A

    2009-05-01

    The tropical agamid lizard, Lophognathus temporalis, has higher metabolic and feeding rates during the wet season compared to the dry season. Also, lizards from urban sites tend to be larger than those from natural sites, partly due to site differences in food availability. Therefore, we hypothesized that activity of membrane-bound intestinal enzymes and masses of organs related to digestion would differ both seasonally and between urban and natural sites. To test this, we measured activities of aminopeptidase-N (APN), maltase, and sucrase, as well as organ masses. APN activity (micromol min(-1) g(-1)) was highest in the middle portion of the intestine (section 2), followed by the proximal portion (section 1) and then the distal portion (section 3). Maltase activity was highest in section 1 and decreased distally. We detected some sucrase activity in section 1 but none in sections 2 or 3. We found similar enzyme activities within each section irrespective of site or season. However, total enzyme activities were higher during the wet season compared to the dry season for both urban and bush L. temporalis. Total wet season enzyme activity in urban and bush L. temporalis was greatest for APN (25.4; 15.8 micromol min(-1); respectively), then maltase (3.9; 3.6 micromol min(-1); respectively) and then sucrase (0.3; 0.2 micromol min(-1); respectively). The higher total enzyme activities was the result of an increase in intestinal mass during the wet season.

  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. Visualization of enzyme activities inside earthworm biopores by in situ soil zymography

    NASA Astrophysics Data System (ADS)

    Thu Duyen Hoang, Thi; Razavi, Bahar. S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    Earthworms can strongly activate microorganisms, increase microbial and enzyme activities and consequently the turnover of native soil organic matter. In extremely dynamic microhabitats and hotspots as biopores made by earthworms, the in situ enzyme activities are a footprint of complex biotic interactions. The effect of earthworms on the alteration of enzyme activities inside biopores and the difference between bio-pores and earthworm-free soil was visualized by in situ soil zymography (Spohn and Kuzyakov, 2014). For the first time, we prepared quantitative imaging of enzyme activities in biopores. Furthermore, we developed the zymography technique by direct application of a substrate saturated membrane to the soil to obtain better spatial resolution. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). Simultaneously, maize seed was sown in the soil. Control soil box with maize and without earthworm was prepared in the same way. After two weeks when bio-pore systems were formed by earthworm, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine aminopeptidase) and phosphatase. Followed by non-destructive zymography, biopore samples and control soil were destructively collected to assay enzyme kinetics by fluorogenically labeled substrates method. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. These differences were further confirmed by fluorimetric microplate enzyme assay detected significant difference of Vmax in four above mentioned enzymes. Vmax of β-glucosidase, chitinase, xylanase and phosphatase in biopores is 68%, 108%, 50% and 49% higher than that of control soil. However, no difference in cellobiohydrolase and leucine aminopeptidase kinetics between biopores and control soil were detected. This indicated little effect of earthworms on protein and cellulose transformation in soil

  7. Enzyme activity in terrestrial soil in relation to exploration of the Martian surface

    NASA Technical Reports Server (NTRS)

    Ardakani, M. S.; Mclaren, A. D.; Pukite, A. H.

    1972-01-01

    An exploration was made of enzyme activities in soil, including abundance, persistence and localization of these activities. An attempt was made to develop procedures for the detection and assaying of enzymes in soils suitable for presumptive tests for life in planetary soils. A suitable extraction procedure for soil enzymes was developed and measurements were made of activities in extracts in order to study how urease is complexed in soil organic matter. Mathematical models were developed, based on enzyme action and microbial growth in soil, for rates of oxidation of nitrogen as nitrogen compounds are moved downward in soil by water flow. These biogeochemical models should be applicable to any percolating system, with suitable modification for special features, such as oxygen concetrations, and types of hydrodynamic flow.

  8. Unidirectional inhibition and activation of "malic' enzyme of Solanum tuberosum by meso-tartrate.

    PubMed

    Do Nascimento, K H; Davies, D D; Patil, K D

    1975-08-01

    A kinetic study of "malic' enzyme (EC 1.1.1.40) from potato suggests that the mechanism is Ordered Bi Ter with NADP+ binding before malate, and NADPH binding before pyruvate and HCO3-. The analysis is complicated by the non-linearity that occurs in some of the plots. meso-Tartrate is shown to inhibit the oxidative decarboxylation of malate but to activate the reductive carboxylation of pyruvate. To explain these unidirectional effects it is suggested that the control site of "malic' enzyme binds organic acids (including meso-tartrate) which activate the enzyme. meso-Tartrate, however, competes with malate for the active site and thus inhibits the oxidative decarboxylation of malate. Because meso-tartrate does not compete effectively with pyruvate for enzyme-NADPH, its binding at the control site leads to a stimulation of the carboxylation of pyruvate. A similar explanation is advanced for the observation that malic acid stimulates its own synthesis.

  9. Effects of phosphorus fertilizer supplementation on antioxidant enzyme activities in tomato fruits.

    PubMed

    Ahn, Taehyun; Oke, Moustapha; Schofield, Andrew; Paliyath, Gopinadhan

    2005-03-01

    The effects of soil and foliar phosphorus supplementation on the activities and levels of superoxide dismutase (SOD), guaiacol peroxidase (POX), and ascorbate peroxidase (APX) in tomato fruits were evaluated by determining enzyme activities and isoenzyme analysis. Both protein levels and enzyme activities varied depending on the variety and season. In general, phosphorus supplementation did not alter SOD, POX, and APX activities significantly;however, some treatments showed season- and stage-specific enhancement in activities as noticed with hydrophos and seniphos supplementation. Three different SOD isozymes were observed, and these isozymes showed very similar staining intensities in response to P application and during the three developmental stages studied. Two major isozymes of POX and two different APX isozymes were observed at all the developmental stages. The results suggest that antioxidant enzyme activities may be influenced by the availability of phosphorus, but are subject to considerable variation depending on the developmental stage and the season. PMID:15740037

  10. Photoswitching of enzyme activity by laser-induced pH-jump.

    PubMed

    Kohse, Stefanie; Neubauer, Antje; Pazidis, Alexandra; Lochbrunner, Stefan; Kragl, Udo

    2013-06-26

    Controlled initiation of biochemical events and in particular of protein activity is a powerful tool in biochemical research. Specifically, optical trigger signals are an attractive approach for remote control of enzyme activity. We present a method for generating optical control of enzyme activity applicable to a widespread range of enzymes. The approach is based on short laser pulses as optical "switches" introducing an instantaneous change of the pH-value for activation of protein function. The pH-jump is induced by proton release from 2-nitrobenzaldehyde. Reaction conditions were chosen to yield a pH-jump of almost 3 units on switching from inactive to active conditions for the enzyme. In this experimental setup, irradiation can be realized without any loss of enzyme activity. Following this change in pH-value, a controlled activation of hydrolytic activity of acid phosphatase is successfully demonstrated. This application provides a general method for photocontrol of enzymatic function for proteins having a significant pH-profile. The kinetic data for the substrate 6-chloro-8-fluoro-4-methylumbelliferone phosphate are determined.

  11. Generation of in vivo activating factors in the ischemic intestine by pancreatic enzymes

    NASA Astrophysics Data System (ADS)

    Mitsuoka, Hiroshi; Kistler, Erik B.; Schmid-Schönbein, Geert W.

    2000-02-01

    One of the early events in physiological shock is the generation of activators for leukocytes, endothelial cells, and other cells in the cardiovascular system. The mechanism by which these activators are produced has remained unresolved. We examine here the hypothesis that pancreatic digestive enzymes in the ischemic intestine may be involved in the generation of activators during intestinal ischemia. The lumen of the small intestine of rats was continuously perfused with saline containing a broadly acting pancreatic enzyme inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, 0.37 mM) before and during ischemia of the small intestine by splanchnic artery occlusion. This procedure inhibited activation of circulating leukocytes during occlusion and reperfusion. It also prevented the appearance of activators in portal venous and systemic artery plasma and attenuated initiating symptoms of multiple organ injury in shock. Intestinal tissue produces only low levels of activators in the absence of pancreatic enzymes, whereas in the presence of enzymes, activators are produced in a concentration- and time-dependent fashion. The results indicate that pancreatic digestive enzymes in the ischemic intestine serve as an important source for cell activation and inflammation, as well as multiple organ failure.

  12. [Effect of pyrazole on the activity of acetaldehyde-producing enzymes in the liver].

    PubMed

    Gerashchenko, D Iu; Gorenshteĭn, B I; Pyzhik, T N; Ostrovskiĭ, Iu M

    1993-01-01

    Influence of pyrazole on the endogenous ethanol level and activities of acetaldehyde-producing enzymes was investigated. Drastic enhancement of the endogenous ethanol level in the blood and tissues was accompanied by an insignificant increase of phosphoethanolamine lyase activity, while activity of threonine aldolase and pyruvate dehydrogenase was unchanged.

  13. Structure-Activity Relations In Enzymes: An Application Of IR-ATR Modulation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Fringeli, Urs P.; Ahlstrom, Peter; Vincenz, Claudius; Fringeli, Marianna

    1985-12-01

    Relations between structure and specific activity in immobilized acetylcholinesterase (ACNE) have been studied by means of pH- and Ca++-modulation technique combined with attenuated total reflection (ATR) infrared (IR) spectroscopy and enzyme activity measurement. Periodic modulation of pH and Ca++-concentration enabled a periodic on-off switching of about 40% of the total enzyme activity. It was found that about 0.5 to 1% of the amino acids were involved in this process. These 15 to 30 amino acids assumed antiparallel pleated sheet structure in the inhibited state and random and/or helical structure in the activated state.

  14. Construction of a Fusion Enzyme Exhibiting Superoxide Dismutase and Peroxidase Activity.

    PubMed

    Sharapov, M G; Novoselov, V I; Ravin, V K

    2016-04-01

    A chimeric gene construct encoding human peroxiredoxin 6 and Mn-superoxide dismutase from Escherichia coli was developed. Conditions for expression of the fusion protein in E. coli cell were optimized. Fusing of the enzymes into a single polypeptide chain with peroxiredoxin 6 at the N-terminus (PSH) did not affect their activities. On the contrary, the chimeric protein with reverse order of enzymes (SPH) was not obtained in a water-soluble active form. The active chimeric protein (PSH) exhibiting both peroxidase and superoxide dismutase activities was prepared and its physicochemical properties were characterized. PMID:27293100

  15. Mineralogical impact on long-term patterns of soil nitrogen and phosphorus enzyme activities

    NASA Astrophysics Data System (ADS)

    Mikutta, Robert; Turner, Stephanie; Meyer-Stüve, Sandra; Guggenberger, Georg; Dohrmann, Reiner; Schippers, Axel

    2014-05-01

    Soil chronosequences provide a unique opportunity to study microbial activity over time in mineralogical diverse soils of different ages. The main objective of this study was to test the effect of mineralogical properties, nutrient and organic matter availability over whole soil pro-files on the abundance and activity of the microbial communities. We focused on microbio-logical processes involved in nitrogen and phosphorus cycling at the 120,000-year Franz Josef soil chronosequence. Microbial abundances (microbial biomass and total cell counts) and enzyme activities (protease, urease, aminopeptidase, and phosphatase) were determined and related to nutrient contents and mineralogical soil properties. Both, microbial abundances and enzyme activities decreased with soil depth at all sites. In the organic layers, microbial biomass and the activities of N-hydrolyzing enzymes showed their maximum at the intermediate-aged sites, corresponding to a high aboveground biomass. In contrast, the phosphatase activity increased with site age. The activities of N-hydrolyzing enzymes were positively correlated with total carbon and nitrogen contents, whereas the phosphatase activity was negatively correlated with the phosphorus content. In the mineral soil, the enzyme activities were generally low, thus reflecting the presence of strongly sorbing minerals. Sub-strate-normalized enzyme activities correlated negatively to clay content as well as poorly crystalline Al and Fe oxyhydroxides, supporting the view that the evolution of reactive sec-ondary mineral phases alters the activity of the microbial communities by constraining sub-strate availability. Our data suggest a strong mineralogical influence on nutrient cycling par-ticularly in subsoil environments.

  16. Response of enzyme activities and microbial communities to soil amendment with sugar alcohols.

    PubMed

    Yu, Huili; Si, Peng; Shao, Wei; Qiao, Xiansheng; Yang, Xiaojing; Gao, Dengtao; Wang, Zhiqiang

    2016-08-01

    Changes in microbial community structure are widely known to occur after soil amendment with low-molecular-weight organic compounds; however, there is little information on concurrent changes in soil microbial functional diversity and enzyme activities, especially following sorbitol and mannitol amendment. Soil microbial functional diversity and enzyme activities can be impacted by sorbitol and mannitol, which in turn can alter soil fertility and quality. The objective of this study was to investigate the effects of sorbitol and mannitol addition on microbial functional diversity and enzyme activities. The results demonstrated that sorbitol and mannitol addition altered the soil microbial community structure and improved enzyme activities. Specifically, the addition of sorbitol enhanced the community-level physiological profile (CLPP) compared with the control, whereas the CLPP was significantly inhibited by the addition of mannitol. The results of a varimax rotated component matrix demonstrated that carbohydrates, polymers, and carboxylic acids affected the soil microbial functional structure. Additionally, we found that enzyme activities were affected by both the concentration and type of inputs. In the presence of high concentrations of sorbitol, the urease, catalase, alkaline phosphatase, β-glucosidase, and N-acetyl-β-d-glucosaminidase activities were significantly increased, while invertase activity was decreased. Similarly, this increase in invertase, catalase, and alkaline phosphatase and N-acetyl-β-d-glucosaminidase activities was especially evident after mannitol addition, and urease activity was only slightly affected. In contrast, β-glucosidase activity was suppressed at the highest concentration. These results indicate that microbial community diversity and enzyme activities are significantly affected by soil amendment with sorbitol and mannitol. PMID:27005019

  17. The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth.

    PubMed

    Liu, Ying-Li; Zheng, Jie; Tang, Li-Juan; Han, Wei; Wang, Jian-Min; Liu, Dian-Wu; Tian, Qing-Bao

    2015-11-01

    Ubiquitin-specific protease 22 (USP22) can regulate the cell cycle and apoptosis in many cancer cell types, while it is still unclear whether the deubiquitinating enzyme activity of USP22 is necessary for these processes. As little is known about the impact of USP22 on the growth of HeLa cell, we observed whether USP22 can effectively regulate HeLa cell growth as well as the necessity of deubiquitinating enzyme activity for these processes in HeLa cell. In this study, we demonstrate that USP22 can regulate cell cycle but not apoptosis in HeLa cell. The deubiquitinating enzyme activity of USP22 is necessary for this process as confirmed by an activity-deleted mutant (C185S) and an activity-decreased mutant (Y513C). In addition, the deubiquitinating enzyme activity of USP22 is related to the levels of BMI-1, c-Myc, cyclin D2 and p53. Our findings indicate that the deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, and it promotes cell proliferation via the c-Myc/cyclin D2, BMI-1 and p53 pathways in HeLa cell.

  18. Enzymes extracted from apple peels have activity in reducing higher alcohols in Chinese liquors.

    PubMed

    Han, Qi'an; Shi, Junling; Zhu, Jing; Lv, Hongliang; Du, Shuangkui

    2014-10-01

    As the unavoidable byproducts of alcoholic fermentation, higher alcohols are unhealthy compounds widespread in alcoholic drinks. To investigate the activity of apple crude enzymes toward higher alcohols in liquors, five kinds of apple peels, namely, Fuji, Gala, Golden Delicious, Red Star, and Jonagold, were chosen to prepare enzymes, and three kinds of Chinese liquors, namely, Xifeng (containing 45% ethanol), Taibai (containing 50% ethanol), and Erguotou (containing 56% ethanol), were tested. Enzymes were prepared in the forms of liquid solution, powder, and immobilized enzymes using sodium alginate (SA) and chitosan. The treatment was carried out at 37 °C for 1 h. The relative amounts of different alcohols (including ethanol, 1-propanol, isobutanol, 1-butanol, isoamylol, and 1-hexanol) were measured using gas chromatography (GC). Conditions for preparing SA-immobilized Fuji enzymes (SA-IEP) were optimized, and the obtained SA-IEP (containing 0.3 g of enzyme) was continuously used to treat Xifeng liquor eight times, 20 mL per time. Significant degradation rates (DRs) of higher alcohols were observed at different degrees, and it also showed enzyme specificity according to the apple varieties and enzyme preparations. After five repeated treatments, the DRs of the optimized Fuji SA-IEP remained 70% for 1-hexanol and >15% for other higher alcohols.

  19. The assembly of the FtsZ ring at the mid-chloroplast division site depends on a balance between the activities of AtMinE1 and ARC11/AtMinD1.

    PubMed

    Fujiwara, Makoto T; Hashimoto, Haruki; Kazama, Yusuke; Abe, Tomoko; Yoshida, Shigeo; Sato, Naoki; Itoh, Ryuuichi D

    2008-03-01

    Chloroplast division comprises a sequence of events that facilitate symmetric binary fission and that involve prokaryotic-like stromal division factors such as tubulin-like GTPase FtsZ and the division site regulator MinD. In Arabidopsis, a nuclear-encoded prokaryotic MinE homolog, AtMinE1, has been characterized in terms of its effects on a dividing or terminal chloroplast state in a limited series of leaf tissues. However, the relationship between AtMinE1 expression and chloroplast phenotype remains to be fully elucidated. Here, we demonstrate that a T-DNA insertion mutation in AtMinE1 results in a severe inhibition of chloroplast division, producing motile dots and short filaments of FtsZ. In AtMinE1 sense (overexpressor) plants, dividing chloroplasts possess either single or multiple FtsZ rings located at random intervals and showing constriction depth, mainly along the chloroplast polarity axis. The AtMinE1 sense plants displayed equivalent chloroplast phenotypes to arc11, a loss-of-function mutant of AtMinD1 which forms replicating mini-chloroplasts. Furthermore, a certain population of FtsZ rings formed within developing chloroplasts failed to initiate or progress the membrane constriction of chloroplasts and consequentially to complete chloroplast fission in both AtMinE1 sense and arc11/atminD1 plants. Our present data thus demonstrate that the chloroplast division site placement involves a balance between the opposing activities of AtMinE1 and AtMinD1, which acts to prevent FtsZ ring formation anywhere outside of the mid-chloroplast. In addition, the imbalance caused by an AtMinE1 dominance causes multiple, non-synchronous division events at the single chloroplast level, as well as division arrest, which becomes apparent as the chloroplasts mature, in spite of the presence of FtsZ rings. PMID:18204083

  20. Influence of vegetation spatial heterogeneity on soil enzyme activity in burned Mediterranean areas

    NASA Astrophysics Data System (ADS)

    Mayor, Á. G.; Goirán, S.; Bautista, S.

    2009-04-01

    Mediterranean ecosystems are commonly considered resilient to wildfires. However, depending on fire severity and recurrence, post-fire climatic conditions and plant community type, the recovery rate of the vegetation can greatly vary. Often, the post-fire vegetation cover remains low and sparsely distributed many years after the wildfire, which could have profound impacts on ecosystem functioning. In this work, we studied the influence of vegetation patchiness on soil enzyme activity (acid phosphatase, β-glucosidase and urease), at the patch and landscape scales, in degraded dry Mediterranean shrublands affected by wildfires. At the patch scale, we assessed the variation in soil enzyme between bare soils and vegetation patches. At the landscape scale, we studied the relationships between soil enzyme activity and various landscape metrics (total patch cover, average interpatch length, average patch width, and patch density). The study was conducted in 19 sites in the Valencia Region (eastern Spain), which had been affected by large wildfires in 1991. Site selection aimed at capturing a wide range of the variability of post-fire plant recovery rates in Mediterranean areas. The activities of the three enzymes were significantly higher in soils under the vegetation canopies than in adjacent bare areas, which we attributed to the effect of plants on the soil amount of both enzyme substrates and enzymes. The differences between bare and plant microsites were larger in the case of the acid phosphatase and less marked for urease. The activity of acid phosphatase was also higher under patches of resprouter species than under patches of seeder species, probably due to the faster post-fire recovery and older age of resprouter patches in fire-prone ecosystems. Soil enzyme activities of β-glucosidase and urease in both bare soils and vegetation patches showed no relationships with any of the landscape metrics analysed. However, the activity of acid phosphatase increased

  1. Extracellular enzyme activity at the air-water interface of an estuarine lake

    NASA Astrophysics Data System (ADS)

    Mudryk, Z. J.; Skórczewski, P.

    2004-01-01

    Variations in hydrolytic activity of eight extracellular enzymes in surface and subsurface waters in estuarine Lake Gardno were measured. The ranking of potential activity rates of the assayed enzymes was the same in both surface and subsurface water, i.e. esterase > lipase > aminopeptidase > phosphatase > β-glucosidase > α-glucosidase > chitinase > β-lactosidase. The vertical activity profiles show that esterase, aminopeptidase, α-glucosidase, β-glucosidase and β-lactosidase reached the highest values in surface layer, whereas lipase, phosphatase and chitinase showed maximum activity in subsurface water. Significant differences in enzyme activity between different parts of the studied lake were demonstrated, with higher values in the seawater zone, and lower values in the freshwater zone.

  2. Changes in metabolome and in enzyme activities during germination of Trichoderma atroviride conidia.

    PubMed

    Kaliňák, Michal; Simkovič, Martin; Zemla, Peter; Matata, Matej; Molnár, Tomáš; Liptaj, Tibor; Varečka, L'udovít; Hudecová, Daniela

    2014-08-01

    The aim of this work was to study the metabolic changes during germination of Trichoderma atroviride conidia along with selected marker enzyme activities. The increase in proteinogenic amino acid concentrations together with the increase in glutamate dehydrogenase activity suggests a requirement for nitrogen metabolism. Even though the activities of tricarboxylic acid cycle enzymes also increased, detected organic acid pools did not change, which predisposes this pathway to energy production and supply of intermediates for further metabolism. The concentrations of many metabolites, including the main osmolytes mannitol and betaine, also increased during the formation of germ tubes. The activities of H(+)-ATPase and GDPase, the only marker enzymes that did not have detectable activity in non-germinated conidia, were shown with germ tubes.

  3. Novel TPP-riboswitch activators bypass metabolic enzyme dependency

    NASA Astrophysics Data System (ADS)

    Mayer, Günter; Lünse, Christina; Suckling, Colin; Scott, Fraser

    2014-07-01

    Riboswitches are conserved regions within mRNA molecules that bind specific metabolites and regulate gene expression. TPP-riboswitches, which respond to thiamine pyrophosphate (TPP), are involved in the regulation of thiamine metabolism in numerous bacteria. As these regulatory RNAs are often modulating essential biosynthesis pathways they have become increasingly interesting as promising antibacterial targets. Here, we describe thiamine analogs containing a central 1,2,3-triazole group to induce repression of thiM-riboswitch dependent gene expression in different E. coli strains. Additionally, we show that compound activation is dependent on proteins involved in the metabolic pathways of thiamine uptake and synthesis. The most promising molecule, triazolethiamine (TT), shows concentration dependent reporter gene repression that is dependent on the presence of thiamine kinase ThiK, whereas the effect of pyrithiamine (PT), a known TPP-riboswitch modulator, is ThiK independent. We further show that this dependence can be bypassed by triazolethiamine-derivatives that bear phosphate-mimicking moieties. As triazolethiamine reveals superior activity compared to pyrithiamine, it represents a very promising starting point for developing novel antibacterial compounds that target TPP-riboswitches. Riboswitch-targeting compounds engage diverse endogenous mechanisms to attain in vivo activity. These findings are of importance for the understanding of compounds that require metabolic activation to achieve effective riboswitch modulation and they enable the design of novel compound generations that are independent of endogenous activation mechanisms.

  4. [Activities of some yeast flavogenic enzymes in situ].

    PubMed

    Logvinenko, E M; Trach, V M; Kashchenko, V E; Zakal'skiĭ, A E; Koltun, L V; Shavlovskiĭ, G M

    1977-09-01

    Effects of digitonin, dimethylsulfoxide and protamine sulfate on yeast Pichia guilliermondii were studied in order to produce cells with increased permeability and possessing the GTP-cyclohydrolase, riboflavinsynthetase and riboflavinkinase activities. The digitonin-treated cells exhibited a higher cyclohydrolase activity than the cell-free extracts; the activities of riboflavinsynthetase and riboflavinkinase in the cells and cell-free extracts were found to be similar. Treatment of cells with dimethylsulfoxide proved to be most effective to determine the activity of GTP-cyclohydrolase and also helpful to determine that of riboflavinsynthetase. Protamine sulfate had no effect on the cells of P. guilliermondii. The methods developed were used to determine the activities of GTP-cyclohydrolase, riboflavinsynthetase and riboflavinkinase in the cells of flavinogenic (P. guiller-mondii, Torulopsis candida) and non-flavinogenic (Candida utilis, Candida pulcherrima) yeasts grown in iron-rich and iron-deficient media. Derepression of riboflavinsynthetase and GTP-cyclohydrolase syntheses under conditions of Fe deficiency in the flavinogenic yeast cells confirmed previously made assumptions. PMID:199288

  5. Spinach thylakoid polyphenol oxidase isolation, activation, and properties of the native chloroplast enzyme

    SciTech Connect

    Golbeck, J.H.; Cammarata, K.V.

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14,18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. Sonication releases polyphenol oxidase from the membrane largely in the latent state. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time. Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K/sub m/. A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  6. Isolation and characterization of an enzyme with esterase activity from Micropolyspora faeni.

    PubMed Central

    Bannerman, E N; Nicolet, J

    1976-01-01

    The isolation and the characterization of one of the enzymes of Micropolyspora faeni that hydrolyzes the substrate N-benzoyl-DL-phenylalanine-beta-naphthyl ester and that seems to be of medical importance are described. This enzyme (enzyme 1) was isolated with an 86-fold purification by using the following seven steps: ammonium sulfate precipitation, gel filtration through Sephadex G-150, heat treatment, chromatography on diethylaminoethyl-cellulose, rechromatography on diethylaminoethyl-Sephadex, gel filtration through Sephadex G-200, and affinity chromatography. Enzyme 1 has a molecular weight of approximately 500,000 and maximum activity at pH 7.8 to 8.0 and at 20 degrees C. The enzyme is stable between pH 7.5 and 10.5 and at temperatures up to 60 degrees C. Its activity is not inhibited by ethylenediaminetetraacetic acid. It is, however, sensitive to diisopropyl phosphofluoride and phenylmethyl sulfonyl fluoride. These properties and the ability to hydrolyze the esters of phenylalanine, tyrosine, and tryptophan without endopeptidasic activity and no marked proteolytic activity suggest that the enzyme is an esterase. Images PMID:9899

  7. Saccharification of Lignocelluloses by Carbohydrate Active Enzymes of the White Rot Fungus Dichomitus squalens.

    PubMed

    Rytioja, Johanna; Hildén, Kristiina; Mäkinen, Susanna; Vehmaanperä, Jari; Hatakka, Annele; Mäkelä, Miia R

    2015-01-01

    White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification. PMID:26660105

  8. [Photodynamic reaction and oxidative stress - influence of the photodynamic effect on the activity antioxidant enzymes].

    PubMed

    Romiszewska, Anna; Nowak-Stępniowska, Agata

    2014-01-01

    The interaction of light with a photosensitizer, accumulated in a tissue in the presence of oxygen, leads to formation of reactive oxygen species, mainly of singlet oxygen and free radicals. These factors react with biomolecules producing their oxidized states. Reactive oxygen species, such as singlet oxygen and free radicals are able to damage membranes, DNA, enzymes, structural peptides and other cellular structures leading to cell death. An antioxidant protection of cell is formed by enzymes belonging to the family of oxidoreductases: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR). Photodynamic therapy leads to the increased production of oxidizing toxic forms. It is important to analyze impact of PDT on the activity of antioxidant enzymes, such as SOD, CAT, GPx. The activity of antioxidant enzymes during the photodynamic effect is influenced by both the light energy dose and the concentration of photosensitizer. The presence only of the photosensitizer or only the light energy may also result in changes in the activity of these enzymes. The differences in changes in the activity of these enzymes depend on the type of used photosensitizer. A phenomenon of selective accumulation of photosensitizer in tumor tissues is used in the photodynamic method of tumor diagnosis and treatment.

  9. Amy63, a novel type of marine bacterial multifunctional enzyme possessing amylase, agarase and carrageenase activities

    PubMed Central

    Liu, Ge; Wu, Shimei; Jin, Weihua; Sun, Chaomin

    2016-01-01

    A multifunctional enzyme is one that performs multiple physiological functions, thus benefiting the organism. Characterization of multifunctional enzymes is important for researchers to understand how organisms adapt to different environmental challenges. In the present study, we report the discovery of a novel multifunctional enzyme Amy63 produced by marine bacterium Vibrio alginolyticus 63. Remarkably, Amy63 possesses amylase, agarase and carrageenase activities. Amy63 is a substrate promiscuous α-amylase, with the substrate priority order of starch, carrageenan and agar. Amy63 maintains considerable amylase, carrageenase and agarase activities and stabilities at wide temperature and pH ranges, and optimum activities are detected at temperature of 60 °C and pH of 6.0, respectively. Moreover, the heteroexpression of Amy63 dramatically enhances the ability of E. coli to degrade starch, carrageenan and agar. Motif searching shows three continuous glycosyl hydrolase 70 (GH70) family homologs existed in Amy63 encoding sequence. Combining serial deletions and phylogenetic analysis of Amy63, the GH70 homologs are proposed as the determinants of enzyme promiscuity. Notably, such enzymes exist in all kingdoms of life, thus providing an expanded perspective on studies of multifunctional enzymes. To our knowledge, this is the first report of an amylase having additional agarase and carrageenase activities. PMID:26725302

  10. Saccharification of Lignocelluloses by Carbohydrate Active Enzymes of the White Rot Fungus Dichomitus squalens

    PubMed Central

    Rytioja, Johanna; Hildén, Kristiina; Mäkinen, Susanna; Vehmaanperä, Jari; Hatakka, Annele; Mäkelä, Miia R.

    2015-01-01

    White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification. PMID:26660105

  11. Novel biohybrids of layered double hydroxide and lactate dehydrogenase enzyme: Synthesis, characterization and catalytic activity studies

    NASA Astrophysics Data System (ADS)

    Djebbi, Mohamed Amine; Braiek, Mohamed; Hidouri, Slah; Namour, Philippe; Jaffrezic-Renault, Nicole; Ben Haj Amara, Abdesslem

    2016-02-01

    The present work introduces new biohybrid materials involving layered double hydroxides (LDH) and biomolecule such as enzyme to produce bioinorganic system. Lactate dehydrogenase (Lac Deh) has been chosen as a model enzyme, being immobilized onto MgAl and ZnAl LDH materials via direct ion-exchange (adsorption) and co-precipitation methods. The immobilization efficiency was largely dependent upon the immobilization methods. A comparative study shows that the co-precipitation method favors the immobilization of great and tunable amount of enzyme. The structural behavior, chemical bonding composition and morphology of the resulting biohybrids were determined by X-ray diffraction (XRD) study, Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM), respectively. The free and immobilized enzyme activity and kinetic parameters were also reported using UV-Visible spectroscopy. However, the modified LDH materials showed a decrease in crystallinity as compared to the unmodified LDH. The change in activity of the immobilized lactate dehydrogenase was considered to be due, to the reduced accessibility of substrate molecules to the active sites of the enzyme and the partial conformational change of the Lac Deh molecules as a result of the immobilization way. Finally, it was proven that there is a correlation between structure/microstructure and enzyme activity dependent on the immobilization process.

  12. Effects of butachlor on microbial enzyme activities in paddy soil.

    PubMed

    Min, Hang; Ye, Yang-Fang; Chen, Zhong-Yun; Wu, Wei-Xiang; Du, Yu-Feng

    2002-07-01

    This paper reports the influences of the herbicide butachlor (n-butoxymethl-chloro-2', 6'-diethylacetnilide) on microbial respiration, nitrogen fixation and nitrification, and on the activities of dehydrogenase and hydrogen peroxidase in paddy soil. The results showed that after application of butachlor with concentrations of 5.5 micrograms/g dried soil, 11.0 micrograms/g dried soil and 22.0 micrograms/g dried soil, the application of butachlor enhanced the activity of dehydrogenase at increasing concentrations. The soil dehydrogenase showed the highest activity on the 16th day after application of 22.0 micrograms/g dried soil of butachlor. The hydrogen peroxidase could be stimulated by butachlor. The soil respiration was depressed within a period from several days to more than 20 days, depending on concentrations of butachlor applied. Both the nitrogen fixation and nitrification were stimulated in the beginning but reduced greatly afterwards in paddy soil.

  13. Single-stranded DNA binding activity of C1-tetrahydrofolate synthase enzymes.

    PubMed

    Wahls, W P; Song, J M; Smith, G R

    1993-11-15

    In eukaryotes C1-5,6,7,8-tetrahydrofolate (THF) synthase is a trifunctional enzyme that catalyzes the interconversion of reduced forms of folate to supply activated one-carbon units required for a variety of metabolic pathways. The enzymatic activities include 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5). In bacteria separate, monofunctional or bifunctional polypeptides catalyze the same reactions. We have purified C1-THF synthase from the fission yeast Schizosaccharomyces pombe and found its physical and enzymatic properties similar to those of other eukaryotic C1-THF synthase enzymes. Unexpectedly, the S. pombe enzyme bound strongly (Keq = 100 pM) to single-stranded DNA, but not to double-stranded DNA or to RNA. The binding was sequence-independent, apparently not cooperative, and not detectably inhibited by C1-THF synthase substrates or cofactors. Trifunctional cytoplasmic enzyme from Saccharomyces cerevisiae and monofunctional (synthetase) enzyme from Clostridium acidiurici also bound tightly to single-stranded DNA, while bifunctional (dehydrogenase and cyclohydrolase) enzyme from Escherichia coli did not, suggesting that single-stranded DNA binding is a conserved function of the synthetase domain of C1-THF synthase enzymes. PMID:8226914

  14. Alleviation of Waterlogging Damage in Winter Rape by Uniconazole Application: Effects on Enzyme Activity, Lipid Peroxidation, and Membrane Integrity.

    PubMed

    Leul; Zhou

    1999-08-01

    Oilseed rape (Brassica napus L.) seedlings treated with uniconazole [(E)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-l-yl)-l-penten-3-ol] were transplanted at the five-leaf stage into specially designed experimental containers and then exposed to waterlogging for 3 weeks. After waterlogging stress, uniconazole-treated seedlings had significantly higher activities of superoxide dismutase, catalase, and peroxidase enzymes and endogenous free proline content at both the seedling and flowering stages. Uniconazole plus waterlogging-treated plants had a significantly higher content of unsaturated fatty acids than the waterlogged plants. There was a parallel increase in the lipid peroxidation level and electrolyte leakage rate from the leaves of waterlogged plants. Leaves from uniconazole plus waterlogging-treated plants had a significantly lower lipid peroxidation level and electrolyte leakage rate compared with waterlogged plants at both the seedling and flowering stages. Pretreatment of seedlings with uniconazole could effectively delay stress-induced degradation of chlorophyll and reduction of root oxidizability. Uniconazole did not alter the soluble sugar content of leaves and stems, after waterlogging of seedlings. Uniconazole improved waterlogged plant performance and increased seed yield, possibly because of improved antioxidation defense mechanisms, and it retarded lipid peroxidation and membrane deterioration of plants.Key Words. Waterlogging-Uniconazole-Brassica napus L.-Enzymes-Lipid peroxidation-Membrane integrityhttp://link.springer-ny.com/link/service/journals/00344/bibs/18n1p9.html PMID:10467014

  15. Inhibition of Angiotensin-Converting Enzyme Activity by Flavonoids: Structure-Activity Relationship Studies

    PubMed Central

    Guerrero, Ligia; Castillo, Julián; Quiñones, Mar; Garcia-Vallvé, Santiago; Arola, Lluis; Pujadas, Gerard; Muguerza, Begoña

    2012-01-01

    Previous studies have demonstrated that certain flavonoids can have an inhibitory effect on angiotensin-converting enzyme (ACE) activity, which plays a key role in the regulation of arterial blood pressure. In the present study, 17 flavonoids belonging to five structural subtypes were evaluated in vitro for their ability to inhibit ACE in order to establish the structural basis of their bioactivity. The ACE inhibitory (ACEI) activity of these 17 flavonoids was determined by fluorimetric method at two concentrations (500 µM and 100 µM). Their inhibitory potencies ranged from 17 to 95% at 500 µM and from 0 to 57% at 100 µM. In both cases, the highest ACEI activity was obtained for luteolin. Following the determination of ACEI activity, the flavonoids with higher ACEI activity (i.e., ACEI >60% at 500 µM) were selected for further IC50 determination. The IC50 values for luteolin, quercetin, rutin, kaempferol, rhoifolin and apigenin K were 23, 43, 64, 178, 183 and 196 µM, respectively. Our results suggest that flavonoids are an excellent source of functional antihypertensive products. Furthermore, our structure-activity relationship studies show that the combination of sub-structures on the flavonoid skeleton that increase ACEI activity is made up of the following elements: (a) the catechol group in the B-ring, (b) the double bond between C2 and C3 at the C-ring, and (c) the cetone group in C4 at the C-ring. Protein-ligand docking studies are used to understand the molecular basis for these results. PMID:23185345

  16. 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.

  17. Development of in vivo biotransformation enzyme assays for ecotoxicity screening: In vivo measurement of phases I and II enzyme activities in freshwater planarians.

    PubMed

    Li, Mei-Hui

    2016-08-01

    The development of a high-throughput tool is required for screening of environmental pollutants and assessing their impacts on aquatic animals. Freshwater planarians can be used in rapid and sensitive toxicity bioassays. Planarians are known for their remarkable regeneration ability but much less known for their metabolic and xenobiotic biotransformation abilities. In this study, the activities of different phase I and II enzymes were determined in vivo by directly measuring fluorescent enzyme substrate disappearance or fluorescent enzyme metabolite production in planarian culture media. For phase I enzyme activity, O-deethylation activities with alkoxyresorufin could not be detected in planarian culture media. By contrast, O-deethylation activities with alkoxycoumarin were detected in planarian culture media. Increases in 7-ethoxycoumarin O-deethylase (ECOD) activities was only observed in planarians exposed to 1μM, but not 10μM, β-naphthoflavone for 24h. ECOD activity was inhibited in planarians exposed to 10 and 100μM rifampicin or carbamazepine for 24h. For phase II enzyme activity, DT-diaphorase, arylsulfatases, uridine 5'-diphospho (UDP)-glucuronosyltransferase or catechol-O-methyltransferase activity was determined in culture media containing planarians. The results of this study indicate that freshwater planarians are a promising model organism to monitor exposure to environmental pollutants or assess their impacts through the in vivo measurement of phase I and II enzyme activities.

  18. Development of in vivo biotransformation enzyme assays for ecotoxicity screening: In vivo measurement of phases I and II enzyme activities in freshwater planarians.

    PubMed

    Li, Mei-Hui

    2016-08-01

    The development of a high-throughput tool is required for screening of environmental pollutants and assessing their impacts on aquatic animals. Freshwater planarians can be used in rapid and sensitive toxicity bioassays. Planarians are known for their remarkable regeneration ability but much less known for their metabolic and xenobiotic biotransformation abilities. In this study, the activities of different phase I and II enzymes were determined in vivo by directly measuring fluorescent enzyme substrate disappearance or fluorescent enzyme metabolite production in planarian culture media. For phase I enzyme activity, O-deethylation activities with alkoxyresorufin could not be detected in planarian culture media. By contrast, O-deethylation activities with alkoxycoumarin were detected in planarian culture media. Increases in 7-ethoxycoumarin O-deethylase (ECOD) activities was only observed in planarians exposed to 1μM, but not 10μM, β-naphthoflavone for 24h. ECOD activity was inhibited in planarians exposed to 10 and 100μM rifampicin or carbamazepine for 24h. For phase II enzyme activity, DT-diaphorase, arylsulfatases, uridine 5'-diphospho (UDP)-glucuronosyltransferase or catechol-O-methyltransferase activity was determined in culture media containing planarians. The results of this study indicate that freshwater planarians are a promising model organism to monitor exposure to environmental pollutants or assess their impacts through the in vivo measurement of phase I and II enzyme activities. PMID:27062342

  19. Soil Rhizosphere Microbial Communities and Enzyme Activities under Organic Farming

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study investigated the activities of ß-glucosidase (C cycling, ß-glucosaminidase (C and N cycling), acid phosphatase (P cycling) and arylsulfatase (S cycling) under lettuce (Lactuca sativa), potato (Solanum Tuberosum), onion (Allium cepa L), broccoli (Brassica oleracea var. botrytis) and Tall f...

  20. Enzyme Activity and Biomolecule Templating at Liquid and Solid Interfaces

    SciTech Connect

    Harvey W. Blanch

    2004-12-01

    There are two main components of this research program. The first involves studies of the adsorption and catalytic activity of proteins at fluid-fluid and fluid-solid interfaces; the second employs biological macromolecules as templates at the solid-liquid interface for controlled crystallization of inorganic materials, to provide materials with specific functionality.

  1. 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.

  2. Redox enzyme-mimicking activities of CeO2 nanostructures: Intrinsic influence of exposed facets

    PubMed Central

    Yang, Yushi; Mao, Zhou; Huang, Wenjie; Liu, Lihua; Li, Junli; Li, Jialiang; Wu, Qingzhi

    2016-01-01

    CeO2 nanoparticles (NPs) have been well demonstrated as an antioxidant in protecting against oxidative stress-induced cellular damages and a potential therapeutic agent for various diseases thanks to their redox enzyme-mimicking activities. The Ce3+/Ce4+ ratio and oxygen vacancies on the surface have been considered as the major originations responsible for the redox enzyme-mimicking activities of CeO2 NPs. Herein, CeO2 nanostructures (nanocubes and nanorods) exposed different facets were synthesized via a facile hydrothermal method. The characterizations by X-ray photoelectron spectroscopy, Raman spectroscopy, and UV-Vis spectroscopy show that the Ce3+/Ce4+ ratio and oxygen vacancy content on the surfaces of as-synthesized CeO2 nanostructures are nearly at the same levels. Meanwhile, the enzymatic activity measurements indicate that the redox enzyme-mimicking activities of as-synthesized CeO2 nanostructures are greatly dependent on their exposed facets. CeO2 nanocubes with exposed {100} facets exhibit a higher peroxidase but lower superoxide dismutase activity than those of the CeO2 nanorods with exposed {110} facets. Our results provide new insights into the redox enzyme-mimicking activities of CeO2 nanostructures, as well as the design and synthesis of inorganic nanomaterials-based artificial enzymes. PMID:27748403

  3. Redox enzyme-mimicking activities of CeO2 nanostructures: Intrinsic influence of exposed facets

    NASA Astrophysics Data System (ADS)

    Yang, Yushi; Mao, Zhou; Huang, Wenjie; Liu, Lihua; Li, Junli; Li, Jialiang; Wu, Qingzhi

    2016-10-01

    CeO2 nanoparticles (NPs) have been well demonstrated as an antioxidant in protecting against oxidative stress-induced cellular damages and a potential therapeutic agent for various diseases thanks to their redox enzyme-mimicking activities. The Ce3+/Ce4+ ratio and oxygen vacancies on the surface have been considered as the major originations responsible for the redox enzyme-mimicking activities of CeO2 NPs. Herein, CeO2 nanostructures (nanocubes and nanorods) exposed different facets were synthesized via a facile hydrothermal method. The characterizations by X-ray photoelectron spectroscopy, Raman spectroscopy, and UV-Vis spectroscopy show that the Ce3+/Ce4+ ratio and oxygen vacancy content on the surfaces of as-synthesized CeO2 nanostructures are nearly at the same levels. Meanwhile, the enzymatic activity measurements indicate that the redox enzyme-mimicking activities of as-synthesized CeO2 nanostructures are greatly dependent on their exposed facets. CeO2 nanocubes with exposed {100} facets exhibit a higher peroxidase but lower superoxide dismutase activity than those of the CeO2 nanorods with exposed {110} facets. Our results provide new insights into the redox enzyme-mimicking activities of CeO2 nanostructures, as well as the design and synthesis of inorganic nanomaterials-based artificial enzymes.

  4. 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

  5. Relevance of drug metabolizing enzyme activity modulation by tea polyphenols in the inhibition of esophageal tumorigenesis.

    PubMed

    Maliakal, Pius; Sankpal, Umesh T; Basha, Riyaz; Maliakal, Cima; Ledford, Andrea; Wanwimolruk, Sompon

    2011-09-01

    Tea is a popular, socially accepted, drink that is enjoyed by millions of people. A growing body of evidence suggests that moderate consumption of tea may protect against several forms of cancer. It is also known that bioactivation of precarcinogens and detoxification of ultimate carcinogens is carried out mainly by drug metabolizing enzymes such as cytochrome P450 (CYP). The present study investigates the effect of tea consumption on modulating CYP and phase II conjugating enzymes, and their association in the chemopreventive effect against esophageal tumorigenesis using both in vitro and in vivo techniques. Female Wistar rats were given aqueous solutions (2% w/v) of six different teas, standard green tea extract (GTE) (0.5% w/v), and dandelion tea (2% w/v) as the sole source of fluid for two weeks prior to and during the entire period of tumour induction (12 weeks). Animals were gavaged with 0.5 mg/kg N-nitrosomethylbenzylamine (NMBA) twice weekly for 12 weeks for esophageal tumor induction and the activities of different CYP isoforms and phase II enzymes were determined in the liver microsomes or cytosols. GTE, green tea and Dandelion tea caused decrease in tumour multiplication, tumour size and tumour volume; however, none of these tea preparations altered tumour incidence. No appreciable changes in drug metabolizing enzyme activity were observed in the treatment groups. Thus, the modulations in the activities of CYP 1A1/ 1A2 and CYP2E enzymes, by pre-treatment with green and dandelion teas, observed in our earlier experiments, seem to be compensated by the tumor inducing agent, NMBA. The balance between phase I carcinogen-activating enzymes and phase II detoxifying enzymes could be important in determining the risk of developing chemically-induced cancer and the present study in conjunction with the previous observations suggest a possible role of drug metabolizing enzymes in the anticancer effect of tea.

  6. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

    PubMed Central

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W.; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. PMID:27276217

  7. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae.

    PubMed

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. PMID:27276217

  8. Enzyme activities of demersal fishes from the shelf to the abyssal plain

    NASA Astrophysics Data System (ADS)

    Drazen, Jeffrey C.; Friedman, Jason R.; Condon, Nicole E.; Aus, Erica J.; Gerringer, Mackenzie E.; Keller, Aimee A.; Elizabeth Clarke, M.

    2015-06-01

    The present study examined metabolic enzyme activities of 61 species of demersal fishes (331 individuals) trawled from a 3000 m depth range. Citrate synthase, lactate dehydrogenase, malate dehydrogenase, and pyruvate kinase activities were measured as proxies for aerobic and anaerobic activity and metabolic rate. Fishes were classified according to locomotory mode, either benthic or benthopelagic. Fishes with these two locomotory modes were found to exhibit differences in metabolic enzyme activity. This was particularly clear in the overall activity of citrate synthase, which had higher activity in benthopelagic fishes. Confirming earlier, less comprehensive studies, enzyme activities declined with depth in benthopelagic fishes. For the first time, patterns in benthic species could be explored and these fishes also exhibited depth-related declines in enzyme activity, contrary to expectations of the visual interactions hypothesis. Trends were significant when using depth parameters taken from the literature as well as from the present trawl information, suggesting a robust pattern regardless of the depth metric used. Potential explanations for the depth trends are discussed, but clearly metabolic rate does not vary simply as a function of mass and habitat temperature in fishes as shown by the substantial depth-related changes in enzymatic activities.

  9. Probing phosphatase activity using redox active nanoparticles: a novel colorimetric approach for the detection of enzyme activity.

    PubMed

    Hayat, Akhtar; Gonca Bulbul; Andreescu, Silvana

    2014-06-15

    A new colorimetric assay for the detection of alkaline phosphatase (ALP) activity is reported based on the surface reactivity and optical properties of redox active nanoparticles of cerium oxide, or nanoceria. The method takes advantage of nanoceria color changes after interaction with products of the ALP catalyzed reaction, resulting in charge transfer complexes with very strong absorption characteristics. The developed assay is easy-to-use, robust and cost effective and does not involve labeled reagents, secondary enzymes or soluble dyes. Hydrolytic products of more stable substrates (catechol monophosphate, ascorbic 2-phosphate and hydroquinone diphosphate) that could previously not be used in ALP assays can be conveniently colorimetrically detected with this assay. A detection limit of 0.04 U/L ALP with a linear range up to 2U/L was obtained with ascorbic 2-phosphate substrate. The proposed assay can eliminate multistep procedures and minimize problems associated with the poor stability of substrates and enzyme labels of conventional ALP assays. The assay has been adapted to a paper platform and has demonstrated functionality for ALP detection in human serum. This sensing concept can find wide applications as a general approach for improving sensitivity and simplifying detection schemes of colorimetric bioassays, e.g. enzyme, gene, immuno and aptamer assays and related affinity sensing methods. PMID:24531308

  10. Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance

    SciTech Connect

    Beckman, J.S.; Minor, R.L. Jr.; White, C.W.; Repine, J.E.; Rosen, G.M.; Freeman, B.A.

    1988-05-15

    Covalent conjugation of superoxide dismutase and catalase with polyethylene glycol (PEG) increases the circulatory half-lives of these enzymes from <10 min to 40 h, reduced immunogenicity, and decreases sensitivity to proteolysis. Because PEG has surface active properties and can induce cell fusion, the authors hypothesized that PEG conjugation could enhance cell binding and association of normally membrane-impermeable enzymes. Incubation of cultured porcine aortic endothelial cells with /sup 125/I-PEG-catalase or /sup 125/I-PEG-superoxide dismutase produced a linear, concentration-dependent increase in cellular enzyme activity and radioactivity. Fluorescently labeled PEG-superoxide dismutase incubated with endothelial cells showed a vesicular localization. Mechanical injury to cell monolayers, which is known to stimulate endocytosis, further increased the uptake of fluorescent PEG-superoxide dismutase. Addition of PEG and PEG-conjugated enzymes perturbed the spin-label binding environment, indicative of producing an increase in plasma membrane fluidity. Thus, PEG conjugation to superoxide dismutase and catalase enhances cell association of these enzymes in a manner which increases cellular enzyme activities and provides prolonged protection from partially reduced oxygen species.

  11. Oscillatory behavior of US -galactosidase enzyme activity in Escherichia coli during perturbed batch experiments

    SciTech Connect

    Pih, N.P.; Dhurjat, P.

    1987-02-05

    The behaviour of a wild-type and mutant strain of Escherichia coli under batch aerobic conditions were studied. In these experiments the bacteria were initially grown with lactose as the sole carbon source. When exponential growth on lactose was achieved, the batch was perturbed with D-glucose. Periodic off-line samples were taken from the fermentor and analyzed for US -galactosidase enzyme activity, D-glucose, and lactose. Continuous on-line measurements of optical density of fermentation media were also made. Oscillations in the measured enzyme activity were observed. Oscillatory behavior of US -galactosidase enzyme in E. coli was previously reported by Knorre. In his study cells were grown in D-glucose, washed, and then grown on lactose. Oscillations were attributed to the varying enzyme synthesis rate. In the present study the cells were grown initally on lactose, thus assuring high synthesis rates of US -galactosidase from the start. The oscillations observed after perturbation with glucose are pronounced and appear to be the result of combined changes in the substrate transport system and enzyme activity in addition to possible changes in enzyme synthesis rate. 10 references.

  12. A neglected modulator of insulin-degrading enzyme activity and conformation: The pH.

    PubMed

    Grasso, Giuseppe; Satriano, Cristina; Milardi, Danilo

    2015-01-01

    Insulin-degrading enzyme (IDE), a ubiquitously expressed zinc metalloprotease, has multiple activities in addition to insulin degradation and its malfunction is believed to connect type 2 diabetes with Alzheimer's disease. IDE has been found in many different cellular compartments, where it may experience significant physio-pathological pH variations. However, the exact role of pH variations on the interplay between enzyme conformations, stability, oligomerization state and catalysis is not understood. Here, we use ESI mass spectrometry, atomic force microscopy, surface plasmon resonance and circular dichroism to investigate the structure-activity relationship of IDE at different pH values. We show that acidic pH affects the ability of the enzyme to bind the substrate and decrease the stability of the protein by inducing an α-helical bundle conformation with a concomitant dissociation of multi-subunit IDE assemblies into monomeric units and loss of activity. These effects suggest a major role played by electrostatic forces in regulating multi-subunit enzyme assembly and function. Our results clearly indicate a pH dependent coupling among enzyme conformation, assembly and stability and suggest that cellular acidosis can have a large effect on IDE oligomerization state, inducing an enzyme inactivation and an altered insulin degradation that could have an impact on insulin signaling.

  13. Variation in pH optima of hydrolytic enzyme activities in tropical rain forest soils.

    PubMed

    Turner, Benjamin L

    2010-10-01

    Extracellular enzymes synthesized by soil microbes play a central role in the biogeochemical cycling of nutrients in the environment. The pH optima of eight hydrolytic enzymes involved in the cycles of carbon, nitrogen, phosphorus, and sulfur, were assessed in a series of tropical forest soils of contrasting pH values from the Republic of Panama. Assays were conducted using 4-methylumbelliferone-linked fluorogenic substrates in modified universal buffer. Optimum pH values differed markedly among enzymes and soils. Enzymes were grouped into three classes based on their pH optima: (i) enzymes with acidic pH optima that were consistent among soils (cellobiohydrolase, β-xylanase, and arylsulfatase), (ii) enzymes with acidic pH optima that varied systematically with soil pH, with the most acidic pH optima in the most acidic soils (α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase), and (iii) enzymes with an optimum pH in either the acid range or the alkaline range depending on soil pH (phosphomonoesterase and phosphodiesterase). The optimum pH values of phosphomonoesterase were consistent among soils, being 4 to 5 for acid phosphomonoesterase and 10 to 11 for alkaline phosphomonoesterase. In contrast, the optimum pH for phosphodiesterase activity varied systematically with soil pH, with the most acidic pH optima (3.0) in the most acidic soils and the most alkaline pH optima (pH 10) in near-neutral soils. Arylsulfatase activity had a very acidic optimum pH in all soils (pH ≤3.0) irrespective of soil pH. The differences in pH optima may be linked to the origins of the enzymes and/or the degree of stabilization on solid surfaces. The results have important implications for the interpretation of hydrolytic enzyme assays using fluorogenic substrates.

  14. Variation in pH Optima of Hydrolytic Enzyme Activities in Tropical Rain Forest Soils ▿

    PubMed Central

    Turner, Benjamin L.

    2010-01-01

    Extracellular enzymes synthesized by soil microbes play a central role in the biogeochemical cycling of nutrients in the environment. The pH optima of eight hydrolytic enzymes involved in the cycles of carbon, nitrogen, phosphorus, and sulfur, were assessed in a series of tropical forest soils of contrasting pH values from the Republic of Panama. Assays were conducted using 4-methylumbelliferone-linked fluorogenic substrates in modified universal buffer. Optimum pH values differed markedly among enzymes and soils. Enzymes were grouped into three classes based on their pH optima: (i) enzymes with acidic pH optima that were consistent among soils (cellobiohydrolase, β-xylanase, and arylsulfatase), (ii) enzymes with acidic pH optima that varied systematically with soil pH, with the most acidic pH optima in the most acidic soils (α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase), and (iii) enzymes with an optimum pH in either the acid range or the alkaline range depending on soil pH (phosphomonoesterase and phosphodiesterase). The optimum pH values of phosphomonoesterase were consistent among soils, being 4 to 5 for acid phosphomonoesterase and 10 to 11 for alkaline phosphomonoesterase. In contrast, the optimum pH for phosphodiesterase activity varied systematically with soil pH, with the most acidic pH optima (3.0) in the most acidic soils and the most alkaline pH optima (pH 10) in near-neutral soils. Arylsulfatase activity had a very acidic optimum pH in all soils (pH ≤3.0) irrespective of soil pH. The differences in pH optima may be linked to the origins of the enzymes and/or the degree of stabilization on solid surfaces. The results have important implications for the interpretation of hydrolytic enzyme assays using fluorogenic substrates. PMID:20709838

  15. Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

    PubMed

    Buchanan, R L; Lewis, D F

    1984-08-01

    Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes.

  16. 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.

  17. Inhibitory activity of Plantago major L. on angiotensin I-converting enzyme.

    PubMed

    Nhiem, Nguyen Xuan; Tai, Bui Huu; Van Kiem, Phan; Van Minh, Chau; Cuong, Nguyen Xuan; Tung, Nguyen Huu; Thu, Vu Kim; Trung, Trinh Nam; Anh, Hoang Le Tuan; Jo, Sung-Hoon; Jang, Hae-Dong; Kwon, Young-In; Kim, Young Ho

    2011-03-01

    Eight compounds were isolated from methanol extract of Plantago major L. leaves and investigated for their ability to inhibit angiotensin I-converting enzyme activity. Among them, compound 1 showed the most potent inhibition with rate of 28.06 ± 0.21% at a concentration of 100 μM. Compounds 2 and 8 exhibited weak activities. These results suggest that compound 1 might contribute to the ability of P. major to inhibit the activity of angiotensin I- converting enzyme. PMID:21547673

  18. The effect of the presence of globular proteins and elongated polymers on enzyme activity.

    PubMed

    Derham, Barry K; Harding, John J

    2006-06-01

    We have studied the effect of a crowded (macromolecular) solution on reaction rates of the decarboxylating enzymes urease, pyruvate decarboxylase and glutamate decarboxylase. A variety of crowding agents were used including haemoglobin, lysozyme, various dextrans and polyethylene glycol. Enzyme reaction rates of all three enzymes show two different types of effect that separate the globular proteins from the polysaccharides/polymers. Increasing concentration of globular proteins caused a dramatic rise in enzyme activity up to 30% crowding concentration then the activity decreased, whereas the polymers caused a concentration dependent decrease in activity. The viscosities of the globular proteins were low compared to the polymers. The increased activity with proteins may be due to the decreased amount of free water, which leads to higher effective concentration of substrates, or to an increased oligomeric state by self-association. The lower activities of all enzymes with all agents at high concentrations may be explained by a decrease in rates of diffusion. An increase in protein crowding (decrease in cell water content) may contribute to pathological conditions including cataract and Alzheimer's disease.

  19. Relative Activities and Characteristics of Some Oxidative Respiratory Enzymes from Conidia of Verticillium albo-atrum

    PubMed Central

    Throneberry, G. O.

    1967-01-01

    Conidia of Verticillium albo-atrum Reinke and Berthold, collected from shake cultures grown in Czapek broth, were sonified for 4 or 8 minutes or ground frozen in a mortar to obtain cell-free homogenates. These were assayed for certain enzymes associated with respiratory pathways. Malic dehydrogenase was the most active, glucose-6-P and NADH dehydrogenase were less active, NADH-cytochrome c reductase, NADPH dehydrogenase, and cytochrome oxidase were low in activity, and succinic dehydrogenase and succinic cytochrome c reductase were very low to negligible in activity. No NADH oxidase activity was detected. With the exception of NADH-cytochrome c reductase and possibly succinic dehydrogenase and cytochrome c reductase, there was no evident increase in specific activity of the enzymes during germination. Some NADH-cytochrome c reductase and a small amount of succinic-dehydrogenase and cytochrome c reductase were associated with the particulate fraction from 105,000 × g centrifugation. The other enzymes, including cytochrome oxidase, almost completely remained in the supernatant fraction. Menadione and vitamin K-S(II) markedly stimulated NADH-cytochrome c reductase activity in the supernatant fraction but had much less effect on NADPH-cytochrome c reductase in this fraction or on either of these enzyme systems in the particulate fraction. Electron transport inhibitors affected particulate NADH- and NADPH-cytochrome c reductase activity but had no effect on these in the supernatant fraction. PMID:16656681

  20. Poly(2-hydroxyethyl methacrylate) for enzyme immobilization: impact on activity and stability of horseradish peroxidase.

    PubMed

    Lane, Sarah M; Kuang, Zhifeng; Yom, Jeannie; Arifuzzaman, Shafi; Genzer, Jan; Farmer, Barry; Naik, Rajesh; Vaia, Richard A

    2011-05-01

    On the basis of their versatile structure and chemistry as well as tunable mechanical properties, polymer brushes are well-suited as supports for enzyme immobilization. However, a robust surface design is hindered by an inadequate understanding of the impact on activity from the coupling motif and enzyme distribution within the brush. Herein, horseradish peroxidase C (HRP C, 44 kDa), chosen as a model enzyme, was immobilized covalently through its lysine residues on a N-hydroxysuccinimidyl carbonate-activated poly(2-hydroxyethyl methacrylate) (PHEMA) brush grafted chemically onto a flat impenetrable surface. Up to a monolayer coverage of HRP C is achieved, where most of the HRP C resides at or near the brush-air interface. Molecular modeling shows that lysines 232 and 241 are the most probable binding sites, leading to an orientation of the immobilized HRP C that does not block the active pocket of the enzyme. Michaelis-Menten kinetics of the immobilized HRP C indicated little change in the K(m) (Michaelis constant) but a large decrease in the V(max) (maximum substrate conversion rate) and a correspondingly large decrease in the k(cat) (overall catalytic rate). This indicates a loss in the percentage of active enzymes. Given the relatively ideal geometry of the HRPC-PHEMA brush, the loss of activity is most likely due to structural changes in the enzyme arising from either secondary constraints imposed by the connectivity of the N-hydroxysuccinimidyl carbonate linking moiety or nonspecific interactions between HRP C and DSC-PHEMA. Therefore, a general enzyme-brush coupling motif must optimize reactive group density to balance binding with neutrality of surroundings. PMID:21438540

  1. Mechanistic studies on activation of ubiquitin and di-ubiquitin-like protein, FAT10, by ubiquitin-like modifier activating enzyme 6, Uba6.

    PubMed

    Gavin, James M; Chen, Jesse J; Liao, Hua; Rollins, Neil; Yang, Xiaofeng; Xu, Qing; Ma, Jingya; Loke, Huay-Keng; Lingaraj, Trupti; Brownell, James E; Mallender, William D; Gould, Alexandra E; Amidon, Benjamin S; Dick, Lawrence R

    2012-05-01

    Uba6 is a homolog of the ubiquitin-activating enzyme, Uba1, and activates two ubiquitin-like proteins (UBLs), ubiquitin and FAT10. In this study, biochemical and biophysical experiments were performed to understand the mechanisms of how Uba6 recognizes two distinct UBLs and catalyzes their activation and transfer. Uba6 is shown to undergo a three-step activation process and form a ternary complex with both UBLs, similar to what has been observed for Uba1. The catalytic mechanism of Uba6 is further supported by inhibition studies using a mechanism-based E1 inhibitor, Compound 1, which forms covalent adducts with both ubiquitin and FAT10. In addition, pre-steady state kinetic analysis revealed that the rates of UBL-adenylate (step 1) and thioester (step 2) formation are similar between ubiquitin and FAT10. However, distinct kinetic behaviors were also observed for ubiquitin and FAT10. FAT10 binds Uba6 with much higher affinity than ubiquitin while demonstrating lower catalytic activity in both ATP-PP(i) exchange and E1-E2 transthiolation assays. Also, Compound 1 is less potent with FAT10 as the UBL compared with ubiquitin in ATP-PP(i) exchange assays, and both a slow rate of covalent adduct formation and weak adduct binding to Uba6 contribute to the diminished potency observed for FAT10. Together with expression level analysis in IM-9 cells, this study sheds light on the potential role of cytokine-induced FAT10 expression in regulating Uba6 pathways.

  2. Inhibition of digestive enzyme activities by copper in the guts of various marine benthic invertebrates.

    PubMed

    Chen, Zhen; Mayer, Lawrence M; Weston, Donald P; Bock, Michael J; Jumars, Peter A

    2002-06-01

    Digestive systems of deposit and suspension feeders can be exposed to high concentrations of copper (Cu) by ingestion of contaminated sediments. We assessed a potential impact of this Cu exposure on digestive enzyme activities in a wide range of benthic organisms by monitoring enzyme activities in their gut fluids during in vitro titrations with dissolved Cu, which mimics Cu solubilization from sediments. Increasing Cu inhibited digestive protease activities at threshold values, which varied widely among organisms, from 8 microM for an echinoderm to 0.4 M for an echiuran. More Cu was required to inhibit proteases in guts containing higher amino acid concentrations because strong Cu-binding sites on amino acids prevent Cu interaction with the enzymatically active sites. Threshold Cu concentrations were similar for proteases, esterases, lipases, and alpha- and beta-glucosidases, suggesting the same inhibition mechanism. Copper was less effective at inhibiting enzymes at lower pH, suggesting that protons can compete with Cu ion for binding to enzymatically active sites or that enzyme conformation is less vulnerable to Cu inhibition at lower pH. These results lead to the counterintuitive conclusion that deposit feeders with low enzyme activity, low amino acid concentration, and high pH values are most vulnerable to harm from sedimentary Cu by this mechanism, although they solubilize less sedimentary Cu than their counterparts with high enzyme activity, high amino acid concentrations, and low gut pH. In general, digestive systems of echinoderms may therefore be more susceptible to Cu contamination than those of polychaetes, with various other phyla showing intermediate susceptibilities. If threshold Cu values are converted to solid-phase sedimentary Cu concentrations, the thresholds are at least consistent with Cu loadings that have been observed to lead to biological impacts in the field.

  3. Daily rhythms of digestive enzyme activity and gene expression in gilthead seabream (Sparus aurata) during ontogeny.

    PubMed

    Mata-Sotres, José Antonio; Moyano, Francisco Javier; Martínez-Rodríguez, Gonzalo; Yúfera, Manuel

    2016-07-01

    In order to identify daily changes in digestive physiology in developing gilthead seabream larvae, the enzyme activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try, chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2 and α-amylase-amy2a) were measured during a 24h cycle in larvae reared under a 12h light/12h dark photoperiod. Larvae were sampled at 10, 18, 30 and 60days post-hatch. In each sampling day, larvae were sampled every 3h during a complete 24h cycle. The enzyme activity and gene expression exhibited a marked dependent behavior to the light/darkness cycle in all tested ages. The patterns of activity and expression of all tested enzymes were compared to the feeding pattern found in the same larvae, which showed a rhythmic feeding pattern with a strong light synchronization. In the four tested ages, the activities of trypsin, and to a lesser extent lipases and amylase, were related to feeding activity. Molecular expression of the pancreatic enzymes tended to increase during the night, probably as an anticipation of the forthcoming ingestion of food that will take place during the next light period. It follows that the enzymatic activities are being regulated at translational and/or post-translational level. The potential variability of enzyme secretion along the whole day is an important factor to take into account in future studies. A particularly striking consequence of the present results is the reliability of studies based in only one daily sample taken at the same hour of the day, as those focused to assess ontogeny of digestive enzymes.

  4. Activity and dynamics of an enzyme, pig liver esterase, in near-anhydrous conditions

    SciTech Connect

    Lopez, Murielle; Kurkal-Siebert, V; Dunn, Rachel V.; Tehei, M; Finney, J.L.; Smith, Jeremy C; Daniel, R. M.

    2010-10-01

    Water is widely assumed to be essential for life, although the exact molecular basis of this requirement is unclear. Water facilitates protein motions, and although enzyme activity has been demonstrated at low hydrations in organic solvents, such nonaqueous solvents may allow the necessary motions for catalysis. To examine enzyme function in the absence of solvation and bypass diffusional constraints we have tested the ability of an enzyme, pig liver esterase, to catalyze alcoholysis as an anhydrous powder, in a reaction system of defined water content and where the substrates and products are gaseous. At hydrations of 3 ( 2) molecules of water per molecule of enzyme, activity is several orders-of-magnitude greater than nonenzymatic catalysis. Neutron spectroscopy indicates that the fast ( nanosecond) global anharmonic dynamics of the anhydrous functional enzyme are suppressed. This indicates that neither hydration water nor fast anharmonic dynamics are required for catalysis by this enzyme, implying that one of the biological requirements of water may lie with its role as a diffusion medium rather than any of its more specific properties.

  5. Screening of halophilic bacteria and Alteromonas species for organophosphorus hydrolyzing enzyme activity.

    PubMed

    DeFrank, J J; Beaudry, W T; Cheng, T C; Harvey, S P; Stroup, A N; Szafraniec, L L

    1993-06-01

    Previously, a G-type nerve agent degrading enzyme activity was found in a halophilic bacterial isolate designated JD6.5. This organism was tentatively identified as an unknown species of the genus Alteromonas. In order to determine whether this type of enzyme activity was common in other species of Alteromonas, a screening program was initiated. A number of Alteromonas species and five halophilic bacterial isolates were cultured and their crude cell extracts screened for hydrolytic activity against several organophosphorus chemical agents and other related compounds. The samples were also screened for cross-reactivity with a monoclonal antibody raised against the purified enzyme from JD6.5 and for hybridization with a DNA probe based on its N-terminal amino acid sequence A wide spectrum of activities and reactivities were seen, suggesting a significant heterogeneity between the functionally similar enzymes that are present in these bacterial species. Enzymes of the type described here have considerable potential for the decontamination and demilitarization of chemical warfare agents.

  6. Enzyme renaturation to higher activity driven by the sol-gel transition: Carbonic anhydrase

    NASA Astrophysics Data System (ADS)

    Vinogradov, Vladimir V.; Avnir, David

    2015-09-01

    We describe a so-far unknown route for renaturing denatured enzymes, namely subjecting the denatured enzyme to an oxide sol-gel transition. The phenomenon was revealed in a detailed study of denatured carbonic anhydrase which was subjected to an alumina sol-gel transition, up to the thermally stabilizing entrapment in the final xerogel. Remarkably, not only that the killed enzyme regained its activity during the sol-gel process, but its activity increased to 180% of the native enzyme. To the best of our knowledge, this is the first report of enhanced activity following by renaturing (a “Phoenix effect”). Kinetic study which revealed a five-orders of magnitude (!) increase in the Arrhenius prefactor upon entrapment compared to solution. Circular dichroism analysis, differential scanning calorimetry, zeta potential analyses as well as synchronous fluorescence measurements, all of which were used to characterize the phenomenon, are consistent with a proposed mechanism which is based on the specific orienting interactions of the active site of the enzyme with respect to the alumina interface and its pores network.

  7. Redox-initiated hydrogel system for detection and real-time imaging of cellulolytic enzyme activity.

    PubMed

    Malinowska, Klara H; Verdorfer, Tobias; Meinhold, Aylin; Milles, Lukas F; Funk, Victor; Gaub, Hermann E; Nash, Michael A

    2014-10-01

    Understanding the process of biomass degradation by cellulolytic enzymes is of urgent importance for biofuel and chemical production. Optimizing pretreatment conditions and improving enzyme formulations both require assays to quantify saccharification products on solid substrates. Typically, such assays are performed using freely diffusing fluorophores or dyes that measure reducing polysaccharide chain ends. These methods have thus far not allowed spatial localization of hydrolysis activity to specific substrate locations with identifiable morphological features. Here we describe a hydrogel reagent signaling (HyReS) system that amplifies saccharification products and initiates crosslinking of a hydrogel that localizes to locations of cellulose hydrolysis, allowing for imaging of the degradation process in real time. Optical detection of the gel in a rapid parallel format on synthetic and natural pretreated solid substrates was used to quantify activity of T. emersonii and T. reesei enzyme cocktails. When combined with total internal reflection fluorescence microscopy and AFM imaging, the reagent system provided a means to visualize enzyme activity in real-time with high spatial resolution (<2 μm). These results demonstrate the versatility of the HyReS system in detecting cellulolytic enzyme activity and suggest new opportunities in real-time chemical imaging of biomass depolymerization. PMID:25116339

  8. 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.

  9. Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

    PubMed Central

    Santiago, Margarita; Ramírez-Sarmiento, César A.; Zamora, Ricardo A.; Parra, Loreto P.

    2016-01-01

    Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications. PMID:27667987

  10. Shape-Dependent Biomimetic Inhibition of Enzyme by Nanoparticles and Their Antibacterial Activity.

    PubMed

    Cha, Sang-Ho; Hong, Jin; McGuffie, Matt; Yeom, Bongjun; VanEpps, J Scott; Kotov, Nicholas A

    2015-09-22

    Enzyme inhibitors are ubiquitous in all living systems, and their biological inhibitory activity is strongly dependent on their molecular shape. Here, we show that small zinc oxide nanoparticles (ZnO NPs)-pyramids, plates, and spheres-possess the ability to inhibit activity of a typical enzyme β-galactosidase (GAL) in a biomimetic fashion. Enzyme inhibition by ZnO NPs is reversible and follows classical Michaelis-Menten kinetics with parameters strongly dependent on their geometry. Diverse spectroscopic, biochemical, and computational experimental data indicate that association of GAL with specific ZnO NP geometries interferes with conformational reorganization of the enzyme necessary for its catalytic activity. The strongest inhibition was observed for ZnO nanopyramids and compares favorably to that of the best natural GAL inhibitors while being resistant to proteases. Besides the fundamental significance of this biomimetic function of anisotropic NPs, their capacity to serve as degradation-resistant enzyme inhibitors is technologically attractive and is substantiated by strong shape-specific antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), endemic for most hospitals in the world. PMID:26325486

  11. Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

    PubMed Central

    Santiago, Margarita; Ramírez-Sarmiento, César A.; Zamora, Ricardo A.; Parra, Loreto P.

    2016-01-01

    Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications.

  12. Biotransformation of anthelmintics and the activity of drug-metabolizing enzymes in the tapeworm Moniezia expansa.

    PubMed

    Prchal, Lukáš; Bártíková, Hana; Bečanová, Aneta; Jirásko, Robert; Vokřál, Ivan; Stuchlíková, Lucie; Skálová, Lenka; Kubíček, Vladimír; Lamka, Jiří; Trejtnar, František; Szotáková, Barbora

    2015-04-01

    The sheep tapeworm Moniezia expansa is very common parasite, which affects ruminants such as sheep, goats as well as other species. The benzimidazole anthelmintics albendazole (ABZ), flubendazole (FLU) and mebendazole (MBZ) are often used to treat the infection. The drug-metabolizing enzymes of helminths may alter the potency of anthelmintic treatment. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (ABZ, MBZ and FLU) in M. expansa. Activities of biotransformation enzymes were determined in subcellular fractions. Metabolites of the anthelmintics were detected and identified using high performance liquid chromatography/ultra-violet/VIS/fluorescence or ultra-high performance liquid chromatography/mass spectrometry. Reduction of MBZ, FLU and oxidation of ABZ were proved as well as activities of various metabolizing enzymes. Despite the fact that the conjugation enzymes glutathione S-transferase, UDP-glucuronosyl transferase and UDP-glucosyl transferase were active in vitro, no conjugated metabolites of anthelmintics were identified either ex vivo or in vitro. The obtained results indicate that sheep tapeworm is able to deactivate the administered anthelmintics, and thus protects itself against their action.

  13. Shape-Dependent Biomimetic Inhibition of Enzyme by Nanoparticles and Their Antibacterial Activity.

    PubMed

    Cha, Sang-Ho; Hong, Jin; McGuffie, Matt; Yeom, Bongjun; VanEpps, J Scott; Kotov, Nicholas A

    2015-09-22

    Enzyme inhibitors are ubiquitous in all living systems, and their biological inhibitory activity is strongly dependent on their molecular shape. Here, we show that small zinc oxide nanoparticles (ZnO NPs)-pyramids, plates, and spheres-possess the ability to inhibit activity of a typical enzyme β-galactosidase (GAL) in a biomimetic fashion. Enzyme inhibition by ZnO NPs is reversible and follows classical Michaelis-Menten kinetics with parameters strongly dependent on their geometry. Diverse spectroscopic, biochemical, and computational experimental data indicate that association of GAL with specific ZnO NP geometries interferes with conformational reorganization of the enzyme necessary for its catalytic activity. The strongest inhibition was observed for ZnO nanopyramids and compares favorably to that of the best natural GAL inhibitors while being resistant to proteases. Besides the fundamental significance of this biomimetic function of anisotropic NPs, their capacity to serve as degradation-resistant enzyme inhibitors is technologically attractive and is substantiated by strong shape-specific antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), endemic for most hospitals in the world.

  14. Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes.

    PubMed

    Santiago, Margarita; Ramírez-Sarmiento, César A; Zamora, Ricardo A; Parra, Loreto P

    2016-01-01

    Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications.

  15. Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes.

    PubMed

    Santiago, Margarita; Ramírez-Sarmiento, César A; Zamora, Ricardo A; Parra, Loreto P

    2016-01-01

    Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications. PMID:27667987

  16. Adsorption and enzyme activity of sucrose phosphorylase on lipid Langmuir and Langmuir-Blodgett films.

    PubMed

    Rocha, Jefferson Muniz; Caseli, Luciano

    2014-04-01

    The production of bioelectronic devices, including biosensors, can be conducted using enzymes immobilized in ultrathin solid films, for which preserving the enzymatic catalytic activity is crucial for optimal performance. In this sense, nanostructured films that allow for control over molecular architectures are of interest. In this paper, we investigate the adsorption of sucrose phosphorylase onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid, which caused the surface pressure isotherms to expand. With polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), the amide bands from the enzyme could be identified, with the C-N and C=O dipole moments lying parallel to the air-water interface. Structuring of the enzyme into an α-helix was noted, and this structure was preserved when the mixed enzyme-phospholipid monolayer was transferred in the form of a Langmuir-Blodgett (LB) film. The latter was demonstrated with measurements of the catalytic activity of sucrose phosphorylase, which presented the highest enzyme activity for multilayer LB film. The approach presented in this study not only allows for optimized catalytic activity toward sucrose but also permits to explain why certain film architectures exhibit superior performance.

  17. Effect of zinc concentration on the activity of angiotensin converting enzyme in human plasma and serum

    SciTech Connect

    Reeves, P.G.; Carl, G.F.; Smith, D.K.; O'Dell, B.L.

    1986-03-05

    The activity of angiotensin converting enzyme is measured clinically to assist in the diagnosis of sarcoidosis and to monitor therapy with steroids, and with antihypertensive drugs that inhibit the enzyme. Even though it has been known for some time that ACE is a zinc dependent enzyme, it was discovered only recently that zinc, in addition to endogenous levels in the assay mixture, is required for maximal activity of rat serum ACE. The present experiment was designed to determine if additional zinc is required for maximal activation of ACE in plasma and serum of human subjects. Plasma or serum samples were incubated at 37/sup 0/ in a zinc-free medium, pH 7.4, containing hippurylglyclglycine as the substrate. The addition of 20 ..mu..M zinc significantly increased ACE activity in plasma (95.4 +/- 11.9 vs 192.8 +/- 24.3 U/L) and in serum (89.9 +/- 5.6 vs 195.7 +/- 9.3 U/L) compared to samples without added zinc. Enzyme activity was increased 2.4-fold when zinc was added to plasma from a patient with low plasma zinc. These data suggest that the endogenous level of zinc in the assay mixture resulting from the addition of an aliquot of plasma or serum is insufficient to obtain maximal activity of ACE. The addition of zinc to zinc deficient plasma increased ACE activity even more.

  18. Diel changes in stream periphyton extracellular enzyme activity throughout community development on inert and organic substrates

    NASA Astrophysics Data System (ADS)

    Rier, S. T.; Francoeur, S. N.; Kuehn, K. A.

    2005-05-01

    We tested the hypothesis that algal photosynthesis in stream periphyton communities would influence the activities of extracellular enzymes produced by associated heterotrophic bacteria and fungi to acquire organic compounds and inorganic nutrients. We approached this question by looking for diurnal variation in activities of four extracellular enzymes in periphyton communities that were grown on either inert (glass fiber filters) or organic (leaves) substrata that there were incubated in stream-side channels that were either open to full sun or shaded. Substrata were subsampled for β-glucosidase, alkaline phosphotase, leucine-aminopeptidase, and phenol oxidase activities at 3-5 hr. intervals over two consecutive diurnal cycles that were repeated at an early and later stage of periphyton community development. Activities of all enzymes displayed diurnal periodicity but the strength of the diurnal effects depended largely on the substrate type and stage of community development. The most consistent diurnal change was observed with phenol oxidase activity with significantly greater (p<0.05) activities being observed in during the day for both stages of community development and for both substrate types. It is likely that oxygen produced by algal photosynthesis is driving the activity of this oxidative enzyme and that algae might indirectly influence the decomposition of phenolic compounds.

  19. Age-Related Changes in Hepatic Activity and Expression of Detoxification Enzymes in Male Rats

    PubMed Central

    Vyskočilová, Erika; Szotáková, Barbora; Skálová, Lenka; Bártíková, Hana; Hlaváčová, Jitka

    2013-01-01

    Process of aging is accompanied by changes in the biotransformation of xenobiotics and impairment of normal cellular functions by free radicals. Therefore, this study was designed to determine age-related differences in the activities and/or expressions of selected drug-metabolizing and antioxidant enzymes in young and old rats. Specific activities of 8 drug-metabolizing enzymes and 4 antioxidant enzymes were assessed in hepatic subcellular fractions of 6-week-old and 21-month-old male Wistar rats. Protein expressions of carbonyl reductase 1 (CBR1) and glutathione S-transferase (GST) were determined using immunoblotting. Remarkable age-related decrease in specific activities of CYP2B, CYP3A, and UDP-glucuronosyl transferase was observed, whereas no changes in activities of CYP1A2, flavine monooxygenase, aldo-keto reductase 1C, and antioxidant enzymes with advancing age were found. On the other hand, specific activity of CBR1 and GST was 2.4 folds and 5.6 folds higher in the senescent rats compared with the young ones, respectively. Interindividual variability in CBR1 activity increased significantly with rising age. We suppose that elevated activities of GST and CBR1 may protect senescent rats against xenobiotic as well as eobiotic electrophiles and reactive carbonyls, but they may alter metabolism of drugs, which are CBR1 and especially GSTs substrates. PMID:23971034

  20. Optimisation of halogenase enzyme activity by application of a genetic algorithm.

    PubMed

    Muffler, Kai; Retzlaff, Marco; van Pée, Karl-Heinz; Ulber, Roland

    2007-01-10

    A genetic algorithm (GA) was applied for the optimisation of an enzyme assay composition respectively the enzyme activity of a recombinantly produced FADH(2)-dependent halogenating enzyme. The examined enzyme belongs to the class of halogenases and is capable to halogenate tryptophan regioselective in position 5. Therefore, the expressed trp-5-halogenase can be an interesting tool in the manufacturing of serotonin precursors. The application of stochastic search strategies (e.g. GAs) is well suited for fast determination of the global optimum in multidimensional search spaces, where statistical approaches or even the popular classical one-factor-at-a-time method often failures by misleading to local optima. The concentrations of six different medium components were optimised and the maximum yield of the halogenated tryptophan could be increased from 3.5 up to 65%.

  1. A Thiamine-Dependent Enzyme Utilizes an Active Tetrahedral Intermediate in Vitamin K Biosynthesis.

    PubMed

    Song, Haigang; Dong, Chen; Qin, Mingming; Chen, Yaozong; Sun, Yueru; Liu, Jingjing; Chan, Wan; Guo, Zhihong

    2016-06-15

    Enamine is a well-known reactive intermediate mediating essential thiamine-dependent catalysis in central metabolic pathways. However, this intermediate is not found in the thiamine-dependent catalysis of the vitamin K biosynthetic enzyme MenD. Instead, an active tetrahedral post-decarboxylation intermediate is stably formed in the enzyme and was structurally determined at 1.34 Å resolution in crystal. This intermediate takes a unique conformation that allows only one proton between its tetrahedral reaction center and the exo-ring nitrogen atom of the aminopyrimidine moiety in the cofactor with a short distance of 3.0 Å. It is readily convertible to the final product of the enzymic reaction with a solvent-exchangeable proton at its reaction center. These results show that the thiamine-dependent enzyme utilizes a tetrahedral intermediate in a mechanism distinct from the enamine catalytic chemistry.

  2. A Thiamine-Dependent Enzyme Utilizes an Active Tetrahedral Intermediate in Vitamin K Biosynthesis.

    PubMed

    Song, Haigang; Dong, Chen; Qin, Mingming; Chen, Yaozong; Sun, Yueru; Liu, Jingjing; Chan, Wan; Guo, Zhihong

    2016-06-15

    Enamine is a well-known reactive intermediate mediating essential thiamine-dependent catalysis in central metabolic pathways. However, this intermediate is not found in the thiamine-dependent catalysis of the vitamin K biosynthetic enzyme MenD. Instead, an active tetrahedral post-decarboxylation intermediate is stably formed in the enzyme and was structurally determined at 1.34 Å resolution in crystal. This intermediate takes a unique conformation that allows only one proton between its tetrahedral reaction center and the exo-ring nitrogen atom of the aminopyrimidine moiety in the cofactor with a short distance of 3.0 Å. It is readily convertible to the final product of the enzymic reaction with a solvent-exchangeable proton at its reaction center. These results show that the thiamine-dependent enzyme utilizes a tetrahedral intermediate in a mechanism distinct from the enamine catalytic chemistry. PMID:27213829

  3. Epigenetic drugs that do not target enzyme activity.

    PubMed

    Owen, Dafydd R; Trzupek, John D

    2014-06-01

    While the installation and removal of epigenetic post-translational modifications or ‘marks’ on both DNA and histone proteins are the tangible outcome of enzymatically catalyzed processes, the role of the epigenetic reader proteins looks, at first, less obvious. As they do not catalyze a chemical transformation or process as such, their role is not enzymatic. However, this does not preclude them from being potential targets for drug discovery as their function is clearly correlated to transcriptional activity and as a class of proteins, they appear to have binding sites of sufficient definition and size to be inhibited by small molecules. This suggests that this third class of epigenetic proteins that are involved in the interpretation of post-translational marks (as opposed to the creation or deletion of marks) may represent attractive targets for drug discovery efforts. This review mainly summarizes selected publications, patent literature and company disclosures on these non-enzymatic epigenetic reader proteins from 2009 to the present.

  4. [Effects of nitrogen addition on soil physico-chemical properties and enzyme activities in desertified steppe].

    PubMed

    Su, Jie-Qiong; Li, Xin-Rong; Bao, Jing-Ting

    2014-03-01

    To investigate the impacts of nitrogen (N) enrichment on soil physico-chemical property and soil enzyme activities in desert ecosystems, a field experiment by adding N at 0, 1.75, 3.5, 7, or 14 g N x m(-2) a(-1) was conducted in a temperate desert steppe in the southeastern fringe of the Tengger Desert. The results showed that N addition led to accumulations of total N, NO(3-)-N, NH(4+)-N, and available N in the upper soil (0-10 cm) and subsoil (10-20 cm), however, reductions in soil pH were observed, causing soil acidification to some extent. N addition pronouncedly inhibited soil enzyme activities, which were different among N addition levels, soil depths, and years, respectively. Soil enzyme activities were significantly correlated with the soil N level, soil pH, and soil moisture content, respectively.

  5. Arsenic fractions and enzyme activities in arsenic-contaminated soils by groundwater irrigation in West Bengal.

    PubMed

    Bhattacharyya, Pradip; Tripathy, Subhasish; Kim, Kangjoo; Kim, Seok-Hwi

    2008-09-01

    A study for the effect of arsenic (As) contamination on beta-glucosidase, urease, acid-/alkaline-phosphatase, and arylsulphatase activities was conducted in As contaminated soils of West Bengal, India. The studied soils show low total As contents relative to those in other As-contaminated sites. A sequential extraction technique was used to quantify water soluble, exchangeable, carbonate bound, Fe/Mn oxide bound, organic bound, and residual As fractions. Arsenic concentrations in the two most labile fractions (i.e., water soluble and exchangeable fractions) were generally low. The studied enzymes were significantly and negatively correlated with water soluble and exchangeable As forms but did not show any significant correlations with other forms, indicating that water soluble and exchangeable forms exert a strong inhibitory effect on the soil enzyme activities. It is thus suggested that the enzyme activity can be helpful in assessing the effects of As on biochemical quality of soils.

  6. NanoCluster Beacons as Reporter Probes in Rolling Circle Enhanced Enzyme Activity Detection†

    PubMed Central

    Juul, Sissel; Obliosca, Judy M.; Liu, Cong; Liu, Yen-Liang; Chen, Yu-An; Imphean, Darren M.; Knudsen, Birgitta R.; Ho, Yi-Ping; Leong, Kam W.; Yeh, Hsin-Chih

    2015-01-01

    As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp.. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics. PMID:25901841

  7. Rabbit pulmonary angiotensin-converting enzyme: the NH2-terminal fragment with enzymatic activity and its formation from the native enzyme by NH4OH treatment.

    PubMed

    Iwata, K; Blacher, R; Soffer, R L; Lai, C Y

    1983-11-01

    The NH2-terminal sequence of 22 residues of rabbit lung angiotensin-converting enzyme has been determined as (NH2)Thr-Leu-Asp-Pro-Gly-Leu-Leu-Pro-Gly-Asp-Phe-Ala -Ala-Asp-Asn-Ala-Gly-Ala-Arg-Leu-Phe-Ala-. In the course of purification of the enzyme for structural analysis a protein of Mr = 82,000 with angiotensin-converting activity was separated from the major fraction containing the native enzyme (Mr = 140,000). This low-molecular-weight enzyme catalyzed the hydrolysis of the synthetic substrate Hip-His-Leu at a rate 23% of that with the native enzyme, and exhibited a similar Km value as well as behaviors towards various effectors of angiotensin-converting enzyme. Edman degradation of both the native and the 82K enzymes revealed that they contain identical amino acid sequences from the NH2-termini. This result and those of peptide mapping and carbohydrate and amino acid analyses indicate that the 82K enzyme is a fragment derived from the NH2-terminal portion of the native enzyme, and hence contains its catalytic site. Evidence has been obtained indicating that the active fragment was formed from the native enzyme during its elution from the antibody-affinity column with NH4OH: on treatment of the native enzyme (140K Mr) with 1 N NH4OH at room temperature, a cleavage occurred and two proteins with Mr = 82K and Mr = 62K were obtained. The 82K Mr fragment was found to be enzymatically active and to contain the same NH2-terminal sequence as the native enzyme. The other fragment (62K Mr) was devoid of the activity and was shown to derive from the COOH-terminal portion of the native enzyme by the peptide mapping and terminal analyses. Cleavage of a peptide bond with NH4OH is unusual and appears to be specific for the native angiotensin-converting enzyme from rabbit lung.

  8. The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat

    SciTech Connect

    Abdulla, Dalya; Goralski, Kerry B.; Renton, Kenneth W. . E-mail: Ken.Renton@dal.ca

    2006-10-01

    It is well known that inflammatory and infectious conditions differentially regulate cytochrome P450 (P450)-mediated drug metabolism in the liver. We have previously outlined a potential pathway for the downregulation in hepatic cytochrome P450 following LPS-mediated inflammation in the CNS (Abdulla, D., Goralski, K.B., Garcia Del Busto Cano, E., Renton, K.W., 2005. The signal transduction pathways involved in hepatic cytochrome P450 regulation in the rat during an LPS-induced model of CNS inflammation. Drug Metab. Dispos). The purpose of this study was to outline the effects of LPS-induced peripheral and central nervous system inflammation on hepatic cytochrome P450 2E1 (CYP2E1) in vivo, an enzyme that plays an important role in various physiological and pathological states. We report an increase in hepatic mRNA expression of CYP2E1 that occurred as early as 2-3 h following either the intraperitoneal (i.p.) injection of 5 mg/kg LPS or i.c.v. administration of 25 {mu}g of LPS. This increase in CYP2E1 mRNA expression was sustained for 24 h. In sharp contrast to the increase in hepatic CYP2E1 mRNA, we observed a significant reduction in the catalytic activity of this enzyme 24 h following either the i.c.v. or i.p. administration of LPS. Cycloheximide or actinomycin-D did not change the LPS-mediated downregulation in hepatic CYP2E1 catalytic activity. Our results support the idea that LPS acts at two different levels to regulate hepatic CYP2E1: a transcriptional level to increase CYP2E1 mRNA expression and a post-transcriptional level to regulate CYP2E1 protein and activity.

  9. Effect of environmental pH on enzyme activity and growth of Bacteroides gingivalis W50.

    PubMed Central

    McDermid, A S; McKee, A S; Marsh, P D

    1988-01-01

    Since the pH of the gingival crevice increases from below neutrality in health to above pH 8 in disease, we decided to investigate the effect of environmental pH on the growth and enzyme activity of Bacteroides gingivalis W50. Cells were grown in a chemostat under hemin-excess conditions over a range of pH values; stable growth was observed only between pH 6.7 and 8.3, with the maximum yields obtained between pH 7.0 and 8.0. The enzyme profile of cells varied markedly with pH. Enzymes with a specificity for gingival connective tissue (collagenase, hyaluronidase) were produced optimally at or below neutral pH, whereas trypsinlike activity increased with the growth pH and was maximal at pH 8.0. Chymotrypsinlike activity was generally low, although its activity was highest at the extremes of growth pH, i.e., at pH 6.7 and 8.3. Inhibitor studies provided evidence that the breakdown of collagen involved the concerted action of both a collagenase and the trypsinlike enzyme. The ratio of trypsin to collagenolytic activity rose from 1:1 during growth at neutral pH and below to almost 7:1 during growth at pH 8.3. Thus B. gingivalis appears to be uniquely adapted as a periodontopathic organism in that under environmental conditions likely to prevail during the initial stages of pocket development it produces maximally those enzymes with a tissue-damaging potential. Then, as the pH of the pocket rises during the host inflammatory response, the activity of the trypsinlike enzyme increases markedly, which may enable cells to inactivate key components of the host defenses such as immunoglobulins and complement. PMID:3281900

  10. 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.

  11. 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

  12. Structural comparison of cytochromes P450 2A6, 2A13, and 2E1 with pilocarpine

    SciTech Connect

    DeVore, Natasha M.; Meneely, Kathleen M.; Bart, Aaron G.; Stephens, Eva S.; Battaile, Kevin P.; Scott, Emily E.

    2013-11-20

    Human xenobiotic-metabolizing cytochrome P450 (CYP) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally, a single ligand can interact with multiple CYP enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the CYP superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human CYP enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drug pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key residues involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 {angstrom}), CYP2A13 (3.0 {angstrom}), CYP2E1 (2.35 {angstrom}), and the CYP2A6 mutant enzyme, CYP2A6 I208S/I300F/G301A/S369G (2.1 {angstrom}) have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual residues lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine.

  13. Uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by preventing the activation of PI3K/Akt in MC3T3-E1 cells.

    PubMed

    Liu, Jingli; Yang, Jianhong

    2016-01-01

    Uncarboxylated osteocalcin, an osteoblast-derived protein, plays an important role in the regulation of glucose metabolism. It has previously been demonstrated that high glucose levels inhibit osteoblast proliferation and differentiation. However, the mechanisms through which uncarboxylated osteocalcin regulates osteoblast proliferation and differentiation under high glucose conditions remain unclear. Thus, in the present study, we aimed to examine the effects of uncarboxylated osteocalcin on the proliferation and differentiation of MC3T3-E1 cells under high glucose conditions. We demonstrated that high glucose levels induced the production of reactive oxygen species (ROS) in MC3T3-E1 cells, and this production was inhibited by treatment with uncarboxylated osteocalcin and N-acetyl-L-cysteine (NAC), a ROS scavenger. In addition, we found that uncarboxylated osteocalcin reduced high glucose‑induced oxidative stress and increased the mRNA expression of the osteogenic markers, runt-related transcription factor 2 (Runx2), osterix and osteocalcin, as well as the formation of mineralized nodules; it also inhibited adipogenic differentiation, as shown by a decrease in the mRNA expression of the adipogenic markers, peroxisome proliferator‑activated receptor γ (PPARγ), adipocyte fatty acid-binding protein (adipocyte protein 2; aP2) and fatty acid synthase (FAS), and reduced lipid drop accumulation. Furthermore, we found that uncarboxylated osteocalcin inhibited PI3K/Akt signaling which was induced by ROS and facilitated the osteogenic differentiation of MC3T3-E1 cells under high glucose conditions. Taken together and to the best of ou knowledge, our results demonstrate for the first time that uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by inhibiting the activation of PI3K/Akt in